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Clinical Chemistry in Diagnosis and Treatment by Joan Zilva and Peter Pannall PDF
Clinical Chemistry in Diagnosis and Treatment by Joan Zilva and Peter Pannall PDF
CLINICAL BIOCHEMISTRY
& METABOLIC MEDICINE
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Contents
Preface vii
3 The kidneys 36
4 Acid–base disturbances 59
5 Potassium 83
14 Nutrition 216
Index 403
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Preface
Were it not for the textbook Clinical Chemistry in Diagnosis and Treatment by Joan Zilva and Peter Pannall, I would
not be a chemical pathologist. As a medical student, I was so struck by its clarity, depth and clinical relevance that
I decided that theirs was the medical field I wished to work in.
Over the years, the field of clinical biochemistry has changed radically. Confusingly, there is no consensus
on the name for this field of medicine, which is known variously as clinical chemistry, chemical pathology or
clinical biochemistry, to name but a few. Additionally, the field now overlaps with that of metabolic medicine, a
clinical specialty involved with the management and treatment of patients with disorders of metabolism. Clinical
biochemistry laboratories have become further automated, molecular biology technologies have entered the
diagnostic arena, and chemical pathologists have become more clinically orientated towards running out-patient
clinics for a variety of biochemical disturbances. This book aims to address these new changes. Indeed, it is difficult
to imagine a branch of medicine that does not at some time require clinical biochemistry tests, which may not be
too surprising, given the fact that every body cell is composed of chemicals!
Unfortunately, there have been some difficulties in recent times, with a relative shortage of graduates entering
the specialty, which has not been helped by some people’s attitude that clinical biochemistry is merely a laboratory
factory churning out results that anyone can interpret. There are also concerns that medical student clinical
biochemistry teaching may become ‘diluted’ as part of an expanding curriculum. It is hoped that this book will
excite a new generation to enter this fascinating and essential field, as well as benefit patients as their doctors learn
more about their biochemical and metabolic problems.
I am most grateful to Dr Sethsiri Wijeratne, Dr Alam Garrib (particularly for molecular biology expertise) and
Dr Paul Eldridge for constructive criticism of the text. I am also grateful to Professor Philip Mayne for his earlier
contributions and the anonymous medical student reviewer(s) who commented on the text. The book has also
greatly benefited from the wise, helpful and experienced input of Dr Andrew Day – many thanks. Although every
effort has been made to avoid inaccuracies and errors, it is almost inevitable that some may still be present, and
feedback from readers is therefore welcome.
Martin Crook
London, 2012
Disclaimer The publishers and author accept no responsibility for errors in the text or misuse of the material
presented. Drugs and their doses should be checked with a pharmacy, and the investigation protocols with an
appropriate clinical laboratory. Dynamic test protocols should be checked with an accredited clinical investigation
unit and may require different instructions in the elderly, children and the obese.
List of abbreviations
REQUESTING LABORATORY TESTS rapidly in patients given large doses of diuretics and
There are many laboratory tests available to the clinician. these alterations may indicate the need to instigate or
Correctly used, these may provide useful information, change treatment (see Chapter 5).
but, if used inappropriately, they are at best useless and Laboratory investigations are very rarely needed
at worst misleading and dangerous. more than once daily, except in some patients receiving
In general, laboratory investigations are used: intensive therapy. If they are, only those that are
● to help diagnosis or, when indicated, to screen for essential should be repeated.
metabolic disease, WHEN IS A LABORATORY
● to monitor treatment or detect complications, INVESTIGATION ‘URGENT’?
● occasionally for medicolegal reasons or, with due
The main reason for asking for an investigation to be
permission from the patient, for research.
performed ‘urgently’ is that an early answer will alter
Overinvestigation of the patient may be harmful, the patient’s clinical management. This is rarely the
causing unnecessary discomfort or inconvenience, case and laboratory staff should be consulted and the
delaying treatment or using resources that might be sample ‘flagged’ as clearly urgent if the test is required
more usefully spent on other aspects of patient care. immediately. Laboratories often use large analysers
Before requesting an investigation, clinicians should capable of assaying hundreds of samples per day
consider whether its result would influence their clinical (Fig. 1.1). Point-of-care testing can shorten result
management of the patient. turnaround time and is discussed in Chapter 30.
Close liaison with laboratory staff is essential; they
may be able to help determine the best and quickest
procedure for investigation, interpret results and
discover reasons for anomalous findings.
Plasma 140
Bilirubin 14 µmol/L (< 20) 120
Alanine transaminase 14 U/L (< 42)
Number of subjects
laboratory carrying out the assay. Some analytes have the patient fasting. This is not usually possible, and
risk limits for treatment, such as plasma glucose (see these variations should be taken into account when
Chapter 12), or target or therapeutic limits, such as serial results are interpreted.
plasma cholesterol (see Chapter 13). Some constituents vary monthly, especially in
Various non-pathological factors may affect the women during the menstrual cycle. These variations
results of investigations, the following being some of can be very marked, as in the results of sex hormone
the more important ones. assays, for example plasma oestradiol, which can only be
interpreted if the stage of the menstrual cycle is known;
Between-individual differences
plasma iron may fall to very low concentrations just
Physiological factors such as the following affect the before the onset of menstruation. Other constituents
interpretation of results. may also vary seasonally. For example, vitamin D
concentrations may be highest in the summer months.
Age-related differences
Some of these changes, such as the relation between
These include, for example, bilirubin in the neonate plasma glucose and meals, have obvious causes.
(see Chapter 26) and plasma alkaline phosphatase
activity, which is higher in children and the elderly (see Random
Chapter 18). Day-to-day variations, for example in plasma iron
concentrations, can be very large and may swamp regular
Sex-related differences
cycles. The causes of these are not clear, but they should be
Examples of sex-related differences include plasma allowed for when serial results are interpreted – for example
urate, which is higher in males, and high-density the effect of ‘stress’ on plasma cortisol concentrations.
lipoprotein cholesterol, which is higher in pre- The time of meals affects plasma glucose
menopausal women than in men (see Chapters 13 and concentrations, and therefore correct interpretation
20). Obviously, sex hormone concentrations also differ is often only possible if the blood is taken when the
between the sexes (see Chapter 9). patient is fasting or at a set time after a standard dose of
glucose (see Chapter 12).
Ethnic differences
These may occur because of either racial or environmental Methodological differences between
laboratories
factors, for example plasma creatine kinase may be higher
in black than in white people (see Chapter 18). It has been pointed out that, even if the same method
is used throughout a particular laboratory, it is
Within-individual variations difficult to define normality clearly. Interpretation
There are biological variations of both plasma may sometimes be even more difficult if the results
concentrations and urinary excretion rates of many obtained in different laboratories, using different
constituents, and test results may be incorrectly analytical methods, are compared. Agreement between
interpreted if this is not taken into consideration. laboratories is close for many constituents partly due
Biological variations may be regular or random. to improved standardization procedures and because
many laboratories belong to external quality control
Regular schemes. However, for others, such as plasma enzymes,
Such changes occur throughout the 24-h period different methods may give different results. For various
(circadian or diurnal rhythms, like those of body technical reasons, the results would still vary unless the
temperature) or throughout the month. The daily substrate, pH and all the other variables were the same.
(circadian) variation of plasma cortisol is of diagnostic
value, but, superimposed on this regular variation, IS THE ABNORMALITY OF DIAGNOSTIC
‘stress’ will cause an acute rise (see Chapter 8). Plasma VALUE?
iron concentrations may fall by 50 per cent between Relation between plasma and cellular
morning and evening (see Chapter 21). To eliminate concentrations
the unwanted effect of circadian variations, blood Intracellular constituents are not easily sampled,
should ideally always be taken at the same time of day, and plasma concentrations do not always reflect the
preferably in the early morning and, if indicated, with situation in the cells; this is particularly true for those
4 Requesting laboratory tests and interpreting the results
Sensitivity
a test being negative when a certain disease is absent,
that is, the percentage of true-negative (TN) results.
Ideally, a test would have 100 per cent specificity and
100 per cent sensitivity.
The usefulness of tests can be expressed visually as
receiver operating characteristic (ROC) curves (Fig. 1.3).
Unfortunately, in population screening, some 0
subjects with a disorder may have a negative test (false- 0 1
negative, FN); conversely, some subjects without the 1 – Specificity
condition in question will show an abnormal or positive Figure 1.3 Receiver operating characteristic (ROC)
result (false-positive, FP). curve. The greater the area under the curve, the more
The predictive value of a negative result is the percentage useful the diagnostic test. Test B is less useful than
of all negative results that are true negatives, that is, the test A, which has greater sensitivity and specificity. C
frequency of subjects without the disorder in all subjects depicts chance performance (area under the curve 0.5).
with negative test results. A high negative predictive
value is important in screening programmes if affected specificity decline. Conversely, if a diagnostic test has its
individuals are not to be missed. This can be expressed as: cut-off or action limit set too high, fewer falsely positive
TN individuals will be encompassed, but more individuals
¥ 100% (1.2)
TN + FN will be falsely defined as negative, that is, its sensitivity
The predictive value of a positive result is the will decrease and its specificity will increase.
percentage of all positive results that are true positives: Likelihood ratios of laboratory tests
in other words, the proportion of screening tests that
Some may find predictive values confusing, and the
are correct. A high positive predictive value is important
likelihood ratio (LR) may be preferable. This can be
to minimize the number of false-positive individuals
defined as the statistical odds of a factor occurring
being treated unnecessarily. This can be expressed as:
in one individual with a disorder compared with it
TP occurring in an individual without that disorder.
¥ 100% (1.3)
TP + FP The LR for a negative test is expressed as:
The overall efficiency of a test is the percentage of
1 – sensitivity
patients correctly classified by the test. This should be (1.5)
specificity
as high as possible and can be expressed as:
The LR for a positive test is expressed as:
TP + TN
¥ 100% (1.4) sensitivity (1.6)
TP + FP + TN + FN
If the cut-off, or action, limit of a diagnostic test 1 – specificity
is set too low, more falsely positive individuals will The greater the LR, the more clinically useful is the
be included, and its sensitivity will increase and its test in question.
SUMMARY
● Careful thought is required when it comes to ● The laboratory reference range should be consulted
requesting and interpreting clinical biochemistry when interpreting biochemical results, and results
tests. should be interpreted in the light of the clinical findings.
● Communication with the laboratory is essential to ● Just because a result is ‘abnormal’ does not mean
ensure optimal interpretation of results and patient that the patient has an illness; conversely, a ‘normal’
management. result does not exclude a disease process.
2 Water and sodium
It is essential to understand the linked homeostatic mainly in the ECF (Table 2.2). Water and electrolyte
mechanisms controlling water and sodium balance intake usually balance output in urine, faeces, sweat
when interpreting the plasma sodium concentration and expired air.
and managing the clinically common disturbances of
Water and sodium intake
water and sodium balance. This is of major importance
in deciding on the composition and amount, if any, of The daily water and sodium intakes are variable, but in
intravenous fluid to give. It must also be remembered an adult amount to about 1.5–2 L and 60–150 mmol,
that plasma results may be affected by such intravenous respectively.
therapy, and can be dangerously misunderstood. Water and sodium output
Water is an essential body constituent, and Kidneys and gastrointestinal tract
homeostatic processes are important to ensure that
the total water balance is maintained within narrow The kidneys and intestine deal with water and electrolytes
limits, and the distribution of water among the in a similar way. Net loss through both organs depends
vascular, interstitial and intracellular compartments is on the balance between the volume filtered proximally
maintained. This depends on hydrostatic and osmotic Table 2.1 Approximate contributions of solutes to
forces acting across cell membranes. plasma osmolality
Sodium is the most abundant extracellular cation
and, with its associated anions, accounts for most of Osmolality (mmol/kg) Total (%)
the osmotic activity of the extracellular fluid (ECF); it Sodium and its anions 270 92
is important in determining water distribution across Potassium and its anions 7
cell membranes. Calcium (ionized) and its anions 3
Osmotic activity depends on concentration, and
Magnesium and its anions 1
therefore on the relative amounts of sodium and water 8
Urea 5
in the ECF compartment, rather than on the absolute
quantity of either constituent. An imbalance may cause Glucose 5
hyponatraemia (low plasma sodium concentration) or Protein 1 (approx.)
hypernatraemia (high plasma sodium concentration), Total 292 (approx.)
and therefore changes in osmolality. If water and sodium
are lost or gained in equivalent amounts, the plasma Table 2.2 The approximate volumes in different body
sodium, and therefore the osmolal concentration, is compartments through which water is distributed in a
unchanged; symptoms are then due to extracellular 70-kg adult
volume depletion or overloading (Table 2.1). As the
metabolism of sodium is so inextricably related to that Volume (L)
of water, the two are discussed together in this chapter. Intracellular fluid compartment 24
Extracellular fluid compartment 18
TOTAL SODIUM AND WATER BALANCE
Interstitial (13)
In a 70-kg man, the total body water (TBW) is about
Intravascular (blood volume) (5)
42 L and contributes about 60 per cent of the total body
weight; there are approximately 3000 mmol of sodium, Total body water 42
Control of water and sodium balance 7
and that reabsorbed more distally. Any factor affecting Control of antidiuretic hormone secretion
either passive filtration or epithelial cellular function The secretion of ADH is stimulated by the flow of
may disturb this balance. water out of cerebral cells caused by a relatively high
Approximately 200 L of water and 30 000 mmol of extracellular osmolality. If intracellular osmolality
sodium are filtered by the kidneys each day; a further is unchanged, an extracellular increase of only
10 L of water and 1500 mmol of sodium enter the 2 per cent quadruples ADH output; an equivalent
intestinal lumen. The whole of the extracellular water fall almost completely inhibits it. This represents a
and sodium could be lost by passive filtration in little change in plasma sodium concentration of only about
more than an hour, but under normal circumstances 3 mmol/L. In more chronic changes, when the osmotic
about 99 per cent is reabsorbed. Consequently, the gradient has been minimized by solute redistribution,
net daily losses amount to about 1.5–2 L of water and there may be little or no effect. In addition, stretch
100 mmol of sodium in the urine, and 100 mL and receptors in the left atrium and baroreceptors in the
15 mmol, respectively, in the faeces. aortic arch and carotid sinus influence ADH secretion
Fine adjustment of the relative amounts of water in response to the low intravascular pressure of severe
and sodium excretion occurs in the distal nephron and hypovolaemia, stimulating ADH release. The stress
the large intestine, often under hormonal control. The due to, for example, nausea, vomiting and pain may
effects of antidiuretic hormone (ADH) or vasopressin also increase ADH secretion. Inhibition of ADH
and the mineralocorticoid hormone aldosterone on secretion occurs if the extracellular osmolality falls,
the kidney are the most important physiologically, for whatever reason.
although natriuretic peptides are also important.
Actions of antidiuretic hormone
Sweat and expired air Antidiuretic hormone, by regulating aquaporin
About 1 L of water is lost daily in sweat and expired 2, enhances water reabsorption in excess of solute
air, and less than 30 mmol of sodium a day is lost in from the collecting ducts of the kidney and so
sweat. The volume of sweat is primarily controlled dilutes the extracellular osmolality. Aquaporins are
by skin temperature, although ADH and aldosterone cell membrane proteins acting as water channels
have some effect on its composition. Water loss in that regulate water flow. When ADH secretion is a
expired air depends on the respiratory rate. Normally, response to a high extracellular osmolality with the
losses in sweat and expired air are rapidly corrected by danger of cell dehydration, this is an appropriate
changes in renal and intestinal loss. However, neither response. However, if its secretion is in response to
of these losses can be controlled to meet sodium and a low circulating volume alone, it is inappropriate to
water requirements, and thus they may contribute the osmolality. The retained water is then distributed
considerably to abnormal balance when homeostatic throughout the TBW space, entering cells along the
mechanisms fail. osmotic gradient; the correction of extracellular
depletion with water alone is thus relatively inefficient
CONTROL OF WATER AND SODIUM
in correcting hypovolaemia. Plasma osmolality
BALANCE
normally varies by less than 1–2 per cent, despite
Control of water balance
great variation in water intake, which is largely due to
Both the intake and loss of water are controlled by the action of ADH.
osmotic gradients across cell membranes in the brain’s In some circumstances, the action of ADH is
hypothalamic osmoreceptor centres. These centres, opposed by other factors. For example, during an
which are closely related anatomically, control thirst osmotic diuresis the urine, although not hypo-osmolal,
and the secretion of ADH. contains more water than sodium. Patients with severe
hyperglycaemia, as in poorly controlled diabetes
Antidiuretic hormone (arginine vasopressin)
mellitus, may show an osmotic diuresis.
Antidiuretic hormone is a polypeptide with a half-life
of about 20 min that is synthesized in the supraoptic Control of sodium balance
and paraventricular nuclei of the hypothalamus and, The major factors controlling sodium balance are renal
after transport down the pituitary stalk, is secreted blood flow and aldosterone. This hormone controls
from the posterior pituitary gland (see Chapter 7). loss of sodium from the distal tubule and colon.
8 Water and sodium
intake and output; this is particularly pertinent for may be affected by pre-existing abnormalities of
unconscious patients. ‘Insensible loss’ is usually protein or red cell concentrations.
assumed to be about 1 L/day, but there is endogenous ● Haemoconcentration ECF is usually lost from
water production of about 500 mL/day as a result the vascular compartment first and, unless the
of metabolic processes. Therefore the net daily fluid is whole blood, depletion of water and small
‘insensible loss’ is about 500 mL. The required daily molecules results in a rise in the concentration of
intake may be calculated from the output during the large molecules, such as proteins and blood cells,
previous day plus 500 mL to allow for ‘insensible with a rise in blood haemoglobin concentration and
loss’; this method is satisfactory if the patient is haematocrit, raised plasma urea concentration and
normally hydrated before day-to-day monitoring reduced urine sodium concentration.
is started. Serial patient body weight determination
Table 2.4 shows various intravenous fluid regimens
can also be useful in the assessment of changes in
that can be used clinically. A summary of the British
fluid balance.
Consensus Guidelines on Intravenous Fluid Therapy
Pyrexial patients may lose 1 L or more of fluid in sweat
for Adult Surgical Patients (GIFTASUP) can be found
and, if they are also hyperventilating, respiratory water
at www.bapen.org.uk/pdfs/bapen_pubs/giftasup.pdf.
loss may be considerable. In such cases an allowance
of about 500 mL for ‘insensible loss’ may be totally DISTRIBUTION OF WATER AND SODIUM
inadequate. In addition, patients may be incontinent IN THE BODY
of urine, and having abnormal gastrointestinal losses In mild disturbances of the balance of water and
makes the accurate assessment of fluid losses very electrolytes, their total amounts in the body may be of
difficult. less importance than their distribution between body
Inaccurate measurement and charting are useless and compartments (see Table 2.2).
may be dangerous. Water is distributed between the main body
Keeping a cumulative fluid balance record is a useful fluid compartments, in which different electrolytes
way of detecting a trend, which may then be corrected contribute to the osmolality. These compartments are:
before serious abnormalities develop.
In the example shown in Table 2.3, 500 mL has been ● intracellular, in which potassium is the predominant
allowed for as net ‘insensible daily loss’; calculated losses cation,
are therefore more likely to be underestimated than ● extracellular, in which sodium is the predominant
overestimated. This shows how insidiously a serious cation, and which can be subdivided into:
deficit can develop over a few days. – interstitial space, with very low protein
The volume of fluid infused should be based on the concentration, and
calculated cumulative balance and on clinical evidence – intravascular (plasma) space, with a relatively
of the state of hydration, and its composition adjusted high protein concentration.
to maintain normal plasma electrolyte concentrations. Electrolyte distribution between cells and
Assessment of the state of hydration of a patient relies interstitial fluid
on clinical examination and on laboratory evidence of Sodium is the predominant extracellular cation, its
haemodilution or haemoconcentration. intracellular concentration being less than one-tenth
● Haemodilution Increasing plasma volume with of that within the ECF. The intracellular potassium
protein-free fluid leads to a fall in the concentrations concentration is about 30 times that of the ECF. About
of proteins and haemoglobin. However, these findings 95 per cent of the osmotically active sodium is outside
Table 2.3 Hypothetical cumulative fluid balance chart assuming an insensible daily loss of 500 mL
Measured intake (mL) Measured output (mL) Total output (minimum mL) Daily balance (mL) Cumulative balance (mL)
Day 1 2000 1900 2400 –400 –400
Day 2 2000 2000 2500 –500 –900
Day 3 2100 1900 2400 –300 –1200
Day 4 2200 2000 2500 –300 –1500
10 Water and sodium
greater than the volume of solvent only (water) in contributes much more than 6 per cent to the measured
which the small molecules are dissolved. At a protein plasma volume, the calculated osmolarity may be
concentration of 70 g/L, the volume of water is about significantly lower than the true osmolality in the plasma
6 per cent less than the total volume of the solution (that water. A hypothetical example is shown in Figure 2.2.
is, the molarity should theoretically be about 6 per cent Many formulae of varying complexity have been
less than the molality). Most methods for measuring proposed to calculate plasma osmolarity. None of them
individual ions assess them in molarity (mmol/L). If can predict the osmotic effect, but the following formula
the concentration of proteins in plasma is markedly (in which square brackets indicate concentration) gives
increased, the volume of solvent is significantly reduced a close approximation to plasma osmolality (although
but the volume of solution remains unchanged. some equations omit the potassium, which may be
Therefore the molarity (in mmol/L) of certain ions preferable):
such as sodium will be reduced but the molality will be
Plasma osmolality = 2[Na+] + 2[K+] + [urea]
unaltered. This apparently low sodium concentration is + [glucose] in mmol/L (2.1)
known as pseudohyponatraemia.
The factor of 2, which is applied to the sodium and
Measured plasma osmolality
potassium concentrations, allows for the associated
Osmometers measure changes in the properties of a anions and assumes complete ionization. This
solution, such as freezing point depression or vapour calculation is not valid if gross hyperproteinaemia
pressure, which depend on the total osmolality or hyperlipidaemia is present or an unmeasured
of the solution – the osmotic effect that would be osmotically active solute, such as mannitol, methanol,
exerted by the sum of all the dissolved molecules and ethanol or ethylene glycol, is circulating in plasma.
ions across a membrane permeable only to water. A significant difference between measured
These properties are known as colligative properties. and calculated osmolality in the absence of
Sodium and its associated anions (mainly chloride) hyperproteinaemia or hyperlipidaemia may suggest
contribute 90 per cent or more to this measured plasma alcohol or other poisoning. For example, a plasma
osmolality, the effect of protein being negligible. As the alcohol concentration of 100 mg/dL contributes about
only major difference in composition between plasma 20 mmol/kg to the osmolality. This osmotic difference is
and interstitial fluid is in protein content, the plasma known as the osmolar gap and can be used to assess the
osmolality is almost identical to the osmolality of the presence in plasma of unmeasured osmotically active
interstitial fluid surrounding cells. particles. In such cases the plasma sodium concentration
Calculated plasma osmolarity may be misleading as a measure of the osmotic effect. It
It is the osmolam, rather than the osmolar, concentration is not possible to calculate urinary osmolarity because
that exerts an effect across cell membranes and that of the considerable variation in the concentrations of
is controlled by homeostatic mechanisms. However, different, sometimes unmeasured, solutes; the osmotic
as discussed below, the calculated plasma osmolarity pressure of urine can be determined only by measuring
is usually as informative as the measured plasma the osmolality.
osmolality.
Although, because of the space-occupying effect of Distribution of water across cell membranes
protein, the measured osmolality of plasma should be Osmotic pressure gradient
higher than the osmolarity, calculated from the sum of Because the hydrostatic pressure difference across the
the molar concentrations of all the ions, there is usually cell membrane is negligible, cell hydration depends on
little difference between the two figures. This is because the effective osmotic difference between intracellular
there is incomplete ionization of, for example, NaCl to and extracellular fluids. The cell membranes are freely
Na+ and Cl–; this reduces the osmotic effect by almost permeable to water and to some solutes, but different
the same amount as the volume occupied by protein solutes diffuse (or are actively transported) across cell
raises it. membranes at different rates, although always more
Consequently, the calculated plasma osmolarity is a slowly than water. In a stable state, the total intracellular
valid approximation to the true measured osmolality. osmolality, due mostly to potassium and associated
However, if there is gross hyperproteinaemia or anions, equals that of the interstitial fluid, due mostly
hyperlipidaemia such that either protein or lipid to sodium and associated anions; consequently, there is
12 Water and sodium
Plasma [Na+]
144 mmol/kg H2O
Raised protein
Normal protein 0.12 L or lipid
concentration concentration
0.06 L
No lipid
Figure 2.2 The consequence of gross hyperproteinaemia or hyperlipidaemia on the plasma water volume and its
effect on the calculated plasma osmolarity and the true plasma osmolality.
no net movement of water into or out of cells. In some increased osmotic gradient alters cell hydration, but
pathological states, rapid changes of extracellular solute in chronic uraemia, although the measured plasma
concentration affect cell hydration; slower changes may osmolality is often increased, the osmotic effect of urea
allow time for the redistribution of solute and have is reduced as the concentrations gradually equalize on
little or no effect. the two sides of the membrane.
Sodium In normal subjects sodium and its associated Glucose Like urea, the normally low extracellular
anions account for at least 90 per cent of extracellular concentration of glucose does not contribute significantly
osmolality. Rapid changes in their concentration therefore to the osmolality. However, unlike urea, glucose is actively
affect cellular hydration. If there is no significant change transported into many cells, but once there it is rapidly
in the other solutes, a rise causes cellular dehydration metabolized, even at high extracellular concentrations,
and a fall causes cellular overhydration. and the intracellular concentration remains low. Severe
Urea Normal extracellular concentrations are so hyperglycaemia, whether acute or chronic, causes a
low as to contribute very little to the measured plasma marked osmotic effect across cell membranes, with
osmolality. However, concentrations 15-fold or more movement of water from cells into the extracellular
above normal can occur in severe uraemia and can compartment causing cellular dehydration.
then make a significant contribution (see Chapter 3). Although hyperglycaemia and acute uraemia can
However, urea does diffuse into cells very much more cause cellular dehydration, the contribution of normal
slowly than water. Consequently, in acute uraemia, the urea and glucose concentrations to plasma osmolality
Urinary sodium estimation 13
is so small that reduced levels of these solutes, unlike is the most important protein contributing to the
those of sodium, do not cause cellular overhydration. colloid osmotic pressure. It is present intravascularly
Solutes such as potassium, calcium and magnesium at significant concentration but extravascularly only at
are present in the ECF at very low concentrations. a very low concentration because it cannot pass freely
Significant changes in these are lethal at much lower across the capillary wall.
concentrations than those that would change osmolality. The osmotic gradient across vascular walls cannot be
Mannitol is an example of an exogenous substance estimated by simple means. The total plasma osmolality
that remains in the extracellular compartment because it gives no information about this. Moreover, the plasma
is not transported into cells, and may be infused to reduce albumin concentration is a poor guide to the colloid
cerebral oedema. Ethanol is only slowly metabolized, osmotic pressure. Although other proteins, such as
and a high concentration in the ECF may lead to cerebral globulins, are present in the plasma at about the same
cellular dehydration; this may account for some of the concentration as albumin, their estimation for this
symptoms of a hangover. High glucose concentrations purpose is even less useful: their higher molecular weights
account for the polyuria of severe diabetes mellitus. mean that they have even less effect than albumin.
Large rises in the osmotic gradient across cell
membranes may result in the movement of enough Relation between sodium and water
homeostasis
water from the intracellular compartment to dilute
extracellular constituents. Consequently, if the change In normal subjects, the concentrations of sodium and
in osmolality has not been caused by sodium and its its associated anions are the most important osmotic
associated anions, a fall in plasma sodium concentration factors affecting ADH secretion. Plasma volume, by its
is appropriate to the state of osmolality. If, under such effect on renal blood flow, controls aldosterone secretion
circumstances, the plasma sodium concentration is not and therefore sodium balance. The homeostatic
low, this indicates hyperosmolality. mechanisms controlling sodium and water excretion
Generally, plasma osmolarity calculated from sodium, are interdependent. (A simplified scheme is shown
potassium, urea and glucose concentrations is at least as in Fig. 2.4.) Thirst depends on a rise in extracellular
clinically valuable as measured plasma osmolality. It has osmolality, whether due to water depletion or sodium
the advantage that the solute responsible, and therefore excess, and also on a very large increase in the activity
its likely osmotic effect, is often identified. of the renin–angiotensin system.
A rise in extracellular osmolality reduces water
Distribution of water across capillary membranes loss by stimulating ADH release and increases intake
The maintenance of blood pressure depends on by stimulating thirst; both these actions dilute the
the retention of fluid within the intravascular extracellular osmolality. Osmotic balance (and therefore
compartment at a higher hydrostatic pressure than that cellular hydration) is rapidly corrected.
of the interstitial space. Hydrostatic pressure in capillary Assessment of sodium status
lumina tends to force fluid into the extravascular space.
As already discussed, the plasma sodium concentration
In the absence of any effective opposing force, fluid
is important because of its osmotic effect on fluid
would be lost rapidly from the vascular compartment.
distribution. Plasma sodium concentrations should be
Unlike other cell membranes, those of the capillaries are
monitored while volume is being corrected to ensure
permeable to small ions. Therefore sodium alone exerts
that the distribution of fluid between the intracellular
almost no osmotic effect and the distribution of water
and extracellular compartments is optimal. The
across capillary membranes is little affected by changes in
presence of other osmotically active solutes should be
electrolyte concentration.
taken into account.
Colloid osmotic pressure
The very small osmotic effect of plasma protein URINARY SODIUM ESTIMATION
molecules produces an effective osmotic gradient Urinary sodium excretion is not related to body content
across capillary membranes; this is known as the but to renal blood flow.
colloid osmotic, or oncotic, pressure. It is the most Estimation of the urinary sodium concentration in
important factor opposing the net outward hydrostatic a random specimen may be of value in the diagnosis of
pressure (Fig. 2.3). Albumin (molecular weight 65 kDa) the syndrome of inappropriate antidiuretic hormone
14 Water and sodium
INTRACELLULAR
COMPARTMENT
INTERSTITIAL
COMPARTMENT
INTRAVASCULAR
COMPARTMENT
Capillary membrane
ARTERIOLE VENULE
Figure 2.3 Osmotic factors that control the distribution of water between the fluid compartments of the body.
(Posterior pituitary)
ADH Plasma volume
Thirst
Renal blood flow
Hypothalamic
osmolality
Renin release
Angiotensin II
Plasma [Na+]
secretion (SIADH) and may help to differentiate renal urine [sodium] plasma [creatinine]
FENa% = ¥ ¥ 100%
circulatory insufficiency (pre-renal) from intrinsic plasma [sodium] urine [creatinine]
renal damage (see Chapter 3). (2.2)
The fractional excretion of sodium (FENa%) may
also be useful in helping to assess renal blood flow and A value of less than 1 per cent may be found in poor
can be measured using a simultaneous blood sample renal perfusion, for example pre-renal failure, and of
and spot urine sample: more than 1 per cent in intrinsic renal failure.
Disturbances of water and sodium metabolism 15
+
ADH ISOSMOTIC VOLUME
–
+
+ Thirst Renal blood flow
+ +
Hypothalamic
osmolality Renin release
+
+ Angiotensin II
+
Figure 2.5 Homeostatic correction of isosmotic volume depletion. The reduced intravascular volume impairs renal blood
flow and stimulates renin and therefore aldosterone secretion. There is selective sodium reabsorption from the distal
tubules and a low urinary sodium concentration. (Shading indicates primary change.) ADH, antidiuretic hormone.
–
ADH H2O VOLUME
+
–
Hypothalamic –
osmolality Renin release
–
– Angiotensin II
–
Figure 2.6 Infusion of hypotonic fluid as a cause of predominant sodium depletion. Increased circulating volume
with reduction in plasma osmolality inhibits aldosterone and antidiuretic hormone (ADH) secretion. (Shading
indicates primary change.)
16 Water and sodium
+
ADH H2O VOLUME
–
+
Hypothalamic
osmolality Renin release
+
Angiotensin II
–
======== BLOCK
Figure 2.7 Initial effect of aldosterone deficiency is impaired sodium retention and hypovolaemia; later, severe
hypovolaemia stimulates increased antidiuretic hormone (ADH) secretion with water retention, sometimes
causing a dilutional hyponatraemia. (Shading indicates primary change.)
the composition of the fluid lost or to that of the fluid Isosmolar volume depletion
given to replace it. The initial effects depend on the Causes of isosmolar fluid loss
composition of the fluid lost compared with that of The sodium concentrations of all small intestinal
plasma. secretions, and of urine when tubular function is grossly
● Isosmolar volume depletion results if the sodium impaired, are between 120 mmol/L and 140 mmol/L.
concentration of the fluid lost is similar to that of Clinical conditions causing approximately isosmolar
plasma; changes in plasma sodium concentration are loss are as follows:
then unusual. ● blood loss,
● Predominant sodium depletion is usually the result ● small intestinal fistulae and ileostomy,
of inappropriate treatment, as only bile, secreted in ● small intestinal obstruction and paralytic ileus, in
small volumes, has a significantly higher sodium which the fluid accumulating in the gut lumen has,
concentration than that of plasma. Hyponatraemia like urine in the bladder, been lost from the ECF,
(a low plasma sodium concentration) usually results. ● severe renal tubular damage with minimal glomerular
● Predominant water depletion results if the sodium dysfunction, for example the recovery phase of acute
concentration of the lost fluid is much less than that oliguric renal dysfunction, or polyuric chronic renal
of plasma. Hypernatraemia (an abnormally high dysfunction (see Chapter 3).
concentration of sodium) may occur, and indicates
loss of relatively more water than sodium, even if Results of isosmolar fluid loss
there is little evidence of volume depletion.
Hypovolaemia reduces renal blood flow and causes renal
The term ‘dehydration’ can be misleading. It is often circulatory insufficiency with oliguria with uraemia.
used interchangeably to describe the conditions listed Sodium and water are lost in almost equivalent
above, although the clinical and biochemical findings amounts, and the plasma sodium concentration is
are very different. The consequent confusion may lead usually normal; for this reason the patient may not
to inappropriate and possibly dangerous treatment; complain of thirst despite some volume depletion.
therefore, an attempt should be made to assess the Haemoconcentration confirms considerable loss of
approximate composition of fluid lost by identifying its fluid other than blood, although its absence does not
origin. exclude such loss.
Disturbances of water and sodium metabolism 17
Postural hypotension (a fall in blood pressure on concentration greater than 20 mmol/L in a patient with
standing) is a relatively early sign of volume depletion, adequate tubular function suggests overcorrection and
and tachycardia may also occur. the need to slow the rate of infusion. In cases in which
all losses, other than in sweat, faeces and expired air, can
Changes produced by homeostatic mechanisms be measured, further maintenance of normal balance
The ability to respond to hormonal homeostatic should be based on accurate fluid balance charts.
changes depends mainly on renal tubular function For crystalloid resuscitation or replacement,
and cannot occur if the isosmolar volume depletion ‘balanced’ solutions, such as Ringer’s lactate/acetate or
is due to tubular damage. The reduced intravascular Hartmann’s solution, should replace 0.9 per cent saline,
volume impairs renal blood flow and stimulates renin, except in cases of hypochloraemia, due for example
and therefore aldosterone, secretion. There is selective to vomiting or gastric drainage or if lactic acidosis is
sodium reabsorption from the distal tubules and present. Losses from diarrhoea/ileostomy/small bowel
therefore a low urinary sodium concentration. fistula/ileus/obstruction should be replaced volume for
The tendency of the retained sodium to increase volume with Hartmann’s or Ringer’s lactate/acetate-type
plasma osmolality stimulates ADH secretion, and solutions. ‘Saline depletion’, for example due to excessive
water is reabsorbed; this tends to correct the circulating diuretic exposure, is best managed with a balanced
volume and keep the plasma sodium concentration electrolyte solution such as Hartmann’s solution.
normal. Severe intravascular volume depletion may also Intravenous solutions such as 4 per cent dextrose/
stimulate ADH secretion and therefore water retention, 0.18 per cent saline and 5 per cent dextrose are
causing mild hyponatraemia. This additional water is important sources of free water for maintenance, but
distributed throughout the total body water and moves should be used with caution as excessive amounts may
from the depleted, and now slightly hypo-osmolar, cause dangerous hyponatraemia, especially in children
ECF into the relatively well-hydrated intracellular and the elderly and post-operatively. These solutions are
compartment. not suitable for resuscitation or replacement therapy,
Even maximal renal water and sodium retention except in conditions of significant free water deficit,
cannot correct extrarenal losses that exceed those of such as diabetes insipidus (see Actions of antidiuretic
a normal urine output. Water and sodium must be hormone above).
replaced in adequate amounts. To meet maintenance requirements, adult patients
should receive sodium 50–100 mmol/day, and
Effects of intravenous volume replacement
potassium 40–80 mmol/day in 1.5–2.5 L water by the
Patients unable to absorb adequate amounts of oral oral, enteral or parenteral route (or a combination of
fluid because of gastrointestinal loss usually need routes). Additional amounts should only be given to
intravenous replacement. correct deficit or continuing losses. It is essential to
Fluid replacement in a patient who presents with carefully monitor patients by clinical examination, fluid
hypovolaemia can be monitored by clinical observation balance charts, and regular body weighing if possible.
and by measurement of urine output, plasma
electrolytes and urea. Predominant sodium depletion
The infusion of protein-free fluid increases the Incorrect intravenous fluid administration
hydrostatic gradient and reduces the opposing An important and common cause of sodium depletion
colloid osmotic gradient by diluting plasma proteins. is infusion of intravenous fluid of inappropriate
The increase in glomerular filtration caused by the composition. No bodily secretion (other than bile,
overcorrection of hypovolaemia results in an increase which is secreted in very small amounts) has a sodium
in urine output, and is a common cause of a low plasma concentration significantly higher than that of plasma.
urea concentration. The composition of the fluid is even more important
Measurement of urinary sodium concentration may than the volume.
sometimes help. If tubular function is adequate, a urinary Patients with isosmolar fluid depletion, or those
sodium concentration of less than about 20 mmol/L recovering from major surgery, may be infused
suggests that renal blood flow is still low enough to with fluid such as ‘dextrose saline’, which contains
stimulate maximal renin and aldosterone secretion, about 30 mmol/L of sodium. Glucose in the infused
and infusion should be increased. A urinary sodium fluid renders it isosmolar despite the low sodium
18 Water and sodium
concentration, but the glucose is metabolized, and both Sodium depletion due to failure of homeostatic mechanisms
plasma sodium concentration and osmolality are diluted Aldosterone deficiency, such as occurs in Addison’s
by the remaining hypo-osmolar fluid. Homeostatic disease, is a rare cause of sodium depletion. Initial
mechanisms tend to correct this hypo-osmolality but homeostatic reactions tend to maintain osmolality at
may be overwhelmed if the infusion rate is high. Severe, the expense of volume (see Chapter 8). Although less
and even life-threatening, hyponatraemia can result than 1.5 per cent of the filtered sodium is reabsorbed
from the imprudent administration of hypotonic fluid, in the renal distal convoluted tubules, this is where the
such as 5 per cent dextrose. fine adjustment is made in the ratio of sodium to water
Excess hypo-osmolar infused fluid dilutes the and therefore to plasma osmolality, and thus normal
plasma sodium, causing a dilutional hyponatraemia cell hydration is safeguarded. If aldosterone cannot
with hypo-osmolality. The homeostatic mechanisms be secreted normally in response to appropriately
that tend to correct this hypo-osmolality involve the increased amounts of renin and angiotensin, this
inhibition of ADH secretion. The excess water is lost adjustment cannot be made (Fig. 2.7). Under such
in the urine until the restoration of normal plasma circumstances, although a greater proportion of
osmolality again stimulates normal ADH secretion. sodium may be reabsorbed from the proximal tubules,
Correction of osmolality may occur at the expense of there may still be relative sodium deficiency and
intravascular volume. This would stimulate renin and hypovolaemia. Initially plasma osmolality and therefore
aldosterone secretion and sodium would be retained, plasma sodium concentration are maintained by water
with the consequent restoration of osmolality and loss; loss of relatively more sodium than water reduces
normal ADH secretion. plasma osmolality and cuts off ADH secretion. Later,
However, if intravascular volume is maintained by hypovolaemia stimulates ADH secretion, with water
replacing the urinary volume with effectively hypotonic retention in excess of sodium; dilutional hyponatraemia
fluid, hypo-osmolality with hyponatraemia persists and may occur despite intravascular volume depletion.
sodium depletion is aggravated (Fig. 2.6). Restoration The clinical features include circulatory insufficiency
of the plasma volume inhibits renin and aldosterone with postural hypotension and the following findings:
secretion and sodium is lost in the urine despite hypo-
osmolality. The net effect of this procedure is the ● haemoconcentration due to fluid depletion,
restoration of circulating volume at the expense of ● renal circulatory insufficiency with mild uraemia
sodium depletion and cellular overhydration. due to volume depletion,
The findings include: ● an inappropriately high urinary sodium
concentration in the presence of volume depletion,
● hypo-osmolality, ● dilutional hyponatraemia and hyperkalaemia.
● a large volume of dilute urine due to the inhibition
of ADH secretion, Predominant water depletion
● hyponatraemia. Predominant water depletion is caused by loss of
water in excess of sodium. It is usually due to loss of
If fluid intake is excessive, the following may also
fluid that has a sodium concentration less than that
occur:
of plasma, deficient water intake, or both. The rise in
● haemodilution, extracellular osmolality stimulates both ADH secretion
● a low plasma urea concentration due to the high GFR (which minimizes water loss) and thirst. Laboratory
(excessive intravenous infusion is one of the com- abnormalities are most marked if the patient is unable
monest causes of a low plasma urea concentration), to respond to thirst.
● high urinary sodium concentration due to the The causes of predominant water depletion can be
inhibition of aldosterone secretion. divided into the following groups.
During the immediate post-operative period, Predominant water depletion with normal homeostatic
pain and stress also stimulate ADH secretion and mechanisms (Fig. 2.8)
therefore water retention; this effect is short lived. Such ● Excessive loss of fluid that has a sodium concentration
hyponatraemia is rarely a problem, but may become so less than that of plasma. The causes include:
if hypo-osmolar fluid is being infused. In rare cases it – loss of excessive fluid stools of low sodium
can then be lethal due to cerebral damage. concentration, usually in infantile gastroenteritis,
Disturbances of water and sodium metabolism 19
– excessive respiratory loss due to hyperventilation ● Excess water loss due to polyuria:
in, for example, pneumonia, – osmotic diuresis, which can be caused by
– loss of gastric fluid, hypertonic intravenous infusions, such as
– loss of large amounts of sweat, such as in parenteral nutrition tissue damage and hence
pyrexial patients, increased production of urea from protein,
– loss of fluid from the body surface after and glycosuria in severe diabetes mellitus with
extensive burns. a very high plasma glucose concentration.
● Deficiency of water intake as a result of inadequate
Failure of homeostatic mechanisms involving antidiuretic
water supply, or mechanical obstruction to its hormone (Fig. 2.9)
intake.
These syndromes are relatively rare and include the
Failure of homeostatic mechanisms controlling water retention following:
● Inadequate response to thirst: ● Cranial diabetes insipidus, a syndrome associated
– in comatose or confused patients, with impairment of ADH secretion. It may be
– in infants, idiopathic in origin or due to either pituitary or
– caused by damage to the cerebral thirst centre hypothalamic damage caused by head injury or by
(rare). invasion of the region by tumour or infiltration.
+
ADH H2O VOLUME
+ –
+ Angiotensin II
+
Figure 2.8 Homeostatic correction of predominant water depletion. Reduced circulating water volume and
hypernatraemia, due to water depletion, stimulate aldosterone and antidiuretic hormone (ADH) secretion.
(Shading indicates primary change.)
–
ADH ISOSMOTIC VOLUME
====== BLOCK –
+
Thirst Renal blood flow
+
+
Hypothalamic
osmolality Renin release
+
+ Angiotensin II
+
Figure 2.9 Consequences of antidiuretic hormone (ADH) deficiency (diabetes insipidus). Impaired water retention
results in an increased plasma osmolality with stimulation of thirst and hypovolaemia with increased aldosterone
secretion. (Shading indicates primary change.)
20 Water and sodium
Diabetes insipidus following a head injury may ● thirst, increasing water intake, if water is available
present with polyuria, and then pass through a and if the patient can respond to it,
temporary ‘recovery’ phase following transient ● ADH secretion: urinary volume falls and water loss
release of ADH from the remaining granules in the through the kidney is reduced.
pituitary stalk; this results in water retention and
If an adequate amount of water is available, depletion
occasionally causes a dilutional hyponatraemia.
is rapidly corrected. If there is an inadequate intake
Some patients with diabetes insipidus due to trauma
to replace the loss, hypernatraemia may occur before
recover partially or completely as cerebral oedema
clinical signs of volume depletion are detectable. The
resolves. There is a hereditary autosomal dominant
clinical features of predominant water depletion include:
form of cranial diabetes insipidus due to mutations
in the arginine vasopressin–neurophysin II gene. ● thirst,
In addition, there is the autosomal recessive form ● oliguria and concentrated urine due to ADH
DIDMOAD (diabetes insipidus, diabetes mellitus, secretion,
optic atrophy and deafness – see Chapter 12), due to ● later – signs of volume depletion: the signs of
mutations in the wolframin gene. intravenous volume depletion are relatively less than
● Nephrogenic diabetes insipidus is caused by the isotonic fluid loss as the deficit is distributed across
reduced action of ADH on the renal collecting ducts. TBW space.
The disorder may be either familial or acquired. Conversely, the clinical features differ if there is ADH
There is an X-linked recessive form due to mutations deficiency, and include polyuria and dilute urine. The
in the vasopressin type 2 (V2) receptor gene and also laboratory findings are:
an autosomal recessive form due to mutations in the
aquaporin 2 gene (chromosome 12), which codes ● hypernatraemia,
for the vasopressin-dependent water channel in the ● haemoconcentration due to fluid depletion,
renal collecting ducts. Causes of secondary acquired ● mild uraemia due to volume depletion and therefore
diabetes insipidus include: low GFR,
– drugs, such as lithium carbonate, amphotericin ● high urinary osmolality and urea concentration due
or demeclocycline, which interfere with the to the action of ADH,
action of ADH causing the clinical picture of ● low urinary sodium concentration in response to
nephrogenic diabetes insipidus, high aldosterone levels stimulated by the low renal
– hypercalcaemia or hypokalaemia, both of which blood flow.
impair the urine-concentrating mechanism
and may present with polyuria. Water and sodium excess
An excess of water or sodium is usually rapidly
Water deficiency without thirst is called adipsic, or
corrected. Syndromes of excess are usually associated
hypodipsic, hypernatraemic syndrome or essential
with impaired homeostatic mechanisms. These can be
hypernatraemia, and is thought to be due to a
conveniently classified into those with:
hypothalamic disorder. In this syndrome there is a lack
of thirst without polyuria. ● oedematous states,
Features of predominant water depletion
● predominant sodium excess, which may be caused
by hyperaldosteronism or, in mentally disturbed
The immediate effects of water depletion result in:
subjects, by excessive salt intake or by deliberate salt
● loss of water in excess of sodium: the plasma poisoning as infants; there may be hypernatraemia,
sodium concentration, and therefore osmolality, ● predominant water excess, which may result from
increases, excessive ADH secretion or increased water intake
● reduction of circulating volume, which reduces and usually results in hyponatraemia.
renal blood flow and stimulates aldosterone
secretion; sodium is retained and hypernatraemia is Oedematous states (increased interstitial fluid)
aggravated.
Volume excess not primarily due to an osmotic difference
Compensatory effects occur because the increase in across cell membranes may cause hypertension and
the plasma osmolality stimulates: cardiac failure. Oedema occurs only if:
Disturbances of water and sodium metabolism 21
● the capillary hydrostatic pressure is increased, as in ● Redistribution of ECF, leading to a reduced plasma
congestive cardiac failure, volume despite a normal or even high total ECF
● the capillary colloid osmotic pressure is reduced due volume. The resulting conditions may be caused by
to hypoalbuminaemia. a reduction in plasma colloid osmotic pressure, and
are therefore associated with low plasma albumin
Laboratory findings are characteristic of
concentrations. Oedema is present. Persistent
haemodilution and, unless there is glomerular
hypoalbuminaemia may be due to liver disease,
dysfunction, plasma urea concentrations tend to be low.
nephrotic syndrome or protein undernutrition.
Some factors that increase fluid accumulation in the
● Cardiac failure, in which two factors may reduce
interstitial space are summarized in Box 2.1.
renal blood flow and a third factor aggravates the
Secondary aldosteronism hyperaldosteronism:
By convention, the term secondary aldosteronism – a low cardiac output results in poor renal
usually refers to the clinical condition in which long- perfusion,
standing aldosterone secretion is stimulated by the – an increased capillary hydrostatic pressure in
renin–angiotensin system following a low renal blood cardiac failure may cause the redistribution of
flow (Fig. 2.10). This may be due to local abnormalities fluid into the interstitial space, with oedema,
in renal vessels or to a reduced circulating volume and – both impaired catabolism of aldosterone
is therefore usually associated with the following: and elevated plasma atriuretic peptide
concentration may aggravate the condition.
Box 2.1 Some factors that increase the ● Damage to renal vessels, reducing renal blood flow.
accumulation of fluid within the interstitial These conditions are rarely associated with oedema
and include:
space (oedema)
– essential hypertension,
Decrease in colloid osmotic pressure gradient – malignant hypertension,
Decrease in plasma albumin concentration – renal hypertension, such as that due to renal
Increase in capillary permeability to albumin artery stenosis.
‘Shock’ and any severe illness
Infection with inflammation and therefore increased The reduced renal blood flow stimulates aldosterone
Permeability of the capillary endothelium secretion, with enhanced sodium reabsorption
from the distal convoluted tubules and subsequent
Increase in hydrostatic pressure gradient
Heart failure with sodium retention water retention. These processes tend to restore the
intravascular volume. If there is hypoalbuminaemia
+
ADH ISOSMOTIC VOLUME
+ –
+
Hypothalamic
osmolality + Renin release
+
+ Angiotensin II
+
Figure 2.10 Effect of secondary aldosterone secretion. Decreased effective intravascular volume increases renin and
aldosterone secretion; if pronounced, antidiuretic hormone (ADH) secretion may be increased and thirst stimulated,
resulting in hyponatraemia despite an increase in total body sodium. (Shading indicates primary change.)
22 Water and sodium
or heart failure, more fluid passes into the interstitial Predominant excess of sodium
fluid as the retained water dilutes the plasma albumin Predominant sodium excess is rare. It is usually caused
concentration and therefore lowers the colloid by inappropriate secretion of aldosterone, such as in
osmotic pressure and raises the capillary hydrostatic primary hyperaldosteronism (Conn’s syndrome), or by
pressure. The circulating volume can be maintained glucocorticoids, as in Cushing’s syndrome (see Chapter
only by further fluid retention. A vicious cycle leads 8). In these syndromes, sodium retention stimulates
to the accumulation of excess fluid in the interstitial the retention of water, minimizing changes in plasma
compartment (oedema). sodium concentration. Sodium excess can also be
Initially, this cycle stimulates a parallel increase in water caused by excessive sodium intake.
and sodium retention, with normonatraemia. However,
stimulation of ADH secretion by hypovolaemia, if long Primary hyperaldosteronism (Conn’s syndrome)
standing, may cause enough sodium-free water retention About 50 per cent of cases of this syndrome are due to
to produce mild hyponatraemia despite the excess of a single benign adenoma of the glomerulosa cells of
total body sodium. Angiotensin II also stimulates thirst the adrenal cortex; about 10 per cent of adenomas are
and therefore increases water intake. multiple, and most of the remaining cases are associated
Hypokalaemia is less common in secondary than with bilateral nodular hyperplasia of the adrenal
in primary hyperaldosteronism, perhaps because the cortex. Rarer causes include aldosterone-producing
low GFR reduces the amount of sodium reaching the carcinoma of the adrenal gland or ectopic aldosterone-
distal tubules and therefore the amount available for secreting tumours. Another rare form is glucocorticoid-
exchange with either hydrogen or potassium ions (see suppressible hyperaldosteronism. In all these conditions,
Chapters 4 and 5). However, if reabsorption of sodium aldosterone secretion is relatively autonomous (Fig. 2.11):
without water is inhibited by loop diuretics, potassium
● Aldosterone excess causes urinary sodium retention.
depletion occurs more readily than in subjects without
● The rise in plasma sodium concentration stimulates
hyperaldosteronism.
ADH secretion and water retention.
The clinical features found in these patients are
● Water retention tends to restore plasma sodium
those of the underlying primary condition. Laboratory
concentrations to normal.
findings include:
● Aldosterone secretion is not subject to normal
● normonatraemia or hyponatraemia, feedback suppression. Its action causes sodium and
● a low urinary sodium concentration, water retention at the expense of potassium loss;
● those due to the primary abnormality, such as hypokalaemic alkalosis is common (see Chapters 3,
hypoalbuminaemia. 4 and 5).
+
ADH VOLUME
+ +
====== BLOCK
Plasma [Na+] ALDOSTERONE
+
Figure 2.11 Effect of primary aldosteronism (Conn’s syndrome). Aldosterone secretion is relatively autonomous,
causing sodium retention and increasing the plasma osmolality. This stimulates antidiuretic hormone (ADH)
secretion. The increase in intravascular volume inhibits renin secretion. (Shading indicates primary change.)
Disturbances of water and sodium metabolism 23
The typical clinical features of primary hyper- iatrogenically in patients treated with glucocorticoids
aldosteronism are those of hypervolaemia (patients (see Chapter 8).
have mild to moderate hypertension but are rarely
Increased sodium intake
oedematous) and those associated with hypokalaemia.
Sodium excess may be due to increased sodium intake,
The laboratory findings include the following:
such as excessive salt intake or overenthusiastic infusion
● A plasma sodium concentration that may be of saline or sodium-containing solutions, such as sodium
within the higher reference range but may show bicarbonate, or infusion of sodium salts of antibiotics.
hypernatraemia. Hyperosmolar saline is dangerous if used as an emetic
● Hypokalaemic alkalosis (low plasma potassium and in cases of poisoning. The movement of water into the
raised plasma bicarbonate concentrations) due to gut along the osmotic gradient and absorption of some
excess aldosterone secretion: of the sodium can cause marked hypernatraemia.
– a urinary potassium concentration greater than Death has occurred as a consequence of this practice,
about 20 mmol/L, indicating kaliuria (urinary particularly when patients could not respond to
potassium loss) in the face of hypokalaemia hyperosmolality by drinking because of vomiting or
(see Chapter 5), because they were unconscious.
– a low urinary sodium concentration in the early
stages; later, sodium excretion may rise, possibly Predominant excess of water
because of hypokalaemic tubular damage. Predominant water overload may occur in
These findings in a patient without an obvious circumstances in which normal homeostasis has failed,
reason for potassium loss and with mild to moderate for example in the following:
hypertension suggest the diagnosis of primary ● If fluid of low sodium concentration, such as
hyperaldosteronism. The finding of high plasma 5 per cent dextrose, has been infused, especially in
aldosterone with low renin activity confirms the the post-operative period or if there is glomerular
diagnosis; in secondary aldosteronism both the plasma dysfunction (see Fig. 2.12 and Chapter 3).
aldosterone concentration and renin activity are high, ● If there is ‘inappropriate’ ADH secretion. Antidiuretic
although various antihypertrensive drugs may interfere hormone release may be stimulated by stress, such as
with these tests (see Chapter 8, Investigation of a patient that due to pain, nausea, chest infection and cerebral
with suspected primary hyperaldosteronism). trauma (Box 2.2).
Cushing’s syndrome Hormone secretion is defined as inappropriate
This results from glucocorticoid excess, such as is if it continues when it should be cut off by negative
found with adrenal or pituitary adenomas, ectopic feedback control, in this case by a low plasma
adrenocorticotrophic hormone (ACTH) secretion or osmolality. Like many other peptide hormones, ADH
–
ADH H2O VOLUME
+
–
Hypothalamic –
osmolality Renin release
–
– Angiotensin II
–
Figure 2.12 Homeostatic correction of water excess. Increased intravascular water volume, with decreased plasma
osmolality, inhibits aldosterone and antidiuretic hormone (ADH) secretion. (Shading indicates primary change.)
24 Water and sodium
The infusion of isosmolal sodium-containing fluids ● Hypovolaemic hyponatraemia The TBW decreases
with the aim of ‘correcting’ artefactual, appropriate although the total body sodium decreases to a larger
or pseudohyponatraemia is dangerous. When extent. Thus the ECF volume is decreased.
hyponatraemia reflects true hypo-osmolality, cerebral ● Euvolaemic hyponatraemia Total body sodium is
cellular overhydration may cause headache, confusion, normal, while TBW increases. The ECF volume is
fits and even death. If the plasma sodium concentration slightly increased but there is no oedema. One cause
falls slowly over days or weeks, the brain can compensate is SIADH (Box 2.2).
by the redistribution of water and solute between the ● Hypervolaemic hyponatraemia The total body
intracellular and extracellular fluid. However, if the sodium increases but TBW increases to a greater
plasma sodium concentration declines rapidly over extent. The ECF is markedly increased and oedema
24–48 h, these processes are inefficient and cerebral is present.
oedema can occur. Cerebral pontine myelinolysis can
occur if the plasma sodium concentration is corrected Investigation of hyponatraemia (Fig. 2.13)
too rapidly, before redistribution can be reversed. The ● Exclude artefactual hyponatraemia when blood is
rapid change in osmolality causes focal demyelination taken from the vein into which fluid is being infused.
26 Water and sodium
Hyponatraemia
No Yes
Measure plasma
osmolality
Normal/elevated Decreased
Hyperglycaemia – Alcohol?
– Infusion
(e.g. mannitol)?
Measure spot
urine sodium concentration
This occurs whether the blood is taken close to the and can result in neurological damage or death.
site of infusion, where the infused fluid is at a higher Menopausal women, children and the elderly are
concentration than in the rest of the circulation. especially susceptible to hyponatraemia, particularly
● Is there hyperlipidaemia or hyperproteinaemia? if they are taking diuretics or certain antidepressant
Exclude pseudohyponatraemia when plasma drugs. Furthermore, there is also misunderstanding
osmolality is normal. about the most appropriate treatment of severe
● Assess the patient’s medication history, including hyponatraemia: rapid correction can cause cerebral
intravenous fluid therapy and also alcohol intake. pontine myelinolysis, and correction that is too slow
● Hyperglycaemia can result in excessive extracellular may allow cerebral oedema to develop; because of these
solute and water shift out of cells: risks, treatment should be instigated by experts on a
high-dependency unit.
Corrected sodium concentration = [glucose]
Severe hyponatraemia known to have been present
4 (2.3)
for less than 48 h should be treated as a medical
+ measured [sodium] (mmol/L)
emergency; because of the danger of brainstem
● Abnormal electrolyte shifts may occur very rarely in herniation, the aim should be to abolish symptoms
critically ill patients (the sick cell syndrome). or to raise the plasma sodium concentration to about
● It is important to exclude adrenal insufficiency 125 mmol/L. Some people recommend infusion of
(Addison’s disease) and severe hypothyroidism (see sodium at the rate of 1 or 2 mmol/h. Patients with
Chapters 8 and 11). These are rare causes. signs of acute brainstem herniation such as coma or
● Clinically assess the state of the ECF volume;
is there obvious hypovolaemia, euvolaemia or CASE 2
hypervolaemia?
– If there is obvious hypovolaemia, check the A 74-year-old woman underwent a left total hip
sodium concentration in a random urine replacement and had received 5 L of 5 per cent
specimen (assuming not on a diuretic): if it is dextrose intravenously over one day. She had the
less than or equal to 20 mmol/L, it is suggestive following post-operative results:
of extrarenal sodium loss; if it is more than Plasma
20 mmol/L, it suggests renal sodium loss. Sodium 117 mmol/L (135–145)
– If the patient is clinically euvolaemic, check a Potassium 3.7 mmol/L (3.5–5.0)
random urinary sodium concentration: if it Urea 3.4 mmol/L (2.5–7.0)
is less than or equal to 20 mmol/L, consider Creatinine 76 µmol/L (70–110)
acute water overload, for example due to The pre-operative results were as follows:
the administration of too much fluid post-
operatively; if it is more than 20 mmol/L, Plasma
consider SIADH (see Box 2.2). Sodium 138 mmol/L (135–145)
– If the patient is oedematous and is not in Potassium 4.2 mmol/L (3.5–5.0)
cardiac or renal tubular failure, consider Urea 5.6 mmol/L (2.5–7.0)
nephrotic syndrome. Creatinine 90 µmol/L (70–110)
Hypernatraemia
Yes No
Clinical assessment of
extracellular fluid volume
Hypovolaemic/euvolaemic Hypervolaemic
1 1
Consider Consider
diabetes osmotic
insipidus diuresis
(see Box 2.5) (see Box 2.5)
● Is there polyuria? If so, look for evidence of diabetes insipidus is usually treated with desmopressin. Hereditary
insipidus. Determine the ratio of urinary to plasma nephrogenic diabetes insipidus can be difficult to treat,
osmolality (see later). although thiazide diuretics or prostaglandin inhibitors
● If there is mild hypernatraemia with hypokalaemia such as indometacin have been used.
and hypertension, consider mineralocorticoid excess,
Investigation of polyuria (Fig. 2.16)
due, for example, to administration of steroids or to
Cushing’s syndrome or primary hyperaldosteronism. The causes of polyuria are given in Box 2.5. Polyuria is
usually defined as more than 3 L of urine per day. The
Other causes of hypernatraemia are shown in Box causes and investigation of anuria (no urine) or oliguria
2.4, and may be clinically obvious. (less than 400 mL/day) are discussed in Chapter 3.
● Take a clinical history and examine the patient:
CASE 3 – Distinguish between true polyuria (check 24-h
urinary volume) and frequency (a normal 24-h
A 5-year-old girl was admitted to hospital because volume but abnormally frequent micturition).
of a 4-day history of diarrhoea and vomiting. A midstream urine specimen may be useful to
On examination she was found to be clinically exclude urinary tract infection as a cause of
‘dehydrated’ with loss of skin turgor; her pulse was urinary frequency.
120 beats/min and her blood pressure 74/50 mmHg. – Is the patient taking diuretics? Consider other
Her admission results were as follows: drugs, for example lithium or demeclocycline.
Plasma
Sodium 167 mmol/L (135–145) Box 2.5 Some causes of polyuria
Potassium 3.0 mmol/L (3.5–5.0)
Urea 19 mmol/L (2.5–7.0) Increased fluid intake (oral or intravenous)
Oral or intravenous therapy
Creatinine 110 µmol/L (70–110)
Thyrotoxicosis
Urine Psychogenic polydipsia
Spot sodium < 10 mmol/L Osmotic diuresis
DISCUSSION Endogenous substances
The severe hypernatraemia is in keeping with Glycosuria
hypotonic fluid loss due to the diarrhoea and Uraemia
Exogenous substances
vomiting. The loss of skin turgor, tachycardia and
Mannitol infusion (used therapeutically)
hypotension suggest hypovolaemia. The low urinary
Impaired antidiuretic hormone production (cranial
sodium concentration indicates renal sodium
diabetes insipidus)
conservation. Children are particularly susceptible Congenital
to hypernatraemia induced by hypotonic fluid loss. Acquired
Cerebral trauma
Infection
Treatment of hypernatraemia Cerebral tumours and other infiltrations
If the hypernatraemia is due to water depletion, oral Drugs and other agents, e.g. alcohol
repletion is the treatment of choice. If this is not possible, Impaired renal tubular response to antidiuretic
fluid of low sodium concentration, such as 5 per cent hormone (nephrogenic diabetes insipidus)
dextrose saline, should be infused slowly. The aim Congenital
should be to lower the plasma sodium concentration at Acquired
no more than 1–2 mmol/L per hour or less, especially if Chronic kidney disease with predominant tubular
dysfunction, e.g. interstitial nephritis, medullary
the hypernatraemia is long standing.
cystic disease
In hypervolaemic hypernatraemia, the Severe hypercalcaemia or hypokalaemia
hypernatraemia may improve if patients reduce their Drugs – lithium, demeclocycline
voluntary salt intake. The management of primary Other drugs
hyperaldosteronism (Conn’s syndrome) and Cushing’s Diuretics
syndrome is discussed in Chapter 8. Cranial diabetes
Disturbances of water and sodium metabolism 31
Yes No
Yes No
Yes No
Evidence of hypokalaemia?
Yes No
Evidence of hypercalcaemia?
Yes No
Urine/plasma 1 Urine/plasma 1
Figure 2.16 Summary algorithm for polyuria investigation. DDAVP, 1-desamino-8-D-arginine vasopressin.
32 Water and sodium
– If there is a recent history of oliguric renal ● In cases of cranial diabetes insipidus, a neurological
failure, the patient has probably entered opinion may be necessary. Is there a possible obvious
the polyuric phase, and should be treated cause for diabetes insipidus, such as a head injury?
accordingly (see Chapter 3). (See Chapter 7.) Consider brain imaging such as
● Exclude chronic kidney disease, hypokalaemia, computerized tomography or magnetic resonance
hypercalcaemia, adrenal insufficiency and thyrotoxi- imaging.
cosis.
The causes of nephrogenic diabetes insipidus are
● Check for causes of an osmotic diuresis such as:
given in Box 2.5 (see also Chapter 3).
– severe glycosuria due to diabetes mellitus or
infusion of dextrose, Water deprivation test
– tissue damage, leading to a high urea load,
This section should be read in conjunction with Chapter
– infusion of amino acids, for example during
7, in which pituitary hormonal function is discussed.
parenteral nutrition,
The restriction of water intake for some hours should
– in infants, consider inborn errors of metabolism
stimulate posterior pituitary ADH secretion. Solute-
(see Chapter 27),
free water is reabsorbed from the collecting ducts
– osmotic diuretics, for example mannitol.
and concentrated urine is passed. Maximal water
● Accurately monitor fluid intake and output. The
reabsorption is impaired if:
patient should be carefully watched for secret fluid
ingestion (psychogenic polydipsia). Note also that ● the countercurrent multiplication mechanism is
some patients may even add fluid to their urine or impaired,
drink ‘toilet’ water. ● ADH activity is low.
● Measure plasma urea and creatinine and electrolyte
concentrations: CASE 4
– Elevated plasma urea and/or creatinine
concentrations suggest renal impairment. Below are the pre-operative blood results of a
– Low or low-normal plasma urea or creatinine 44-year-old man. The sample was reported by the
concentrations, especially if there is mild laboratory to be grossly ‘lipaemic’.
hyponatraemia, suggest that polydipsia is Plasma
the primary cause and that the polyuria is Sodium 118 mmol/L (135–145)
appropriate. Question the patient about his Potassium 4.0 mmol/L (3.5–5.0)
or her intake in relation to true thirst. Patients Urea 6.0 mmol/L (2.5–7.0)
with true psychogenic polydipsia may give Creatinine 95 µmol/L (70–110)
misleading answers. Glucose 4.0 mmol/L (3.5–5.5)
● Measure the urinary and plasma osmolality. In an Cholesterol 17.3 mmol/L (3–5)
osmotic diuresis the urine to plasma osmolality ratio Triglycerides 68 mmol/L (<1.5)
is usually more than 1. Osmolality 290 mmol/kg (275–295)
● If urinary osmolality is more than 750 mmol/kg, Further tests excluded other common causes of
a water deprivation test is not usually indicated. A hyponatraemia.
useful screening test is an early morning urine test.
DISCUSSION
● If it is still not possible to distinguish between
The plasma osmolality was not low despite
psychogenic polydipsia and diabetes insipidus, contact
hyponatraemia, suggesting pseudohyponatraemia.
the laboratory to arrange a water deprivation test, if
This is probably due to the gross lipaemia when
necessary followed by administering 1-desamino-8-
measured by indirect sodium ion-detecting electrodes
D-arginine vasopressin (DDAVP) (see below). This
which some laboratories use to assay sodium (direct
may help distinguish cranial diabetes insipidus from
electrodes do not usually detect pseudohypo-
nephrogenic diabetes insipidus (see below).
natraemia). Note that calculated plasma osmolality
● In difficult cases when diabetes insipidus is suspec-
is 2[118 + 4] + 6.0 + 4.0 mmol/L = 254 mmol/kg,
ted and the water deprivation test has not been
which differs markedly from the measured value of
helpful, a hypertonic saline test may be indicated
290 mmol/kg.
(see below).
Disturbances of water and sodium metabolism 33
If the feedback mechanism is intact, plasma ADH ● If the urinary osmolality is more than 750 mmol/
levels will already be high and the administration kg, neither significant tubular disease nor diabetes
of exogenous hormone will not improve the renal insipidus is likely and the test may be stopped.
concentrating power. If diabetes insipidus is the ● If the urinary osmolality is less than 750 mmol/kg,
cause, tubular function will be normal and therefore and plasma osmolality is normal, fluid restriction
the administration of ADH will increase the renal should be continued and the estimations repeated at
concentrating ability and increase the urinary hourly intervals. The test should be stopped as soon
osmolality. as the urinary osmolality exceeds 750 mmol/kg.
Warning The test is potentially dangerous, and ● Failure of concentration of three consecutive urine
should not be performed if the patient is volume specimens indicates either tubular disease or diabetes
depleted or has even mild hypernatraemia as fatality insipidus. If the diagnosis of diabetes insipidus is
could result. In such cases the finding of a low urinary considered, continue with the DDAVP test.
to plasma osmolality ratio (see below) should be
diagnostic, and further fluid restriction may be DDAVP test
dangerous. If the test is necessary, it must be stopped if: DDAVP is a potent synthetic analogue of vasopressin.
● the patient becomes distressed, Procedure
● the plasma osmolality (or sodium concentration) Give 2 µg DDAVP intramuscularly; continue to collect
rises to high or high-normal concentrations – usually urine and plasma samples.
taken as more than 300 mmol/kg; urine and blood
specimens should be collected at once, Interpretation
● the patient loses more than 3 per cent of his or her If there is tubular dysfunction or nephrogenic diabetes
body weight. insipidus, there will be no change in the urinary
osmolality, but the plasma osmolality may increase
Procedure owing to water loss during the test. In cranial diabetes
Always contact the laboratory before starting the test, insipidus the urinary osmolality increases, usually to a
both to ensure efficient and speedy analysis and to value of more than 750 mmol/kg.
check local variations in the protocol. The test should be After prolonged overhydration, usually due to
performed by a clinician experienced in the technique. psychogenic polydipsia, concentrating power may be
Patients are not allowed food or water after about impaired, even after the administration of exogenous
20.00 h on the night before the test. They must be in hormone. This is due to ‘washing out’ of medullary
hospital and closely observed during the period of fluid hyperosmolality. The test should be repeated after
restriction and weighed regularly during the test. The several days of relative water restriction. The patient
duration of water deprivation depends on the clinical should be kept under close observation, both for signs
presentation and the degree of polyuria. of genuine distress associated with a rise in plasma
osmolality and for surreptitious drinking.
On the day of the test
08.00 h The bladder is emptied. Blood and urine are
collected and plasma and urinary osmolalities are
measured. If the plasma osmolality is low-normal or
low, water depletion is unlikely and polyuria is probably Table 2.5 Summary of the results of water deprivation/
due to an appropriate response to a high intake. If the DDAVP tests
plasma osmolality is high-normal or high and the
Post-water restriction Post-DDAVP
urinary osmolality is more than 750 mmol/kg, the test
should be stopped. Plasma Urine Urine Interpretation
osmolality osmolality osmolality
09.00 h Blood and urine are again collected and the (mmol/kg) (mmol/kg) (mmol/kg)
plasma and urinary osmolalities are measured hourly.
285–295 > 750 > 750 Normal
Interpretation (Table 2.5) > 295 < 300 > 750 Cranial diabetes insipidus
The duration of the test depends on changes in the > 295 < 300 < 300 Nephrogenic diabetes insipidus
urinary osmolalities. DDAVP, 1-desamino-8-D-arginine vasopressin.
34 Water and sodium
Figure 2.17 Fluid balance chart. It is essential to keep these accurate and regularly updated and reviewed. I
am grateful for the permission of Marian Davies and Dr Marthin Mostert, of University Hospital Lewisham, who
helped design the chart.
Disturbances of water and sodium metabolism 35
SUMMARY
● Water and sodium homeostasis are intimately related, ECF osmolality stimulate ADH release from
and it is essential that doctors understand them posterior pituitary gland.
when investigating and managing hyponatraemia ● Polyuria (daily urine output more than 3 L) may be
and hypernatraemia. Summary algorithms for the due to failures of renal concentrating ability or lack
investigation of hyponatraemia and hypernatraemia of ADH (Fig. 2.16).
are depicted in Figures 2.13 and 2.15. ● Patient fluid balance must be monitored carefully,
● Sodium is the main ECF cation and is controlled with attention to the input and output, particularly
by adrenal aldosterone (renal sodium retention if for those on ‘drip’ therapy (Fig. 2.17).
ECF falls) and atrial natrial peptides. ● Polyuria (daily urine output more than 3 L) may be
● Body water excretion is controlled by ADH due to failures of renal concentrating ability or lack
(vasopressin). Decreased ECF volume and increased of ADH (Fig. 2.16).
3 The kidneys
In this chapter kidney function and how it can be membranes into the glomerular spaces (Bowman’s
altered in disease states is discussed. It is best read in capsules).
conjunction with Chapters 2, 4 and 5. Interpretation The proximal convoluted tubules, also in the cortex,
of renal function tests is also discussed. receive filtrate from the glomerular spaces. Convolution
The kidneys excrete metabolic waste products, increases the tubular length and therefore contact
and have an essential homeostatic function in that between the luminal fluid and the proximal tubular
they control the body solute and water status and cells.
the acid–base balance. There are about one million The loops of Henle extend down into the renal
nephrons per kidney, each of which is made up of medulla and ascend again after forming the loop.
five main functional segments (Fig. 3.1). The distal convoluted tubules, situated in the
The glomeruli, in the cortex of the kidney, are cortex, are important for fine adjustment of luminal
invaginated and surround a capillary network of fluid. They lie near the afferent arterioles, with the
blood vessels derived from the afferent, and draining juxtaglomerular apparatus between them. The enzyme
into the efferent, arterioles. Small molecules and renin is produced by the latter and its release is
water are passively filtered during the passage of controlled by local blood flow.
blood through these capillaries, the ultrafiltrate The collecting ducts start as the distal tubules lead
passing through the vessel walls and the glomerular down into the medulla and end by opening into the
Efferent arteriole
Glomerulus
Juxtaglomerular
apparatus
Distal tubule
Afferent arteriole
Proximal tubule
CORTEX
MEDULLA
Figure 3.1 The anatomical relation between the nephron and the juxtaglomerular apparatus.
Renal tubular function 37
renal pelvis. The modified fluid from the original filtrate The filtrate contains diffusible constituents at
flows from the collecting ducts into the renal tract. almost the same concentrations as in plasma. About
Normal function of the kidneys depends on the 30 000 mmol of sodium, 800 mmol of potassium,
following: 800 mmol of urea, 300 mmol of free ionized calcium
and 1000 mmol of glucose are filtered daily. Proteins
● an adequate blood supply, which under normal
(mainly low-molecular-weight proteins) and protein-
circumstances is about 20 per cent of the cardiac
bound substances are filtered in only small amounts by
output, flowing through the kidneys,
normal glomeruli and most are reabsorbed. The huge
● normal secretion and feedback control of hormones
volume of filtrate allows adequate elimination of waste
acting on the kidney,
products such as urea; death from water and electrolyte
● the integrity of the glomeruli and the tubular cells.
depletion would occur within a few hours were the bulk
In addition to the excretory function and acid– of this water containing essential solutes not reclaimed.
base control, the kidneys have important endocrine
functions, including: RENAL TUBULAR FUNCTION
● production of 1,25-dihydroxyvitamin D, the active Changes in filtration rate alter the total amount of water
metabolite of vitamin D, which is produced following and solute filtered, but not the composition of the filtrate.
hepatic hydroxylation of 25-hydroxyvitamin by the From the 200 L of plasma filtered daily, only about 2 L
renal enzyme 1-hydroxylase, of urine are formed. The composition of urine differs
● production of erythropoietin, which stimulates markedly from that of plasma, and therefore of the
erythropoiesis. filtrate. The tubular cells use adenosine triphosphate-
dependent active transport, sometimes selectively, against
RENAL GLOMERULAR FUNCTION physicochemical gradients. Transport of charged ions
About 200 L of plasma ultrafiltrate usually enter the tends to produce an electrochemical gradient that inhibits
tubular lumina daily, mainly by glomerular filtration further transport. This is minimized by two processes.
into glomerular capsules but also through the spaces Isosmotic transport This occurs mainly in the
between cells lining the tubules (tight junctions). proximal tubules and reclaims the bulk of filtered
Production of ultrafiltrate depends on the blood essential constituents. Active transport of one ion leads
flow through normal glomeruli and on the difference to passive movement of an ion of the opposite charge in
between the hydrostatic pressure gradient and the the same direction, along the electrochemical gradient.
plasma effective colloid osmotic (oncotic) pressure The movement of sodium (Na+) depends on the
gradient across the membranes (Fig. 3.2) and tight availability of diffusible negatively charged ions, such
junctions. The colloid osmotic effect is weak relative as chloride (Cl–). The process is ‘isosmotic’ because the
to the hydrostatic gradient but does facilitate some active transport of solute causes equivalent movement
reabsorption of fluid from the proximal renal tubules. of water reabsorption in the same direction. Isosmotic
transport also occurs to a lesser extent in the distal part
of the nephron.
Ion exchange This occurs mainly in the more
Afferent Efferent
arteriole arteriole distal parts of the nephrons and is important for fine
adjustment after bulk reabsorption has taken place.
Ions of the same charge, usually cations, are exchanged
Blood
flow and neither electrochemical nor osmotic gradients are
created.
COLLOID Therefore, during cation exchange there is
OSMOTIC insignificant net movement of anions or water. For
HYDROSTATIC PRESSURE
PRESSURE example, Na+ may be reabsorbed in exchange for
Bowman’s potassium (K+) or hydrogen (H+) ions. Na+ and H+
capsule exchange also occurs proximally, but at that site it is
Figure 3.2 The relationship between flow of blood through more important for bicarbonate reclamation than for
the glomerulus and the factors that affect the rate of fine adjustment of solute reabsorption (see Chapter 4).
filtration across the glomerular basement membrane. In the cells lining the renal tubules, the intestine and
38 The kidneys
many secretory organs, the pumps are located on the to produce this gradient (see also Chapter 2). Two main
membrane on one side of the cell only and therefore processes are involved in water reabsorption:
solute flows in one direction.
● Isosmotic reabsorption of water from the proximal
Other substances, such as phosphate and urate, are
tubules. The nephrons reabsorb 99 per cent of the
secreted into, as well as reabsorbed from, the tubular
filtered water, about 70–80 per cent (140–160 L/day)
lumen. The tubular cells do not deal actively with waste
of which is returned to the body from the proximal
products such as urea and creatinine to any significant
tubules. Active solute reabsorption from the filtrate
degree. Most filtered urea is passed in urine (which
is accompanied by passive reabsorption of an
accounts for most of the urine’s osmolality), but some
osmotically equivalent amount of water. Therefore,
diffuses back passively from the collecting ducts with
fluid entering the lumina of the loops of Henle,
water; by contrast, some creatinine is secreted into the
although much reduced in volume, is still almost
tubular lumen.
isosmotic.
Reclamation of solute from the proximal ● Dissociation of water reabsorption from that of
tubule solute in the loops of Henle, distal tubules and
Almost all the potassium is actively reabsorbed from collecting ducts. Normally between 40 and 60 L of
the proximal tubules, as is more than 70 per cent water enter the loops of Henle daily. This volume
of the filtered sodium, free ionized calcium and is reduced to about 2 L as varying amounts of
magnesium. Some free ionized calcium is reabsorbed water are reabsorbed, helping to correct for
at more distal sites, possibly from the loops of Henle. changes in extracellular osmolality. At extremes
This reabsorption may be stimulated by parathyroid of water intake, urinary osmolality can vary from
hormone (PTH) and inhibited by loop diuretics such about 40 to 1400 mmol/kg. The proximal tubules
as furosemide. Only about 2 per cent of filtered calcium cannot dissociate water and solute reabsorption,
appears in the urine. and the adjustment must occur between the
Many inorganic anions follow an electrochemical end of the proximal tubule and the end of the
gradient; the reabsorption of sodium is limited by the collecting duct.
availability of chloride, the most abundant diffusible Two mechanisms are involved:
anion in the filtrate. Bicarbonate is almost completely
recovered following exchange of sodium and hydrogen ● Countercurrent multiplication is an active process
ions (see Chapters 2 and 4). Specific active transport occurring in the loops of Henle, whereby a high
mechanisms result in the almost complete reabsorption osmolality is created in the renal medulla and
of glucose, urate and amino acids. Some urate is secreted urinary osmolality is reduced. This can occur in the
into the lumina, mainly in the proximal tubules, but absence of antidiuretic hormone (ADH), also called
most of this is reabsorbed. arginine vasopressin or vasopressin, and a dilute
Phosphate reabsorption is incomplete; phosphate hypo-osmolal urine is produced.
in tubular fluid is important for buffering hydrogen ● Countercurrent exchange is a passive process,
ions. Inhibition of phosphate reabsorption by PTH occurring only in the presence of ADH. Water
occurs in both the proximal and the distal convoluted without solute is reabsorbed from the collecting
tubules, and accounts for the hypophosphataemia of ducts into the ascending vasa recta along the osmotic
PTH excess. Thus almost all the reusable nutrients and gradient created by countercurrent multiplication
the bulk of electrolytes are reclaimed from the proximal and by the high osmolality in the medulla, producing
tubules, with fine homeostatic adjustment taking place a concentrated urine.
more distally. Almost all the filtered metabolic waste
products, such as urea and creatinine, which cannot be Countercurrent multiplication
reused by the body, remain in the luminal fluid. This occurs in the loops of Henle. It depends on the
close apposition of the descending and ascending
WATER REABSORPTION: URINARY limbs of the loops to the vasa recta. The vasa recta
CONCENTRATION AND DILUTION make up a capillary network derived from the efferent
Water is always reabsorbed passively along an osmotic arterioles and, like the loops of Henle, pass deep into
gradient. However, active solute transport is necessary the medulla.
Water reabsorption: urinary concentration and dilution 39
The descending limbs are permeable to water but the the loops and the adjacent medullary tissue would be
thick ascending limbs are impermeable to water and about 300 mmol/kg (Fig. 3.3a).
solute. Chloride is actively pumped from the thick Suppose the fluid column remained stationary and
ascending to the descending limbs as fluid flows through 1 mmol of solute per kilogram were pumped from the
the lumina of the loops; positively charged sodium ions ascending into the descending limb, the result would be
follow along the electrochemical gradient. Thus, the as in Figure 3.3b. If this pumping continued and there
osmolality progressively increases in the descending were no flow, the fluid in the descending limb would
limbs and renal medullary interstitium; it decreases in become hyperosmolal and that in the ascending limb
the ascending limbs, but, as these are impermeable to correspondingly hypo-osmolal.
water, this change is not transmitted to the interstitium. Suppose that the fluid flowed so that each figure ‘moved
The almost isosmolal fluid enters the descending limbs two places’ (Fig. 3.3c). As this happened, more solute
having the same osmolality as the plasma, just under would be pumped from the ascending to the descending
300 mmol/kg. If the fluid in the loops was stationary and limbs (Fig. 3.3d). If the fluid again flowed ‘two places’, the
no pumping had taken place, the osmolality throughout situation would be as shown in Figure 3.3e.
D A D A
Cortex Impermeable Cortex
300 300 to water 300 300
Medulla
300 300
Medulla
301 ¨ 299
300 300 301 ¨ 299
300 300 301 ¨ 299
300 300 301 ¨ 299
300 300 301 ¨ 299
(a) (b)
D A D A
Cortex Cortex
300 299 300 299
Medulla
300 299
Medulla
301 ¨ 298
300 299 301 ¨ 298
301 299 302 ¨ 298
301 301 302 ¨ 300
301 301 302 ¨ 300
(c) (d)
D A D A
Cortex Cortex
300 298 300 200
(e) (f)
Figure 3.3 The renal counter-regulatory system. D, descending loop of Henle; A, ascending loop of Henle.
40 The kidneys
If these steps occurred simultaneously and then moves passively along the osmotic gradient
continuously, the consequences would be as follows: created by multiplication. Consequently luminal fluid
is concentrated as the collecting ducts pass into the
● Increasing osmolality in the tips of the loops of
increasingly hyperosmolal medulla.
Henle Because the walls of most of the loops are
The increasing concentration of the fluid would
permeable to water and solute, osmotic equilibrium
reduce the osmotic gradient as it passes down the
would be reached with the surrounding tissues in the
ducts if it did not meet even more concentrated plasma
deeper layers of the medulla, including the plasma
flowing in the opposite (countercurrent) direction.
within the vasa recta.
The gradient is thus maintained, and water continues
● Hypo-osmolal fluid leaving the ascending limbs
to be reabsorbed until the fluid reaches the deepest
(Fig. 3.3f) In the absence of ADH, the walls of the
layers, where the osmolality is about four or five times
collecting ducts are impermeable to water, and
that of plasma (Fig. 3.3f). The low capillary hydrostatic
therefore no further change in osmolality occurs,
pressure at this site and the osmotic effect of plasma
and hypo-osmolal urine would be passed.
proteins ensure that much of the reabsorbed water
within the interstitium enters the vascular lumina.
Countercurrent exchange (Fig. 3.4) The diluted blood is carried towards the cortex and
Countercurrent exchange is essential, together ultimately enters the general circulation and helps to
with multiplication, for regulating the osmolal dilute the extracellular fluid.
concentration of urine. It can only occur in the presence The osmotic action of urea in the medullary
of ADH and depends on the ‘random’ apposition interstitium may potentiate the countercurrent
of the collecting ducts and the ascending vasa recta. multiplication. As water is reabsorbed from the
Antidiuretic hormone increases the permeability of collecting ducts under the influence of ADH, the
the cell membranes (via the aquaporins) lining the luminal urea concentration increases. Because the distal
distal parts of the collecting ducts to water, which collecting ducts are permeable to urea, it enters the
MULTIPLICATION
Urine from
Isosmotic zone
proximal tubule
Distal tubule
Ascending vessel
+ –
NaCl Increasing
osmolality
Descending vessel
H2O
+ –
NaCl
H2O
Site of action of
+ –
ADH EXCHANGE
NaCl
H2O
Collecting duct
To renal pelvis
Hyperosmotic zone
Connecting capillary
Blood
Direction of flow of blood or urine
Direction of movement of solute (multiplication)
Figure 3.4 The countercurrent mechanism, showing the relationship between the renal tubules and the vasa
recta. ADH, antidiuretic hormone.
Water reabsorption: urinary concentration and dilution 41
deeper layers of the medullary interstitium, increasing Thus, not only is more water than usual lost in the
the osmolality and drawing water from the lower parts urine, more solute is ‘reclaimed’. Because medullary
of the descending limbs of the loops. The amount of hyperosmolality, and therefore the ability to concentrate
urea reabsorbed depends on: the urine maximally, is dependent on medullary blood
flow, under normal circumstances urinary osmolality
● the amount filtered,
will be fully restored only several days after a prolonged
● the rate of flow of tubular fluid: as much as
water load has stopped (see Chapter 2).
50 per cent of filtered urea may be reabsorbed when
flow is significantly reduced. Osmotic diuresis
In summary, both concentration and dilution An excess of filtered solute in the proximal tubular
of urine depend on active processes, which may be lumina impairs the bulk water reabsorption from
impaired if tubules are damaged. this site by its osmotic effect. Unabsorbed solute
concentration rises progressively as water is reabsorbed
Renal homeostatic control of water excretion with other solute during passage through the proximal
In this section, the mechanisms involved in the normal tubules, and this opposes further water reabsorption.
homeostatic control of urinary water excretion in Thus a larger volume than usual reaches the loops of
the extremes of water intake are discussed. It may be Henle. Moreover, fluid leaving the proximal tubules,
helpful to read it in conjunction with Chapter 2, which although still isosmotic with plasma, has a lower
deals with sodium and water balance. sodium concentration than plasma. The relative lack
of the major cation (sodium) to accompany the anion
Water restriction chloride along the electrochemical gradient inhibits
By increasing the plasma osmolality, water restriction the pump in the loops. The resulting impairment of
increases ADH secretion and allows countercurrent build-up of medullary osmolality inhibits distal water
exchange with enhanced water reabsorption. Reduced reabsorption, under the influence of ADH from the
circulatory volume results in a sluggish blood flow in the collecting ducts, resulting in a diuresis (see Chapter 2).
vasa recta and increased urea reabsorption, allowing a Normally most filtered water leaves the proximal
build-up of the medullary hyperosmolality produced by tubular lumina with reabsorbed solute. For example,
multiplication. This potentiates water reabsorption in glucose (with an active transport system) and urea
the presence of ADH. The reduced capillary hydrostatic (which diffuses back passively) are sometimes filtered
pressure and increased colloid osmotic pressure, due to at high enough concentration to exceed the proximal
the haemoconcentration following non-protein fluid tubular reabsorptive capacity. They can then act as
loss, ensure that much of the reabsorbed water enters osmotic diuretics and cause water depletion. This
the vascular compartment. is important, for example, in diabetes mellitus or in
uraemia.
Water load The most effective osmotic diuretics are substances
A high water intake dilutes the extracellular fluid, and that cannot cross cell membranes to any significant
the consequent fall in plasma osmolality reduces ADH degree; therefore, they must be infused, as they cannot
secretion. The walls of the collecting ducts therefore be absorbed from the gut. One example is mannitol, a
remain impermeable to water and the countercurrent sugar alcohol, which is sometimes used therapeutically
multiplication produces a dilute urine and a high as a diuretic.
osmolality within the medulla and medullary vessels.
Blood from the latter flows into the general circulation, Homeostatic solute adjustment in the distal
so helping to correct the fall in systemic osmolality. tubule and collecting duct
During maximal water diuresis the osmolality at Sodium reabsorption in exchange for hydrogen ions
the tips of the medullary loops may be 600 mmol/kg occurs throughout the nephrons. In the proximal
or less, rather than the maximum of about 1400 mmol/ tubules the main effect of this exchange is on
kg. Increasing the circulating volume increases renal reclamation of filtered bicarbonate. In the distal
blood flow; the rapid flow in the vasa recta ‘washes tubules and collecting ducts, the exchange process is
out’ medullary hyperosmolality, returning some of usually associated with net generation of bicarbonate
the solute, without extra water, to the circulation. to replace that lost in extracellular buffering. Potassium
42 The kidneys
and hydrogen ions compete for secretion in exchange ● reduced hydrogen ion secretion throughout the
for sodium ions. The possible mechanism stimulated nephron: bicarbonate can be reclaimed only if
by aldosterone is discussed in Chapter 2. The most hydrogen ions are secreted; plasma bicarbonate
important stimulus to aldosterone secretion is mediated concentrations will fall,
by the effect of renal blood flow on the release of renin ● reduced potassium secretion in the distal tubule,
from the juxtaglomerular apparatus; this method of with potassium retention (potassium can still be
reabsorption is part of the homeostatic mechanism reabsorbed proximally).
controlling sodium and water balance.
If there is a low GFR accompanied by a low renal
BIOCHEMISTRY OF RENAL DISORDERS blood flow:
Pathophysiology ● Systemic aldosterone secretion will be maximal: in
Different parts of the nephrons are in close anatomical such cases, any sodium reaching the distal tubule will
association and are dependent on a common blood be almost completely reabsorbed in exchange for H+
supply. Renal dysfunction of any kind affects all parts of and K+, and the urinary sodium concentration will
the nephrons to some extent, although sometimes either be low.
glomerular or tubular dysfunction is predominant. The ● ADH secretion will be increased: ADH acting on
net effect of renal disease on plasma and urine depends the collecting ducts allows water to be reabsorbed
on the proportion of glomeruli to tubules affected and in excess of solute, further reducing urinary volume
on the number of nephrons involved. and increasing urinary osmolality well above that of
To understand the consequences of renal disease it plasma and reducing plasma sodium concentration.
may be useful to consider the hypothetical individual This high urinary osmolality is mainly due to
nephrons, first with a low glomerular filtration rate substances not actively dealt with by the tubules. For
(GFR) and normal tubular function, and then with example, the urinary urea concentration will be well
tubular damage but a normal GFR. It should be above that of plasma. This distal response will occur
emphasized that these are hypothetical examples, as only in the presence of ADH; in its absence, normal
in clinical reality a combination of varying degree may nephrons will form a dilute urine.
exist. If the capacity of the proximal tubular cells to
Uraemia is the term used to describe a raised plasma reabsorb solute, and therefore water, is normal, a larger
urea concentration and is almost always accompanied proportion than usual of the reduced filtered volume
by an elevated creatinine concentration: in North will be reclaimed by isosmotic processes, thus further
America this is usually referred to as azotaemia (a raised reducing urinary volume.
nitrogen concentration). In summary, the findings in venous plasma and
urine from the affected nephrons will be as follows.
Reduced glomerular filtration rate with normal tubular
function
Plasma
The total amounts of urea and creatinine excreted
are affected by the GFR. If the rate of filtration fails ● High urea (uraemia) and creatinine concentrations.
to balance that of production, plasma concentrations ● Low bicarbonate concentration, with low pH
will rise. (acidosis).
Phosphate and urate are released during cell ● Hyperkalaemia.
breakdown. Plasma concentrations rise because less ● Hyperuricaemia and hyperphosphataemia.
than normal is filtered. Most of the reduced amount
reaching the proximal tubule can be reabsorbed, and Urine
the capacity for secretion is impaired if the filtered ● Reduced volume (oliguria).
volume is too low to accept the ions; these factors ● Low (appropriate) sodium concentration – only
further contribute to high plasma concentrations. if renal blood flow is low, stimulating aldosterone
A large proportion of the reduced amount of filtered secretion.
sodium is reabsorbed by isosmotic mechanisms; less ● High (appropriate) urea concentration and
than usual is then available for exchange with hydrogen therefore a high osmolality – only if ADH secretion
and potassium ions distally. This has two main outcomes: is stimulated.
Biochemistry of renal disorders 43
Reduced tubular function with normal glomerular There may also be tubular proteinuria, which
filtration rate usually refers to low-molecular-weight proteins that
Damage to tubular cells impairs adjustment of the are normally produced in the body, filtered across the
composition and volume of the urine. Impaired solute glomerular membrane and reabsorbed in the proximal
reabsorption from proximal tubules reduces isosmotic tubule, but appear in the urine as a result of proximal
water reabsorption. Countercurrent multiplication tubular damage, for example a1-microglobulin and
may also be affected, and therefore the ability of the retinol binding protein. However, tubular proteinuria
collecting ducts to respond to ADH is reduced. A large also occurs when proximal tubular enzymes and
volume of inappropriately dilute urine is produced. proteins, such as N-acetyl-b-D-glucosaminidase
The tubules cannot secrete hydrogen ions and (NAG), are released into the urine due to tubular cell
therefore cannot reabsorb bicarbonate normally injury. See Chapter 19.
or acidify the urine. The response to aldosterone
and therefore the exchange mechanisms involving Clinical and biochemical features of renal
disease
reabsorption of sodium are impaired; the urine contains
an inappropriately high concentration of sodium for The biochemical findings and urine output in renal
the renal blood flow. Potassium reabsorption from the disease depend on the relative contributions of
proximal tubule is impaired and plasma potassium glomerular and tubular dysfunction. When the GFR
concentrations may be low. Reabsorption of glucose, falls, substances that are little affected by tubular action
phosphate, magnesium, urate and amino acids is (such as urea and creatinine) are retained. Although
impaired. Plasma phosphate, magnesium and urate their plasma concentrations start rising above the
concentrations may be low. baseline for that individual soon after the GFR falls,
Thus, the findings in venous plasma and urine from they seldom rise above the reference range for the
the affected nephrons will be as follows. population until the GFR is below about 60 per cent
of normal, although in individual patients they do rise
Plasma
above baseline.
● Normal urea and creatinine concentrations (normal Plasma concentrations of urea and creatinine
glomerular function). depend largely on glomerular function (Fig. 3.5). By
● Due to proximal or distal tubular failure: contrast, urinary concentrations depend almost entirely
– low bicarbonate concentration and low pH, on tubular function. However little is filtered at the
– hypokalaemia. glomeruli, the concentrations of substances in the initial
● Due to proximal tubular failure: filtrate are those of a plasma ultrafiltrate. Any difference
– hypophosphataemia, hypomagnesaemia and between these concentrations and those in the urine is
hypouricaemia. due to tubular activity. The more the tubular function is
Urine impaired, the nearer the plasma concentrations will be
● Due to proximal and/or distal tubular failure: to those of urine. Urinary concentrations inappropriate
– increased volume, to the state of hydration suggest tubular damage,
– pH inappropriately high compared with that in whatever the degree of glomerular dysfunction.
plasma. The plasma sodium concentration is not primarily
● Due to proximal tubular failure: affected by renal disease. The urinary volume depends
– generalized amino aciduria, on the balance between the volume filtered and the
– phosphaturia, proportion reabsorbed by the tubules. As 99 per cent
– glycosuria. of filtered water is normally reabsorbed, a very small
● Due to distal tubular failure: impairment of reabsorption causes a large increase in
– even if renal blood flow is low, an urine volume. Consequently, if tubular dysfunction
inappropriately high sodium concentration predominates, impairment of water reabsorption
(inability to respond to aldosterone), causes polyuria, even though glomerular filtration is
– even if ADH secretion is stimulated, an reduced (see Chapter 2).
inappropriately low urea concentration and The degree of potassium, phosphate and urate
therefore osmolality (inability of the collecting retention depends on the balance between the degree of
ducts to respond to ADH). glomerular retention and the loss as a result of a reduced
44 The kidneys
Urea and water retained Increased urea and Normal urea load
normal water load
Figure 3.5 The effects of glomerular and tubular dysfunction on urinary output and on plasma concentrations of
retained ‘waste’ products of metabolism, the volume depending on the proportion of nephrons involved.
most common cause. It is known as renal circulatory in an inappropriately dilute urine for the degree of
insufficiency (‘pre-renal uraemia’) and may be due to: hypovolaemia. Fluid must be given with caution, and
only until volume depletion has been corrected; there is
● intravascular depletion of whole blood
a danger of overloading the circulation.
(haemorrhage) or plasma volume (usually due to
During recovery, oliguria is followed by polyuria.
gastrointestinal loss), or reduced intake,
When cortical blood flow increases, and as tubular
● reduced pressure as a result of the vascular dilatation
oedema resolves, glomerular function recovers before
caused by ‘shock’, causes of which include myocardial
that of the tubules. The biochemical findings gradually
infarction, cardiac failure and intravascular haemolysis,
progress to those of tubular dysfunction until they
including that due to mismatched blood transfusion.
approximate those for ‘pure’ tubular lesions. Urinary
The patient is usually hypotensive and clinically output is further increased by the osmotic diuretic effect
volume depleted. If renal blood flow is restored within of the high load of urea. The polyuria may cause water
a few hours, the condition is reversible, but, the longer it and electrolyte depletion. The initial hyperkalaemia may
persists, the greater the danger of intrinsic renal damage. be followed by hypokalaemia. Mild acidosis (common to
As most glomeruli are involved and tubular function is both glomerular and tubular disorders) persists until late.
relatively normal, the biochemical findings in plasma and Recovery of the tubules may restore full renal function.
urine are those described earlier. Uraemia due to renal
dysfunction may be aggravated if there is increased protein Acute oliguria due to renal outflow obstruction (post-
breakdown as a result of tissue damage, a large haematoma renal)
or the presence of blood in the gastrointestinal lumen. Oliguria or anuria (absence of urine) may occur in
Intravenous amino acid infusion may have the same post-renal failure. The cause is usually, but not always,
effect because the urea is derived, by hepatic metabolism, clinically obvious and may be due to the following:
from the amino groups of amino acids. Increased
● Intrarenal obstruction, with blockage of the tubular
tissue breakdown may also aggravate hyperkalaemia,
lumina by haemoglobin, myoglobin and, very rarely,
hyperuricaemia and hyperphosphataemia.
urate or calcium. Obstruction caused by casts and
Acute oliguria due to intrinsic renal damage oedema of tubular cells is usually the result of true
renal damage.
This may be due to:
● Extrarenal obstruction, due to calculi, neoplasms,
● prolonged renal circulatory insufficiency, for example prostate or cervix, urethral strictures
● acute glomerulonephritis, usually in children – the or prostatic hypertrophy, any of which may cause
history of a sore throat and the finding of red cells in sudden obstruction. The finding of a palpable
the urine usually make the diagnosis obvious, bladder indicates urethral obstruction, and in males
● septicaemia, which should be considered when the is most likely to be due to prostatic hypertrophy,
cause of oliguria is obscure, although there are other, rarer, causes.
● ingestion of a variety of poisons or drugs,
Early correction of outflow obstruction may rapidly
● myoglobulinuria (see Chapters 18 and 19),
increase the urine output. The longer it remains
● Bence Jones proteinuria (see Chapter 19).
untreated, the greater the danger of ischaemic or
One problem in the differential diagnosis of acute pressure damage to renal tissue. Imaging studies such
oliguria is distinguishing between renal circulatory as renal tract ultrasound may be useful to confirm post-
insufficiency and intrinsic renal damage that may have renal obstruction (Box 3.1).
followed it. Acute oliguric renal dysfunction often
follows a period of reduced GFR and renal circulatory Investigation of acute kidney injury
insufficiency. ● A careful clinical history, especially of taking
The oliguria is due to reduced cortical blood flow nephrotoxic drugs, and examination may give
with glomerular damage, aggravated by back-pressure clues to the cause of acute kidney injury (AKI). It
on the glomeruli due to obstruction to tubular flow is essential to exclude reversible causes of pre-renal
by oedema. At this stage, the concentrations of many failure, including hypovolaemia or hypotension,
constituents in plasma, such as urea and creatinine, and also post-renal urinary tract obstruction (renal
are raised with hyperkalaemia; tubular damage results tract imaging may be useful, such as abdominal
46 The kidneys
Anuria present?
Yes No
Figure 3.6 Algorithm for the investigation of acute kidney injury (AKI). FENa%, fractional excretion of sodium.
In chronic kidney disease (CKD) the functional Other abnormal findings in chronic kidney
adaptive effects can be divided into three main disease
categories: diminished renal reserve, renal insufficiency, Apart from uraemia, hyperkalaemia and metabolic
and end-stage uraemia. The loss of 75 per cent of renal acidosis, other abnormalities that may occur in CKD
tissue produces a fall in GFR of 50 per cent. Although include the following:
there is a loss of renal function, homeostasis is initially
preserved at the expense of various adaptations ● Plasma phosphate concentrations rise and
such as glomerulotubular changes and secondary plasma total calcium concentrations fall. The
hyperparathyroidism. increased hydrogen ion concentration increases
Chronic renal dysfunction may pass through two the proportion of free ionized calcium, the plasma
main phases: concentration of which does not fall in parallel with
the fall in total calcium concentration. Impaired
● an initially polyuric phase,
renal tubular function and the raised phosphate
● subsequent oliguria or anuria, sometimes needing
concentration inhibit the conversion of vitamin D
dialysis or renal transplantation.
to the active metabolite and this contributes to
the fall in plasma calcium concentration. Usually,
Polyuric phase
hypocalcaemia should be treated only after correction
At first, glomerular function may be adequate to of hyperphosphataemia. After several years of CKD,
maintain plasma urea and creatinine concentrations secondary hyperparathyroidism (see Chapter 6)
within the reference range. As more glomeruli are may cause decalcification of bone, with a rise in
involved, the rate of urea excretion falls and the plasma the plasma alkaline phosphatase activity. Some
concentration rises. This causes an osmotic diuresis in of these features of CKD can also evoke renal
functioning nephrons; in other nephrons the tubules osteodystrophy, associated with painful bones. The
may be damaged out of proportion to the glomeruli. increase in plasma PTH occurs early when the GFR
Both tubular dysfunction in nephrons with functioning falls below 60 mL/min per 1.73 m2.
glomeruli and the osmotic diuresis through intact ● Plasma urate concentrations rise in parallel with
nephrons contribute to the polyuria, other causes of plasma urea. A high plasma concentration does not
which should be excluded (see Chapter 2). necessarily indicate primary hyperuricaemia; clinical
During the polyuric phase, the plasma concentration gout is rare unless hyperuricaemia is the cause of the
of many substances, other than urea and creatinine, renal damage (see Chapter 20).
may be anywhere between the glomerular and tubular ● Hypermagnesaemia can also occur (see Chapter 6).
ends of the spectrum, although metabolic acidosis is ● Normochromic, normocytic anaemia due to
usually present. erythropoietin deficiency is common and, because
haemopoiesis is impaired, does not respond to
Oliguric phase iron therapy; this can be treated with recombinant
If nephron destruction continues, the findings become erythropoietin.
more like those of pure glomerular dysfunction. ● One of the commonest causes of death in patients
Glomerular filtration decreases significantly and urine with CKD is cardiovascular disease, in part
output falls; oliguria precipitates a steep rise in plasma explained by hypertension and a dyslipidaemia
urea, creatinine and potassium concentrations; and the of hypertriglyceridaemia and low high-density
metabolic acidosis becomes more severe. lipoprotein cholesterol. Some of these effects may be
The diagnosis of CKD is usually obvious. In due to reduced lipoprotein lipase activity.
the early phase, before plasma urea and creatinine ● Abnormal endocrine function, such as
concentrations have risen significantly, there may hyperprolactinaemia, insulin resistance, low plasma
be microscopic haematuria or proteinuria. However, testosterone and abnormal thyroid function, may
haematuria may originate from either the kidney also be seen in chronic renal dysfunction.
or the urinary tract, and may therefore indicate ● Some of the features of CKD may be explained by the
the presence of other conditions, such as urinary presence of ‘middle molecules’ – compounds that the
tract infections, renal calculi or tumours (see Box kidneys would normally excrete. These compounds,
3.2).
Diagnosis of renal dysfunction 49
Measurement of plasma concentrations of urea and If the plasma concentration of either urea or
creatinine creatinine is significantly raised, and especially if it is
Urea is derived in the liver from amino acids and rising, impaired glomerular function is likely. Serial
therefore from protein, whether originating from the changes may be used to monitor changes in the GFR
diet or from tissues. The normal kidney can excrete and changes greater than 10–15 per cent are likely to be
large amounts of urea. If the rate of production exceeds clinically significant.
the rate of clearance, plasma concentrations rise. The With a reduced GFR, plasma urea concentrations
rate of production is accelerated by: tend to rise faster than those of creatinine and tend to
be disproportionately higher with respect to the upper
● a high-protein diet,
reference limit. The rate at which urea is reabsorbed
● absorption of amino acids and peptides from digested
from the collecting ducts is dependent on the amount
blood after haemorrhage into the gastrointestinal
filtered by the glomerulus and by the rate of luminal
lumen or soft tissues,
fluid flow (see Table 3.2).
● increased catabolism due to starvation, tissue
damage, sepsis or steroid treatment. Clearance as an assessment of glomerular filtration
In catabolic states, glomerular function is often rate (Fig. 3.7)
impaired due to circulatory factors and this contributes For a substance (S) that is filtered by the glomerulus,
more to the uraemia than does increased production. but not reabsorbed from or secreted into the tubules,
Conversely, the plasma urea concentration may be the amount filtered (GFR ¥ plasma[S]) must equal the
lower than 1.0 mmol/L, the causes of which include the amount excreted in the urine (urinary[S] ¥ volume per
following: unit time):
● those due to increased GFR or haemodilution GFR ¥ plasma[S]
(common): = urinary[S] ¥ urine volume per unit time (3.2)
– pregnancy (the commonest cause in young Thus, rearranging gives:
women),
– overenthusiastic intravenous infusion (the urinary[S] ¥ urine volume per unit time
GFR = (3.3)
commonest cause in hospital patients), plasma[S]
– ‘inappropriate’ ADH secretion (syndrome of
inappropriate ADH secretion, SIADH).
● those due to decreased synthesis:
400
– use of amino acids for protein anabolism
during growth, especially in children,
– low protein intake,
– very severe liver disease (low amino acid 300
Plasma creatinine (µmol/L)
deamination),
– inborn errors of the urea cycle are rare and
usually only occur in infants.
200
Creatinine is largely derived from endogenous sources
by muscle creatine breakdown. Plasma creatinine usually
correlates with muscle mass, with 95 per cent of creatine
occurring in skeletal muscle. The plasma creatinine 100
The GFR thus measured is referred to as the clearance Creatinine clearance is higher than inulin clearance
– the volume of plasma that could theoretically be because some creatinine is secreted by the tubules.
completely cleared of a substance in 1 min. Only Urea clearance is lower than inulin clearance as some
substances freely filtered by glomeruli and not acted on urea is reabsorbed into the tubules (urea and inulin
by the tubules can be used to give true measurement clearance are now essentially obsolete in clinical
of GFR. There is no such endogenous substance, but practice).
inulin, a polysaccharide, fulfils the criteria closely. However, there are various factors that make the
Inulin is not produced by the body; it must be given measurement of creatinine clearance inaccurate:
either by constant infusion in order to maintain steady
● All laboratory assays have an inherent imprecision.
plasma concentrations during the period of the test, or
The combined imprecision of two assays is greater
by a single injection followed by serial blood sampling
than that of one. Urine as well as plasma is assayed
to enable the concentration at the midpoint of the
for clearance measurements.
collection to be calculated.
● The most significant error of any method depending
Radiochromium-labelled ethylenediamine tetra-
on a timed urine collection is in the measurement
acetic acid (EDTA) is another exogenous compound
of urine volume. Inaccurate urine collection
that some consider the ‘gold standard’ for calculating
may yield misleading results. The difficulties are
patient GFR, although this requires the use of nuclear
increased in infants and young children, and in
medicine tests and is rarely used.
patients who have difficulty in bladder emptying
For endogenously produced substances such as
or are incontinent.
creatinine, with its relatively constant production, the
● Both creatinine and urea may be partly destroyed by
following equation can be used to calculate a clearance
bacterial action in infected or old urine.
that acts as an approximation for GFR:
For an individual patient, plasma creatinine
Creatinine clearance (mL/min)
concentrations may rise above the baseline level but
urinary [creatinine] ¥ urine volume (mL)
= remain within the population reference range despite a
plasma [creatinine] ¥ urine collection period (min)
deterioration in glomerular function. The reciprocal of
(3.4)
the plasma creatinine concentration is called the renal
The modification of diet in renal disease (MDRD) index.
formula can be used to estimate GFR (eGFR) and The plasma creatinine concentration may not
has generally superseded the need to use creatinine exceed the upper limit of the reference range until the
clearances in clinical practice and is also used to titrate GFR, and therefore the creatinine clearance, has been
drug dosing in patients with renal impairment. This is reduced by approximately 60 per cent (see Fig. 3.7).
calculated by the isotope dilution mass spectrometry Thus the measurement of creatinine clearance should
(IDMS) traceable MDRD equation recommended be a more sensitive but less accurate indicator of early
in the UK as: 175 ¥ ([plasma creatinine]/88.4)–1.154 ¥ glomerular dysfunction than of plasma creatinine
(age)–0.203 ¥ (0.742 if female) ¥ (1.210 if black). concentration.
The equation has not been validated in the following Clearance values will be low whether the reduced
groups: those under 18 years old, those acutely ill, GFR is due to renal circulatory insufficiency, intrinsic
patients with limb amputations, pregnant women, renal damage or ‘post-renal’ causes, and it cannot
the very elderly and the obese and malnourished. In distinguish among them. Creatinine clearance has
some of these situations there may be differences in been said to be useful in deciding the dose of a renally
muscle mass and hence in creatine concentrations and excreted drug.
ultimately creatinine. Those with muscle breakdown
may show higher plasma creatinine concentration Cystatin C
and the converse may be seen in those with reduced Another endogenous substance that can be used as a
muscle bulk. There may be increased muscle bulk in marker of GFR is plasma cystatin C (Cys C), and its
black compared with white people. Individuals taking use may alleviate some of the problems associated
creatine supplements for body building may show with creatinine clearance determinations. This is a
increased plasma creatinine and also plasma creatine 13-kDa protein that is a member of the family of
kinase (CK). cystine proteinase inhibitors. Unlike other endogenous
52 The kidneys
compounds such as creatinine, Cys C is not secreted are increased in the urine because of reduced tubular
by the renal tubules and does not return to the reabsorption and increased renal tubular secretion. If
bloodstream after glomerular filtration. It has been there is detectable glycosuria, phosphaturia and non-
suggested that plasma Cys C may approximate to selective amino aciduria, the condition is known as
the ‘ideal’ endogenous marker for GFR, as blood Fanconi’s syndrome.
concentrations are independent of patient age and sex,
although currently this test is not routinely available in Distal tubular function tests
most laboratories. Impaired distal tubular function primarily affects
urine acidification, with a failure to excrete hydrogen
Renal tubular function tests
ions; the urinary pH rarely falls below 5.5. There
Reduced tubular function, with normal glomerular is an impaired response to aldosterone involving
function, impairs the adjustment of the composition and reabsorption of sodium, and the urine contains an
volume of the urine with minimal effect on the plasma inappropriately high concentration of sodium for the
urea or creatinine concentration. The investigations renal blood flow. The associated findings may include
used to diagnose tubular disorders can be divided into the following.
those that predominantly identify proximal tubular
dysfunction and those that predominantly identify Plasma
distal tubular dysfunction. ● Low bicarbonate and high chloride concentration with
low pH (hyperchloraemic acidosis), hypokalaemia.
Proximal tubular function tests
Urine
Impaired solute reabsorption from the proximal tubules
● Increased volume.
reduces isosmotic water reabsorption. Countercurrent
● pH inappropriately high.
multiplication may also be affected, and hence the
● An inappropriately high sodium concentration, even
ability to respond to ADH is reduced. A large volume of
if renal blood flow is low (inability to respond to
inappropriately dilute urine is produced.
aldosterone).
The tubules cannot secrete hydrogen ions and so
● An inappropriately low urea concentration, and
cannot reabsorb bicarbonate normally and therefore
therefore osmolality, even if ADH secretion is
the urine is inappropriately alkaline for the degree of
stimulated.
acidosis in the blood.
The reabsorption of potassium, phosphate, The ability to form concentrated urine in response
magnesium, urate, glucose and amino acids is impaired. to fluid deprivation depends on normal tubular
The following findings may be present, and measurement function (countercurrent multiplication) and on
may occasionally be useful. the presence of ADH. Failure of this ability is usually
Plasma
due to renal disease, or cranial diabetes insipidus (see
Chapter 2 for discussion of the fluid deprivation test).
● Normal urea and creatinine concentrations (normal The investigation of renal tubular acidosis is covered in
glomerular function). Chapter 4.
● Low bicarbonate concentration with low pH
(metabolic acidosis). URINARY SODIUM AND OSMOLALITY
● Hypokalaemia, hypophosphataemia, hypomagnes- Urinary sodium estimation
aemia and hypouricaemia.
Urinary sodium estimation may be used to differentiate
Urine
acute oliguria due to renal damage from that due to
renal circulatory insufficiency. Aldosterone secretion
● Increased volume (polyuria). will be maximal only if renal blood flow is reduced;
● pH may be inappropriately high. in such circumstances, functioning tubules respond
● Phosphaturia, glycosuria, uricosuria. appropriately by selectively reabsorbing sodium by
● Generalized amino aciduria. distal tubular exchange mechanisms. A urinary sodium
Tubular proteinuria can be diagnosed by measuring concentration of less than about 20 mmol/L is usually
specific low-molecular-weight proteins such as retinol- taken to indicate that tubular function is not significantly
binding protein, NAG or a1-microglobulin, which impaired.
Biochemical principles of the treatment of renal dysfunction 53
Negative pressure on the dialysate side of the membrane – a high rate of excretion of the metabolic
can be varied to adjust the amount of water removed. product forming the stone, due either to
● In intermittent and continuous ambulatory high plasma and therefore filtrate levels or to
peritoneal dialysis, the folds of the peritoneum are impairment of normal tubular reabsorption
used as the dialysing membrane with capillaries from the filtrate.
on one side, and an appropriate fluid of higher ● Changes in pH of the urine, often due to bacterial
osmolality is infused into the peritoneal cavity on the infection, which favour precipitation of different
other. After a suitable time to allow for equilibration salts at different hydrogen ion concentrations.
of diffusible solutes, depending on the type of ● Urinary stagnation due to obstruction to urinary
peritoneal dialysis, the peritoneal cavity is drained outflow or renal tract structural abnormality.
and the cycle is repeated. ● Lack of normal inhibitors: urine normally contains
inhibitors, such as citrate, pyrophosphate and
Dialysis is used in some cases of acute kidney injury
glycoproteins, which inhibit the growth of calcium
until renal function improves, or as a regularly repeated
phosphate and calcium oxalate crystals respectively.
procedure in suitable cases of end-stage kidney disease.
Hypocitraturia may partly explain the renal calculi
It may also be used to prepare patients for renal
found in distal or type 1 renal tubular acidosis (see
transplantation.
Chapter 4).
RENAL CALCULI
Renal calculi are usually composed of products of Constituents of urinary calculi
metabolism present in normal glomerular filtrate, often at Renal calculi may consist of the following (Box 3.3):
concentrations near their maximum solubility (Fig. 3.8).
● calcium-containing salts:
Conditions favouring renal calculus – calcium oxalate,
formation – calcium phosphate,
● A high urinary concentration of one or more ● urate,
constituents of the glomerular filtrate, due to: ● cystine,
– a low urinary volume with normal renal ● xanthine.
function, because of restricted fluid intake or
excessive fluid loss over a long period of time Calculi composed of calcium salts
(particularly common in hot climates) – this About 80 per cent of all renal stones contain calcium.
favours formation of most types of calculi, Precipitation is favoured by hypercalciuria, and the type
especially if one of the other conditions listed of salt depends on urinary pH and on the availability
below is also present, of oxalate. Any patient presenting with calcium-
containing calculi should have plasma calcium and
phosphate estimations performed, and, if the results are
normal, they should be repeated at regular intervals to
exclude primary hyperparathyroidism.
Hypercalcaemia causes hypercalciuria if glomerular
function is normal. The causes and differential
Yes No
Evidence of hyperuricaemia?
Yes No
Yes No
Assess 24-h
urine oxalate
High Low/normal
Hyperoxaluria Measure
urine cystine
High Low/normal
Cystinuria Consider
rare calculi
(see Box 3.3)
SUMMARY
● The kidneys are vital organs for the excretion and, as renal reserve declines, there is further
of various waste products as well as for acid– hyperkalaemia, hyperphosphataemia, metabolic
base balance, fluid volume control, hormone acidosis, hypocalcaemia and anaemia. This may
production and metabolic function, such as necessitate renal support such as dialysis.
calcium homeostasis. ● Renal calculi can be the result of urinary stasis or
● Plasma creatinine determination is a useful test of infection associated with urinary supersaturation.
renal function, but plasma creatinine concentration The commonest calculi are calcium containing.
can still remain within the reference range in the ● Nephrotic syndrome is defined as gross proteinuria
presence of a significant decline in renal function. associated with oedema and hypoproteinaemia
● Acute kidney injury (AKI) can be due to pre-renal, (discussed further in Chapter 19). This is a disorder
renal or post-renal causes. Raised plasma urea of the renal glomerular membrane.
and creatinine concentrations occur along with ● Renal tubular disease can result in Fanconi’s
fluid retention, anuria or oliguria, hyperkalaemia, syndrome associated with acid–base and
hyperphosphataemia and metabolic acidosis. potassium disturbance, glycosuria, amino aciduria,
● End-stage chronic kidney disease (CKD5) implies hypouricaemia and hypophosphataemia.
slow, irreversible renal disease. Raised plasma ● Renal replacement therapy, such as dialysis
urea and creatinine concentrations occur initially (Fig. 3.10), may be indicated in AKI and CKD5.
58 The kidneys
Figure 3.10 A renal dialysis machine used to give renal replacement therapy in some patients with end-stage
chronic kidney disease (CKD5). Reproduced with kind permission of Pemed.
4 Acid–base disturbances
The importance of acid–base homeostasis cannot A strong acid is almost completely dissociated
be overstated because of its importance in keeping in aqueous solution, and so produces many H+. For
hydrogen ion (H+) balance under control. Acid–base example, hydrochloric acid is a strong acid and is
abnormalities are relatively common medically, and it almost entirely dissociated in water to form H+ and
is therefore essential for clinicians to be fully conversant chloride (Cl–). Weak acids dissociate less, although
with their interpretation. very small changes in [H+] may have important
Our cells release between 50 and 100 mmol of H+ consequences.
into the extracellular fluid (ECF) daily. Despite this, the Buffering is a process by which a strong acid (or
extracellular H+ concentration ([H+]) is maintained at base) is replaced by a weaker one, with a consequent
about 40 nmol/L (pH 7.4). reduction in the number of free H+, and therefore the
The predominant sources of H+ are: change in pH, after addition of acid, is less than it would
be in the absence of the buffer. For example:
● Anaerobic carbohydrate metabolism produces
lactate, and anaerobic metabolism of fatty acids H+Cl– + NaHCO3 ´ H2CO3 + NaCl
by b-hydroxylation and of ketogenic amino acids (strong acid) (buffer) (weak acid) (neutral salt)
produces acetoacetate, which releases equimolar (4.1)
amounts of H+. Lactic acidosis or ketoacidosis can
The pH is a measure of H+ activity. It is log 10 of the
occur if the release of H+ by these reactions exceeds
reciprocal of [H+] in mol/L. The log 10 of a number is
the compensatory capacity.
the power to which 10 must be raised to produce that
● Release of H+ can occur during conversion of amino
number:
nitrogen to urea in the liver, or of the sulphydryl
groups of some amino acids to sulphate. log 100 = log 102 = 2, and log 107 = 7 (4.2)
Hydrogen ion balance is largely dependent upon If [H+] is 10–7 (0.0000001) mol/L, then –log [H+] = 7.
the secretion of H+ from the body into the urine due But:
to renal tubular action. Aerobic metabolism of the
carbon skeletons of organic compounds converts 1
pH = log [H+] = – log [H+] (4.3)
carbon, hydrogen and oxygen into carbon dioxide
and water. Carbon dioxide is an essential component
Therefore pH = 7.
of the extracellular buffering system. Thus the body is
A change of 1 pH unit represents a 10-fold change
dependent upon healthy function of the kidneys and
in [H+]. Changes of this magnitude do not normally
the lungs for normal acid–base homeostasis.
occur in tissues. However, in pathological conditions
the blood pH can change by more than 0.3 of a unit;
DEFINITIONS a decrease of pH by 0.3, from 7.4 to 7.1, represents a
Acids can dissociate to produce H+ (protons), which doubling of the [H+] from 40 to 80 nmol/L. Thus, the
can be accepted by a base. An alkali dissociates to use of the pH notation makes a very significant change
produce hydroxyl ions (OH–). Acidosis is commoner in [H+] appear deceptively small.
than alkalosis because metabolism tends to produce H+ A blood pH of 7.0 indicates a severe acidosis. A blood
rather than OH–. pH of 7.7 similarly indicates a severe alkalosis. Urinary
60 Acid–base disturbances
pH is much more variable than that of blood, and [H+] of the weak acid of the buffer pair causes only a
can vary 1000-fold (a change of 3 pH units). small change in pH (see Henderson–Hasselbalch
The Henderson–Hasselbalch equation expresses the equation). If H+ are not completely neutralized
relation between pH and a buffer pair – that is, a weak or eliminated from the body, and if production
acid and its conjugate base. The equation is valid for continues, buffering power will eventually be so
any buffer pair, the pH being dependent on the ratio depleted that the pH will change significantly.
of the concentration of base to acid. Note that pKa is ● There are two main ways by which H+ can be lost
the negative logarithm of the acid dissociation constant from the body: through the kidneys or some of the
(Ka), and, the larger the value of pKa, the smaller the intestine, mainly the stomach. This mechanism is
extent of acid dissociation: coupled with the generation of HCO3–. In the kidney
[base] this is the method by which secretion of excess H+
pH = pKa + log [acid] (4.4) ensures regeneration of buffering capacity.
4. The rate of respiration, and therefore the rate of CO2 Therefore, an increase in intracellular PCO2 or a
elimination, is controlled by chemoreceptors in the decrease in intracellular [HCO3–] in the erythrocytes
respiratory centre in the medulla of the brainstem and renal tubular cells maintains the extracellular
and by those in the carotid and aortic bodies. The [HCO3–] by accelerating the production of HCO3–. This
receptors respond to changes in the [CO2] or [H+] minimizes changes in the ratio of [HCO2–] to PCO2 and
of plasma or of the cerebrospinal fluid. If the PCO2 therefore changes in pH.
rises much above 5.3 kPa, or if the pH falls, the rate In the normal subject, at a plasma PCO2 of 5.3 kPa
of respiration increases. (a [CO2] of about 1.2 mmol/L), erythrocytes and
renal tubular cells maintain the extracellular HCO3–
Normal lungs have a very large reserve capacity for
at about 25 mmol/L. The extracellular ratio of
elimination of CO2. Not only is there a plentiful supply
[HCO3–] to [CO2] (both in mmol/L) is just over 20:1.
of CO2, the denominator in the Henderson–Hasselbalch
It can be calculated from the Henderson–Hasselbalch
equation, but the normal respiratory centre and lungs
equation, and with a pKa of 6.1 this represents a pH
can control its concentration within narrow limits
very near 7.4. An increase of intracellular PCO2, or a
by responding to changes in the [H+] and therefore
decrease in intracellular [HCO3–], accelerates HCO3–
compensate for changes in acid–base disturbances.
production and minimizes changes in the ratio and
Bicarbonate buffer system therefore in pH.
The control of bicarbonate by the kidneys and
erythrocytes
Bicarbonate generation by the erythrocytes (Fig. 4.1)
The erythrocytes and renal tubular cells generate
Haemoglobin is an important blood buffer. However,
HCO3–, the buffer base in the bicarbonate system, from
it only works effectively in cooperation with the
CO2. Under physiological conditions:
bicarbonate system:
● the erythrocyte mechanism makes fine adjustments [Hb–]
to the plasma [HCO3–] in response to changes in pH = pKa + log [HHb] (4.9)
PCO2 in the lungs and tissues,
● the kidneys play the major role in maintaining the Erythrocytes produce little CO2 as they lack aerobic
circulating [HCO3–] and in eliminating H+ from the pathways. Plasma CO2 diffuses along a concentration
body. gradient into erythrocytes, where CD catalyses its
reaction with water to form carbonic acid (H2CO3),
The carbonate dehydratase system which then dissociates. Much of the H+ is buffered by
Bicarbonate is produced following the dissociation Hb, and the HCO3– diffuses out into the extracellular
of carbonic acid formed from CO2 and H2O. This is fluid along a concentration gradient.
catalysed by carbonate dehydratase (CD; carbonic Electrochemical neutrality is maintained by
anhydrase), which is present in high concentrations in diffusion of Cl– in the opposite direction into cells. This
erythrocytes and renal tubular cells: movement of ions is known as the ‘chloride shift’.
CD
Bicarbonate reclamation
Normal urine is nearly HCO3– free. An amount Cellular CO2
action of CD, both in the brush border on the luminal ● A fall of [HCO3–]: reduction of extracellular [HCO3–],
surfaces and within tubular cells, and on H+ secreted by increasing the concentration gradient across renal
into the lumina in exchange for sodium (Na+). This tubular cell membranes, increases the loss of HCO3–
occurs predominantly in the proximal tubules but also from cells.
in the first part of the distal tubules:
Normally almost all the filtered HCO3– is reabsorbed.
● Bicarbonate is filtered through the glomeruli at a Once the luminal fluid is HCO3– free, continued
plasma concentration of about 25 mmol/L. secretion of H+ and the intracellular generation of
● Filtered HCO3– combines with H+, secreted by HCO3– depend on the presence of other filtered buffer
tubular cells, to form H2CO3. bases (B–). In their absence, the luminal acidity would
● The H2CO3 dissociates to form CO2 and water. In the increase so much that further H+ secretion would be
proximal tubules this reaction is catalysed by CD in inhibited. These buffers, unlike HCO3–, do not form
the brush border. In the distal tubules, where the pH is compounds capable of diffusing back into tubular cells,
usually lower, H2CO3 probably dissociates spontaneously. nor is H+ incorporated into water.
● As the luminal PCO2 rises, CO2 diffuses into tubular There is net loss of H+ in urine as HB. The HCO3–
cells along a concentration gradient, and as the formed in the cell is derived from cellular CO2 and
intracellular concentration of CO2 rises, CD catalyses therefore represents a net gain in HCO3–. Whenever
its combination with water to form H2CO3, which 1 mmol of H+ is secreted into the tubular lumen,
dissociates into H+ and HCO3–. 1 mmol of HCO3– passes into the ECF with Na+. The
● Hydrogen ions are secreted in the tubular lumina mechanism of HCO3– generation is very similar to that
in exchange for Na+ and so the HCO3–-generating in erythrocytes. However, unlike red cells, renal tubular
reactions start again from the second stage. As the cells are exposed to a relatively constant PCO2. Bicarbonate
intracellular concentration of HCO3– rises, HCO3– generation, coupled with H+ secretion, becomes very
diffuses into the ECF accompanied by Na+, which important in acidosis, when it is stimulated by either a
has been reabsorbed in exchange for H+. fall in extracellular [HCO3–] (metabolic acidosis) or a
rise in extracellular PCO2 (respiratory acidosis).
This self-perpetuating cycle reclaims buffering
capacity that would otherwise have been lost from the
Urinary buffers
body by glomerular filtration. The secreted H+ is derived
from cellular water, and is incorporated into water in the Apart from HCO3–, the two other major urinary buffers
lumina. Because there is no net change in H+ balance and are phosphate and ammonia (NH3); they are also
no net gain of HCO3–, this mechanism cannot correct an involved in HCO3– generation.
acidosis but can maintain a steady state.
Phosphate buffer pair
Bicarbonate generation At pH 7.4, most of the phosphate in plasma, and also
The mechanism in renal tubular cells for generating in the glomerular filtrate, is monohydrogen phosphate
HCO3– is identical to that of HCO3– reabsorption, but (HPO42–), which can accept H+ to become dihydrogen
there is net loss of H+ from the body as well as a net gain phosphate (H2PO4–). Bicarbonate can continue to
of HCO3–. Therefore this mechanism is well suited to be generated within tubular cells, with H+, and to be
correcting any type of acidosis. returned to the body after all that in the filtrate has
Within tubular cells, CD may be stimulated by the been reabsorbed. Therefore it can help to replace that
following. used in extracellular buffering. The pKa of this buffer
pair is about 6.8:
● A rise in PCO2: in this case, the rise in [CO2] is the
[HPO42–]
indirect result of a rise in the extracellular PCO2. Renal pH = 6.8 + log (4.10)
tubular cells, unlike erythrocytes with anaerobic [H2PO4–]
pathways, constantly produce CO2 aerobically. This Phosphate is normally the most important buffer in
diffuses out of cells into the extracellular fluid along the urine because its pKa is relatively close to the pH of
a concentration gradient. An increase in extracellular the glomerular filtrate and because the concentration
PCO2, by reducing the gradient, slows this diffusion of phosphate increases 20-fold, to nearly 25 mmol/L, as
and the intracellular PCO2 rises. water is reabsorbed from the tubular lumen.
64 Acid–base disturbances
Even in a mild acidosis, more phosphate ions are Explanations for the role of NH3 in the correction of
released from bone than at normal pH; the need for acidosis include the following:
increased urinary H+ secretion is linked with increased
● The fate of the GluCOO– produced at the same
buffering capacity in the glomerular filtrate owing to the
time as the NH4+. After further deamination to
increase of phosphate. At a urinary pH below 5.5, most
2-oxoglutarate, it can be converted to glucose;
of the filtered phosphate is converted to dihydrogen
gluconeogenesis uses an amount of H+ equivalent to
phosphate. Therefore, at low pH, urinary phosphate
that used in the generation of NH4+ produced from
cannot maintain the essential buffering of continued H+
glutamine. Therefore, the H+ liberated into the cell
secretion. The predominant urinary anion is Cl–, but,
may be incorporated into glucose.
because hydrochloric acid is almost completely ionized
● A shift from urea synthesis to glutamine production
in aqueous solution, it cannot act as a buffer.
in the liver with a fall in systemic H+ production in
The role of ammonia the presence of an acidosis. This is a minor factor.
As the urine becomes more acid, it can be shown to The rate of gluconeogenesis and glutamine synthesis
contain increasing amounts of ammonium ion (NH4+). and glutaminase activity all increase in an acidosis.
Urinary ammonia probably allows H+ secretion, and CI– Na+ –
Na+ HCO3
therefore HCO3– formation, to continue after other
buffers have been depleted.
Ammonia, produced by hepatic deamination of
amino acids, is rapidly incorporated into urea, with a net
production of H+. However, as the systemic [H+] increases,
there is some shift from urea to glutamine (GluCONH2)
synthesis, with a slight fall in hepatic H+ production. Na+ HCO3
–
CI– Na+
Glutamine is taken up by renal tubular cells, where it
is hydrolysed by glutaminase to glutamate (GluCOO–) H+ H+
and NH4+:
H2O + GluCONH2 Æ GluCOO – + NH4+ (4.11)
H2CO3
Ammonia and NH4+ form a buffer pair with a pKa of
about 9.8: CD
● Na+ exchange for H+, by a mechanism that is the Net effect on plasma – gain of bicarbonate and loss of chloride
opposite of that in renal tubular cells,
● passage of Cl– with H+.
Plasma Pancreatic or Proximal small
biliary cell intestinal lumen
Acid secretion by the stomach Na+ Na+
The parietal cells of the stomach secrete H into the
+
More than 80 per cent of plasma anions are accounted In renal glomerular dysfunction (see Chapter 3),
for by Cl– and HCO3–; the remaining 20 per cent or so even if tubular function is normal, HCO3– generation
(sometimes referred to as unmeasured anion) is made is impaired because the amount of Na+ available
up of protein, and the normally low concentrations of for exchange with H+ and the amount of filtered
urate, phosphate, sulphate, lactate and other organic buffer A– available to accept H+ are both reduced.
anions. These buffer anions contribute to the unmeasured
The protein concentration remains relatively A–. For each milliequivalent of buffer A– retained,
constant, but the concentrations of other unmeasured 1 mEq fewer H+ can be secreted, and therefore 1 mEq
anions can vary considerably in disease. fewer HCO3– is generated. The retained A– therefore
The anion gap, represented as A– in the following replaces HCO3–.
equations, is the difference between the total In the following example, the plasma [HCO3–] will
concentration of measured cations (Na+ and K+) and be assumed to have fallen by 10 mmol(mEq)/L:
measured anions (Cl– and HCO3–); it is normally about [Na+] + [K+] = [HCO–3] + [Cl–] + [A–]
15–20 mEq/L. Therefore: 140 + 4 = 15 + 100 + 29 mEq/L
[Na+] + [K+] = [HCO–3] + [Cl–] + [A–] (4.14)
140 + 4 = 25 + 100 +19 mEq/L The [HCO ] has fallen from 25 to 15 mEq/L and the
–
3
(4.13) anion gap, entirely due to [A–], has risen by the same
As we will see later, the urinary anion gap can also be amount, from 19 to 29 mEq/L, that is, a high anion gap.
measured and is useful in the diagnosis of renal tubular If renal HCO3– generation is so impaired that it cannot
acidosis (Fig. 4.6). keep pace with its peripheral utilization, the pH will fall.
It is worth noting that the plasma anion gap can be
High anion gap acidosis decreased by an increase in unmeasured cations, e.g.
A useful mnemonic to help remember some of the causes hyperkalaemia, hypercalcaemia, hypermagnesaemia,
of a high anion gap metabolic acidosis is DR MAPLES: high immunoglobulin G (IgG), bromide or lithium
D = diabetic ketoacidosis, R = renal, M = methanol, intoxication, or by a decrease in unmeasured anions,
A = alcoholic ketoacidosis, P = paracetamol, L = lactic for example hypoalbuminaemia.
acidosis, E = ethylene glycol, S = salicylates.
If the number of negatively charged ions (in this Lactic acidosis
case HCO3–) is reduced, electrochemical neutrality
Lactic acidosis is a common form of high anion gap
is maintained by replacing them with an equivalent
metabolic acidosis; some of the causes are shown in
number of other anion(s) or with ketone bodies
Box 4.2. One definition used is an arterial pH of less
(acetoacetate and 3-hydroxybutyrate) in ketoacidosis
than 7.2 with a plasma lactate concentration greater
or lactate in lactic acidosis. Other causes of unmeasured
than 5.0 mmol/L. The normal fasting blood lactate
anion (A–) are salicylate in aspirin overdose and
is between 0.4 and 1.0 mmol/L, which can rise to
metabolites from methanol, paracetamol or ethylene
1.5 mmol/L on prolonged exercise (hyperlactataemia).
glycol poisoning.
Most forms of lactic acidosis are due to increased
L-lactate, although in some malabsorption states, with
bacterial overgrowth, D-lactic acidosis may occur.
ECF RENAL TUBULAR CELL The Cohen and Woods classification of lactic acidosis
‘Added’ H+ + HCO3– –
HCO3 + H+ Urine is as follows. The commonest type of lactic acidosis is
type A, due to poor tissue perfusion or hypoxia. Type
B is lactic acidosis not due to tissue underperfusion.
H2CO3 H2CO3 Type B1 is due to organ or tissue dysfunction including
severe hepatic disturbances as the liver is involved in
lactate metabolism. Metastatic malignant disease
Lungs CO2 + H2O H2O Metabolic CO2
can also evoke a lactic acidosis, probably because the
Figure 4.6 Hydrogen ion ‘shuttle’ between the site of carcinoma tissue produces lactic acid. Type B2 lactic
production and buffering and the site of elimination in acidosis is due to drugs and toxins. Type B3 is due to
the kidneys. ECF, extracellular fluid. inborn errors of metabolism.
68 Acid–base disturbances
permeable to H+, and this impairs the ability of distal In renal tubular acidosis type II there is impairment
tubules to build up a [H+] gradient between the tubular of HCO3– reabsorption in the proximal tubule. Loss
lumina and cells. The re-entry of H+ into distal tubular of HCO3– may cause systemic acidosis, but the ability
cells inhibits CD activity at that site; proximal HCO3– to form acid urine when acidosis becomes severe is
reabsorption is normal. The inability to acidify the retained; the response to NH4Cl loading may therefore
urine normally can be demonstrated by using the be normal. Type II renal tubular acidosis is also
furosemide test or an NH4Cl load. associated with amino aciduria, phosphaturia and
glycosuria, as in Fanconi’s syndrome.
Renal tubular acidosis type IV is associated with
CASE 2 hyporeninism hypoaldosteronism (see Chapter 8)
and with a hyperkalaemic hyperchloraemic acidosis.
A 52-year-old woman with Sjögren’s syndrome and
Sometimes plasma renin and aldosterone measurement
distal or type I renal tubular acidosis attended a renal
may be useful to confirm this.
out-patient clinic. Her blood results were as follows:
Plasma Investigation of renal tubular acidosis
Sodium 144 mmol/L (135–145) ● Measure the urinary anion gap and pH in a fresh
Potassium 3.0 mmol/L (3.5–5.0) spot urine sample along with the plasma anion gap.
Bicarbonate 13 mmol/L (24–32) In practice, the urinary anion gap is the difference
Chloride 118 mmol/L (95–105) between the sum of the urinary [Na+] and [K+]
DISCUSSION minus the urinary [Cl–].
The results are suggestive of a normal anion gap ● A urinary anion gap of less than 100 mmol/L implies
metabolic acidosis or hyperchloraemic acidosis. low urinary [NH4+].
Hypokalaemia is unusual in the face of an acidosis, ● In such cases a urinary pH of more than 5.5 in the
one of the exceptions being renal tubular acidosis presence of a hyperchloraemic metabolic acidosis
type I or II. Note that the anion gap here is and hypokalaemia is suggestive of type I, or distal,
(144 + 3) – (13 + 118) = 16 mmol/L, which is a normal renal tubular acidosis. Conversely, a urinary pH of
gap although with high plasma [Cl–] and low [HCO3–]. less than 5.5 in the presence of hyperchloraemic
metabolic acidosis and hyperkalaemia is suggestive
of renal tubular acidosis type IV. Plasma aldosterone
and renin concentrations may show hyporeninism
Box 4.4 Some causes of type I and type II hypoaldosteronism in renal tubular acidosis type IV.
renal tubular acidosis ● A urinary anion gap of more than 100 mmol/L
implies high urinary [NH4+].
Type I (failure of distal tubule secretion of hydrogen
● Measure fractional excretion of HCO3– (FE% HCO3–),
ions)
which equals:
Primary: sporadic/hereditary
Genetic: Wilson’s disease urine[HCO3–] ¥ plasma[creatinine]
Dysproteinaemia: amyloidosis, cryoglobulinaemia
¥ 100% (4.16)
plasma[HCO3–] ¥ urine[creatinine]
Renal diseases: chronic pyelonephritis, renal
transplants which is normally less than 5 per cent.
Autoimmune disorders: Sjögren’s syndrome, chronic ● If the fractional excretion of HCO3– is increased,
active hepatitis this is suggestive of renal tubular acidosis type II,
Hyperparathyroidism particularly if glycosuria, hypophosphaturia and
Drugs/toxins: lithium, amphotericin hypophosphataemia are also present.
Type II (defect of proximal tubule bicarbonate ● Sometimes a diagnosis of acidosis is difficult to
reabsorption) establish and additional tests are indicated. See
Primary: familial Table 4.1.
Secondary
Myeloma Furosemide screening test
Amyloidosis
Heavy metal poisons The test relies on the fact that increased Na+ delivery
to the distal tubules results in exchange for H+ and K+,
70 Acid–base disturbances
Table 4.1 Biochemical features of renal tubular does not occur. Urinary acidification is normal in
acidosis (RTA) proximal tubular acidosis.
This test can cause nausea and vomiting and is
Classic Proximal Type
dRTA type I RTA type II IV RTA contraindicated in liver disease.
Urine pH > 5.5 < 5.5 < 5.5
Management of metabolic acidosis
Urine anion gap Positive Negative Positive
The biochemical findings in plasma in a metabolic
FE% HCO3– < 5–10% > 15% 5–15%
acidosis are:
Furosemide test Abnormal Normal Normal
Urine calcium Normal/high Normal Normal
● plasma [HCO3–] is always low,
● PCO2 is usually low (compensatory change),
Urine citrate Low Normal Normal
● the pH is low (uncompensated or partly
Nephrocalcinosis Common Rare Rare
compensated) or near normal (fully compensated),
Metabolic bone disease Rare Common Rare ● plasma [Cl–] is unaffected in most cases unless
Other tubular defects Rare Common Rare there is hyperchloraemic acidosis, for example it is
Plasma potassium Normal/low Normal/low High raised after ureteric transplantation, saline excess,
dRTA, distal renal tubular acidosis; FE% HCO3–, fractional excretion diarrhoea/fistulae, in renal tubular acidosis or during
of bicarbonate. acetazolamide treatment.
In many cases the diagnosis may be obvious from
resulting in a decrease in urinary pH to less than 5.5 the clinical history (particularly drug history) and
under normal conditions. examination (Fig. 4.7).
Furosemide 40 mg is given orally and urine is Tests that may help to elucidate the cause of the
collected for pH every half-hour. However, like the metabolic acidosis are:
ammonium chloride test, a urinary pH of less than 5.5
● plasma urea and creatinine estimation,
usually precludes its need. Generally this is simpler and
● determination of plasma anion gap,
safer than the ammonium chloride test and may be
● plasma glucose estimation,
preferable, assuming that there are no contraindications
● blood lactate determination,
to the use of furosemide.
● tests for ketones in urine,
Ammonium chloride load test for type I renal tubular acidosis ● tests for blood gases,
This test is not necessary if the pH of a urinary specimen, ● tests for drugs or poisons, for example ethanol,
collected overnight, is already less than 5.5. paracetamol, salicylate (if indicated),
The NH4+ is potentially acid because it can dissociate ● specialized tests for renal tubular acidosis.
to NH3 and H+. After ingestion of ammonium chloride, Treatment of a metabolic acidosis is usually that of
the kidneys usually secrete the H+, and the urinary pH the underlying condition. Dialysis may be necessary in
falls. the face of uraemia. Overdoses may also require dialysis,
Procedure for example ethylene glycol, methanol and salicylate.
● No food or fluid is taken after midnight. Infusion of sodium bicarbonate (usually small boluses
● 08.00 h: gelatine-encapsulated ammonium chloride containing about 50–100 mmol) is only rarely indicated
is given orally in a dose of 0.1 g/kg body weight. This in severe metabolic acidosis of pH less than 7.1 and can
can act as a gastric irritant. cause complications such as hypernatraemia, volume
● Urine specimens are collected hourly and the pH of overload, hypokalaemia, ‘overshoot’ alkalosis and
each is measured immediately in the laboratory. paradoxical central nervous system acidosis probably
due to the rapid diffusion of CO2 across cell membranes
Interpretation in comparison with slower HCO3– movement.
In normal subjects, the urinary pH falls to 5.5 or below In distal type I and type II renal tubular acidosis,
between 2 and 8 h after the dose. In generalized tubular oral HCO3– therapy may be necessary. In type IV, if due
disease there may be a sufficient number of functioning to hyporeninism hypoaldosteronism, fludrocortisone
nephrons to achieve a normal level of acidification. In (sometimes in conjunction with furosemide) may be
distal renal tubular acidosis this degree of acidification useful.
Acid–base disorders 71
Metabolic acidosis
Yes No
Yes No
Yes No Yes No
Elevated urine/plasma
ketones?
Acute kidney injury (AKI) or Ureteric diversion
Diabetic ketoacidosis
chronic kidney disease (CKD)?
Yes No
Yes No
Yes No
Gastrointestinal
Consider lactic acidosis
Consider RTA type IV disorders?
(see Box 4.2)
(see Table 4.1)
Yes No
Figure 4.7 Algorithm for the investigation of a metabolic acidosis. RTA, renal tubular acidosis.
72 Acid–base disturbances
Metabolic alkalosis
Yes No Yes No
Normotensive Hypertension
Evidence of
hypomagnesaemia?
It may be difficult to distinguish clinically between centre directly, and overdosage initially causes respiratory
the overbreathing due to metabolic acidosis, in which alkalosis. They also uncouple oxidative phosphorylation,
the fall in plasma [HCO3–] is the primary biochemical and may evoke a lactic acidosis. Both these effects lower
abnormality, and that due to respiratory alkalosis, in plasma [HCO3–], but the pH may be high if respiratory
which it is compensatory. In doubtful cases, estimation alkalosis is predominant, normal if the two cancel each
of arterial pH and PCO2 is indicated. other out, or low if metabolic acidosis is predominant.
The arterial blood findings in respiratory alkalosis
Management of respiratory alkalosis
are:
The cause of the hyperventilation may be obvious from
● PCO2 is always reduced, the clinical history and examination (Fig. 4.10). A chest
● [HCO3–] is low-normal or low, radiograph and lung function tests may be indicated.
● pH is raised (uncompensated or partly compensated) Laboratory tests could include:
or near normal (fully compensated).
● blood gases,
Acute hypocapnia (low PCO2) can cause hypokalaemia ● plasma [K+] and ionized [Ca2+] may be low,
due to increased intracellular uptake of K+. A reduction in ● elevated white cell count and relevant cultures may
free or ionized calcium can lead to tetany and paraesthesiae. point to sepsis as a cause of hyperventilation,
The causes of respiratory alkalosis are shown in Box ● full blood count to exclude anaemia,
4.7. One of the most common causes is anxiety-induced ● liver function tests when hepatic failure is suspected,
hyperventilation. Salicylates stimulate the respiratory ● exclude salicylate overdose and respiratory stimulants.
76 Acid–base disturbances
CASE 5 Yes No
pH 7.61 (7.35–7.45)
Evidence of central respiratory stimulation (see Box 4.7)?
PaCO2 2.7 kPa (4.6–6.0)
PaO2 13.3 kPa (9.3–13.3)
Bicarbonate 18 mmol/L (24–32) Yes No
DISCUSSION
The patient is showing a respiratory alkalosis. The Evidence of specific conditions that can
cause a respiratory alkalosis (see Box 4.7)?
panic attack had resulted in hyperventilation. The
peri-oral paraesthesia is due to a lowering of plasma Figure 4.10 Algorithm for the investigation of a
ionized calcium concentration as a result of the respiratory alkalosis.
alkalosis. Compensation is by the kidneys, which
increase HCO3– excretion and reduce acid excretion.
Compensatory changes in acid–base disturbances
In either metabolic or respiratory acidosis the ratio of
Treatment is usually of the underlying condition, [HCO3–] to PCO2, and therefore the pH, can be corrected
which is rarely life threatening unless arterial pH is by a change in concentration of the other member of
more than 7.5. During hyperventilation syndrome, the buffer pair in the same direction as the primary
patients may benefit from reassurance, the treatment of abnormality. This compensation may be either partial
underlying psychological stress and rebreathing into a or complete, but never overcompensates, that is, there
paper bag during acute episodes. is no pH overshoot.
Blood gases 77
[H] mmol/L
Table 4.2 Summary of findings in arterial blood in
disturbances of hydrogen ion homeostasis 10 1.93,110 20 30 mM
15
pH Pco2 [HCO3–] Plasma [K+] 15
Acidosis 10
Metabolic 20 A B
8
Acute state Ø N Ø Usually ≠ (Ø in C
[HCO3] mmol/L
RTA I or II or 6
P CO2 (kPa)
acetazolamide) G 5
Compensation N Ø* Ø ≠ 25
4
Respiratory F
D 3
Acute change Ø ≠ N or ≠ ≠ E
Compensation N ≠ ≠≠* ≠
2
Alkalosis
Metabolic
Acute state ≠ N ≠ Ø 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7
Compensation N ≠* ≠ Ø pH
Respiratory A – Acute respiratory acidosis
Acute change ≠ Ø N or Ø Ø B – Chronic respiratory acidosis
C – Chronic metabolic alkalosis
Compensation N Ø Ø* Ø D – Acute respiratory alkalosis
Arrows, primary change; *arrows, compensatory change; N, normal; E – Chronic respiratory alkalosis
RTA, renal tubular acidosis. F – Chronic metabolic acidosis
Potassium depletion can cause alkalosis, or alkalosis can cause G – Acute metabolic acidosis
hypokalaemia. The clinical history may differentiate the cause of the Central shaded area = Normal
combination of hypokalaemia and alkalosis.
Overbreathing causes a low [HCO3–] in respiratory alkalosis.
Figure 4.11 Siggaard–Andersen acid–base chart for
Metabolic acidosis, with a low [HCO3–], causes overbreathing. arterial blood. Copyright Radiometer Medical Aps.
Measurement of blood pH and PCO2 can differentiate these two. Adapted with permission.
78 Acid–base disturbances
● pH of blood: as the pH falls, the affinity for the rate of O2 transport through the fluid cannot be
O2 decreases. This is known as the Bohr effect. increased enough to restore normal arterial PO2. This
Deoxygenated Hb binds H+ more avidly and results in a low or normal PCO2 and a low PO2. Only
accounts for the increased buffering capacity of Hb in very severe cases is the PCO2 raised.
in venous blood. ● Haemoglobin in arterial blood is normally 95 per cent
● 2,3-diphosphoglycerate (2,3-DPG) is formed during saturated with O2, and the small amount of O2 in
glycolysis and is plentiful within the erythrocyte. simple solution in the plasma is in equilibrium with
It binds to Hb, liberating more O2, so that the oxyhaemoglobin. Increased air entry at atmospheric
dissociation curve shifts to the right. A fall in pH PO2 cannot significantly increase O2 carriage in
decreases 2,3-DPG levels, thus potentially reducing blood leaving normal alveoli, but can reduce the
tissue oxygenation. Erythrocyte levels of 2,3- PCO2. Breathing pure O2 increases arterial PO2, but
DPG increase in anaemia and in some conditions not Hb saturation.
associated with chronic hypoxia. ● In conditions such as lobar pneumonia, pulmonary
● Fetal Hb has a greater affinity for O2 than adult Hb, collapse and pulmonary fibrosis or infiltration, not
thus facilitating the transfer of O2 across the placenta. all alveoli are affected equally. At first the gaseous
composition of blood leaving them is normal.
Factors affecting blood gas results Increasing the rate or depth of respiration may later
In respiratory acidosis it is often important to know the lower the PCO2 considerably, but does not alter either
partial pressure of oxygen (PO2) as well as the pH, PCO2 the PO2 or the Hb saturation.
and [HCO3–]. ● Obstruction of small airways means that air cannot
Normal gaseous exchange across the pulmonary reach those alveoli supplied by them; the composition
alveoli involves loss of CO2 and gain of O2. However, of blood leaving them will be near that of venous
in disease a fall in PO2 and a rise in PCO2 do not always blood and there is a ventilation/perfusion mismatch
coexist. The reasons for this are as follows: with a right-to-left shunt. Only if gaseous flow due
to increased respiratory exertion can overcome the
● Carbon dioxide is much more soluble in water than obstruction will it help to correct the low PO2 and
O2 and its rate of diffusion is about 20 times as high. high PCO2.
For example, in pulmonary oedema, diffusion of O2 ● Some alveoli may have normal air entry, but much
across alveolar walls is hindered by oedema fluid and reduced blood supply. This is ‘dead space’. Increased
arterial PO2 falls. The hypoxia and alveolar distension ventilation will have no effect, because there is little
stimulate respiration and CO2 is excreted. However, blood to interact with the increased flow of gases.
Blood from unaffected alveoli and from those with
100 obstructed airways mixes in the pulmonary vein before
entering the left atrium. The high PCO2 and low PO2
O2 saturation of haemoglobin (%)
blood supply; the poor aeration will cause a high PCO2 is breathing room air. Common causes include
and low PO2 in the blood draining them, and therefore chest wall abnormalities, neuromuscular disease,
in systemic arterial blood. Occasionally, stimulation drug overdose or severe airway disease, for example
of respiration by alveolar stretching can maintain a asthma or COPD. This may be acute (within a few
normal, or even low, PCO2 early in an acute asthmatic hours), when pH may be less than 7.3, because there
attack. A raised PCO2 in an asthmatic indicates a severe is not time for renal compensation to take place, or
attack (Box 4.8). chronic, when arterial pH is only slightly low.
Respiratory failure As well as treatment for the cause of the condition,
Respiratory failure can be classified as either hypoxaemic some patients may need mechanical ventilation. It is
or hypercapnic: important to remember that CO2 narcosis can occur in
some patients with hypercapnia who are given O2. This
● Hypoxaemic respiratory failure (type I) is defined is because they may lose their hypoxic respiratory drive,
as hypoxaemia with a normal or low PaCO2. This is which can lead to rapid increases in PCO2.
the most common form of respiratory failure and is
associated with most forms of pulmonary disease,
Pulse oximeters
particularly when there is collapse of or fluid in
the alveoli. Examples are pulmonary oedema or Pulse oximeters give non-invasive estimation of the
pneumonia. It may be difficult to determine from arterial Hb O2 saturation. They are useful in anaesthesia,
blood gases whether hypoxaemic failure is acute or recovery or intensive care (including neonatal units)
chronic, although in chronic failure polycythaemia and during patient transport. There are two main
or cor pulmonale may occur. principles involved:
● Hypercapnic respiratory failure (type II) is ● differential light absorption by Hb and
characterized by both hypoxaemia and hypercapnia. oxyhaemoglobin,
Hypoxaemia may occur, particularly if the patient ● identification of the pulsatile blood component of
the signal.
Box 4.8 Blood gases and respiratory Oximetry gives a good estimation of adequate
disease oxygenation, but no direct information about
ventilation, including CO2 status.
In the list of examples given below the conditions
marked * may fall into either group, depending on Inaccuracies of oximetry
the severity of the disease.
Bright overhead lighting may give pulsatile waveforms
Low arterial PO2 with low or normal PCO2 (hypoxia) and saturation values when there is no pulse. Dyes
Pulmonary oedema (diffusion defect) and pigments, including nail varnish, may give
Lobar pneumonia
artificially low values. Abnormal haemoglobins, such
Pulmonary collapse*
Pulmonary fibrosis or infiltration* as methaemoglobinaemia, cause readings to tend
towards 85 per cent (see Fig. 4.12). Furthermore,
Low arterial PO2 with a high PCO2 (hypoxia and
hypercapnia)
carboxyhaemoglobin, caused by carbon monoxide (CO)
Impairment of movement of the chest poisoning, causes saturation values to tend towards
Chest injury 100 per cent. A pulse oximeter is thus misleading in
Gross obesity cases of CO poisoning for this reason and should not
Ankylosing spondylitis be used. Oxygen saturation values less than 70 per cent
Neurological conditions affecting the respiratory drive can be inaccurate.
Neurological conditions, e.g. poliomyelitis, affecting Cardiac arrhythmias may interfere with the oximeter
innervation of respiratory muscles picking up the pulsatile signal properly and with
Extensive airway obstruction: chronic obstructive calculation of the pulse rate. Vasoconstriction and
pulmonary disease; severe asthma; laryngeal spasm
hypothermia cause reduced tissue perfusion and failure
Bronchopneumonia
Pulmonary collapse*
to register a signal. Cardiac valve defects may cause
Pulmonary fibrosis or infiltration* venous pulsation, and therefore venous O2 saturation is
recorded by the oximeter.
80 Acid–base disturbances
Collection of specimens for blood gas In such conditions, the estimations are required only
estimations if the results will influence treatment.
● Arterial specimens are usually preferable to capillary ● There is an acute exacerbation of COPD or acute,
specimens. Venous samples are avoided as the carbon potentially reversible, lung disease. In such cases,
dioxide is raised compared with arterial samples as a vigorous therapy or artificial respiration may tide
result of tissue metabolism. the patient over until lung function improves; more
● The heparin and specimen in the syringe must be precise information than the plasma [HCO3–] is
well mixed. Warning Excess heparin may dilute the needed to monitor and control treatment.
specimen and cause haemolysis. ● Blood is being taken for estimation of PO2 and to
● If sodium heparin is used, do not estimate [Na+] guide O2 therapy or artificial ventilation.
using the same specimen as a resultant factitious ● To complement oximetry, particularly if low
hypernatraemia can occur. saturations are found or hypercapnia is suspected.
● Expel any air bubbles at once. The nozzle should then
be stoppered. Leaving the specimen in the syringe INVESTIGATION OF HYDROGEN ION
should minimize gas exchange with the atmosphere. DISTURBANCES
● Performing the assay as soon as possible should
The following may be used to assess acid–base
minimize the effect on pH of anaerobic erythrocyte
disturbances in patients (see Fig. 4.11.)
metabolism. The specimen should be kept cool and
not sent through a vacuum tube system because of Blood gases
potential shaking. Measurement of blood pH
In newborn infants, arterial puncture may be used or Measurement of blood pH determines whether there
capillary samples taken as an alternative. However, the is an acidosis or alkalosis. If the pH is abnormal, the
following precautions are essential: primary abnormality may be in the control of CO2 by
the lungs or respiratory centre, or in the balance between
● In order for the composition of the blood to be as
HCO3– utilization in buffering and its reabsorption and
near arterial as possible, the area from which the
regeneration by renal tubular cells and erythrocytes.
specimen is taken should be warm and pink. If there
However, a normal pH does not exclude a disturbance
is peripheral cyanosis, results may be dangerously
of these pathways: compensatory mechanisms may be
misleading.
maintaining it.
● The blood should flow freely. Squeezing the skin
Assessment of these factors can be made only by
while sampling may dilute the specimen with
measuring components of the bicarbonate buffer
interstitial fluid.
system. There is reasonable correlation between arterial
● The capillary tubes must be heparinized, and mixing
and venous pH (the latter being about 0.04 units less)
with the blood must be complete (see above).
but of course there are usually considerable PO2 and
● The tubes must be completely filled with blood. Air
PCO2 differences.
bubbles invalidate the results.
● The ends of the tubes should be sealed immediately. Measurement of blood [HCO3–]
There are two methods that may be used to estimate
Indications for arterial blood gas the circulating [HCO3–], although it can of course be
determination measured directly:
Arterial blood gas estimations may be indicated in the
● Plasma total CO2 (TCO2) or sometimes termed
following situations:
by some laboratories as bicarbonate on their
● There is doubt about the cause of the abnormal reports. The plasma [HCO3–] is probably the most
plasma HCO3– (for example to differentiate metabolic commonly measured index of H+ homeostasis. If pH
acidosis from respiratory alkalosis or metabolic and PCO2 estimations are not needed, the assay has
alkalosis from respiratory acidosis). certain advantages: venous blood can be used and it
● An acid–base disturbance is suspected, for example can be performed together with assays for urea and
after cardiopulmonary arrest, in renal failure electrolytes. It is an estimate of the sum of plasma
complicated by lung disease or in salicylate overdose. HCO3–, carbonic acid and dissolved CO2. At pH 7.4
Investigation of hydrogen ion disturbances 81
the ratio of [HCO3–] to the other two components is log [SID] – Kb [Atotal]/(Kb + 10 – pH)
pH = pKa +
about 20:1, and at pH 7.1 it is still 10:1. Thus, if the aPCO2
TCO2 were 21 mmol/L, [HCO3–] would contribute (4.17)
20 mmol/L at pH 7.4 and just over 19 mmol/L at
where [SID] = [Na+] + [K+] – [Cl–] – [lactate],
pH 7.1. Only 1 mmol/L and just under 2 mmol/L,
[Atotal] = all plasma weak acids mainly albumin, and
respectively, would be due to H2CO3 and CO2
a, b, and k are all constants.
respectively. Thus, TCO2 is effectively a measure of
This theory may help to explain the metabolic
the plasma [HCO3–].
alkalosis seen in severely ill hypoalbuminaemic patients
● The [HCO3–] is calculated from the Henderson–
(see Chapter 19) and the hyperchloraemic acidosis seen
Hasselbalch equation, using the measured values
with excess saline administration.
of pH and PCO2 (in kPa) in whole arterial blood. It
is a measure of the whole blood [HCO3–] and, for
the reasons discussed above, usually agrees well with Indications for plasma and urinary chloride
estimation
plasma TCO2. It is the estimate of choice if the other
two parameters are being measured. Hyperchloraemia occurs in a normal anion gap
● Base excess is defined as the amount of acid that metabolic acidosis. Conversely, hypochloraemia is seen
must be added to 1 L of fully oxygenated blood in the metabolic alkalosis of pyloric stenosis or chronic
to return the pH to 7.40 at 37°C and PCO2 at vomiting.
40 mmHg (5.3 kPa). The usual reference range is Plasma [Cl–] estimation may help in two main
– 2 to + 2 mEq/L. It is a measure of the metabolic situations:
component that is independent of the respiratory ● If a patient who is vomiting has a high plasma
component. In a metabolic alkalosis base excess is [HCO3–], the finding of a low [Cl–] favours the
more than + 2 mEq/L, and in a metabolic acidosis diagnosis of pyloric stenosis or chronic vomiting.
base excess is less than – 2 mEq/L. ● If there is a low plasma [HCO3–], the finding of a
● Standard bicarbonate is provided by some blood gas high plasma [Cl–] (and therefore a normal anion
machines and defined as the bicarbonate concentration gap) strengthens the suspicion of hyperchloraemic
under standard conditions: PCO2 = 40 mmHg acidosis. In metabolic acidosis due to most other
(5.3 kPa), at 37°C, and saturated with oxygen and is a causes the plasma [Cl–] is normal and the anion gap
good measure of the metabolic component. is increased.
Concentration of dissolved CO2 A spot urinary [Cl–] can be useful in determining
The concentration of dissolved CO2 is calculated by the cause of a metabolic alkalosis. A [Cl–] less than
multiplying the measured PCO2 by the solubility constant 20 mmol/L is indicative of a saline-responsive
of the gas: 0.23 if PCO2 is in kPa or 0.03 if it is in mmHg. metabolic alkalosis, for example chronic vomiting, and
a urinary [Cl–] more than 20 mmol/L of a saline-non-
Stewart’s strong ion difference (SID) hypothesis responsive form of metabolic alkalosis, for example as
Although this chapter focuses on the Siggaard– in mineralocorticoid excess syndromes such as Bartter’s
Andersen ‘classic’ theory of acid–base disturbance, there syndrome. Diuretics can cause variable urine chloride
is another approach. Stewart takes into consideration concentrations, which are usually raised initially and
the buffering capacity of albumin. Here: then reduce with chronic use.
82 Acid–base disturbances
SUMMARY
● Tight homeostatic control of acid–base balance is
essential, otherwise cell malfunction and death can
occur. The kidneys excrete non-volatile acid via the
renal tubules into the urine, while the lungs excrete
volatile acid as CO2.
● The major extracellular buffer system involves HCO3–.
● Blood pH is inversely proportional to the PCO2 and
directly proportional to the [HCO3–]. This can be
determined on a blood gas analyser (Fig. 4.13).
● Respiratory acidosis results from disorders of the
respiratory system and is caused by CO2 retention.
Conversely, respiratory alkalosis is due to excess
CO2 loss, as in hyperventilation. In the case of the
former, compensation is by the renal excretion of
non-volatile acid and reclamation of HCO3–. In the
latter, the kidneys compensate by losing HCO3–.
● Metabolic (non-respiratory) acidosis results from
increased non-volatile acid such as lactic acid or
certain ketones. Compensation is by the lungs, which
increase CO2 excretion by hyperventilation. Metabolic
(non-respiratory) alkalosis is caused by HCO3– excess Figure 4.13 Analyser used to determine patient arterial
blood gases. Reproduced with kind permission of
and acid loss, such as in prolonged vomiting, and its
Medica Corporation.
compensation is via the lungs, which hypoventilate,
thereby retaining volatile acid as CO2.
5 Potassium
The intracellular potassium ion concentration ([K+]) Potassium is normally almost completely reabsorbed
is large and provides a reservoir for the extracellular in the proximal tubules. Damage to these may cause
compartment. Consequently changes in water balance potassium depletion. Potassium is secreted in the
have little direct effect on the plasma [K+], unlike distal tubules and collecting ducts in exchange for Na+;
plasma [Na+]. hydrogen ions (H+) compete with potassium ions (K+).
The normal potassium intake is about 60–100 mmol/ Aldosterone stimulates both exchange mechanisms.
day. Potassium enters and leaves the extracellular If the proximal tubules are functioning normally,
compartment by three main routes: potassium loss in the urine depends on three factors:
secrete H+ in exchange for Na+: this may be impaired infarction. b-adrenergic stimulation increases cellular
during treatment with carbonate dehydratase potassium uptake by stimulating the Na+/K+-ATPase
(CD) inhibitors and in some types of renal tubular ‘pump’. b-blockade increases plasma potassium
acidosis (Chapter 4). concentration and b-agonists decrease it, an effect that
is independent of body potassium stores.
The cell membranes Synthesis of Na+/K+-ATPase is stimulated by
The Na+/K+ adenosine triphosphatase (ATPase) ‘pump’ thyroxine, which may contribute to the hypokalaemia
on cell surfaces maintains a high intracellular [K+]. sometimes associated with hyperthyroidism.
This exchanges three Na+ ions from cells in exchange
Relationship between hydrogen and
for two K+ ions in the extracellular fluid (ECF), thus potassium ions (Fig. 5.2)
establishing an electrochemical gradient across the cell
membrane, with a net positive charge in the ECF. The The extracellular [H+] affects the entry of potassium
loss of K+ from cells down the concentration gradient is into all cells. Changes in the relative proportions of K+
opposed by this electrochemical gradient. Potassium is and H+ in distal renal tubular cells affect the urinary
also exchanged for H+ (Fig. 5.1). loss of potassium. There is a reciprocal relationship
A small shift of K+ out of cells may cause a significant between K+ and H+:
rise in plasma concentrations, whether in vivo or in vitro. ● In acidosis, increased loss of potassium from cells
In the latter situation, the artefactual hyperkalaemia due into the ECF coupled with reduced urinary secretion
to haemolysed or old specimens may be misinterpreted of potassium causes hyperkalaemia.
and should be avoided. Usually the shift of K+ across ● In alkalosis, net increased uptake of potassium into
cell membranes is accompanied by a shift of Na+ in cells and increased urinary potassium loss cause
the opposite direction, but the percentage change in hypokalaemia.
extracellular [Na+] is much less than that of K+.
In the kidney, Na+ derived from the luminal fluid
Increased uptake and net gain of K+ by cells may
is pumped through the cell in exchange for either K+
occur in alkalosis due to increased uptake by cells and
or H+. If K+ is lost from the ECF, it passes down the
increased urinary loss. Insulin enhances the cellular
increased concentration gradient, so reducing its
uptake of glucose and potassium. Hyperkalaemia
stimulates insulin secretion and hypokalaemia inhibits
it. This effect may be used to treat hyperkalaemia B– Na+
CO2
H2O
H+
K+ Renal tubular cell
intracellular content. Unless there is renal tubular cell Potassium-sparing diuretics can be used together
damage, Na+ are reabsorbed in exchange for fewer K+ with those causing potassium loss when hypokalaemia
and more H+ than usual. For each H+ formed within the cannot be controlled by potassium supplementation.
tubular cell by the CD mechanism, one bicarbonate ion Used alone, they have only a weak diuretic action. Beware
(HCO3–) is produced: of giving potassium supplements to patients taking
potassium-sparing diuretics or angiotensin-converting
H2O + CO2 Æ H+ + HCO3– (5.1)
CD enzyme (ACE) inhibitors or angiotensin II receptor
blockers (ARBs) because of the risk of hyperkalaemia.
As more H+ is secreted into the urine, the reaction
is increased and more HCO3– is generated, passing into Measurement of plasma and urinary
the ECF accompanied by the reabsorbed Na+. The result potassium
is an extracellular alkalosis and acid urine. Therefore, In rapidly changing clinical states frequent estimation
chronic potassium depletion is usually accompanied of plasma potassium is the best way of assessing therapy.
by a high plasma bicarbonate concentration. The Plasma potassium is best measured in a freshly collected,
combination of hypokalaemia and a high plasma unhaemolysed venous sample as a corresponding
bicarbonate concentration is more likely to be due to serum concentration is higher owing to the release of
K+ depletion than to metabolic alkalosis. intracellular potassium as part of the clotting process.
In chronic potassium depletion, measurement of the
Potassium and diuretic therapy plasma bicarbonate concentration ([HCO3–]) may help
Diuretics may be used to treat hypertension as well as to indicate the state of cellular repletion (intracellular
oedema, for example in cardiac failure. They can be potassium depletion causes extracellular alkalosis).
divided into two principal groups, based upon their site Urinary potassium (kaluria) estimations to
of action. determine the primary cause of potassium depletion
● Potassium-losing diuretics increase the sodium load may be useful diagnostically to help find the cause of
on the distal tubules and collecting ducts with hypokalaemia. Most extrarenal causes of potassium loss
enhanced Na+/K+ exchange. This may result in are associated with volume depletion and therefore with
hypokalaemia. secondary hyperaldosteronism. Excretion of less than
– Loop diuretics, such as furosemide or about 20 mmol/day can be expected only in the well-
bumetanide, inhibit sodium reabsorption from hydrated hypokalaemic patient with extrarenal losses,
the ascending limb of the loops of Henle. in whom aldosterone secretion is inhibited. Therefore
– Thiazides act at the junction of the loops and a low potassium excretion confirms extrarenal loss, but
the distal tubules, which are sometimes called a high one does not prove that it is the primary cause.
the ‘cortical diluting segments’. High urinary potassium concentration (for example
– Carbonate dehydratase inhibitors, such as more than 20 mmol/L) in the face of hypokalaemia
acetazolamide, are rarely used as diuretics but suggests inappropriate K+ renal loss as a cause of
are still used to treat glaucoma. They may cause the hypokalaemia, for example due to renal tubular
a hypokalaemic hyperchloraemic acidosis. problems or mineralocorticoid excess syndromes.
● Potassium-sparing diuretics. The transtubular potassium gradient (TTKG) is an
– Diuretics that inhibit either aldosterone directly estimate of the potassium concentration at the end
or the exchange mechanisms in the distal of the cortical collecting duct beyond the site where
tubules and collecting ducts cause potassium aldosterone influences potassium secretion. This can
retention and may lead to hyperkalaemia, be calculated on a spot urine (which should have an
especially if glomerular function is impaired. osmolality greater than that of the plasma) and plasma
Potassium-sparing/-retaining diuretics include samples from the following equation:
spironolactone, a competitive aldosterone urinary [potassium] ¥ plasma osmolality (5.2)
antagonist. plasma [potassium] ¥ urine osmolality
– Other potassium-sparing diuretics include The TTKG may be useful diagnostically in the
amiloride and triamterene, which are direct investigation of hyperkalaemia, when a TTKG less than
inhibitors of the Na+/K+ exchange mechanism 3 implies that the kidneys are not excreting potassium
in the distal tubules. appropriately (i.e. a lack of aldosterone effect).
86 Potassium
ABNORMALITIES OF PLASMA
POTASSIUM Box 5.1 Some causes of hypokalaemia
Hypokalaemia classified into predominantly non-renal
and renal causes
Hypokalaemia is often the result of potassium depletion,
but if the rate of loss of potassium from cells equals or Renal causes
exceeds that from the ECF, potassium depletion may Increased Na+/K+ exchange
exist without hypokalaemia. Hypokalaemia can also Primary hyperaldosteronism (Conn’s syndrome)
occur without depletion if there is a shift of K+ into cells. Secondary hyperaldosteronism
The causes of hypokalaemia (summarized in Box 5.1) Cushing’s syndrome
Steroid therapy
may be classified according to the predominant cause as ‘Ectopic’ adrenocorticotrophic hormone (ACTH)
either renal or non-renal. Pseudohypokalaemia is defined secretion
as the in vitro uptake of K+ from the plasma into blood Bartter’s syndrome or Gitelman’s syndrome
cells, primarily erythrocytes. This sometimes occurs if Carbenoxolone therapy
the blood sample is kept in a warm environment. This Liquorice excess
contrasts with the more usual hyperkalaemia found in Liddle’s syndrome (pseudohyperaldosteronism)
11-hydroxylase and 17-hydroxylase deficiencies
specimens stored in the refrigerator. (rare)
Renal causes of hypokalaemia Excess Na+ available for exchange
Infusion of saline
● Enhanced renal secretion of potassium due to Diuretics (‘loop’ diuretics)
increased activity of the pump in distal renal tubules Decreased Na+/H+ exchange
by mineralocorticoid activity. This is associated with Carbonate dehydratase inhibitors
a hypokalaemic alkalosis. Renal tubular acidosis (types I and II)
– Primary hyperaldosteronism (Conn’s syndrome) Impaired proximal tubular reabsorption
Renal tubular dysfunction
due to adrenal adenoma or hyperplasia (see
Fanconi’s syndrome
Chapter 8). Hypomagnesaemia
– Secondary hyperaldosteronism often aggravates Non-renal causes
other causes of potassium depletion (see Redistribution
Chapter 2). Glucose and insulin
– Cushing’s syndrome and steroid therapy. Catecholamines
Patients secreting excess of, or on prolonged Familial hypokalaemic periodic paralysis (rare)
therapy with, glucocorticoids tend to become Barium intoxication (rare)
Vitamin B12 therapy
hypokalaemic due to the mineralocorticoid Certain rapidly growing tumours
effect on the distal renal tubules (see Chapter 8). Intestinal loss
– ‘Ectopic’ adrenocorticotrophic hormone Prolonged vomiting
(ACTH) secretion, which stimulates cortisol Diarrhoea
secretion (see Chapter 8). Loss through intestinal fistula
– Bartter’s syndrome is a rare autosomal recessive Purgative abuse
Reduced intake
condition in which there is hyperplasia of the
Poor diet
renal juxtaglomerular apparatus with increased Geophagia (rare)
renin and therefore aldosterone secretion. Combined causes
Angiotensin II activity is impaired and therefore Alkalosis
there is no vasoconstriction; consequently the Pyloric stenosis
patient is normotensive. This latter finding
helps to distinguish the syndrome from that of those of high-dose loop diuretic intake. Treatment
primary hyperaldosteronism, in which there can consists of potassium supplementation, often
also be a hypokalaemic alkalosis. Individuals may with a potassium-sparing diuretic such as
have polyuria, polydipsia, short stature, learning spironolactone or amiloride. By suppressing
difficulties and maternal polyhydramnios. The prostaglandin-stimulated renin release, non-
defect is in the epithelial ion channels of the steroidal anti-inflammatory drugs may also
ascending limb. The biochemical features mimic improve the condition.
Abnormalities of plasma potassium 87
– The very rare Gitelman’s syndrome, in contrast – a number of drugs can cause increased renal
to Bartter’s syndrome, is often asymptomatic loss of potassium, usually from distal tubules,
until adulthood. It is an autosomal recessive including diuretics (non-potassium sparing),
condition due to a mutation in the thiazide- cisplatin, aminoglycosides, amphotericin and
sensitive Na+/Cl– co-transporter in the foscarnet, and also CD inhibitors, for example
distal convoluted tubules. Unlike Bartter’s, acetazolamide.
there is more severe hypomagnesaemia and ● Decreased renal Na+/H+ exchange because of
hypocalciuria. Once thiazide use has been impaired generation of H+ within the renal tubular
excluded, a hypokalaemic hypomagnesaemic cells, which favours Na+/K+ exchange associated
alkalosis in the presence of a urinary calcium to with a hypokalaemia. Causes include renal tubular
creatinine molar ratio less than 0.2 is suggestive acidosis types I and II (see Chapter 4).
of Gitelman’s syndrome. ● Excess sodium available for exchange in the distal
– Liddle’s syndrome, also very rare, is an renal tubules can also increase renal potassium loss.
autosomal dominant condition with These include:
hypertension and hypokalaemic metabolic – prolonged infusion of saline,
alkalosisis, in which suppressed plasma – diuretics inhibiting the pump in the loops
aldosterone and renin concentrations are of Henle – the activity of the distal pump is
associated with cerebrovascular disease. enhanced by secondary hyperaldosteronism.
Mutations in the selective sodium channels
cause increased sodium reabsorption through Non-renal causes of hypokalaemia
the collecting duct epithelial sodium channel, ● Redistribution within the body, by entry into cells.
causing hypertension. Treatment involves – Glucose and insulin therapy. This may be used
sodium restriction and potassium-sparing to treat severe hyperkalaemia.
diuretics such as amiloride or triamterene; – Increased secretion of catecholamines, such
spironolactone is ineffective. as may occur from the stress of myocardial
– Carbenoxolone therapy is occasionally used infarction. The use of b-agonists such as
to accelerate the healing of peptic ulcers. It salbutamol in inhalers may also stimulate this
potentiates the action of aldosterone and can secretion.
cause hypokalaemia by inhibiting the enzyme – Rapidly growing tumours, such as certain
11-b-hydroxysteroid dehydrogenase (see leukaemias, can evoke hypokalaemia, probably
Chapter 8). by potassium uptake into the proliferating
– Liquorice and tobacco contain glycyrrhizinic acid, cells.
which also inhibits the enzyme 11-b-hydroxysteroid – Familial hypokalaemic periodic paralysis is
dehydrogenase. Overindulgence in liquorice- rare and is an autosomal dominant defect of
containing sweets or habitual tobacco chewing the dihydropyridine receptor, a voltage-gated
can rarely cause hypokalaemia (see Chapter 8). calcium channel. In this condition episodic
● Increased renal potassium loss may also result from paralysis is associated with entry of potassium
impaired proximal tubular potassium reabsorption into cells, which can be provoked by a high
as in: carbohydrate intake. Thyrotoxic periodic
– renal tubular dysfunction (for example the paralysis is a similar condition, but genetically
recovery phase of acute oliguric kidney injury), different, that is found in hyperthyroidism.
– Fanconi’s syndrome (see Chapter 3), – Barium ingestion blocks potassium exit from
– hypercalcaemia, which may impair the cells.
reabsorption of potassium even if there is no – Treatment of pernicious anaemia with vitamin
renal damage, B12, by rapid cellular uptake of potassium into
– hypomagnesaemia can also reduce the cells (see Chapter 15).
intracellular potassium concentration and ● Reduced potassium intake.
causes renal potassium wasting – it may be a – Chronic starvation. If water and salt intake are
cause of a refractory hypokalaemia unless the also reduced, secondary hyperaldosteronism
plasma magnesium is corrected, may aggravate the hypokalaemia.
88 Potassium
Hypokalaemia
On potassium-lowering medication?
Yes No
Measure spot
urine potassium
No Yes No Yes
(see Box 5.1)
No Yes No Yes
(see Box 5.1)
Consider poor
potassium intake Assess acid–base status
Primary hyperaldosteronism and ectopic ACTH more than 20 mmol/L in Bartter’s or Gitelman’s
production are very rare. syndrome. This is useful diagnostically to distinguish
them from other causes of hypokalaemia such as
● Take a careful drug history, with special reference to
gastrointestinal loss of potassium, for example
potassium-losing diuretics, purgatives and steroids.
vomiting or diarrhoea in which urinary chloride is
Rare causes of a steroid-like effect are ingestion of
usually less than 20 mmol/L. If purgative abuse is
carbenoxolone or liquorice.
suspected, an estimation of the drug levels in stool
● Check the potassium concentration in a spot urine
samples may be indicated.
sample. If less than or equal to 20 mmol/L, it is suggestive
● Exclude hypomagnesaemia, which may be associated
of gastrointestinal loss or transcellular potassium shift
with hypokalaemia (see Chapter 6).
or poor potassium intake. If it is more than 20 mmol/L,
● For the investigation of primary hyperaldosteronism
it is indicative of renal potassium loss such as occurs
(Conn’s syndrome), Cushing’s syndrome and
in the very rare Bartter’s and Gitelman’s syndromes
other mineralocorticoid excess syndromes, see
and other mineralocorticoid excess disorders.
Chapter 8.
● The urinary chloride concentration is usually
90 Potassium
Investigation of hyperkalaemia (Fig. 5.6) ● What are the plasma urea and creatinine
● Exclude artefactual (pseudohyperkalaemia) causes of concentrations? Severe renal glomerular dysfunction
hyperkalaemia such as haemolysis, thrombocytosis, is a common cause of hyperkalaemia.
leucocytosis or delayed separation of plasma from ● Measure the plasma bicarbonate concentration.
blood cells or potassium-EDTA-contaminated tubes. An acidosis, whether metabolic (with a low plasma
● Is the patient taking drugs, such as: bicarbonate concentration) or respiratory (with a
– potassium supplements? normal or high plasma bicarbonate concentration),
– potassium-retaining diuretics? may cause mild hyperkalaemia even if glomerular
– an ACE inhibitor or angiotensin II receptor function is normal. A cause of respiratory acidosis is
antagonist? usually obvious clinically (see Chapter 4).
● Is there a cause for cell damage or transcellular ● Measurement of the TTKG (described above) may
potassium shifts, such as hypoxia, rhabdomyolysis help to determine whether there is a potassium renal
or severe trauma? excretory effect.
● Is there evidence of mineralocorticoid deficiency?
Hyperkalaemia This should not be forgotten, as adrenal insufficiency
can be life threatening. If there is any doubt, consider
Artefact or pseudohyperkalaemia?
performing a short Synacthen test (see Chapter 8 for
details of the investigation of adrenal insufficiency).
● Rare causes of hyperkalaemia, such hyporeninaemic
No Yes hypoaldosteronism, pseudohypoaldosteronism and
type IV renal tubular acidosis, are shown in Box
Patient on potassium-raising 5.2. The investigation of renal tubular acidosis is
medication? described in Chapter 4.
Treatment of hyperkalaemia
No Yes Emergency treatment
Emergency treatment may be necessary if there is life-
Acute kidney injury (AKI) or
threatening hyperkalemia, for example more than
chronic kidney disease (CKD)?
7.0 mmol/L or significant ECG changes. Calcium
gluconate, 10 mL of a 10 per cent solution, is given
No Yes
slowly intravenously with ECG monitoring. This 8.4 per cent sodium bicarbonate is hyperosmolar.
antagonizes the effect of hyperkalaemia on heart muscle Infusing bicarbonate increases the rate of potassium
but does not alter potassium concentrations. However, entry into cells.
calcium should never be added to bicarbonate solutions Sometimes, if the hyperkalaemia is not too severe, 10–
because calcium carbonate is poorly soluble in water. If 20 mg nebulized salbutamol can be used to lower plasma
too much calcium is given, hypercalcaemia may cause potassium as b2-catecholamines cause redistribution of
cardiac arrest. potassium into cells. Reductions in plasma potassium of
Intravenous 50 mL glucose 50 per cent with 10 0.5–1.0 mmol/L can occur and last for 2–3 h. In severe
units of soluble insulin by intravenous injection hyperkalaemia with renal dysfunction haemofiltration
over 15–30 min starts to lower plasma potassium or dialysis may be indicated.
concentrations for a couple of hours by increasing
potassium entry into cells. Insulin enhances the activity Long-term treatment of chronic hyperkalaemia
of the pump and must be given with glucose to prevent Sodium or calcium polystyrene sulphonate
hypoglycaemia. (Resonium-A or Calcium Resonium) 15 g orally three
More rarely, 50–100 mL of 8.4 per cent sodium or four times a day can remove potassium from the
bicarbonate, usually via a central line, may be infused body but may take at least 24 h to work. Type IV renal
over 30 min if a severe acidosis (for example pH less tubular acidosis is a chronic cause of hyperkalaemia
than 7.1) is present, or 1.26 per cent solution in isotonic and may be treated with fludrocortisone and a thiazide
saline if the situation is less urgent. Remember that diuretic or furosemide.
SUMMARY
● Disorders of potassium homeostasis are common ● Hypokalaemia can cause weakness, paralytic ileus
in clinical practice and can be life threatening. and ECG changes; common causes are potassium
● Potassium is a predominantly intracellular cation, loss from the kidneys or gastrointestinal tract,
and thus plasma levels do not adequately reflect cellular redistribution and poor intake.
total body potassium stores. ● Hyperkalaemia can cause lethal cardiac arrhythmias
● Potassium ions are controlled by renal excretion, and is commonly due to renal retention, movement
which is increased by aldosterone and reduced by H+. out of cells or increased intake.
6 Calcium, phosphate and magnesium
Disorders of calcium metabolism are common in may form insoluble, poorly absorbed complexes with
clinical practice and may result in hypocalcaemia or oxalate, phosphate or fatty acids. An excess of fatty acids
hypercalcaemia as well as bone abnormalities. Intimately in the intestinal lumen in steatorrhoea may contribute
associated with calcium disorders are disorders involving to calcium malabsorption.
phosphate and magnesium metabolism.
CONCEPT OF PLASMA CALCIUM AND
ALBUMIN CORRECTION (ADJUSTED)
CALCIUM METABOLISM
The mean plasma calcium concentration in healthy
TOTAL BODY CALCIUM subjects is tightly controlled, at around 2.15–
The total body calcium depends upon the calcium 2.55 mmol/L, and is present in two main forms:
absorbed from dietary intake and that lost from the ● Calcium bound to proteins, mainly albumin: this
body (Fig. 6.1). Ninety-eight per cent of body calcium accounts for a little less than half the total calcium
is found in the skeleton. The extraosseous fraction, concentration as measured by routine analytical
although amounting to only 1 per cent of the total, methods and is the physiologically inactive form.
is essential because of its effect on neuromuscular ● Free ionized calcium (Ca2+), which comprises most
excitability and cardiac muscle. An important mediator of the rest. This is the physiologically active fraction.
of intracellular calcium is calmodulin, a calcium-
Changes in plasma protein concentration,
binding regulatory protein.
particularly of albumin, the principal plasma protein,
Factors affecting calcium intake alter the most commonly measured concentration,
About 25 mmol (1 g) of calcium is ingested per day, that of plasma total calcium, but not that of the free
of which there is a net absorption of 6–12 mmol ionized fraction. The plasma total (but not free
(0.25–0.5 g). The active metabolite of vitamin D, ionized) calcium concentration is lower in the supine
1,25-dihydroxycholecalciferol (1,25-(OH)2D3, also than in the erect position because of the effect of
called calcitriol), is needed for calcium absorption. posture on fluid distribution and therefore on plasma
protein concentration. The direct measurement of the
Factors affecting calcium loss physiologically active free calcium ionized fraction is,
Calcium is lost in urine and faeces. Urinary calcium for technical reasons, confined to special cases such as
excretion depends on the amount of calcium reaching acid–base disturbance.
the glomeruli, the glomerular filtration rate (GFR) Formulae incorporating the albumin concentration
and renal tubular function. Parathyroid hormone and have been devised in an attempt to calculate the active
1,25-dihydroxyvitamin D increase urinary calcium fraction of the plasma total calcium concentration,
reabsorption. but, because binding is not simple, these are not always
Faecal calcium is derived from the diet and that reliable, particularly if extremes of plasma albumin
portion of the large amount of intestinal secretions concentration occur. The following is a commonly used
that has not been reabsorbed. Calcium in the intestine formula:
96 Calcium, phosphate and magnesium
DIETARY INTAKE
SOFT TISSUES
25 mmol/day
25 mmol
Intestine FILTRATE
Kidneys
Figure 6.2 Formation of the active vitamin D metabolite from 7-dehydrocholesterol. UV, ultraviolet.
diet. It is the main circulating form and store of the hypocalcaemia is prolonged, more efficient absorption
vitamin. Other hydroxylated metabolites are found, becomes important. Once the plasma free ionized
such as 24,25-(OH)2D3. calcium concentration is adjusted, the secretion of both
In the proximal renal tubular cells of the kidney, PTH and 1,25-(OH)2D3 is suppressed.
25-OHD3 undergoes a second hydroxylation, catalysed Thus, 25-OHD3 is the circulating, inactive form of
by the enzyme 1-a-hydroxylase to form the active vitamin D and plasma concentrations fall in deficiency
metabolite 1,25-(OH)2D3. states. The measurement of the biologically active
The activity of 1-a-hydroxylase, and hence the metabolite, 1,25-(OH)2D3, which circulates in plasma
production of 1,25-(OH)2D3, may be stimulated by: bound to vitamin D-binding protein (VDBP) in very
low concentrations, is rarely indicated unless a defect in
● a low plasma phosphate concentration,
the vitamin metabolic pathway is suspected, as it does
● an increase in plasma PTH concentration, possibly
not reflect body stores.
because of its phosphate-lowering effect.
The vitamin D receptor (VDR) is found in almost
Its activity is inhibited by: all cell nuclei with various effector systems such as
● hyperphosphataemia, endocrine, paracrine or autocrine. Calcitriol activates
● high levels of free ionized calcium. the receptor, which forms a heterodimer with the
retinoid-X receptor and binds to hormone response
The kidney is an endocrine organ, synthesizing and elements on deoxyribonucleic acid (DNA) and is
releasing the hormone 1,25-(OH)2D3; impairment of involved in the expression of various gene products.
the final hydroxylation helps explain the hypocalcaemia These pathways not only involve bone metabolism
of renal disease. This hormone increases calcium but also have implications for the immune system and
absorption by intestinal mucosal cells. In conjunction carcinogenesis.
with PTH, it stimulates osteoclastic activity, releasing
calcium from bone. Calcium-sensing receptor
The action of PTH on bone is impaired in the The calcium-sensing receptor (CaSR) is a G protein-
absence of 1,25-(OH)2D3. A fall in plasma free ionized coupled receptor. This allows the parathyroid cells and
calcium concentration stimulates PTH secretion. the ascending loop of Henle epithelial cells to respond
The PTH enhances 1-a-hydroxylase activity and to changes in extracellular calcium. The parathyroid
therefore stimulates 1,25-(OH)2D3 synthesis. The two cell surface is rich in CaSR, which allows PTH secretion
hormones act synergistically on the osteoclasts of bone, to be adjusted rapidly depending on the calcium
releasing calcium into the circulation; 1,25-(OH)2D3 concentration.
also increases calcium absorption from the intestinal Defects in the CaSR gene are responsible for
lumen. In the short term, the homeostatic mechanisms various rare defects of calcium homeostasis.
involving the effects on bone are the more important; if Inactivating mutations include familial benign
Disorders of calcium metabolism 99
the Q–T interval and broadening of the T waves. If hyperparathyroidism or malignancy. In the case of the
plasma concentrations exceed about 3.5 mmol/L, latter, 80 per cent are due to bony metastases, with the
there is a risk of sudden cardiac arrest or ventricular remainder being mainly due to ectopic PTHRP. Some
arrhythmias. For this reason severe hypercalcaemia causes of hypercalcaemia are depicted in Box 6.1.
should be treated as a matter of urgency. True free ionized or albumin-adjusted hyper-
● Hypercalcaemia is also associated with bone and calcaemia with hypophosphataemia is usually caused
joint pain. by inappropriate secretion of PTH or PTHRP. The term
‘inappropriate secretion’ is used in this book to indicate
‘Bones, moans, groans and stones’ is a useful
that the release of hormone into the circulation is not
mnemonic to remember some of these clinical
adequately inhibited by negative feedback control.
consequences of hypercalcaemia.
Inappropriate PTH secretion occurs in the following
Causes of hypercalcaemia (Box 6.1) clinical situations:
Overall, thiazides are one of the most common causes ● production of PTH by the parathyroid glands due to:
of mild hypercalcaemia. However, most causes of – primary hyperparathyroidism,
severe hypercalcaemia are related to either primary – tertiary hyperparathyroidism.
If renal glomerular function is adequate, the high
circulating PTH or PTHRP concentrations cause
Box 6.1 Some causes of hypercalcaemia hypercalcaemia, which is associated with a low-normal
or low plasma phosphate concentration in relation
Malignancy
Bony metastases, e.g. breast, lung, prostate, kidney,
to GFR, and to phosphaturia. If glomerular damage
thyroid develops due to hypercalcaemia, the kidneys cannot
Solid tumours with humoral effects respond normally to the phosphaturic effect of PTH
Haematological tumours, e.g. multiple myeloma and, because of impaired hydroxylation of 25-OHD3,
Parathyroid hormone abnormalities plasma calcium concentrations may fall towards or
Primary hyperparathyroidism (adenoma, hyperplasia, within the reference range as renal failure progresses.
carcinoma or associated with multiple endocrine Because plasma phosphate concentrations tend to rise,
neoplasia) diagnosis may be difficult at this stage.
Tertiary hyperparathyroidism
Lithium-induced hyperparathyroidism
High bone turnover
Thyrotoxicosis CASE 2
Immobilization, e.g. with Paget’s disease
A 53-year-old man saw his general practitioner
High levels of vitamin D because of bone pain and constipation. A number
Vitamin D toxicity
of laboratory tests were requested, the results for the
Granulomatous disease, e.g. sarcoidosis, tuberculosis
most relevant of which were as follows:
Drugs
Thiazides (reduced renal calcium excretion) Plasma
Vitamin A toxicity Albumin-adjusted calcium 2.96 mmol/L (2.15–2.55)
Milk–alkali syndrome Phosphate 0.62 mmol/L (0.80–1.35)
Familial hypocalciuric hypercalcaemia Parathyroid hormone 157 ng/L (20–65)
Other endocrine causes
Adrenal insufficiency DISCUSSION
Acromegaly The patient has hypercalcaemia. Note also the
Rarer causes hypophosphataemia and inappropriately raised
Williams’ syndrome PTH concentration. The diagnosis was subsequently
Human immunodeficiency virus (HIV) infection found to be primary hyperparathyroidism due to
Leprosy a parathyroid adenoma associated with multiple
Histoplasmosis endocrine neoplasia (MEN) type I. His symptoms
Berylliosis are typical of chronic hypercalcaemia.
Disorders of calcium metabolism 101
The clinical features of PTH- or PTHRP-induced the terminal phalanges. However, extensive, severe
hypercalcaemia are due to: bone disease, osteitis fibrosa cystica, is now a rare
presenting feature, as patients are usually diagnosed
● excess circulating concentration of free ionized
before the disorder is extensive, and consequently
calcium that is the direct consequence of increased
plasma alkaline phosphatase activity is usually
osteoclastic activity and release of calcium from
normal or only slightly increased. There are increased
bone, and enhanced absorption of calcium from the
numbers of osteoclasts and an increased risk of bone
intestinal lumen by vitamin D; PTH increases the
fracture.
formation of 1,25-(OH)2D3,
● Medical emergency Occasionally patients are
● the effects of persistent PTH or PTHRP activity on
admitted as an emergency with abdominal pain,
bone in the presence of a normal supply of vitamin
vomiting and constipation. Severe hypercalcaemia is
D and calcium (see Fig. 6.1).
a recognized cause of acute pancreatitis and should
The differences between the clinical presentations be considered as one cause of an ‘acute abdomen’.
associated with inappropriately high plasma PTH
Recently, it has been reported that an incipient form
concentrations depend on the duration of the disease.
of primary hyperparathyroidism exists in which there
The following effects on bone become evident only in
is initially normal plasma calcium but elevated PTH.
long-standing cases. Prolonged decalcification of bone
The treatment of primary hyperparathyroidism is
causes a secondary increase in osteoblastic activity.
often surgical, with removal of the parathyroid gland(s).
Alkaline phosphatase-rich osteoblasts release the enzyme
However, this can render the patient hypocalcaemic,
into the circulation and, if the number of cells is greatly
and asymptomatic patients are sometimes treated
increased, plasma alkaline phosphatase activity rises.
conservatively.
Primary hyperparathyroidism
Tertiary hyperparathyroidism
This is caused by inappropriate secretion of PTH by
the parathyroid glands, causing hypercalcaemia. It is This may occur if the parathyroid glands have been
usually due to one or more parathyroid adenomas, subjected to long-standing and sustained positive feedback
but occasionally to hyperplasia of all four parathyroid by low plasma free ionized calcium concentrations
glands or to carcinoma of one of the glands. Ectopic (hypocalcaemia) of secondary hyperparathyroidism
parathyroid tumours do also occur. Primary which have been subsequently corrected.
hyperparathyroidism may be associated with other The parathyroid glands hypertrophy; PTH secretion
multiple endocrine neoplasias (MENs), such as becomes partly autonomous and is not suppressed
pituitary and pancreatic adenomas (MEN type I), or by negative feedback by the hypercalcaemia. The
with phaeochromocytomas and medullary carcinoma diagnosis is usually made when the cause of the original
of the thyroid (MEN type II). The incidence of primary hypocalcaemia is removed, for example by renal
hyperparathyroidism increases with age, being most transplantation or correction of long-standing calcium
common in elderly females. or vitamin D deficiency as in malabsorption. A history
The majority of cases of primary hyperthyroidism of previous hypocalcaemia and the finding of a very
are diagnosed after the chance finding of high high plasma alkaline phosphatase activity due to the
plasma calcium, usually with low plasma phosphate prolonged osteomalacia distinguish it from primary
concentrations. hyperparathyroidism. In some cases, the glandular
Where there are clinical symptoms and signs at hypertrophy gradually regresses and the plasma calcium
presentation, these are due to hypercalcaemia and concentration returns to normal.
include the following: Unlike primary or tertiary hyperparathyroidism, in
which plasma PTH concentration is increased, there are
● Generalized ill health Depression, nausea, anorexia other causes of hypercalcaemia where plasma levels of
and abdominal pain and polyuria. PTH are reduced or suppressed. These are now discussed.
● Renal calculi About 10 per cent of patients
who present with renal calculi have primary Hypercalcaemia of malignancy
hyperparathyroidism. Malignant disease of bone
● Bone pain In most patients, subperiosteal bone Some patients with multiple bony metastases (from,
erosions or cysts may be seen on radiography of for example, breast, lung, prostate, kidney and
102 Calcium, phosphate and magnesium
● Is the plasma phosphate concentration low in relation relies on the fact that hypercalcaemia of primary
to the renal function? Hypophosphataemia suggests hyperparathyroidism is not usually suppressed by
the diagnosis of primary hyperparathyroidism. steroids, unlike many of the other causes of a raised
calcium concentration such as malignant disease.
Apart from thiazide usage, the most common
causes of hypercalcaemia are either primary Treatment of hypercalcaemia
hyperparathyroidism or malignancy; the latter may be Mild to moderate hypercalcaemia
obvious following clinical examination and radiological
If the plasma albumin-adjusted calcium concentration
and haematological tests, for example anaemia, and
is below about 3.5 mmol/L, and if there are no
raised erythrocyte sedimentation rate (ESR) and
significant clinical symptoms or signs such as changes
biochemical investigations.
attributable to hypercalcaemia, there is no need for
It is essential that primary hyperparathyroidism
urgent treatment. However, therapy should be started
and malignant hypercalcaemia are distinguished.
as soon as the abnormality is found and preliminary
In the case of malignancy, pay special attention for
investigations have been performed because of the
breast, kidney, lung or prostate carcinoma. A raised
danger of renal damage. If possible, the primary cause
plasma PTH concentration is usually seen in primary
should also be diagnosed and treated.
hyperparathyroidism; conversely, suppressed levels are
The patient should be fluid volume repleted, if
found in malignant states and indeed in hypercalcaemia
necessary by intravenous infusion of saline. The
of many other causes. Very rarely PTHRP should be
plasma total calcium concentration will often fall as
measured if ectopic secretion of this is suspected, for
the plasma albumin concentration is diluted, but the
example by a tumour.
plasma free ionized calcium concentration is probably
If primary hyperparathyroidism due to an adenoma
little affected. Correcting haemoconcentration enables
is found, exclude MEN syndrome (see Chapter 24).
a more realistic assessment to be made of the degree of
Imaging of the parathyroid glands is often needed
true hypercalcaemia.
to distinguish adenoma from hyperplasia of the
Bisphosphonates are first-line agents in the medical
parathyroid glands. Isotope subtraction scanning or
management of hypercalcaemia. These are structurally
ultrasound of the neck may help to localize the adenoma,
similar to pyrophosphate. They bind to hydroxyapatite
as may venous sampling for PTH levels.
in bone, thus inhibiting bone turnover and the
Very high plasma alkaline phosphatase activity
mobilization of calcium. They are poorly absorbed
is unlikely to be due to uncomplicated primary
from the intestinal tract and may have to be given
hyperparathyroidism; near-normal activity is usual,
intravenously to patients with severe hypercalcaemia.
although it may be raised if there is radiological evidence
Cyclical administration may prevent the long-term
of bone involvement. If it is very high, it suggests either
complication of osteomalacia.
malignancy or some concurrent disease such as Paget’s
The management of apparently asymptomatic mild
disease.
hypercalcaemia due to primary hyperparathyroidism
Perform serum and urinary protein electrophoresis
is controversial. It has been suggested that prolonged
if a multiple myeloma is suspected (see Chapter 19).
hypercalcaemia does not always cause obvious renal
● Look for evidence of sarcoidosis; plasma angiotensin- dysfunction and that the risk of parathyroidectomy
converting enzyme (ACE) concentration is often (e.g. rendering the patient hypocalcaemic) may be
raised (see Chapter 18) and a chest radiograph may greater than that of mild hypercalcaemia. The decision
be useful. whether to operate must be made on clinical grounds;
● Is there acromegaly, Addison’s disease or of particular importance are the fitness of the patient
thyrotoxicosis (see Chapters 7, 8 and 11)? for operation, plasma calcium concentration greater
● A urinary calcium determination (CaE) is useful to than 3.0 mmol/L, deteriorating renal function, renal
help exclude hypocalciuric hypercalcaemia. calculi, poor bone mineral density or 24-h urinary
● Rarer causes of hypercalcaemia are listed in Box 6.1. calcium concentration greater than 10 mmol/L.
A steroid suppression test is rarely necessary to Severe hypercalcaemia
identify the cause of hypercalcaemia because of the The plasma albumin-adjusted calcium concentration
development of robust PTH assays. Briefly, this test at which urgent treatment is indicated because of the
Disorders of calcium metabolism 105
to long-standing deficiency of calcium, phosphate is high, renal dysfunction is the likely cause (see
and vitamin D. Predisposing factors include: Chapter 3).
– reduced dietary intake of vitamin D, calcium ● Is the plasma phosphate concentration low? If so,
and phosphate in undernutrition, calcium deficiency with normal secretion of PTH in
– impaired absorption of vitamin D, for example response to feedback is likely. At this point a plasma
in steatorrhoea or hepatobiliary disease, PTH assay may be useful. Is the plasma alkaline
– impaired metabolism of vitamin D to 1,25- phosphatase activity high? This finding may suggest
(OH)2 D3 due to renal disease, prolonged secondary hyperparathyroidism due to
– increased inactivation of vitamin D due to calcium deficiency.
anticonvulsant therapy, ● If indicated, do relevant bone radiographs show
– renal tubular disorders of phosphate signs of rickets or osteomalacia? These may confirm
reabsorption. prolonged calcium deficiency. Check plasma
● Without osteomalacia or rickets: if PTH action is 25-hydroxyvitamin D levels; if the plasma levels
inadequate to correct the abnormality, the plasma are low, look for causes of undernutrition and
calcium concentration remains low and bone disorders malabsorption states.
are not present. Predisposing factors include:
In the rare hypocalcaemia of type 1 vitamin-D-
– early calcium and vitamin D deficiency,
dependent rickets there are low plasma 1,25-(OH)2D3
– the rare pseudohypoparathyroidism.
concentrations, whereas type 2 vitamin-D-dependent
In secondary hyperparathyroidism the plasma rickets causes a vitamin D resistance and plasma
calcium concentration is never high, and usually 1,25-(OH)2D3 concentrations are elevated.
the plasma calcium and phosphate concentrations Raised plasma phosphate in the face of
tend to be low. High-normal or high plasma calcium hypocalcaemia and low plasma PTH concentration
concentrations in renal glomerular dysfunction suggest suggests hypoparathyroidism.
either that primary hyperparathyroidism has caused the Is there a history of neck surgery which has led to
renal disease or that prolonged calcium deficiency has hypoparathyroidism? Hypoparathyroidism may also
led to the development of tertiary hyperparathyroidism. be of autoimmune origin and associated with other
autoimmune disorders. It needs to be distinguished
Investigation of hypocalcaemia (Fig. 6.4) from the even more rare ‘pseudohypoparathyroidism’
As in the case of hypercalcaemia, the causes of by measuring plasma PTH concentrations. Parathyroid
hypocalcaemia fall into two main groups: hormone concentrations will be low in true
hypoparathyroidism but high if there is the end-organ
● reduced albumin-adjusted calcium concentration unresponsiveness of pseudohypoparathyroidism.
due to primary PTH deficiency and associated with Check plasma magnesium, as severe hypomagnesaemia
hyperphosphataemia, can cause hypocalcaemia by reducing the action of PTH.
● reduced albumin-adjusted calcium concentration A raised urinary calcium concentration may help
due to other causes and associated with diagnose the rare autosomal dominant hypercalciuric
appropriately high PTH concentrations and usually hypocalcaemia.
hypophosphataemia.
Determine first if the fall in plasma total calcium Treatment of hypocalcaemia
concentration (albumin-adjusted calcium) is due to a Asymptomatic hypocalcaemia
low protein-bound fraction.
Apparent hypocalcaemia, due to low plasma albumin
Patients with a low albumin concentration should
concentrations, should not be treated. Always look at
not be given calcium and/or vitamin D unless there is
the albumin-adjusted calcium value.
clinical evidence of a low albumin-adjusted calcium
Asymptomatic true hypocalcaemia, or that causing
concentration.
only mild clinical symptoms, can usually be treated
● Is the patient on drugs or chemicals that may cause effectively with oral calcium supplements and vitamin
hypocalcaemia (see Box 6.2)? D supplements. It is difficult to give enough oral calcium
● Is the plasma phosphate concentration high? If by itself to make a lasting and significant difference
the plasma urea and/or creatinine concentration to plasma calcium concentrations. If a normal diet is
Disorders of calcium metabolism 109
Hypocalcaemia
No Yes
Yes No
No Yes
(see Box 6.2)
No Yes
Evidence of hypomagnesaemia?
No Yes
(see Box 6.6)
Low/normal High
Yes No
Low High/normal
Figure 6.4 Algorithm for the investigation of hypocalcaemia. EDTA, ethylenediamine tetra-acetic acid.
being taken, vitamin D, by increasing the absorption of commonly used as they have short half-lives, particularly
calcium from the intestine, is usually adequate without if there is hypoparathyroidism or a defect in vitamin
calcium supplements. 1,25-Dihydroxycholecalciferol D metabolism. It is important to monitor the plasma
and alfacalcidol (1-a-hydroxycholecalciferol) are most calcium closely to avoid inducing hypercalcaemia and
110 Calcium, phosphate and magnesium
hypercalciuria by ensuring a normal urinary excretion Hypercalciuria can, of course, also occur in the face
of calcium. of hypercalcaemia, such as resorptive hypercalciuria
Because of the danger of ectopic calcification by associated with primary hyperparathyroidism.
precipitation of calcium phosphate, hypocalcaemia Estimation of urinary CaE is rarely diagnostic in
with hyperphosphataemia in renal disease should be the differential diagnosis of hypercalcaemia except
treated cautiously. The plasma phosphate concentration familial hypocalciuric hypercalcaemia. If glomerular
should first be lowered by giving a phosphate-binding function is normal, all other causes of hypercalcaemia
agent that binds phosphate in the intestinal lumen. usually increase the calcium load on the glomeruli
1,25-Dihydroxycholecalciferol and alfacalcidol, the and evoke hypercalciuria. If renal glomerular function
active vitamin D metabolites, have been used to treat is impaired, calcium excretion is low even if there is
hypocalcaemia in renal disease. hypercalcaemia.
Hypocalcaemia with life-threatening symptoms Disorders of bone not usually affecting the
If there are cardiac arrhythmias, seizures or severe plasma calcium concentration
tetany including laryngospasm shown to be due to Some disorders of bone rarely alter plasma calcium
hypocalcaemia, intravenous calcium, usually as 10 mL concentrations but are important in the differential
of 10 per cent calcium gluconate, should be given diagnosis of changes in mineral metabolism.
over about 5 min. Treatment can then begin as above,
depending upon the aetiology of the hypocalcaemia. Osteoporosis
Osteoporosis is not a primary disorder of calcium
Post-operative hypocalcaemia
metabolism. The reduction of bone mass is due to
Hypocalcaemia during the first week after a thinning of the protein on which the calcium is usually
thyroidectomy or parathyroidectomy should be deposited. A slight increase in urinary calcium loss
treated only if there is tetany, and usually with calcium is secondary to this. Disorders associated with an
replacement, which, unlike vitamin D supplements, increased incidence include the following:
has a rapid effect and a short half-life. Persistent
hypocalcaemia may indicate that the parathyroid glands ● low plasma oestrogen concentrations, such as after
are permanently damaged and that long-standing, or the female menopause (the most common cause)
even life-long, vitamin D supplementation is necessary. and prolonged amenorrhoea; also low testosterone
Parathyroid bone disease may result in ‘hungry bones’ concentrations in males,
and prolonged post-operative hypocalcaemia. ● elderly subjects in whom there may also be mild
osteomalacia because of impaired renal production
Hypercalciuria of 1,25-(OH)2D3,
● other endocrine disorders and other miscellaneous
Hypercalciuria in the absence of hypercalcaemia
causes:
(hypercalciuria normocalcaemia) may predispose to
– hypercortisolism, hyperthyroidism, long-term
the formation of renal calculi (see Chapter 3) and may
glucocorticoid use,
occur in:
– drugs such as heparin, and anticonvulsants
● some cases of osteoporosis in which calcium cannot such as phenytoin and carbamazepine,
be deposited in normal amounts because the bone – prolonged immobilization,
matrix is reduced, – smoking, alcohol abuse,
● acidosis, in which the release of free ionized calcium – calcium deficiency,
from bone is increased. – gastrointestinal causes, including malabsorption,
anorexia nervosa.
Hypercalciuria can broadly be divided into absorptive
hypercalciuria, type I (hyperabsorption of calcium), In osteoporosis plasma calcium and phosphate
type II (diet-responsive hypercalciuria) and type III concentrations do not fall and, because there is no
(renal phosphate leak resulting in decreased calcium increase in osteoblastic activity, the plasma total alkaline
resorption), and renal hypercalciuria (decreased renal phosphatase activity does not rise. These findings are
calcium resorption). These can be distinguished by invaluable in distinguishing between osteomalacia and
tests of oral calcium absorption. osteoporosis.
Disorders of calcium metabolism 111
Concentrations of new bone markers, such as bone- bone agent (DABA) that stimulates osteoblasts and
specific alkaline phosphatase (bone formation), plasma inhibits osteoclasts. Calcitonin inhibits osteoclast
osteocalcin (bone formation), type 1 procollagen reabsorption and recombinant PTH analogues such as
peptides (bone formation), urinary deoxypyridinoline teriparatide stimulate osteoblasts. In women, hormone
and cross-linked N-terminal telopeptide and C-terminal replacement therapy is also used if indicated, but this
telopeptide of type 1 collagen (bone resorption), may have side effects. Raloxifene has been used and is a
urinary hydroxyproline (bone resorption) and bone- selective estrogen receptor modulator (SERM).
resistant or tartrate-resistant acid phosphatase (bone
resorption) are raised in osteoporosis and may be Paget’s disease of bone
useful markers of the disease process. However, these Paget’s disease is more common in the elderly, possibly
bone markers do not give information about exact being present in about 5 per cent of people over 60 years
bone anatomy, for which imaging studies are necessary. old. There is increased bone turnover and remodelling
Sometimes radiographs are useful, especially if due to increased osteoclastic and osteoblastic function.
fractures are suspected (Fig. 6.5). Bone mineral density It may be asymptomatic or may present with bone
(BMD) is often used and reported as a T-score that pain, pathological fractures, deafness (due to bone
compares the patient’s BMD with that of a healthy overgrowth) and high-output cardiac failure due to
control. Normal BMD is within –1 standard deviation increased vascularity within the bone. Enlargement of
(SD) of this, whereas osteopenia is defined as between bones such as the skull (osteoporosis circumscripta),
–1 and –2.5 SD and osteoporosis has a T-score of less femur and tibia (sabre tibia) can occur.
than –2.5 SD. Diagnosis may necessitate radiographs and/or bone
Treatment consists of adequate calcium and scanning. Plasma calcium and phosphate concentrations
vitamin D intake. The bisphosphonates increase BMD are rarely affected unless the patient is immobilized,
and inhibit bone resorption, probably by inhibiting in which case the plasma calcium concentrations may
osteoclast activity. Strontium ranelate is a duel action rise. Plasma alkaline phosphatase activity is typically
very high. Less than 1 per cent of patients may develop
osteosarcomas, and this complication may be associated
with rapidly rising plasma alkaline phosphatase activity.
Bisphosphonates are often used to treat Paget’s disease;
although calcitonin has been tried, it is probably less
effective.
concentrations differ from the findings in the ‘classic’ THE FUNCTION OF PHOSPHATE IN VIVO
syndrome. As the free ionized calcium concentration is Phosphate is an important intracellular buffer as well
normal, the parathyroid glands are not overstimulated as being essential for buffering hydrogen ions in urine.
and therefore evidence in the bone of high PTH In addition, it has a structural role as a component of
concentrations is rare. The conditions respond to large phospholipids, nucleoproteins and nucleic acids.
doses of oral phosphate and to a small dose of the active Phosphate plays a central role in cellular metabolic
metabolite of vitamin D. pathways, including glycolysis and oxidative
phosphorylation. A by-product of glycolysis is
PHOSPHATE METABOLISM 2,3-diphosphoglycerate (2,3-DPG). This is a regulator
of haemoglobin oxygen dissociation. Nucleotides such
Phosphate is a divalent anion, approximately as adenosine triphosphate consist of phosphate. Other
80 per cent of which is found in the bony skeleton actions include excitation–stimulus response coupling
and 20 per cent is distributed in the soft tissues and and nervous system conduction. Phosphate also has a
muscle. Phosphate is the major intracellular anion role in the optimal function of leucocytes, for example
and shifts between the intracellular and extracellular chemotaxis and phagocytosis, and for platelets in clot
compartments. Such transcellular movement can retraction.
result from the ingestion of carbohydrate or lipid,
as well as from acid–base alterations – for example, ABNORMALITIES OF PLASMA
PHOSPHATE CONCENTRATION
acidosis can result in shifts of phosphate out of cells
into the plasma. Hyperphosphataemia
The daily phosphate intake is about 30 mmol, The causes of hyperphosphataemia are listed in Box
with approximately 80 per cent being absorbed in 6.3. The majority of the clinical effects are the result of
the jejunum. Protein-rich food is a major source of hypocalcaemia, particularly if the plasma phosphate
phosphate intake, as are cereals and nuts. The output concentration is more than 3.0 mmol/L. The reason
is largely renal, with more than 90 per cent being for this is that calcium/phosphate precipitation into
excreted by this route. Most of the phosphate filtered the tissues can ensue when the phosphate and calcium
at the glomeruli is reabsorbed by the proximal tubules. plasma concentrations exceed their solubility product.
Gastrointestinal loss of phosphate accounts for only Thus, metastatic calcification is a clinical consequence
10 per cent of the body’s phosphate excretion. of hyperphosphataemia.
Urinary phosphate excretion falls as the plasma The treatment for hyperphosphataemia is with oral
phosphate, and therefore glomerular filtrate, phosphate-binding agents, for example magnesium
concentrations decrease in response to reduced hydroxide or calcium carbonate. These agents have
dietary phosphate intake. The measurement of urinary been used in the management of patients with chronic
phosphate concentration may occasionally be useful to
distinguish hypophosphataemia due to true depletion Box 6.3 Some causes of
(low urinary phosphate) and the increased urinary hyperphosphataemia
phosphate excretion found in renal tubular disorders,
such as X-linked hypophosphataemic rickets. Artefact due to in vitro haemolysis or old blood sample
The urinary phosphate excretion threshold can be Inappropriately high phosphate intake, usually
derived from nomograms, and a low value implies renal intravenously
phosphate loss. Another way to assess renal phosphate Acute kidney injury or chronic kidney disease
loss is to calculate the fractional phosphate excretion Increased tissue breakdown
(FEPi%): Tumour lysis syndrome
Malignant hyperpyrexia
urinary[phosphate] Crush injuries
¥ plasma[creatinine] (6.3) Acidaemia (metabolic or respiratory acidosis)
FEPi% =
plasma[phosphate] Diabetic ketoacidosis
¥ urinary[creatinine] Hypoparathyroidism
Acromegaly
An FEPi% of more than 10 per cent implies a renal Excess vitamin D intake
phosphate loss.
Plasma magnesium and its control 113
recommended daily allowance of magnesium for adults Clinical management of severe hypermagnesaemia
is about 4.5 mg/kg; rich dietary sources include cereal, If there is severe hypermagnesaemia, 10 mL of
nuts and vegetables. 10 per cent calcium gluconate given slowly intravenously
Magnesium is largely absorbed in the upper small may relieve symptoms. Analogous to the treatment
intestine but the large intestine may also be important; of hyperkalaemia, insulin and glucose infusion can
unlike calcium, its absorption is not vitamin D be used in severe hypermagnesaemia (see Chapter 5).
dependent. As much as 70 per cent of magnesium Failing this, and if there is impaired renal function,
from dietary intake is not absorbed but eliminated dialysis may be indicated (see Chapter 3).
in the faeces. The major excretory route is via the
kidneys, and about 65 per cent of glomerular-filtered Hypomagnesaemia
magnesium is reabsorbed in the loop of Henle. The Some causes of hypomagnesaemia are shown in
exact mechanisms of magnesium homeostatic control Box 6.6. The symptoms of hypomagnesaemia are
are unclear, although PTH, insulin and calcitonin very similar to those of hypocalcaemia. If the plasma
are important. Parathyroid hormone can increase calcium concentrations (allowing for that of albumin)
magnesium reabsorption, although hypercalcaemia and blood pH are normal in a patient with tetany, the
can increase the renal excretion of magnesium. About plasma magnesium concentration should be assayed.
35 per cent of plasma magnesium is protein bound, and Hypomagnesaemia can result in cardiac arrhythmias,
the plasma concentration is normally 0.7–1.2 mmol/L. including torsade de pointes, and digoxin sensitivity.
In addition, abdominal discomfort and anorexia have useful: 30 mmol of magnesium (usually as sulphate)
been described, as well as neuromuscular sequelae is infused intravenously in 500 mL 5 per cent dextrose
including tremor, paraesthesiae, vertigo, tetany, over 4 h and urine is collected over 24 h for magnesium
seizures, irritability, confusion, weakness and ataxia. determination. Magnesium depletion is unlikely if
Severe hypomagnesaemia can lead to hypocalcaemia more than 24 mmol of magnesium is excreted in 24 h.
due to decreased PTH release and activity. Severe hypomagnesaemia, less than 0.5 mmol/L
Long-term magnesium deficiency may be a risk or if it is symptomatic, can be corrected by oral
factor for coronary artery disease, perhaps increasing magnesium salts, but these may be poorly absorbed
atherosclerosis and platelet aggregation. There are and lead to gastrointestinal upset. A possible regimen
data suggesting that reduced magnesium intake is would be oral magnesium gluconate 12 mmol/day
associated with hypertension and insulin resistance. to a maximum of 48 mmol/L, if required, in three or
four divided doses. Intravenous replacement is often
Treatment of hypomagnesaemia given as magnesium sulphate. Generally, 0.5 mmol/kg
Sometimes the symptoms and signs of magnesium per day can be given by intravenous infusion. Close
deficiency occur in the face of borderline magnesium monitoring of plasma magnesium is necessary.
plasma concentrations, as plasma levels poorly reflect The treatment of hypomagnesaemia may facilitate
intracellular magnesium stores. In such circumstances the treatment of refractory hypokalaemia and
the intravenous magnesium-loading test may be hypocalcaemia.
SUMMARY
● Calcium, phosphate and magnesium metabolism hyperparathyroidism, certain drugs such as thiazides,
are closely related and abnormalities are clinically granulomatous disease such as sarcoidosis, milk–
relatively common. alkali syndrome, thyrotoxicosis, Addison’s disease,
● Plasma calcium levels are controlled by PTH (raises hypocalciuric hypercalcaemia and acromegaly.
plasma calcium), vitamin D activity and optimal ● Hypocalcaemia can present with paraesthesiae,
renal and intestinal function. tetany, osteomalacia and seizures. The causes may
● Hypercalcaemia can result in various symptoms: be due to poor diet, including vitamin D deficiency,
‘bones, stones, moans and groans’. The causes chronic kidney disease, malabsorption, certain drugs,
include malignant disease, primary or tertiary such as loop diuretics, and hypoparathyroidism.
7 The hypothalamus and pituitary gland
General principles of endocrine diagnosis 116 Disorders of posterior pituitary hormone secretion 123
Hypothalamus and pituitary gland 116 Hypopituitarism 123
Disorders of anterior pituitary hormone secretion 119
hormone (ADH) – also called vasopressin or arginine ● Corticotrophs synthesize a large polypeptide
vasopressin (AVP) – and oxytocin, are synthesized in (pro-opiomelanocortin), which is a precursor
the hypothalamus and transported down the nerve of both adrenocorticotrophic hormone (ACTH;
fibres of the pituitary stalk attached to specific carrier corticotrophin) and b-lipotrophin (Fig. 7.1).
proteins – neurophysins. The hormones are stored in the Secretion of these hormones occurs in parallel.
posterior pituitary gland and are released independently ● Adrenocorticotrophic hormone stimulates the
of each other into the bloodstream under hypothalamic synthesis and secretion of steroids, other than
control, together with neurophysin. Neurophysin has aldosterone, from the adrenal cortex and maintains
no apparent biological function and is rapidly cleared adrenal cortical growth. Part of the molecule has
from plasma. melanocyte-stimulating activity, and high circulating
Antidiuretic hormone (arginine vasopressin) is mainly concentrations of ACTH are often associated with
synthesized in the supraoptic nuclei of the hypothalamus pigmentation.
and enhances water reabsorption from the collecting ● b-Lipotrophin is inactive until rapidly converted
ducts in the kidneys (see Chapters 2 and 3). to endorphins. These are neurotransmitters which,
Oxytocin is synthesized in the paraventricular nuclei because they have opiate-like effects, help control
of the hypothalamus. It controls the ejection of milk pain.
from the lactating breast and may have a role in initiating ● Gonadotrophs secrete the gonadotrophins, follicle-
uterine contractions, although normal labour can stimulating hormone (FSH) and luteinizing
proceed in its absence. It may be used therapeutically hormone (LH), which act on the gonads.
to induce labour. ● Thyrotrophs secrete TSH (thyrotrophin), which acts
on the thyroid gland.
● These hormones are structurally similar glycoproteins
Anterior pituitary hormones consisting of two subunits, a and b. The a-subunit
There is no direct neural connection between the is common to all three hormones; the b-subunit is
hypothalamus and the anterior pituitary gland. The important for receptor recognition and therefore in
hypothalamus synthesizes small molecules (regulating specific biological activity.
hormones or factors) that are carried to the cells of
Chromophobes, once thought to be inactive, do
the anterior pituitary lobe by the hypothalamic portal
contain secretory granules. Chromophobe adenomas
system. This network of capillary loops in the median
often secrete hormones, particularly prolactin.
eminence forms veins, which, after passing down the
pituitary stalk, divide into a second capillary network in Control of anterior pituitary hormone secretion
the anterior pituitary gland, from where hypothalamic
Neural and feedback controls are the two most
hormones stimulate or inhibit pituitary hormone
important physiological factors influencing the
secretion into the systemic circulation.
secretion of the anterior pituitary hormones (Fig. 7.2).
The cells of the anterior pituitary lobe can be
classified simply by their staining reactions as acidophils,
ACTH (1–39) -LPH (42–134)
basophils or chromophobes. Immunohistochemistry
can identify specific hormone-secreting cells.
Acidophils are of two cell types:
-LPH (42–101) -endorphin (104–134)
● lactotrophs, which secrete prolactin,
● somatotrophs, which secrete GH (somatotrophin).
These hormones, which are simple polypeptides -MSH -endorphin
(84–101) (104–117)
with similar amino acid sequences, mainly affect
peripheral tissues directly. Stimulation and inhibition
of secretion via the hypothalamus is influenced by
Figure 7.1 The products of pro-opiomelanocortin
neural stimuli. (POMC): adrenocorticotrophic hormone (ACTH),
Basophils secrete hormones that affect other b-lipotrophin (LPH), g-LPH, b-melanocyte-stimulating
endocrine glands. The hypothalamic control is mainly hormone (MSH) and b- and g-endorphin. The numbers
stimulatory. There are three cell types: indicate the amino acid sequence in POMC.
118 The hypothalamus and pituitary gland
gonadotrophin-releasing hormone (GnRH) is less clear elsewhere in the brain, but also in the gastrointestinal
cut (see Chapter 9). tract and pancreatic islet cells, where it inhibits the
secretion of many gastrointestinal hormones. Insulin-
DISORDERS OF ANTERIOR PITUITARY
like growth factor 1 (IGF-1) acts by feedback to inhibit
HORMONE SECRETION
GHRH action.
The main clinical syndromes associated with excessive Growth hormone secretion may be stimulated by:
or deficient anterior pituitary hormone secretion are
shown in Table 7.1. Excessive secretion usually involves ● stress, one cause of which is hypoglycaemia,
a single hormone, but deficiencies are often multiple. ● glucagon,
However, many pituitary tumours are non-secretory ● some amino acids, for example arginine,
and may present clinically with eye signs or headaches. ● drugs such as levodopa and clonidine.
Figure 7.3 Patient with acromegaly; note large hands and prominent mandible and supraorbital ridges. Reproduced
with kind permission from Rees PJ and Williams DG. Principles of Clinical Medicine. London: Hodder Arnold, 1995.
Disorders of anterior pituitary hormone secretion 121
fail to distinguish those with only moderately raised glucose and GH assays at 30, 60, 90 and 120 min after
plasma concentrations from normal subjects. Random the glucose load has been taken.
GH measurements are often not diagnostic owing to
episodic secretion and a short half-life. Interpretation
The diagnosis is confirmed by demonstrating a In normal subjects, plasma GH concentrations fall
raised plasma GH concentration that is not suppressed to undetectable levels. Although failure to suppress
by a rise in plasma glucose concentration. In normal suggests acromegaly or gigantism, it may be found
subjects, plasma GH concentrations fall to very low in some patients with severe liver or renal disease, in
levels – to below 1 mg/L after a 75 g oral glucose load. heroin addicts or in those taking levodopa. Fasting
In acromegaly, the secretion of GH is autonomous and plasma GH can be normal in 8 per cent of acromegalic
this fall may not occur or be only slight, or there may patients. The plasma glucose concentrations may
even be a paradoxical rise. Growth hormone secretion demonstrate impaired glucose tolerance or diabetes
is inhibited by hyperglycaemia in the normal subject. mellitus in acromegaly. Note that the test is usually
Glucose suppression test for suspected acromegaly
unnecessary in patients who are diabetic, as GH should
already be suppressed.
Procedure
If acromegaly is confirmed, it is wise to investigate
After an overnight fast, insert an indwelling intravenous for other pituitary hormone defects, for example TSH,
cannula. After at least 30 min, take basal samples for LH, FSH and ACTH. Acromegaly can also be associated
plasma glucose and GH estimation. The patient should with the MEN syndrome (see Chapter 24).
drink 75 g of glucose dissolved in about 300 mL of Plasma IGF-1 has a long half-life and may be used
water, or an equivalent glucose load. Take samples for in screening for acromegaly. Plasma concentrations
correlate with the activity of the disease. Measurement of
the plasma concentrations of GH, or of IGF-1, may be
used to monitor the efficacy of treatment. Remember that
CASE 1 pregnancy increases IGF-1 concentration, and starvation,
A 48-year-old man noticed that his hat size had obesity and diabetes mellitus decrease it. Insulin-like
increased, and his wife thought that his appearance growth-factor-binding protein-3 is the main binding
had changed since their marriage, his features protein for IGF-1 and its concentration is also increased
becoming coarser and his hands larger. Plasma in acromegaly. Sometimes plasma GHRH concentrations
insulin-like growth factor 1 (IGF-1) concentration are useful and can be raised where there is an ectopic
was raised and an oral glucose suppression test was source or may be suppressed in pituitary disease.
performed. The results were as follows: Computerized tomography or MRI body scanning may
help to find an ectopic source of GH or GHRH.
Plasma
0 minutes: GH 24.5 mg/L Treatment
30 minutes: GH 24.6 mg/L
There are various approaches to treatment, often with
60 minutes: GH 23.7 mg/L
surgery to remove the adenoma, usually by trans-
90 minutes: GH 20.5 mg/L
sphenoidal hypophysectomy. Residual disease requires
120 minutes: GH 25.8 mg/L
medical therapy, usually with either bromocriptine
or cabergoline (dopamine receptor agonists) or
DISCUSSION
somatostatin analogues (somatostatin itself has
The plasma growth hormone (GH) concentration
too short a half-life for effective therapeutic use).
was not suppressed during the test in any of the
Octreotide or lanreotide, which bind to somatostatin
samples. These findings are indicative of acromegaly;
receptors, can be used or pegvisomant (GH receptor
the clinical features are typical of acromegaly. This
antagonist). Radiation therapy is sometimes used as an
case illustrates the principle of using a suppression
adjuvant for large invasive tumours or when surgery is
test when considering a condition involving a
contraindicated.
hormone excess. In healthy individuals, plasma GH
The aim of treatment is to ameliorate symptoms
concentration would be suppressed to less than
and to obtain an oral glucose suppressed GH
1 mg/L by the glucose intake.
concentration of less than 1 mg/L (this cut-off can be
122 The hypothalamus and pituitary gland
CASE 2
Box 7.1 Some causes of growth retardation
A 10-year-old boy was referred to the paediatric out- and short stature
patient clinic because of short stature. His height
was 1.08 m and he had normal body proportions. Familial short stature
Physical examination and preliminary biochemical Social/emotional deprivation
tests showed no obvious explanation for his small Undernutrition and chronic disease
stature. A random plasma GH was less than 2 mg/L. Coeliac disease
After a glucagon stimulation test, the plasma GH Rickets
concentration failed to increase above 2 mg/L. Chronic kidney disease
However, other pituitary hormone concentrations Endocrine disorders
Growth hormone deficiency, congenital or acquired
were normal on biochemical testing.
Hypothyroidism
Cushing’s syndrome
DISCUSSION Congenital adrenal hyperplasia
A diagnosis of isolated GH deficiency was made. Chromosomal abnormalities
Note the failure of GH concentration to increase Turner’s syndrome (45,X0)
after stimulation by glucagon. This illustrates well Skeletal disorders
the concept of using stimulation dynamic tests when Achondroplasia,
considering a hormone deficiency state. Laron-type dwarfism and Pygmies
Hypopituitarism 123
Ross Laboratories
39 75
deficiency unlikely after the presentation of provocative
Provided as a
38 38
service of
50
95 95
37
36
25
10
cm in stimuli on two occasions. Other such stimuli include
90 5
35
34
L
e
41 arginine, clonidine or the GHRH test. (See below for a brief
40
85 n
33
g
18
39 summary of some of these tests.) An unequivocally normal
32 t 38
31
80
h
95 17 37 response to a stimulation test excludes the diagnosis, and a
30 90 36
29
75 16 35 clearly impaired one confirms it. Once GH deficiency has
75 34
28
27
70 15 33 been established, a cause should be sought by appropriate
50 32
26
65 14 31 clinical and imaging means.
25 30
24
60
25
13 29 The following are second-line dynamic tests
23 28
22
55
10
5 12
27 sometimes used for suspected GH deficiency. Clonidine
21 26
20 25 at 0.15 mg/m2 body surface area is given orally after
50 11 24
19
18
23 an overnight fast. Blood samples for plasma GH
45 10 22
17
21 are collected at 0, 30, 60, 90, 120 and 150 min. The
16 20
40 9
W patient should be closely monitored for hypotension.
WM. Physical growth: National Center for Health Statistics percentiles AM J
15
CLIN NUTR 32: 607–629.1979. Data from the Fels Research Institute, Wright
19
•Adapted from Hamill PVV, Drizd TA, Johnson CL, Reed RB, Roche AF, Moore
e
18
in cm i
g
8
17 Arginine hydrochloride, like clonidine, is another agent
h 16
15 7
t
7
15 used in provocative dynamic tests for suspected GH
State University School of Medicine, Yellow Springs, Ohio
14 14
13 6 6 13 deficiency. Arginine should not be given to patients
12 12
11 5 5 11
with renal, hepatic or acid–base disorders or diabetes
10
9 4 4
10
9
mellitus. After an overnight fast, 0.5 g/kg body weight
8 8
to a maximum of 30 g is intravenously infused. Blood
© 1980 Ross Laboratories
7 3 7
3
6
5
6
5
samples for plasma GH are collected at 0, 30, 60, 90 and
4 2 2
Ib kg Age (months) kg
4
Ib
120 min. Arginine may evoke allergic reactions and the
B 3 6 9 12 15 18 21 24 27 30 33 36 necessary precautions should be in place in case of this.
Figure 7.4 Graph showing extreme failure to thrive in a Figure 7.5 shows an algorithm for the investigation
young child; note growth retardation and flattening of of short stature.
growth. Reproduced with kind permission from Nyhan
WL and Barshop BA. Atlas of Inherited Metabolic DISORDERS OF POSTERIOR PITUITARY
Diseases, 3rd edition. London: Hodder Arnold, 2012. HORMONE SECRETION
Disorders of the posterior pituitary are rare compared
conditions rarely exclude the diagnosis. A low plasma with those of the anterior pituitary. Deficiency of ADH in
IGF-1 concentration may be a useful screening test. diabetes insipidus may present as polyuria. In the syndrome
Urinary GH excretion, either in 24-h collections or timed of inappropriate ADH, hyponatraemia due to water excess
overnight, may offer a relatively safe screening test. occurs. (This is discussed in Chapters 2 and 3.)
If blood is taken at a time when physiologically
high concentrations are expected, the need for the HYPOPITUITARISM
more unpleasant stimulation tests may be avoided, Hypopituitarism is a syndrome of deficiency of pituitary
for example 60–90 min after the onset of sleep and hormone production that may result from disorders of
about 20 min after vigorous exercise. An adequate GH the hypothalamus, pituitary or surrounding structures.
response occurs with an absolute response of greater The anterior pituitary gland has considerable functional
than 20 mU/L (7 µg/L), making GH deficiency unlikely. reserve. Clinical features of deficiency are usually absent
It should be noted that these GH cut-offs may be age until about 70 per cent of the gland has been destroyed,
and assay dependent. unless there is associated hyperprolactinaemia, when
If GH deficiency is not excluded by the above amenorrhoea and infertility may be early symptoms.
measurements, it is necessary to perform one or more Panhypopituitarism alludes to the involvement of all
stimulation tests. The response of GH to insulin may be pituitary hormones; alternatively, only one or more
the most reliable to detect GH deficiency, but it is not may be involved, as in partial hypopituitarism.
124 The hypothalamus and pituitary gland
Evidence of dysmorphic
features or syndrome?
Yes No
(e.g. Turner’s syndrome)
Chronic disease or
social deprivation present?
Yes No
Disproportionate Proportionate
Yes No Yes No
Consider Consider
constitutional malabsorption/
or familial short nutritional
stature disorder
Yes No
Growth hormone
deficiency?
Yes No
Consider
rare causes
(see Box 7.1)
Some of the causes of hypopituitarism are shown in ● Secondary hypogonadism This is due to
Box 7.2. gonadotrophin deficiency, presenting as
Panhypopituitarism with the full clinical picture amenorrhoea, infertility and atrophy of secondary
described below is uncommon. Suspicion of anterior sexual characteristics with loss of axillary and pubic
pituitary hypofunction usually arises in patients presenting hair and impotence or loss of libido. Puberty is
with various features such as clinical and radiological delayed in children.
evidence of a pituitary or localized brain tumour, ● Secondary adrenocortical hypofunction (ACTH
hypogonadism, adrenocortical insufficiency, short stature deficiency) In contrast to the primary form
caused by GH deficiency, and hypothyroidism. (Addison’s disease), patients are not hyperpigmented
Although isolated hormone deficiency, particularly of because ACTH secretion is not raised. The
GH, may occur, several hormones are usually involved. If a sodium and water deficiency and hyperkalaemia
deficiency of one hormone is demonstrated, it is important characteristic of Addison’s disease do not usually
to establish whether the secretion of others is also occur because aldosterone secretion (which is
abnormal. Gonadotrophins are often the first to decrease controlled by angiotensin and not by ACTH) is
in hypopituitarism and it is unusual for the post-pituitary normal. However, cortisol is needed for normal free
hormones such as ADH and oxytocin to be affected. water excretion, and consequently there may be a
dilutional hyponatraemia due to cortisol deficiency.
Consequences of pituitary hormone
Cortisol is also necessary for the maintenance
deficiencies
of normal blood pressure. Hypotension may be
Progressive pituitary damage usually presents with associated with ACTH deficiency. Cortisol and/or
evidence of deficiencies of gonadotrophins and GH. GH deficiency may cause increased insulin sensitivity
Plasma ACTH and/or TSH concentrations may remain with fasting hypoglycaemia.
normal, or become deficient months or even years ● Secondary hypothyroidism (TSH deficiency) This
later. The clinical and biochemical consequences of the may sometimes be clinically indistinguishable from
target-gland failure include the following: primary hypothyroidism.
● Growth retardation in children This may be due to ● Prolactin deficiency Associated with failure to lactate,
deficiency of GH; deficiency of TSH, and therefore this may occur after post-partum pituitary infarction
of thyroid hormone, may contribute. (Sheehan’s syndrome). However, in hypopituitarism
due to a tumour, plasma prolactin concentrations are
often raised and may cause galactorrhoea (secretion
Box 7.2 Some causes of hypopituitarism of breast fluid).
Patients with hypopituitarism, like those with
Tumours
Craniopharyngiomas Addison’s disease, may die because of an inability to
Pituitary adenomas (microadenoma < 10 mm, secrete an adequate amount of cortisol in response to
macroadenoma > 10 mm) stress caused by, for example, infection or surgery. Other
Secondary tumour deposits life-threatening complications are hypoglycaemia and
Infections hypothermia.
Tuberculosis
Meningitis Pituitary tumours
Syphilis The clinical presentation of pituitary tumours
Infiltrations depends on the type of cells involved and on the size
Sarcoidosis
of the tumour (microadenomas less than 10 mm and
Haemochromatosis
Histiocytosis X
macroadenomas more than 10 mm).
Vascular Tumours of secretory cells may produce the clinical
Sheehan’s syndrome effects of excess hormone secretion:
Apoplexy ● excess prolactin causes infertility, amenorrhoea and
Empty sella syndrome
varying degrees of galactorrhoea (see Chapter 9),
Autoimmune – lymphocytic hypophysitis
Iatrogenic – radiation, surgery
● excess GH causes acromegaly or gigantism,
● excess ACTH causes Cushing’s syndrome (see Chapter 8).
126 The hypothalamus and pituitary gland
note that treatment with thyroxine can precipitate an If hypoglycaemia has been adequate, plasma
adrenal crisis in such patients and thus corticosteroid cortisol concentrations should rise by more than
replacement is also necessary. 200 nmol/L and exceed 580 nmol/L, and an adequate
Indications of the insulin stimulation test may include: GH response occurs with an absolute response of
greater than 20 mU/L (7 µg/L). In Cushing’s syndrome,
● assessment of GH in growth deficiency,
neither plasma cortisol nor GH concentrations
● assessment of ACTH/cortisol reserve (although the
rise significantly, although they usually do in cases
development of plasma ACTH assays has made such
of pseudo-Cushing’s syndrome (Chapter 8). See
testing less necessary),
Chapters 9 and 11 for details of GnRH and TRH tests
● differentiation of Cushing’s syndrome from pseudo-
if the combined pituitary test is used. After the test,
Cushing’s syndrome, for example depression or
a supervised meal should be given and the patient
alcohol excess.
should not drive for at least 2 hours.
Both ACTH and GH are released in response to the
stress of hypoglycaemia. Glucagon stimulation test of the hypothalamus–
Fifty millilitres of 20 per cent glucose for intravenous pituitary axis
administration must be immediately available in case This test is useful if the insulin hypoglycaemic test is
severe symptomatic hypoglycaemia develops. Care contraindicated. However, it is essential that the test is
should be taken not to induce severe hyperglycaemia carried out in a specialist unit by experienced staff.
during infusion, as it may cause hyperosmolality, which The basic principle is that glucagon stimulates GH
can be dangerous. Plasma cortisol is usually measured and ACTH release probably via a hypothalamic route.
as an index of ACTH secretion. If glucose needs to be The test is contraindicated if there is severe adrenal
given, continue with the sampling. failure, for example if cortisol at 09.00 h is less than
Procedure 100 nmol/L or in hypothyroidism. It is also unreliable
After an overnight fast, insert an indwelling intravenous in the presence of diabetes mellitus. Hypoglycaemia is
cannula, for example 19 gauge. After at least 30 min, not normally provoked by the test.
take basal samples at time 0 min for cortisol, GH and Procedure
glucose.
Patients should fast overnight, although they can drink
Inject soluble insulin in a dose sufficient to
water. An indwelling intravenous cannula, for example
lower plasma glucose concentrations to less than
gauge 19, is inserted. For adults, 1 mg of glucagon is
2.5 mmol/L and evoke symptomatic hypoglycaemia.
injected subcutaneously at 09.00 h.
The recommended dose of insulin must be adjusted
Blood samples are taken at 0, 90, 120, 150, 180, 210
for the patient’s body weight and for the suspected
and 240 min for cortisol and GH.
clinical condition under investigation. The usual
dose is 0.15 U/kg body weight. If either pituitary or Interpretation
adrenocortical hypofunction is suspected, or if a low Plasma cortisol should normally rise by at least
fasting glucose concentration has been found, reduce 200 nmol/L to more than 580 nmol/L, and an adequate
the dose to 0.1 or 0.05 U/kg. If there is likely to be GH response occurs with an absolute response of
resistance to the action of insulin because of Cushing’s greater than 20 mU/L (7 µg/L).
syndrome, acromegaly or obesity, 0.2 or 0.3 U/kg may
be needed. Treatment of hypopituitarism
Take blood samples at 30, 45, 60, 90 and 120 min
This consists of specific therapy depending on
after the injections for cortisol, GH and glucose assays.
its cause and may include surgical removal of a
Interpretation large adenoma. If the ACTH axis is impaired, it is
Methods of hormone assay vary, and results should essential to prescribe a glucocorticoid, for example
not be compared with reference values issued by hydrocortisone in the acute situation or prednisolone
other laboratories. Interpretation is not possible if for maintenance. Secondary hypothyroidism will need
hypoglycaemia is not attained, and the dose of insulin thyroid replacement.
can cautiously be repeated if this is not attained in the Adrenal replacement should precede T4 therapy
45-min blood sample. to avoid an Addisonian crisis (see Chapter 8).
128 The hypothalamus and pituitary gland
Gonadotrophin deficiency may require testosterone in without progesterone as appropriate. In children and
males and oestrogen replacement in women, with or sometimes in adults, GH may be indicated.
SUMMARY
● The anterior pituitary gland releases a number of results in a deficiency of all (panhypopituitarism)
peptide hormones, which are themselves regulated or some of the pituitary hormones.
by hypothalamus hormones that reach the pituitary ● Conversely, excess release of certain anterior
via the portal blood system. The anterior pituitary pituitary hormones can occur because of pituitary
hormones include ACTH, TSH, LH and FSH; their tumours. For example, acromegaly is due to excess
respective target organs are the adrenal and thyroid GH, and Cushing’s disease to excess ACTH release.
glands and the ovaries/testes. Growth hormone ● The posterior pituitary releases oxytocin and ADH.
(GH) is also an anterior pituitary hormone but The former is involved in uterine contraction
does not have a specific target organ – instead it during labour. Antidiuretic hormone controls
influences most tissues. water elimination by changing the renal collecting
● Hypopituitarism can be due to many conditions, ducts’ permeability. Deficiency of ADH results in
such as pituitary infiltration or destruction, and diabetes insipidus (discussed in Chapter 2).
8 The adrenal cortex
Chemistry and biosynthesis of steroids 129 Primary adrenocortical hypofunction (Addison’s disease) 136
Physiology 129 Investigation of suspected adrenal hypofunction 138
The hypothalamic–pituitary–adrenal axis 131 Corticosteroid therapy 140
Factors affecting plasma cortisol concentrations 132 Congenital adrenal hyperplasia 141
Disorders of the adrenal cortex 132 Primary hyperaldosteronism (Conn’s syndrome) 143
Adrenocortical hyperfunction 132
A number of endocrine abnormalities involve the diverge from it (Fig. 8.1). The final product is dependent
adrenal glands, and some of the more common upon the tissue and its enzymes.
conditions are discussed in this chapter, which should The zona glomerulosa secretes aldosterone,
perhaps be read in conjunction with Chapter 7 (on produced by 18-hydroxylation. Synthesis of this steroid
pituitary function) and Chapter 9 (which deals with is controlled by the renin–angiotensin system and not
reproductive endocrinology). normally by adrenocorticotrophic hormone (ACTH).
The adrenal glands are divided into two Although ACTH is important for maintaining growth of
embryologically and functionally distinct parts. The the zona glomerulosa, deficiency does not significantly
adrenal cortex is part of the hypothalamic–pituitary– reduce output.
adrenal endocrine system. Morphologically, the adult The zonae fasciculata and reticularis synthesize and
adrenal cortex consists of three layers. The outer thin secrete two groups of steroid:
layer (zona glomerulosa) secretes only aldosterone.
● Cortisol, a glucocorticoid (the most important
The inner two layers (zona fasciculata and zona
C21 steroid), is formed by progressive addition of
reticularis) form a functional unit and secrete most
hydroxyl groups at C-17, C-21 and C-11.
of the adrenocortical hormones. In the fetus there is a
● Androgens (for example androstenedione) are
wider fourth layer, which disappears soon after birth.
formed after the removal of the side chain to produce
One of its most important functions during fetal life is,
C19 steroids.
together with the adrenal cortex, to synthesize oestriol,
in association with the placenta. The adrenal medulla is Adrenocorticotrophic hormone secreted by the
part of the sympathetic nervous system. anterior pituitary gland stimulates synthesis of these two
steroid groups. Its secretion is influenced by negative
CHEMISTRY AND BIOSYNTHESIS OF feedback from changes in plasma cortisol concentrations.
STEROIDS Impaired cortisol synthesis due, for example, to an
inherited 21-a-hydroxylase or 11-b-hydroxylase
Steroid hormones are derived from the lipid cholesterol.
deficiency (congenital adrenal hyperplasia) results in
Figure 8.1 shows the internationally agreed numbering
increased ACTH stimulation with increased activity
of the 27 carbon atoms of steroid molecules and the
of both pathways. The resultant excessive androgen
lettering of the four rings. The products of cholesterol
production may cause hirsutism or virilization.
are also indicated. If the molecule contains 21 carbon
atoms, it is referred to as a C21 steroid. The carbon PHYSIOLOGY
atom at position 21 of the molecule is written as C-21. The adrenocortical hormones can be classified into
The side chain on C-17 is the main determinant of the groups depending on their predominant physiological
type of hormonal activity (Fig. 8.1), but substitutions effects.
in other positions modify activity within a particular
group. Glucocorticoids
The first hormonal product of cholesterol is Cortisol and corticosterone are naturally occurring
pregnenolone. Several important synthetic pathways glucocorticoids. They stimulate gluconeogenesis and
130 The adrenal cortex
21 22
23
CH3 20 26 O
25 18 24
25 24
C D 12
11 13
17 16 27
17 O–
19 C D
15 BILE ACIDS
1 9 14
2 10 8 AND SALTS
3
A B
5 7
4 6
HO
B CH2
CHOLESTEROL
A 1 CH3
HO
C=O
CALCIFEROL
Pregnenolone
HO
CH2OH CH2OH
O
C=O C=O O O
HO CH HO OH
O O O HO
TESTIS
OVARY
Figure 8.1 Numbering of the steroid carbon atoms of cholesterol and the synthetic pathway of steroid hormones;
the chemical groups highlighted determine the biological activity of the steroid.
the breakdown of protein and fat, that is, they antagonize inactive until it has been converted in vivo to cortisol
some of insulin’s action. Glucocorticoids in excess may (hydrocortisone).
impair glucose tolerance and alter the distribution of Glucocorticoids are conjugated with glucuronate
adipose tissue. Cortisol helps maintain the extracellular and sulphate in the liver to form inactive metabolites,
fluid volume and normal blood pressure. which, because they are more water soluble than
Circulating cortisol is bound to cortisol-binding the mainly protein-bound parent hormones, can be
globulin (CBG; transcortin) and to albumin. At normal excreted in the urine.
concentrations, only about 5 per cent of the total is
unbound and physiologically active. Plasma CBG Mineralocorticoids
is almost fully saturated, so that increased cortisol In contrast to other steroids, aldosterone is not
secretion causes a disproportionate rise in the free transported in plasma bound to specific proteins. It
active fraction. Cortisone is not secreted in significant stimulates the exchange of sodium for potassium and
amounts by the adrenal cortex. It is biologically hydrogen ions across cell membranes and its renal
The hypothalamic–pituitary–adrenal axis 131
action is especially important for sodium and water There is extensive peripheral interconversion of
homeostasis. It is discussed more fully in Chapters 2, adrenal and gonadal androgens. The end products,
3 and 4. Like the glucocorticoids, it is inactivated by androsterone and aetiocholanolone, together with
hepatic conjugation and is excreted in the urine. DHEA, are conjugated in the liver and excreted as
There is overlap in the actions of C21 steroids. glucuronides and sulphates in the urine.
Cortisol, in particular, may have a significant
THE HYPOTHALAMIC–PITUITARY–
mineralocorticoid effect at high plasma concentrations
ADRENAL AXIS
when the free fraction is significantly increased.
The hypothalamus, anterior pituitary gland and adrenal
Adrenal androgens cortex form a functional unit – the hypothalamic–
The main adrenal androgens are dehydroepiandros- pituitary–adrenal axis (see Chapter 7).
terone (DHEA), its sulphate (DHEAS) and Cortisol is synthesized and secreted in response to
androstenedione. They promote protein synthesis ACTH from the anterior pituitary gland.
and are only weakly androgenic at physiological The secretion of ACTH is dependent on corticotrophin-
concentrations. Testosterone, the most powerful releasing hormone (CRH), released from the
androgen, is synthesized in the testes or ovaries but hypothalamus. High plasma free cortisol concentrations
not in the adrenal cortex. Most circulating androgens, suppress CRH secretion (negative feedback) and alter
like cortisol, are protein bound, mainly to sex- the ACTH response to CRH, thus acting on both the
hormone-binding globulin and albumin. hypothalamus and the anterior pituitary gland (Fig. 8.2).
Insulin
hypoglycaemia
+ Rhythm
Stress
Hypothalamus
CRH CRH
+
Anterior pituitary
gland
Dexamethasone –
ACTH
Tetracosactrin
Cortisol + (Synacthen)
Adrenal
cortex
Figure 8.2 The factors controlling the secretion of cortisol from the adrenal gland, including the site of action
of dynamic function tests (shaded). +, stimulates; –, inhibits; ACTH, adrenocorticotrophic hormone; CRH,
corticotrophin-releasing hormone.
132 The adrenal cortex
CASE 1
A 45-year-old woman was referred to the endocrine
clinic because of skin pigmentation, bruising, obesity,
hypertension and proximal muscle weakness. The
following biochemical results were returned:
Plasma
Sodium 140 mmol/L (135–145)
Potassium 3.0 mmol/L (3.5–5.0)
Urea 5.5 mmol/L (2.5–7.5)
Creatinine 100 µmol/L (70–110)
Glucose 12.5 mmol/L (3.5–6.0)
Figure 8.3 This depicts a patient before and after
corticosteroid therapy; note cushingoid appearance. Urine
Reproduced with kind permission from Kinirons M and 24-h free cortisol 1700 nmol (100–350)
Ellis H. French’s Index of Differential Diagnosis, 15th
edition. London: Hodder Arnold, 2011.
The following were further results of an overnight
dexamethasone suppression test:
Hypothalamus
Pituitary gland
NEGATIVE
FEEDBACK
Ectopic source
in the lungs
ACTH
Cortisol
ACTH
Adrenal
cortex
Figure 8.4 Cushing’s disease, indicating excess cortisol production caused either by hyperstimulation of the
adrenal gland by adrenocorticotrophic hormone (ACTH), from either the pituitary or an ectopic source, or by
autonomous hormone secretion from an adrenal tumour.
The secretion of ACTH is appropriately suppressed misleading. Indeed, cyclical Cushing’s syndrome may
in primary cortisol-secreting tumours of the adrenal require repeated investigation. The level of cortisol
cortex. The tumours may be benign or malignant and measured in a 24-h urine sample reflects the overall
are usually derived from the zona fasciculata/zona daily secretion.
reticularis of the adrenal cortex. These glucocorticoid- One of the earliest features of Cushing’s syndrome
secreting tumours do not normally secrete aldosterone, is the loss of the diurnal variation in cortisol secretion,
which is produced in the zona glomerulosa layer of with high concentrations in the late evening, when
the adrenal cortex. Benign adenomas occur and also secretion is normally at a minimum. However, it is not
carcinomas. The latter secrete a variety of steroids, a diagnostic finding because it can also be caused by,
including androgens, and thus may cause hirsutism or for example, stress and endogenous depression. The
virilization. In these cases plasma ACTH is suppressed assessment of diurnal rhythm is not a practical out-
by the excess glucocorticoids. patient procedure.
Out-patient screening tests therefore may include
Basis of investigation of suspected Cushing’s syndrome the following.
The following questions should be asked: Estimation of 24-h urinary free cortisol
● Is there abnormal cortisol secretion? Only the unbound fraction of cortisol in plasma is
● If so, does the patient have any other condition that filtered at the glomeruli and excreted in the urine
may cause it? (urinary ‘free’ cortisol). In Cushing’s syndrome, because
● If Cushing’s syndrome is confirmed, what is the of the loss of circadian rhythm, raised plasma values
cause? are present for longer than normal and daily urinary
cortisol excretion is further increased (i.e. there is a
Is there abnormal cortisol secretion? disproportionately raised free fraction of cortisol).
Plasma cortisol concentrations reflect ACTH activity Plasma and urinary cortisol concentrations are
at that moment and, because of the episodic nature usually much higher when Cushing’s syndrome is
of cortisol secretion, such isolated values may be due to adrenocortical carcinoma or overt ectopic
Adrenocortical hyperfunction 135
ACTH secretion. Determinations of 24-h urinary free Is there another cause for the abnormal cortisol secretion?
cortisol have about a 5 per cent false-negative rate, but Various conditions can mimic Cushing’s syndrome
if three separate determinations are normal, Cushing’s (pseudo-Cushing’s) and thus give false-positive results
syndrome is most unlikely. for screening tests.
The following non-Cushing’s causes of abnormal
Low-dose overnight dexamethasone suppression test
cortisol secretion are important to remember:
A small dose, for example 1 mg, of this synthetic steroid
inhibits ACTH, and thereby cortisol secretion by ● Stress over-rides the other mechanisms controlling
negative feedback. This is usually given at midnight and ACTH secretion, with loss of the normal circadian
blood is taken for cortisol assay at 09.00 h the following variation of plasma cortisol and a reduced feedback
morning. response. Urinary free cortisol excretion may be
The overnight dexamethasone suppression test increased even in relatively minor physical illness or
is a sensitive, but not completely specific, test for mental stress.
evaluating such patients. The false-positive rate is about ● Endogenous depression may be associated with
12 per cent and the false-negative rate about 2 per cent. sustained high plasma cortisol and ACTH
A normal fall in plasma cortisol concentrations makes concentrations that may not be suppressed even
the diagnosis of Cushing’s syndrome very unlikely, by a high dose of dexamethasone. However, these
but failure to suppress plasma cortisol to less than patients often have a normal cortisol response to
50 nmol/L does not confirm it with certainty. insulin-induced hypoglycaemia, whereas those with
There are some pitfalls, for example certain Cushing’s syndrome do not (see Chapter 7).
anticonvulsant drugs, such as phenytoin, may interfere ● Severe alcohol abuse can cause hypersecretion of
with dexamethasone suppression tests, inducing liver cortisol that mimics Cushing’s syndrome clinically
enzymes that increase the rate of metabolism of the and biochemically. The abnormal findings revert to
drug. Plasma concentrations may therefore be too low normal when alcohol is stopped.
to suppress the feedback centre. ● Severe obesity can also imitate Cushing’s syndrome.
What is the cause of Cushing’s syndrome?
Additional tests
The following biochemical investigations may help to
In some cases, additional tests are needed to confirm the
elucidate the cause and ideally should be carried out on
diagnosis of excess cortisol production. The 48-h low-
a metabolic ward by experienced staff (Table 8.2):
dose dexamethasone suppression test may be useful as
it gives fewer false-positives than the overnight low-dose ● Plasma ACTH is detectable only in ACTH-dependent
dexamethasone test: 0.5 mg dexamethasone is given Cushing’s syndrome, and plasma concentrations are
orally at 6-h intervals from 09.00 h on day 1 for eight suppressed in patients with secreting adrenocortical
doses, and then plasma cortisol is measured after 48 h tumours. In patients with Cushing’s disease, plasma
at 09.00 h. Plasma cortisol should normally suppress to ACTH concentration may be either high-normal or
less than 50 nmol/L, but not in Cushing’s syndrome. moderately raised but inappropriate for the high
Table 8.2 Some biochemical test results in patients with Cushing’s syndrome
plasma cortisol concentration. Conversely, plasma such as thin-slice MRI of the chest and abdomen.
ACTH concentrations are markedly raised in Seventy per cent of these tumours co-secrete other
patients with ‘ectopic’ ACTH production. peptide hormones such as glucagon, somatostatin,
● The high-dose dexamethasone suppression test calcitonin and bombesin. Ectopic ACTH-induced
may be useful. The principle of this test is the same Cushing’s syndrome is more likely to present with
as that of the low-dose test described above, but a hypokalaemia, skin pigmentation, short clinical history
higher dose (2 mg) given over 48 h at 6-h intervals and severe myopathy. Plasma ACTH concentrations
may suppress the relatively insensitive feedback are also often much higher than those found in
centre of pituitary-dependent Cushing’s disease (this Cushing’s disease. Sometimes venous sampling of
occurs in about 90 per cent of cases). Plasma cortisol ACTH is necessary to locate the tumour. Rarely, a
concentration suppression by about 50 per cent is tumour may secrete CRH and thus mimic Cushing’s
usual. In the other two categories, ectopic ACTH disease. Abdominal imaging, for example CT scanning,
production or adrenal tumours, when pituitary may locate an adrenal tumour. Here, plasma ACTH is
ACTH is already suppressed, even this high dose usually suppressed and the tumour may also secrete
will usually have no effect, although there may be androgens.
some suppression in some cases of ‘ectopic’ ACTH The treatment of Cushing’s syndrome depends
secretion. upon the cause. Surgery is usually the treatment of
● If there is virilization, measure plasma androgens choice. In Cushing’s disease, pituitary removal by
such as testosterone, DHEA and DHEAS. High trans-sphenoidal surgery with pituitary radiation as
concentrations suggest an adrenocortical carcinoma an adjunct may be necessary. Adrenal tumours also
(see Chapter 9). usually require surgery, although the prognosis may
● In cases of Cushing’s syndrome with raised plasma be poor with carcinoma. Bilateral adrenal removal
ACTH concentration, the intravenous CRH can lead to skin pigmentation due to pituitary ACTH
test (100 µg in adults or 1 µg/kg body weight in release – Nelson’s syndrome. Similarly, if the source
children) has gained popularity. In Cushing’s disease of ectopic ACTH is found, surgery may be indicated
(pituitary disorder), an increase in baseline plasma to remove the tumour, for example lung carcinoma.
cortisol concentration of about 25 per cent occurs in Rarely, cortisol-inhibiting drugs are used such as
90 per cent of patients, whereas patients with ectopic ketoconazole, mitotane and metyrapone, which inhibit
ACTH, for example with lung carcinoma, usually fail steroid 11-b-hydroxylase (Fig. 8.5).
to show this rise.
● The insulin tolerance test may be appropriate.
PRIMARY ADRENOCORTICAL
Elevated plasma cortisol concentrations suppress the HYPOFUNCTION (ADDISON’S DISEASE)
stress response to hypoglycaemia, and this test may
be of value in differentiating Cushing’s syndrome Addison’s disease is caused by bilateral destruction of
from pseudo-Cushing’s syndrome due to depression all zones of the adrenal cortex, usually as the result of
or obesity. This test is not without hazards due to an autoimmune process. The association of Addison’s
severe hypoglycaemia. disease with hypoparathyroidism and mucocutaneous
candidiasis is described as polyglandular autoimmune
Confirmation of Cushing’s disease will require syndrome type 1 and has autosomal recessive
pituitary imaging techniques, for example magnetic inheritance. Polyglandular autoimmune syndrome type
resonance imaging (MRI), as well as pituitary hormone 2 occurs when Addison’s disease is associated with type
assessment (see Chapter 7). Specialized units may 1 diabetes mellitus and autoimmune thyroid disease,
perform simultaneous petrosal sinus sampling in an either Hashimoto’s thyroiditis or Graves’ disease, and is
attempt to localize the pituitary tumour and confirm also related to human leucocyte antigen (HLA)-B8 and
differentiation from an ectopic ACTH source. A DR-3 types.
peripheral blood sample to petrosal blood sample Tuberculosis affecting the adrenal glands is an
ACTH ratio of more than 2 is indicative of Cushing’s important cause in countries where this disease is
disease (ratio more than 3 if CRH stimulation used). common. Other causes of bilateral destruction of the
In cases of ectopic ACTH secretion, imaging adrenal glands include amyloidosis, mycotic infections,
techniques are important to try to locate the tumour, acquired immunodeficiency syndrome (AIDS) and
Primary adrenocortical hypofunction (Addison’s disease) 137
Abnormal Normal
Suppression No suppression
Figure 8.5 Algorithm for the investigation of Cushing’s syndrome. ACTH, adrenocorticotrophic hormone.
secondary deposits often originating from a bronchial pigmentation of the skin and buccal mucosa may occur.
carcinoma. The cause of these vague symptoms may only become
An important cause of acute adrenal crisis is bilateral evident if an Addisonian crisis is precipitated by the
adrenal haemorrhage, which can occur on warfarin stress of some other, perhaps mild, illness or surgery.
therapy or in patients with meningococcus septicaemia, Androgen deficiency is not clinically evident because
as in Waterhouse–Friderichsen syndrome. Certain testosterone production by the testes is unimpaired
drugs can inhibit glucocorticoid synthesis, including in males and because androgen deficiency does not
ketoconazole, aminoglutethimide, methadone and produce obvious effects in women. The pigmentation
etomidate. Glucocorticoid deficiency contributes to the that develops in some cases of Addison’s disease is
hypotension and causes marked sensitivity to insulin; due to the high circulating levels of ACTH or related
hypoglycaemia may be a presenting feature. peptides resulting from the lack of cortisol suppression
The clinical presentation of Addison’s disease of the feedback mechanism. Patients with primary
depends on the degree of adrenal destruction. In cases adrenal insufficiency, as in Addison’s disease due to
of massive haemorrhagic adrenal destruction, as in autoimmune disease, may also show vitiligo.
Waterhouse–Friderichsen syndrome of meningo- Patients with acute cortisol deficiency may present
coccaemia, the patient may be shocked, with volume with nausea, vomiting and hypotension. Dilutional
depletion; this adrenal crisis should be treated as a matter hyponatraemia may be present because cortisol is
of urgency. Conversely, sometimes the presentation needed for sodium-free water excretion by the kidneys.
can be surreptitious; a prolonged period of vague ill The biochemical changes therefore resemble those of
health, tiredness, weight loss, mild hypotension and inappropriate ADH secretion.
138 The adrenal cortex
A plasma autoantibody screen including adrenal Prolonged ACTH deficiency causes reversible adrenal
antibodies may point to a primary adrenal autoimmune insensitivity to trophic stimulation.
problem. Send a blood specimen for plasma ACTH. If the plasma
Plasma ACTH assay may be of value when ACTH concentration is high, this confirms primary
inappropriately low plasma cortisol concentrations adrenal hypofunction. A low ACTH concentration
have been found; a raised plasma ACTH concentration suggests secondary adrenal hypofunction, and pituitary
indicates primary insufficiency, whereas a low ACTH assessment is indicated (see Chapter 7).
concentration suggests secondary insufficiency. If the diagnosis is still unclear, admit the patient to
The essential abnormality in adrenocortical a hospital metabolic ward and perform a prolonged
hypofunction is that the adrenal gland cannot adequately Synacthen test. A normal result excludes primary
increase cortisol secretion in response to stress. This adrenal hypofunction.
may be due to adrenal (primary) or hypothalamic– An increasing response over time to the short and
pituitary (secondary) pathology. In the case of the latter, prolonged Synacthen tests indicates gradual recovery
it may be necessary to test the whole axis (see Chapter of the adrenal cortex and suggests hypothalamic or
7). Insulin-induced hypoglycaemia normally causes pituitary hypofunction (Fig. 8.6; see also Chapter 7).
ACTH secretion from the anterior pituitary gland.
This can be assessed by demonstrating a rise in plasma Tetracosactide (Synacthen) test of adrenal
cortisol concentrations. An impaired response indicates function
pituitary dysfunction only if the adrenal cortices have Tetracosactide is marketed as Synacthen.
already been shown to be capable of responding to
exogenous ACTH. Short Synacthen stimulation test
Procedure
Suspected Addisonian crisis (acute) The patient should be resting quietly but not fasting. It
Before starting treatment, take blood for immediate is recommended that the test is done in the morning.
plasma urea, creatinine and electrolyte estimations as Resuscitation facilities should be available in case of an
well as plasma glucose, calcium and cortisol. allergic reaction.
Hyponatraemia, hyperkalaemia, hypoglycaemia Blood is taken for basal cortisol assay. Synacthen
and uraemia are compatible with an Addisonian crisis. 250 µg is given by intramuscular or intravenous
Hypercalcaemia may also occur (see Chapter 6). injection. Blood is taken for cortisol assay at baseline,
Start steroid treatment once blood has been taken, as 30 and 60 min.
the condition is life threatening.
If the plasma cortisol is very high, an Addisonian Interpretation
crisis is unlikely. Conversely, if the plasma cortisol Normally the plasma cortisol concentration increases by at
concentration is very low or undetectable, and if least 200 nmol/L, to a concentration of at least 580 nmol/L.
there is no reason to suspect severe CBG deficiency, The peak is usually at 30 min, although a steady increase
for example due to the nephrotic syndrome, at 60 min may imply secondary hypoadrenalism due to
an Addisonian crisis is likely. Plasma cortisol pituitary or hypothalamic disease.
concentrations, which would be normal under basal
conditions, may be inappropriately low for the degree Depot or prolonged Synacthen stimulation test
of stress. To confirm this, perform a short Synacthen Repeated injections of depot Synacthen are painful and
test (see below). may cause sodium and water retention. Therefore this
test is contraindicated in patients in whom sodium
Suspected chronic adrenal hypofunction retention may be dangerous, such as those with
Measure the plasma cortisol concentration at 09.00 h. congestive cardiac failure. The test is rarely indicated if
If more than 580 nmol/L, Addison’s disease is unlikely, a basal plasma ACTH concentration is known. Its main
but this does not provide information about the adrenal indication is to differentiate primary from secondary
glands’ reserve to respond to a stressful stimulus. adrenal insufficiency (due either to pituitary failure
If the plasma cortisol concentrations are equivocal, or to prolonged corticosteroid therapy) when adrenal
perform a short Synacthen test. A normal result makes failure has previously been confirmed by the short
long-standing secondary adrenal insufficiency unlikely. Synacthen test, as above.
140 The adrenal cortex
Features of hypoadrenalism
Normal Abnormal
Consider pituitary
assessment
Negative feedback
ACTH
Cholesterol
17-Hydroxylase
21-Hydroxylase
Deoxycorticosterone 11-Deoxycortisol
11-Hydroxylase
Salt-losing Virilization
Figure 8.7 The abnormalities occurring in congenital adrenal hyperplasia. The substances highlighted are of diagnostic
importance; those shown in bold are increased in 21-hydoxylase deficiency. ACTH, adrenocorticotrophic hormone.
142 The adrenal cortex
sodium loss during the first few weeks of life. Volume In CYP17 deficiency, ambiguous genitalia or female
depletion may be accompanied by hyponatraemia and genitalia may be observed in male infants. A female
hyperkalaemia. Even if plasma sodium concentrations with CYP17 deficiency appears phenotypically
are within the reference range, demonstrably increased female at birth but will fail to develop breasts or
plasma renin activity may suggest lesser degrees of menstruate due to inadequate oestradiol production.
sodium and water depletion. Hypertension due to raised deoxycorticosterone
CYP11B1 deficiency may also present with female concentration may be present in CYP17 deficiency
ambiguous genitalia and salt loss. The child may, (Fig. 8.7).
however, show hypertension and a hypokalaemic
Diagnosis
alkalosis. The enzyme CYP11B1 catalyses the conversion
of 11-deoxycortisol to cortisol in the glucocorticoid Only the principles of the diagnosis of 21-a-hydroxylase
pathway and the conversion of deoxycorticosterone deficiency are outlined here (see Fig. 8.7).
to corticosterone in the mineralocorticoid pathway. 17-Hydroxyprogesterone can be metabolized
CYP11B2 or aldosterone synthetase deficiency results by the cortisol pathway only in the presence of
in hyponatraemia and hyperkalaemia, although normal 21-a-hydroxylase, and plasma concentrations are raised
sexual differentiation occurs, as sex steroids are normal. in patients with CAH. In some cases a short Synacthen
test may reveal elevated plasma 17-hydroxyprogesterone
concentrations after stimulation. This test may be
CASE 3 useful, as sometimes cortisol precursors may be
within the normal range. Those with abnormalities of
A 14-year-old woman with primary amenorrhoea 21-a-hydroxylase deficiency often show post-Synacthen
presented to the endocrine out-patient clinic. Physical 17-hydroxyprogesterone values more than 35 nmol/L.
examination revealed hirsutism and clitoromegaly. However, those with either 11-b-hydroxylase or other
Some of her biochemistry tests were as follows: enzyme deficiencies may show a normal response.
Plasma Plasma androstenedione concentrations may be
Sodium 132 mmol/L (135–145) raised in those patients with excessive androgen
Potassium 5.6 mmol/L (3.5–5.0) synthesis. Some indication of which enzyme is deficient
Urea 3.7 mmol/L (2.5–7.5) may be suggested by evaluating the pattern of steroid
Creatinine 101 µmol/L (70–110) excretion in a random or 24-h urine sample.
09.00 h cortisol 128 nmol/L (180–720) In 11-b-hydroxylase deficiency there is a raised
Thyroid-stimulating hormone 1.5 mU/L (0.20–5.0) concentration of 24-h urinary tetrahydrocortisol,
Free thyroxine 13.5 pmol/L (12–25) a metabolite of 11-deoxycortisol, and raised
Prolactin 244 mU/L (<470) deoxycorticosterone concentration. In the salt-losing
Luteinizing hormone 5.2 U/L (1–25) forms, plasma renin and aldosterone concentrations
Follicle-stimulating hormone 4.3 U/L (1–15) may assist diagnosis.
Testosterone 6.2 nmol/L (1–3) If a diagnosis of CAH is confirmed, genetic
Sex-hormone-binding globulin 48 nmol/L (20–90) counselling may be necessary, with deoxyribonucleic
Oestradiol 464 pmol/L (70–880) acid (DNA) and family studies.
17-Hydroxyprogesterone 85 nmol/L (<35) Treatment
DISCUSSION Congenital adrenal hyperplasia is treated by giving
The results are suggestive of congenital adrenal a glucocorticoid, for example hydrocortisone,
hyperplasia. Note the raised plasma 17-hydroxypro- and, if necessary, a mineralocorticoid, for example
gesterone and testosterone concentrations. The fludrocortisone. This treatment not only replaces the
patient was eventually found to have 21-a-hydroxylase deficient hormones but, by negative feedback, also
deficiency, which was confirmed with a 24-h urinary suppresses ACTH secretion and therefore androgen
steroid profile and genetic tests. Raised adrenal production.
androgen concentrations help to explain her hirsutism Measuring 17-hydroxyprogesterone and
and clitoromegaly. Note also the hyponatraemia and androstenedione concentrations in plasma or saliva
hyperkalaemia. enables treatment efficacy to be monitored. Salivary
Primary hyperaldosteronism (Conn’s syndrome) 143
steroid concentrations correlate well with those in doses of glucocorticoids in addition to antihypertensives.
plasma, and saliva collection may be more acceptable This condition is due to a chimeric gene product that
to some patients than repeated venepuncture. combines the promoter of the 11-b-hydroxylase gene
Measuring the plasma renin activity may help assess with the coding region of the aldosterone synthetase
mineralocorticoid replacement. gene and is associated with haemorrhagic stroke.
The treatment of IAH is usually medical, and the
PRIMARY HYPERALDOSTERONISM mineralocorticoid antagonist spironolactone has
(CONN’S SYNDROME) proved useful, sometimes in conjunction with a thiazide
Primary hyperaldosteronism (PH) is considered an diuretic. The treatment of choice for unilateral variants
important cause of secondary hypertension in perhaps of PH such as APA is usually surgical by adrenalectomy.
as many as 5–15 per cent of cases, particularly in the face Investigation of a patient with suspected
of hypokalaemia and kaliuria (e.g. urinary potassium primary hyperaldosteronism
more than 20 mmol/L). (See Chapter 5 for a discussion
Screening tests
of hypokalaemia.)
The majority of cases of PH are due to adrenal Hypertension and a hypokalaemic metabolic alkalosis
aldosterone-producing adenomas (APAs), which in the face of kaliuria (urinary potassium more than
produce 18-oxocortisol and 18-hydroxycortisol steroids 20 mmol/L) are suggestive of mineralocorticoid excess
in excess, although 45 per cent of cases may be due to such as Conn’s syndrome. A urinary potassium is thus a
bilateral idiopathic adrenal hyperplasia (IAH). There useful screening test.
is also a genetic–familial variety of PH. Type 1 familial In PH, plasma aldosterone concentration will be
PH is glucocorticoid remediable aldosteronism (GRA), raised, with suppressed renin levels. Remember that, in
in which the associated hypertension responds to small renal artery stenosis, Bartter’s or Gitelman’s syndromes,
renin-secreting tumours and pseudohypoaldosteron-
ism, both plasma aldosterone and renin concentrations
may be raised. Antihypertensive drugs may alter
renin and aldosterone levels but in some cases it may
CASE 4 not be safe to stop them, although, if there is severe
A 35-year-old man attended the hospital hypertension hypertension, a-adrenergic blockers may interfere less
clinic (presenting blood pressure 184/100 mmHg) than other antihypertensives.
with the following results (off antihypertensive A random plasma aldosterone to renin ratio of more
medication): than 750, where aldosterone is expressed in pmol/L and
Plasma renin activity in µg/L per hour, is suggestive of PH and
Sodium 144 mmol/L (135–145) may be used as a screening test. This ratio is increased
Potassium 2.9 mmol/L (3.5–5.0) by b-blockers and decreased by angiotensin-converting
Urea 3.4 mmol/L (2.5–7.5) enzyme (ACE) inhibitors, diuretics, calcium channel
Creatinine 102 µmol/L (70–110) blockers and angiotensin II receptor blockers. A raised
Bicarbonate 40 mmol/L (24–32) 24-h urinary aldosterone excretion may also be a useful
screening tool.
A spot urinary potassium was 63 mmol/L. In summary ACE inhibitors increase renin and reduce
A random plasma aldosterone to renin activity ratio aldosterone, b-blockers reduce renin and aldosterone,
was 984 (more than 750 is suggestive of Conn’s calcium channel blockers increase renin and reduce
syndrome). aldosterone, and diuretics increase renin and aldosterone,
although a-blocking drugs may have minimal effect
DISCUSSION
upon renin and aldosterone concentrations.
The results suggest Conn’s syndrome (primary
hyperaldosteronism). Note the hypertension, Further tests for the diagnosis of
hypokalaemia and urinary potassium greater than hyperaldosteronism
20 mmol/L. The patient also had a metabolic alkalosis. Sometimes dynamic tests are needed to confirm the
Further investigations, including adrenal imaging, diagnosis of PH, although in many cases the diagnosis
showed an adrenal adenoma. may be obvious. The basis of these additional tests is
144 The adrenal cortex
SUMMARY
This chapter discusses adrenal cortex steroid adrenal cortex hormones and can be due to excess
biochemistry and abnormalities of adrenal cortical ACTH from the pituitary (Cushing’s disease) or
function and how clinical biochemistry tests can be ectopic ACTH release from certain tumours. Other
used in their diagnosis and management. Disorders causes include adrenal tumours, hyperplasia and
of the adrenal medulla are discussed in Chapter 24. excess glucocorticoid intake. Cushing’s syndrome
The algorithms in Figures 8.5, 8.6 and 8.8 summarize may result in hypertension, glucose intolerance,
the investigation of Cushing’s syndrome, adrenal hypokalaemia, weight increase and mood changes.
insufficiency and hyperaldosteronism, respectively. ● Adrenal insufficiency is due to reduced adrenal
cortex hormone production; this can be due to
● The adrenal cortex produces three major groups of autoimmune disease or to adrenal gland infiltration.
hormones: glucocorticoids, mineralocorticoids and Adrenal failure may be associated with weakness,
androgens. hypotension, hyponatraemia and hyperkalaemia
● Cortisol, a glucocorticoid, is a stress hormone and is life threatening.
involved in metabolism. It is controlled by pituitary ● Congenital adrenal hyperplasia, most commonly due to
adrenocorticotrophic hormone (ACTH). 21-a-hydroxylase deficiency, results in glucocorticoid
● Aldosterone is a mineralocorticoid involved in and mineralocorticoid underproduction but an
sodium retention and potassium excretion via the excess of adrenal androgens.
kidneys. Aldosterone is controlled by the renin– ● Excess adrenal mineralocorticoid production can
angiotensin axis. result in hypertension, metabolic alkalosis and
● Cushing’s syndrome is due to the overproduction of hypokalaemia such as Conn’s syndrome.
9 The reproductive system
The study of the endocrinology of the male and female Gonadotrophin-releasing hormone analogues, for
reproductive systems is a specialized field and overlaps example goserelin, after an initial stimulation phase,
with urology, obstetrics and gynaecology. This chapter down-regulate gonadotrophin secretion and have
looks at some of the more common conditions that been used therapeutically for prostate carcinoma and
you are likely to encounter clinically and that have endometriosis.
particular relevance to clinical biochemistry. Pregnancy Prolactin, secreted by acidophil cells, is important
and infertility are discussed in Chapter 10. during pregnancy and the post-partum period. It
stimulates breast epithelial cell proliferation and
HYPOTHALAMIC–PITUITARY–GONADAL induces milk production. Prolactin differs from all
AXIS other pituitary hormones in its method of control.
The reproductive system is responsible not only for the Secretion is inhibited, not stimulated, by dopamine
production of hormones, but also for maturation of the (prolactin inhibitory factor); therefore, impairment of
germ cells in the gonads. It is important to understand the hypothalamic control causes hyperprolactinaemia. Its
relationship between hormones with respect to these two secretion is regulated by a short feedback loop between
functions if the results of reproductive endocrinology pituitary prolactin and hypothalamic dopamine via
tests are to be interpreted correctly; see also Chapter 7. dopamine-2-type receptors.
Oestrogens stimulate the proliferation of pituitary
Hypothalamic hormones
lactotroph cells, although high oestrogen and
The hypothalamus secretes: gonadotrophin-releasing progesterone concentrations inhibit secretion, as in
hormone (GnRH), which regulates the secretion of pregnancy. Circulating prolactin concentrations are
the pituitary gonadotrophins, luteinizing hormone normally higher in pregnancy and increase during
(LH) and follicle-stimulating hormone (FSH); and suckling as a result of the action of vasoactive intestinal
dopamine, a neurotransmitter, which also controls peptide and also high oestrogen concentrations in
prolactin secretion (see Chapter 7). pregnancy. Although thyrotrophin-releasing hormone
Anterior pituitary hormones (TRH) stimulates the secretion of prolactin, as well
as of thyroid-stimulating hormone (TSH), this action
The gonadotrophins (LH and FSH), secreted by
does not seem to be of physiological importance;
pituitary basophil cells, control the function and
it may, however, be important in pathological
secretion of hormones by the testes and ovaries. The
conditions. Similar factors affect prolactin and growth
secretion of GnRH is pulsatile and thus so, in turn, is
hormone secretion. Secretion of both is pulsatile and
that of LH and FSH. Although there is only one releasing
increases during sleep and in response to physical and
hormone, secretion of LH and FSH does not always
psychological stress.
occur in parallel and may be modified by feedback from
the circulating concentrations of gonadal androgens or Ovarian hormones
oestrogens. The actions of the gonadotrophins are:
Oestrogens, progesterone and androgens are secreted
● LH primarily stimulates the production of hormones by the ovarian follicles of the ovaries, which consist of
by the gonads, germ cells (ova) surrounded by granulosa and theca
● FSH stimulates the development of the germ cells. cells. Androgens (C19 steroids), synthesized by theca
Hypothalamic–pituitary–gonadal axis 147
cells, are converted into oestrogens (C18 steroids) in the Testicular hormones
granulosa cells, a process that involves aromatization Testosterone is secreted by the Leydig cells, which
of the A ring and the loss of the C-19 methyl group lie in the interstitial tissue of the testes between the
(Fig. 9.1). Oestradiol is the most important ovarian seminiferous tubules. The production of testosterone is
oestrogen. The liver and subcutaneous fat convert stimulated by LH and it, in turn, inhibits LH secretion
ovarian and adrenal androgens to oestrone. Both by negative feedback. Inhibin is a hormone produced
oestradiol and oestrone are metabolized to the relatively by the Sertoli cells, part of the basement membrane
inactive oestriol. Oestrogens are essential for the of the seminiferous tubules, during germ cell
development of female secondary sex characteristics differentiation and spermatogenesis. These processes
and for normal menstruation, and their concentration require testosterone and are stimulated by FSH. Inhibin
in plasma in children is very low. Androstenedione is the controls FSH secretion by negative feedback (Fig. 9.2)
main androgen secreted by the ovaries. It is converted and activin enhances spermatogenesis. Testosterone
to oestrone and to the more active testosterone in is involved in sexual differentiation, the development
extraovarian tissue. A small amount of testosterone is of secondary sexual characteristics, spermatogenesis
secreted directly by the ovaries. Plasma concentrations and anabolism. In the male, the effects of testosterone
in women are about a tenth of those in men. depend on intracellular conversion to the even more
Progesterone is secreted by the corpus luteum potent androgen dihydrotestosterone by the enzyme
during the luteal phase of the menstrual cycle and by 5-a-reductase in target cells (Fig. 9.3).
the placenta. It prepares the endometrium of the uterus Luteinizing hormone stimulates testosterone
to receive a fertilized ovum and is necessary for the production from the Leydig cells. The Sertoli cells are
maintenance of early pregnancy. It also is pyrogenic and involved in germ cell differentiation and spermatogenesis.
increases the basal body temperature. In plasma, only These functions depend on testosterone and are
about 2 per cent of progesterone is unbound or free, stimulated by FSH.
the majority being bound to albumin and transcortin.
Inhibin from granulosa cells inhibits FSH secretion
Hypothalamus
while activin enhances the action of FSH and LH.
GnRH Pituitary gland
Cholesterol
Dehydroepiandrosterone
–
Androstenedione
+
Testosterone 19-Hydroxyandrostenedione Testosterone LH FSH
Leydig
17--oestradiol + cells
Oestriol Testes
Sertoli
Sperm cells
Figure 9.1 Pathways of sex hormone synthesis. (In this
summary an arrow does not necessarily represent a
single reaction.) Reproduced with kind permission Figure 9.2 The effect of the gonadotrophins luteinizing
from Candlish JK and Crook M. Notes on Clinical hormone (LH) and follicle-stimulating hormone (FSH)
Biochemistry. Singapore: World Scientific Publishing, on testicular function. GnRH, gonadotrophin-releasing
1993. hormone.
148 The reproductive system
Negative feedback control
because of galactorrhoea and oligomenorrhoea. She
Testosterone Testosterone Spermatogenesis was not taking any medication, and her renal function,
Sexual differentiation liver function and blood glucose were normal. Her
5--reductase plasma biochemical results were as follows:
Thyroid-stimulating hormone 1.6 mU/L (0.20–5.0)
Dihydrotestosterone Sexual maturation
at puberty Free thyroxine 13.1 pmol/L (12–25)
Prolactin 2264 mU/L (<470)
Figure 9.3 The relationship between the biological Luteinizing hormone 7.2 U/L (1–25)
actions of testosterone and dihydrotestosterone.
Follicle-stimulating hormone 4.4 U/L (1–15)
Testosterone 2.2 nmol/L (1–3)
Sex-hormone-binding globulin Sex-hormone-binding globulin 58 nmol/L (20–90)
Oestradiol 544 pmol/L (70–880)
Testosterone and, to a lesser extent, oestradiol circulate
bound to a carrier protein, sex-hormone-binding DISCUSSION
globulin (SHBG), as well as to albumin. As with other The patient has hyperprolactinaemia, with a common
hormones, only the free or unbound fraction (about presentation. A pituitary magnetic resonance
3 per cent of the total hormone concentration) is imaging scan was suggestive of a microprolactinoma
metabolically active. Plasma SHBG levels in females (microadenoma). The other pituitary hormones are
are about twice those in males. Changes in SHBG normal, presumably because the microprolactinoma
concentrations change the ratio of free testosterone to had not encroached on the pituitary gland.
free oestrogen (see Table 9.1). Dopamine receptor agonists such as bromocriptine
or cabergoline have been used to lower prolactin
HYPERPROLACTINAEMIA concentration under these circumstances.
This is an important cause of amenorrhoea, sexual
dysfunction, osteoporosis, infertility and possibly
breast cancer. High plasma prolactin concentrations glycol, before extensive investigation is undertaken for
inhibit the normal pulsatile release of GnRH and true hyperprolactinaemia.
inhibit gonadal steroid hormone synthesis directly. Hypothyroidism, polycystic ovary syndrome,chronic
Plasma gonadotrophin and oestrogen concentrations kidney disease and certain drugs, e.g. antipsychotics,
are therefore low, and the symptoms of oestrogen opioids, estrogens, H2 receptor antagonists and
deficiency may occur. About a third of patients with antidepressants, can also evoke hyperprolactinaemia.
hyperprolactinaemia have galactorrhoea. Antipsychotic drugs, such as chlorpromazine,
The finding of hyperprolactinaemia should be haloperidol, clozapine, olanzapine and aripiprazole
interpreted with caution. As mentioned above, plasma generally block dopamine receptors (D2), although
prolactin concentration is raised during pregnancy and the last may be associated with a low rate of
lactation. Plasma prolactin concentrations are higher hyperprolactinaemia. Prolactin plasma concentrations
in females than in males and decrease post menopause. persistently greater than 1000 mU/L may need a change
Samples for prolactin estimations should be taken in antipsychotic medication or a reduction in the dose;
at least 2–3 h after waking in order to eliminate the concentrations over 2000 mU/L probably merit an
misleading elevated plasma concentrations found endocrinologist’s opinion to exclude a prolactinoma.
during sleep; the stress of venepuncture may also The pathological causes of hyperprolactinaemia
cause prolactin secretion. A sustained increase of include a prolactin-secreting tumour of the pituitary
more than about 700 mU/L should be investigated. gland. If a pituitary tumour is found, it is usually
Macroprolactinaemia, in which the raised plasma either a microadenoma (< 10 mm) or a macroadenoma
prolactin concentration is due to a complex with (> 10 mm). The higher the plasma prolactin
immunoglobulins, should be excluded, usually by being concentration, the greater the likelihood that a tumour
precipitated in the plasma sample by polyethylene is present, with concentrations more than 2000 mU/L
Sexual development from conception in females and males 149
U/L
male and female are considered separately below.
20
The female
Development of female characteristics
In the absence of a Y chromosome, the fetus starts to
develop female characteristics at about 12 weeks of 0 Menses
gestation. If androgens are produced at this stage, as, Ovulation
1250
Progesterone (nmol/L)
for example, in congenital adrenal hyperplasia (CAH),
Oestradiol (pmol/L)
Oestradiol
masculinization of the external genitalia may occur, 1000 100
Progesterone
causing female pseudohermaphroditism (see Chapter 750
80
8). Proliferation of fetal germ cells produces several 60
million oocytes. By late fetal life, all the germ cells have 500
40
degenerated and no more oocytes can be produced. 250 20
Those oocytes that are present enter the first stage of
0 0
meiosis and their numbers decline throughout the
rest of the intrauterine period and childhood; the 0 14 28
inability to replenish them explains the limit to the Day of menstrual cycle
span of reproductive life in women, in contrast to Figure 9.5 An example of plasma hormone
the continuous ability of men to produce sperm. An concentrations during the menstrual cycle. FSH,
abnormally high rate of decline leads to premature follicle-stimulating hormone; LH, luteinizing hormone.
menopause.
secretes progesterone and oestradiol. Progesterone high levels may be associated with tachyphylaxis, that
is the principal hormone of the luteal phase and is, tolerance to dose. There may also be a place for its
prepares the endometrium for the implantation of measurement in women on transdermal estradiol HRT
the fertilized ovum. If the ovum is not fertilized, the to ensure adequate absorption. Otherwise, measuring
corpus luteum regresses and plasma ovarian hormone plasma oestradiol is not generally useful, as the results
concentrations fall; the menstrual cycle takes its depend upon the type of estrogen used in the HRT
course, with sloughing of the endometrium and preparation and how this may cause assay cross-
menstrual bleeding. reaction.
As the plasma ovarian hormone concentrations fall,
the concentrations of LH and FSH in plasma begin to Disorders of gonadal function in females
rise and the cycle recommences. If fertilization does Gonadal dysfunction in women usually presents with
occur, pregnancy may supervene (see Chapter 10). any or all of the following:
Interpretation of plasma sex hormone concentrations
must be made in relation to the stage of the cycle. ● amenorrhoea,
It may be important to establish whether a patient ● hirsutism,
who is complaining of infertility has ovulated, either ● virilism,
spontaneously or as a result of treatment to induce ● infertility (see Chapter 10).
ovulation. The plasma progesterone concentration
should be measured in a blood sample taken during Amenorrhoea
the second half of the menstrual cycle; usually day 21. Amenorrhoea is defined as the absence of menstruation;
A value within the reference range for the time of the it may be due to hormonal abnormalities. If
cycle is good presumptive evidence of ovulation (see there is ovarian failure, pituitary gonadotrophin
Fig. 9.5), whereas a value in the range expected in concentrations in plasma are high (hypergonadotrophic
the follicular phase indicates the absence of a corpus hypogonadism); if the cause is in the hypothalamus
luteum, and therefore of ovulation. Ovulation may or anterior pituitary gland, gonadotrophin secretion
also be detected by ultrasound examination of the is reduced (hypogonadotrophic hypogonadism).
ovaries. Progesterone secretion is associated with a rise Amenorrhoea may be classified as either primary or
in body temperature, which may be monitored serially secondary (Box 9.2).
to determine the time of ovulation. Plasma prolactin Primary amenorrhoea occurs when the patient has
concentrations do not significantly change cyclically never menstruated and is most commonly associated
during the menstrual cycle. with delayed puberty. The age of the menarche is very
variable. Extensive investigation should probably be
The menopause postponed until around the age of 16, unless there are
The menopause is defined as the time of permanent other clinical features of either endocrine disturbances,
cessation of menstruation, the average age being such as hirsutism and virilism, or chromosomal
about 50 years. The menopause occurs when all the abnormalities. Turner’s syndrome (45,XO) and
follicles have atrophied. Plasma concentrations of testicular feminization syndrome may present with
oestrogens fall and those of FSH and, to a lesser extent, primary amenorrhoea.
LH increase after removal of the negative feedback Secondary amenorrhoea occurs when previously
to the pituitary. These findings are therefore similar established menstrual cycles have stopped and is
to those of primary gonadal failure. Diagnosis of the most commonly due to physiological factors such as
menopause is a clinical one, although a plasma FSH pregnancy or the menopause. Other causes include
consistently greater than 20–40 IU/L is suggestive of severe illness, excess or rapid weight loss for any
ovarian failure but not a guarantee of sterility, and reason, including anorexia nervosa, or stopping oral
thus suitable contraception is necessary for 1 year of contraceptives. These should be considered before
amenorrhoea in patients over 50 years and for 2 years extensive and potentially dangerous investigations
in those under 50 years. are started. A number of endocrine disorders, such
It may sometimes be useful to measure plasma as hyperprolactinaemia, hyperthyroidism, Cushing’s
oestradiol concentrations in women with estradiol syndrome and acromegaly, may present with
hormone replacement therapy (HRT) implants, as very amenorrhoea.
152 The reproductive system
● to confirm the presence of testes, On day 0, blood is taken for testosterone. Then
● in infants with ambiguous genitalia and palpable 2000 hCG units are given on days 0 and 2 by
gonads, intramuscular injection.
● in males with delayed puberty, On day 4, blood is again taken for testosterone,
● in some cases of undescended testicles. androstenedione and dihydrotestosterone.
Normally there is at least a two-fold increase in
Some patients show an allergic reaction, and testosterone concentration, but in the absence of testes
therefore resuscitation facilities should be at hand and there is no testosterone response.
experienced staff should perform the test.
SUMMARY
● The gonads secrete sex hormones, although there is ● Plasma SHBG carries testosterone and oestrogen,
also some adrenal gland production. In females, the the free forms of which are biologically active.
main sex hormone is the oestrogen 17-b-oestradiol, ● Gonadal dysfunction in females usually presents with
and in males testosterone. any or all of the following: amenorrhoea, hirsutism,
● The anterior pituitary gonadotrophins FSH and virilism, infertility and ambiguous genitalia.
LH stimulate the gonads. ● Gonadal dysfunction in males may present with
● Prolactin is released from the anterior pituitary delayed puberty, decreased libido, gynaecomastia,
gland. Hyperprolactinaemia has many causes and infertility or ambiguous genitalia.
can result in infertility and galactorrhoea.
10 Pregnancy and infertility
This chapter discusses the biochemical changes that plasma or urine to detect fetal abnormalities or to
occur in pregnancy and how clinical biochemistry tests monitor the progress of the pregnancy, for example
can be used in the investigation of infertility. low urine unconjugated oestriol (E3) is associated
with poor pregnancy outcome. Such sampling is
PREGNANCY AND LACTATION relatively safe and simple, but occasionally more
If the ovum is fertilized, it may implant in the invasive testing, such as of amniotic fluid obtained by
endometrium, which has been prepared by progesterone amniocentesis, may be needed. However, the ability to
during the luteal phase. The function of luteinizing visualize the fetus using ultrasound (Fig. 10.1) and the
hormone (LH) is taken over by human chorionic use of cardiotocography for detecting fetal heart rate
gonadotrophin (hCG), produced by the chorion and have reduced the need for such tests.
developing placenta. Human chorionic gonadotrophin
is similar in structure and action to LH and prevents the Human chorionic gonadotrophin
involution of the corpus luteum as circulating pituitary The secretion of hCG by the placenta reaches a peak
gonadotrophin concentrations fall. Consequently, plasma (rising to about 500 000 U/L) at about 13 weeks of
oestrogen and progesterone concentrations continue to pregnancy and then falls. The fetoplacental unit then
rise and endometrial sloughing is prevented. Progesterone takes over hormone production, and the secretion of
is initially produced by the corpus luteum of the ovary for both oestrogen and progesterone rises rapidly. Plasma
the first 8 weeks of pregnancy, then from implantation of or urinary hCG concentrations, which give positive
the embryo the placenta takes over progesterone synthesis. results at 1 or 2 weeks after the first missed menstrual
During pregnancy the predominant oestrogen is oestriol period, are most commonly used to confirm pregnancy.
produced by the placenta. Prolactin concentration
gradually increases during the first two trimesters and then
rises steeply, to about 8000 mU/L, in the third trimester.
Prolactin, oestrogens, progesterone and (human)
placental lactogen stimulate breast development in
preparation for lactation. High plasma oestrogen
concentrations inhibit milk secretion; lactation can
start only when plasma concentrations fall after delivery
of the placenta. Initially lactation depends on prolactin.
Suckling stimulates secretion of the hormone, but, even
during lactation, plasma prolactin concentrations fall
progressively post partum and reach non-pregnant
levels after 2 or 3 months. Apart from the effects on
the breast, the high plasma concentration of prolactin
interferes with gonadotrophin and ovarian function
and produces a period of relative infertility. Figure 10.1 Ultrasound of 12 week pregnancy. Crown–
rump length measurement can be taken and also
Monitoring pregnancy (placental function)
nuchal translucency. Reproduced with kind permission
Substances produced by the fetus or placenta from Baker P. Obstetrics by Ten Teachers, 18th
(fetoplacental unit) may be measured in maternal edition. London: Hodder Arnold, 2006.
158 Pregnancy and infertility
However, by using more sensitive immunoassay order to locate the position of the fetus, placenta and
techniques, plasma hCG may be detected soon after maternal bladder.
implantation of the ovum and before the first missed Analytical results may be misleading if, for example,
period. the specimen is contaminated with maternal or fetal
Measurement of plasma hCG is also useful if an blood or maternal urine, is not fresh or is not properly
ectopic pregnancy is suspected, in conjunction with preserved. Close liaison between the clinician and the
ultrasonography, or if the patient is being treated for laboratory staff helps to ensure the suitability of the
infertility. Serial hCG measurements may be used to specimen and the speed of the assay. Amniotic fluid is
assess the progress of early pregnancy; single values are probably derived from both maternal and fetal sources,
difficult to interpret because of the wide reference range. but its value in reflecting abnormalities arises from its
As a rough guide, plasma concentrations should double intimate contact with the fetus and from the increasing
every 2 days in a normal pregnancy. Raised plasma hCG contribution of fetal urine in later pregnancy.
not due to pregnancy can be due to gestational or non-
gestational trophoblastic neoplasia or the menopause. Detection of neural tube defects
a-Fetoprotein (AFP) is a low-molecular-weight
Human placental lactogen glycoprotein synthesized mainly in the fetal yolk sac
This is a peptide hormone synthesized by the placenta. and liver. Its production is almost completely repressed
It is detectable in maternal plasma after about the in the normal adult. It can diffuse slowly through
eighth week of gestation and has been used to assess the capillary membranes and appears in the fetal urine, and
likelihood of threatened miscarriage and to monitor hence in the amniotic fluid, and in maternal plasma.
late pregnancy, but now is rarely used. Severe fetal neural tube defects, such as open spina
bifida and anencephaly, are associated with abnormally
Detection of fetal abnormalities high concentrations in these fluids. The reason for this
Some fetal abnormalities may be diagnosed by tests is not clear, but the protein may leak from the exposed
carried out on maternal plasma or amniotic fluid. neural tube vessels.
Amniocentesis is a procedure by which amniotic As well as neural tube defects, the causes of raised
fluid is obtained through a needle inserted through AFP concentration in amniotic fluid and maternal
the maternal abdominal wall into the uterus and is plasma include:
usually carried out after about 14 weeks’ gestation.
● multiple pregnancy,
The procedure carries a small risk to the fetus. Both
● serious fetal abnormalities,
the safety and the reliability of the procedure can be
● exomphalos.
improved if combined with ultrasound examination in
In some countries, pregnant women attending for
antenatal care are offered plasma AFP assay at between
CASE 1 16 and 18 weeks’ gestation to screen for the presence
A 20-year-old woman attended the antenatal of a fetus with a neural tube defect (although high-
clinic in the 17th week of pregnancy for a Down’s resolution ultrasound is beginning to replace this test).
syndrome screen test. A plasma a-fetoprotein (AFP) The gestational age should be confirmed by ultrasound,
was 67 kU/L (reference median being 38 kU/L for 17 which should also exclude a multiple pregnancy as a
weeks). Her other blood tests were normal. cause of high concentrations.
Positive results should be confirmed on a fresh
DISCUSSION
sample, which, if still high and if the diagnosis has not
It was feared that the fetus had a neural tube defect
been confirmed by ultrasound, may be followed by AFP
because of the raised plasma AFP concentration.
estimation on amniotic fluid. This is a more precise
However, an ultrasound scan showed a twin
diagnostic test and yields fewer false-positive results than
pregnancy and no evidence of a neural tube defect.
plasma assays if sampling is properly performed. It should
Other causes of a raised plasma AFP concentration
be reserved for subjects known to be at risk, either because
include incorrect gestational dating, fetal renal
of a family history of neural tube defects or because of the
disease, duodenal/oesophageal atresia and ventral
finding of a high concentration in maternal plasma with a
wall defects or exomphalos.
normal or equivocal ultrasound scan.
Pregnancy and lactation 159
Delivery Female
During delivery, blood gases and lactate can be measured Examination should include looking for anorexia
in fetal blood to monitor for hypoxia. Capillary blood nervosa, hirsutism, virilism, galactorrhoea and
samples may be collected from the fetal scalp during ambiguous genitalia. A history should also be taken for
delivery. Transcutaneous oxygen electrodes can medications and drugs (see Chapter 9).
determine fetal PO2.
Investigations
Hypertension in pregnancy and A woman may be infertile despite having a clinically
pre-eclampsia normal menstrual cycle (about 95 per cent of such
Pregnancy-induced hypertension or pre-eclampsia is cycles are ovulatory). Thus, even if the cycle seems to be
associated with convulsions (as occurs in eclampsia), regular, it is important to determine whether ovulation
oedema, impaired renal function, proteinuria and is occurring and if luteal development is normal.
maternal death as well as with placental insufficiency Anovulatory infertility is probably the most common
and intrauterine fetal growth retardation. form of female infertility and is associated with
Pre-eclampsia is defined as a blood pressure taken oligomenorrhoea or amenorrhoea. (Discussion of the
on two occasions, at least 6 h apart, of 140/90 mmHg complete investigation of female infertility, including
or greater (after 20 weeks’ gestation) in a woman tests such as that for tubal patency, is beyond the scope
with previously normal blood pressure and who has of this book.)
proteinuria (defined as ≥ 300 mg protein in 24-h urine
collection). ● If the patient is menstruating regularly, measure
Plasma urate may rise in pre-eclampsia and is plasma progesterone concentration during the
predictive of maternal complications. luteal phase on day 21 of the cycle. A normal
plasma concentration is strong evidence that the
Detection and follow-up of trophoblastic patient has ovulated. A low plasma concentration
tumours of < 30 nmol/L suggests either ovulatory failure or
Trophoblastic tumours (hydatidiform mole,
choriocarcinoma), which may follow abnormal CASE 3
pregnancy or a miscarriage, and some teratomas
secrete hCG, which can be estimated in plasma or A 28-year-old woman attended the gynaecology out-
urine by sensitive tests allowing early detection and patient clinic because of infertility. Her periods were
treatment of recurrence. However, these tests will not noted to be irregular. She was on no medication, and
necessarily differentiate pregnancy or retained products her renal function, liver function and blood glucose
of conception from recurrence of a tumour, because concentration were normal. Her plasma results were
plasma hCG concentrations rise in both situations (see as follows:
Chapter 24). In monitoring trophoblastic tumours Thyroid-stimulating hormone 2.1 mU/L (0.20–5.0)
it is important to monitor plasma hCG down to Free thyroxine 14.3 pmol/L (12–25)
undetectable levels. Prolactin 414 mU/L (< 470)
Luteinizing hormone 7.2 U/L (1–25)
INFERTILITY
Follicle-stimulating hormone 5.4 U/L (1–15)
Infertility can be defined as primary when conception Testosterone 1.7 nmol/L (1–3)
has never occurred despite at least 1 year of unprotected Sex-hormone-binding globulin 33 nmol/L (20–90)
coitus, and secondary when there has been a previous Oestradiol 564 pmol/L (70–880)
pregnancy, either successful or not. Investigation earlier 21-day plasma progesterone 6.6 nmol/L (> 30
than at 1 year may be appropriate if the woman is more suggests ovulation)
than 35 years old or where pregnancy is associated with
DISCUSSION
other risks.
In cases of infertility, both partners should be The low 21-day plasma progesterone concentration
investigated. The history should include coital suggests a poor luteal phase and possible anovulation.
frequency and success, serious illnesses, use of alcohol The patient was having anovulatory menstrual cycles,
and drugs, and sexually transmitted diseases. which explained her infertility.
162 Pregnancy and infertility
impaired luteal function. This investigation should history of mumps, drugs and medications should be
be repeated on more than one occasion. The most obtained.
common cause of a low progesterone concentration
(< 30 nmol/L) is inaccurate sample timing, although, Investigations
if authentic, it suggests lack of ovulation. However, ● Semen analysis: the volume should be at least 2 mL.
a plasma progesterone concentration of more than There should be more than 20 ¥ 109/L spermatozoa,
100 nmol/L suggests pregnancy. more than 50 per cent being motile at 4 h post
● Follicular development and ovulation may be ejaculation and more than 30 per cent normal
monitored by ovarian ultrasound examination. morphology. A post-coital test is useful so that
Polycystic ovary syndrome should be excluded cervical mucus can be examined for the presence
(see Chapter 9). Plasma follicle-stimulating of spermatozoa and their activity. Sperm antibodies
hormone (FSH), LH, oestrogen and testosterone may exist.
concentrations are useful, as is the exclusion of ● Plasma testosterone, LH and FSH concentrations
thyroid disease. should be measured.
● If there is primary amenorrhoea, consider – Raised plasma FSH and LH concentrations
karyotyping the patient, for example Turner’s with a low testosterone concentration
syndrome (45,XO). (hypergonadotrophic hypogonadism) indicate
● Sometimes histological examination of an a testicular problem such as Leydig cell failure.
endometrial biopsy specimen or the appearance of Low plasma FSH and LH and testosterone
cervical mucus can indicate whether luteal function concentrations suggest pituitary or hypothalamic
is normal. An oral progestogen challenge can be disease (hypogonadotrophic hypogonadism).
used: a withdrawal bleed 5–7 days later implies In the case of the latter, a GnRH test may be
adequate endometrial oestrogen, whereas failure to required (see Chapter 9).
bleed despite oestrogen treatment implies uterine – A raised plasma FSH concentration in
disease. comparison with LH may indicate seminiferous
● Hyperprolactinaemia should be excluded by tubular failure, irrespective of the plasma
checking the plasma prolactin concentration (see testosterone concentration. There is usually
Chapter 9). azoospermia or oligospermia. Oligospermia
● Do the plasma FSH, LH and oestrogen results with a low plasma FSH concentration suggests
suggest hypergonadotrophic hypogonadism or pituitary or hypothalamic disease.
hypogonadotrophic hypogonadism? In the presence
● If there is evidence of feminization, karyotype
of amenorrhoea, a plasma FSH of more than 40 U/L
should be considered. This should also be considered
is suggestive of ovarian failure. (See Chapter 9 for the
if azoospermia is present, for example Klinefelter’s
causes of hypergonadotrophic hypogonadism and
syndrome (47,XXY).
hypogonadotrophic hypogonadism.)
● Plasma prolactin should be measured and
● Low concentrations of plasma gonadotrophins may
hyperprolactinaemia and thyroid disease excluded
necessitate a gonadotrophin-releasing hormone
(see Chapter 9).
(GnRH) test to look for pituitary or hypothalamic
● Defects of the male reproductive tract may also be
disease (see Chapter 9).
found and necessitate a urology opinion, but are
● Anti-Müllerian hormone (AMH) is released by
beyond the scope of this book.
granulosa cells of the ovarian follicle and low serum
● An hCG stimulation test may be indicated if absence
concentrations suggest poor ovarian ‘reserve’ (the
of testes is suspected or to assess Leydig cell reserve
size of the ovarian ovum supply). Serum AMH
(see Chapter 9).
may, thus, have a place in the investigation of
● Some tumours can release b-hCG or oestrogens and
infertility.
may induce features of feminization. These can be
Male assayed in plasma if the cause of infertility is unclear.
Systemic illness, for example cystic fibrosis, thyroid Sometimes, despite both partners being investigated,
disease, gynaecomastia, eunuchoid appearance and no cause for the infertility can be found. Some couples
ambiguous genitalia, should be excluded and any decide to try in vitro fertilization (Fig. 10.2).
Some drug effects on the hypothalamic–pituitary–gonadal axis 163
SUMMARY
● A number of physiological changes may take ● Biochemical tests can be used to monitor the
place during pregnancy that may affect laboratory progression of pregnancy as well as to detect fetal
tests. In some cases this is due to increased abnormality.
concentrations of hormone or transport proteins, ● Infertility may be primary or secondary and can
although the free hormone concentrations are be the result of problems with the man or woman,
normal. or both. Biochemical tests can also be useful in the
● Increased urinary (or plasma) hCG concentration diagnosis of infertility in conjunction with other
can be used to confirm pregnancy. investigations.
11 Thyroid function
It is important to understand thyroid biochemistry, mediated by the enzyme TPO. This step is inhibited by
as it helps to interpret thyroid function tests, which carbimazole and propylthiouracil.
are among the most common endocrine requests in Iodotyrosines are coupled to form T4 (DIT and DIT)
clinical practice. Thyroid disease is relatively common, and T3 (DIT and MIT) (Fig. 11.1), which are stored
and therefore is likely to be encountered clinically. in the lumen of the thyroid follicular cells. Normally
Briefly, thyroxine (T4), tri-iodothyronine (T3) and much more T4 than T3 is synthesized, but, if there is
calcitonin are secreted by the thyroid gland. Both T4 and an inadequate supply of iodide, the ratio of T3 to T4
T3 are products of the follicular cells and influence the in the gland increases. The thyroid hormones, still
rate of all metabolic processes. Calcitonin is produced incorporated in thyroglobulin, are stored in the colloid
by the specialized C cells and influences calcium of the thyroid follicle.
metabolism (see Chapter 6). Prior to the secretion of thyroid hormones,
thyroglobulin is taken up by the follicular cells, by a
PHYSIOLOGY process involving endocytosis and then phagocytosis,
Thyroid hormones are synthesized in the thyroid gland and T4 and T3 are released by proteolytic enzymes
by the iodination and coupling of two molecules of the into the bloodstream. This process is stimulated by
amino acid tyrosine, a process that is dependent on an TSH and inhibited by iodide. The thyroid hormones
adequate supply of iodide. Iodide in the diet is absorbed are immediately bound to plasma proteins. Mono-
rapidly from the small intestine. In areas where the iodotyrosine and DIT, released at the same time, are
iodide content of the soil is very low, there used to be de-iodinated and the iodine is reused.
a high incidence of enlargement of the thyroid gland Each step is controlled by specific enzymes, and
(goitre), but the general use of artificially iodized salt congenital deficiency of any of these enzymes can lead
has made this a less common occurrence. Seafoods to goitre and, if severe, hypothyroidism. The uptake of
generally have a high iodide content. Therefore fish iodide, as well as the synthesis and secretion of thyroid
and iodized salt are the main dietary sources of the hormones, is regulated by TSH, secreted from the
element. Normally about a third of dietary iodide is anterior pituitary gland. About 10 times more T4 than
taken up by the thyroid gland and the rest is renally T3 is formed, with most of the latter being formed by
excreted. de-iodination in the liver, kidneys and muscle.
Synthesis of thyroid hormones
Iodide is actively taken up by the thyroid gland under
the control of thyroid-stimulating hormone (TSH) I I
via a sodium/iodide symporter. Uptake is blocked by
HO O CH2CHCOO– Thyroxine (T4)
thiocyanate and perchlorate. The concentration of
iodide in the gland is at least 20 times that in plasma I I NH3+
It is sometimes used as part of the combined pituitary Drug effects on thyroid function tests
stimulation test (see Chapter 7). Drugs may alter plasma T4 and T3 concentrations.
Allergic reactions may occur, and therefore The more common effects are summarized in Table
resuscitation facilities should be available and the test 11.1. If the primary change is in binding protein
should be carried out by experienced staff. concentrations, plasma free hormone concentrations
Procedure are usually normal.
● A basal blood sample is taken. Interference of assays by immunoglobulins
● 200 µg of TRH is injected intravenously over about Anti-T4 or anti-T3 immunoglobulins or heterophilic
a minute. antibodies (induced by external antigens, e.g. derived
● Further blood samples are taken 20 and 60 min after from other species that cross-react with self-antigens)
the TRH injection, and TSH is measured in all samples. can cause a spurious elevation of T4 or T3 (or free
Note that certain drugs, such as dopamine agonists hormones), respectively, when assayed by immunoassay.
and glucocorticoids, reduce the response, and oestrogens, This needs to be remembered when interpreting thyroid
metoclopramide and theophylline enhance it. function test results.
Pathophysiology
As primary hypothyroidism develops, TSH secretion
from the anterior pituitary gland increases as the
negative feedback (associated with the falling plasma
T4 or fT4 concentration) decreases. Plasma T3 or fT3
concentrations may be normal and thus not usually
useful in making the diagnosis.
Generally in primary hypothyroidism the plasma
TSH concentration is high, but it is low in secondary
hypothyroidism due to pituitary or hypothalamic
disease. Initially, the plasma T4 or fT4 concentration
may be within the population reference range, although
abnormally low for the individual. For this reason the Figure 11.3 Congenital hypothyroidism. The head is broad,
plasma TSH concentration is the most sensitive index the eyes wide apart, the tongue protrudes from the mouth
of early hypothyroidism. If the patient is very ill, and all movements and responses are slow and sluggish.
investigations should be deferred (see ‘Sick euthyroid’ Reproduced with kind permission from Browse NL,
below). Black J, Burnand KG and Thomas WEG (eds). Browse’s
Introduction to the Symptoms and Signs of Surgical
Pregnancy and neonatal hypothyroidism Disease, 4th edition. London: Hodder Arnold, 2005.
In about 0.3 per cent of pregnancies the mother has
Hashimoto’s disease. Thyroid hormones are essential fibrillation and osteoporosis; in such cases, plasma TSH
for fetal development, hence the importance of treating concentrations are often low or suppressed.
the mother with thyroxine. If a patient is non-compliant with treatment and only
Routine screening for congenital hypothyroidism takes T4 near to the time of thyroid function testing, a
(Fig. 11.3) is discussed in Chapters 26 and 27. high plasma TSH may be observed with high plasma
fT4 concentrations. This is because there is insufficient
Treatment of hypothyroidism T4 to normalize plasma TSH, and yet the high plasma
This is usually with T4, which can be titrated until the fT4 reflects the recent taking of T4.
plasma TSH is within the reference range. However,
this has recently been challenged, as plasma TSH Subclinical hypothyroidism
concentrations may not adequately reflect tissue Subclinical (compensated hypothyroidism) is the state
hypothyroidism, and it may be better to be guided by in which plasma TSH concentration is raised but the
plasma fT4 concentrations and clinical features. On rare total or fT4 concentration still falls within the reference
occasions, such as in hypothyroid comas, T3 is given range. In individuals over the age of 60 years, the
instead, as its action is more immediate. The response prevalence may be as high as 10 per cent. Some of these
to T4 therapy can be checked every 2–3 months until patients have positive thyroid antibodies, for example
the patient is stable, after which 6- to 12-month blood anti-TPO or anti-Tg, and each year about 2–5 per cent
checks may be useful. of thyroid antibody-positive patients go on to develop
Thyroxine should be used with caution in patients hypothyroidism.
with ischaemic heart disease for fear of worsening angina Some patients may be asymptomatic, whereas
pectoris, and low doses initially plus b-blockers may be others have symptoms suggestive of hypothyroidism.
indicated. Thyroid-stimulating hormone assays are of Thyroxine therapy may be indicated particularly in
little value in monitoring secondary hypothyroidism; pregnancy, when the patient is symptomatic, or with
fT4 is better. Thyroxine therapy may precipitate an positive thyroid antibodies and plasma TSH more than
Addisonian crisis in patients with concomitant adrenal 10 mU/L. It can be associated with increased risk of
insufficiency. Overtreatment with T4 can evoke atrial cardiovascular disease.
170 Thyroid function
Thyroid hormone resistance should be done, if indicated, and the pituitary gland
In generalized thyroid hormone resistance, the plasma assessed (see Chapter 7).
total T4 and fT4 concentrations are elevated, with ● Raised plasma TSH and raised/normal plasma
normal or slightly raised TSH concentration. Some fT4 concentrations in the presence of hypothyroid
patients appear euthyroid, but others may present with symptoms may indicate thyroid hormone resistance.
hypothyroid symptoms, and the defect may be inherited Some causes of hypothyroidism are shown in Box 11.1.
as an autosomal dominant trait in some patients. The
defect is thought to be due to a defect in T4 and/or T3 Hyperthyroidism (thyrotoxicosis)
receptors and may be associated with other end-organ Hyperthyroidism causes sustained high plasma
resistance states. concentrations of T4 and T3. There is often generalized
increase in the metabolic rate, evidenced clinically by,
Laboratory investigation of suspected hypothyroidism for example, heat intolerance, a fine tremor, tachycardia
A careful history (including drugs) should be taken and including atrial fibrillation, weight loss, tiredness,
an examination performed, checking for a goitre. anxiety, sweating and diarrhoea.
The following biochemical features may be associated
● The plasma TSH and total T4 or fT4 concentrations
with hyperthyroidism:
should be measured.
● Slightly elevated plasma TSH and normal fT4 ● Hypercalcaemia is occasionally found in patients
concentrations suggest compensated hypothyroidism. with severe thyrotoxicosis. There is an increased
Measuring circulating thyroid antibodies may be turnover of bone cells, probably due to a direct
useful, that is, anti-TPO. Tests should be repeated action of thyroid hormone.
after 3–6 months as some patients may develop full-
blown hypothyroidism.
● Raised plasma TSH and low fT4 concentrations
Box 11.1 Some causes of hypothyroidism
suggest primary hypothyroidism. The thyroid
Primary hypothyroidism
antibodies should be measured and, if positive, other Iodine deficiency
autoimmune diseases excluded. Autoimmune thyroid disease
● Low plasma TSH and low fT4 concentrations may Hashimoto’s disease
indicate that the hypothyroidism is caused by a Subacute thyroiditis
hypothalamic or pituitary disorder. A TRH test Transient subacute thyroiditis (de Quervain’s)
Following treatment of hyperthyroidism
Post thyroidectomy
CASE 2 Post radioiodine treatment
External irradiation to neck region
A 49-year-old woman was investigated in the medical Surgery or trauma to neck
out-patient department for tiredness. The following Defects of thyroid hormone synthesis
test results were returned: Congenital absence of thyroid gland
Infiltrative disease of the thyroid, e.g. sarcoid,
Plasma thyroid-stimulating hormone (TSH)
haemochromatosis, fibrosis (Riedel’s struma)
10.6 mU/L (0.20–5.0) Drugs
Free thyroxine (fT4) 13.9 pmol/L (12–25) Carbimazole
Propylthiouracil
DISCUSSION Amiodarone
These results are suggestive of compensated Lithium
hypothyroidism, in which the plasma TSH Interferon-a
concentration is raised and the fT4 concentration Tyrosine kinase inhibitors
still remains within the reference range. The patient Secondary hypothyroidism
also had positive thyroid antibodies (anti-thyroid Pituitary or hypothalamic disease
peroxidase). A trial of thyroxine of 50 µg a day Drugs: bexarotene
brought her plasma TSH concentration to within the Thyroid hormone resistance
reference range and improved her symptoms. Generalized thyroid resistance
Disorders of the thyroid gland 171
Autonomous secretion
Graves’ disease
Toxic multinodular goitre (Plummer’s disease) or a
single functioning nodule (occasionally an adenoma)
Subacute thyroiditis
Some metastatic thyroid carcinomas
Excessive ingestion of thyroid hormones or iodine
Amiodarone
Thyrotoxicosis factitia (self-administration of thyroid
hormones)
Administration of iodine to a subject with iodine-
deficiency goitre
Jod–Basedow syndrome
Rare causes
Thyroid-stimulating hormone secretion by tumours,
including pituitary tumours or those of trophoblastic
origin
Struma ovarii (thyroid tissue in an ovarian teratoma) Figure 11.4 A patient with primary hyperthyroidism.
Excess human chorionic gonadotrophin, e.g. molar Note exophthalmos and diffuse thyroid swelling.
pregnancy or choriocarcinoma Reproduced with kind permission from Kinirons M and
Pituitary resistance to thyroid hormone Ellis H. French’s Index of Differential Diagnosis, 15th
edition. London: Hodder Arnold, 2011.
172 Thyroid function
● If the plasma TSH concentration is high, look at thyroid replacement for hypothyroidism, drugs
plasma fT4: such as metoclopramide or domperidone, or
– If fT4 concentration is low, consider primary sick euthyroid.
hypothyroidism (see Box 11.1). – If fT4 concentration is high, consider
– If fT4 concentration is normal, consider generalized thyroid hormone resistance, TSH-
compensated hypothyroidism, inadequate secreting tumour, interfering assay antibodies.
SUMMARY
● Thyroid-stimulating hormone from the anterior ● Thyroid disease is relatively common.
pituitary acts upon the thyroid gland to release Hypothyroidism can be primary or secondary
two iodine-containing hormones, T4 and T3. The and manifest as weight gain, tiredness and
latter is more active and can also be produced from constipation – to name but a few of its clinical
T4 peripherally. These hormones are essential for features. Hyperthyroidism causes thyrotoxicosis
normal growth and development and increase associated with weight loss, sweating and
basal metabolic rate. sometimes thyroid eye disease. Both can present
● Both T4 and T3 are bound to proteins, including with a goitre, although some patients with
TBG, albumin and transthyretin. Only the free or a goitre may be euthyroid (normal thyroid
unbound form is physiologically active, although function tests).
this fraction constitutes less than 1 per cent of the
total hormone concentration.
12 Carbohydrate metabolism
Glucose
Glucose- Ribose- Insulin
6-phosphate 5-phosphate
Insulin is the most important hormone controlling
TPP Transketolase plasma glucose concentrations. A plasma glucose
concentration of greater than about 5 mmol/L acting via
Fructose- Sedoheptulose- the glucose transporter 2 stimulates insulin release from
7-phosphate
6-phosphate the pancreas b-cell. These cells produce proinsulin, which
consists of the 51-amino-acid polypeptide insulin and a
linking peptide (C-peptide, Fig. 12.3). Splitting of the
peptide bonds by prohormone convertases releases via
Fructose-1,
6-diphosphate intermediates (mostly 32–33 split proinsulin) equimolar
amounts of insulin and C-peptide into the ECF.
Insulin binds to specific cell surface receptors on
muscle and adipose tissue, thus enhancing the rate
Pyruvate Lactate of glucose entry into these cells. Insulin-induced
Pyruvate activation of enzymes stimulates glucose incorporation
TPP
dehydrogenase
into glycogen (glycogenesis) in liver and muscle (Fig
Acetyl CoA 12.4). Insulin also inhibits the production of glucose
(gluconeogenesis) from fats and amino acids, partly
by inhibiting fat and protein breakdown (lipolysis and
Oxaloacetate Citrate proteolysis).
The transport of glucose into liver cells is insulin
independent but, by reducing the intracellular glucose
a-Ketoglutarate concentration, insulin does indirectly promote the
Succinate
passive diffusion of glucose into them. Insulin also
a-Ketoglutarate
dehydrogenase directly increases the transport of amino acids,
TPP
potassium and phosphate into cells, especially muscle;
Figure 12.2 Simplification of the tricarboxylic acid (Krebs) these processes are independent of glucose transport.
cycle. CoA, coenzyme A; TPP, thiamine pyrophosphate. In the longer term, insulin regulates growth and
Reproduced with kind permission from Candlish JK and development and the expression of certain genes.
Crook M. Notes on Clinical Biochemistry. Singapore:
World Scientific Publishing, 1993. Glucagon
Glucagon is a single-chain polypeptide synthesized
even after a carbohydrate meal. Significant glycosuria by the a-cells of the pancreatic islets. Its secretion is
usually occurs only if the plasma glucose concentration stimulated by hypoglycaemia. Glucagon enhances
exceeds about 10 mmol/L – the renal threshold. hepatic glycogenolysis (glycogen breakdown) and
gluconeogenesis.
How the body maintains extracellular
glucose concentrations
Control of plasma glucose concentration C-PEPTIDE
During normal metabolism, little glucose is lost
unchanged from the body. Maintenance of plasma
glucose concentrations within the relatively narrow COO–
NH3+
range of 4–10 mmol/L, despite the widely varying input S
S
from the diet, depends on the balance between the
S S
glucose entering cells from the ECF and that leaving S
them into this compartment. INSULIN S
BRAIN
MUSCLE
G6P
Insulin GLYCOGEN
INTESTINE CO2 + H2O
G6P
GLUCOSE
Glucose
Insulin
Insulin
G6P G6P
GLYCOGEN Triose-P
Triose-P
Acetyl CoA
Fatty acid + Glycerol-3-P
Fatty acid + Glycerol-3-P
Glycerol
Triglyceride TRIGLYCERIDE
VLDL
Figure 12.5 Post-prandial metabolism of glucose. CoA, coenzyme A; G6P, glucose-6-phosphate; Glycerol-3-P,
glycerol-3-phosphate; Triose-P, triose phosphate or glyceraldehyde 3-phosphate; VLDL, very low-density lipoprotein.
stimulates the b-cells of the pancreas to secrete insulin. these actions are impaired. Both muscle and adipose
Insulin may further enhance hepatic and muscle tissue store the excess post-prandial glucose, but the
glycogenesis. More importantly, entry of glucose into mode of storage and the function of the two types of
adipose tissue and muscle cells, unlike that into liver and cell are very different, as will be shown later.
brain, is stimulated by insulin and, under physiological
conditions, the plasma glucose concentration falls to Ketosis
near fasting levels. Conversion of intracellular glucose Adipose tissue and the liver
to G6P in adipose and muscle cells is catalysed by the Adipose tissue triglyceride is the most important long-
enzyme hexokinase, which, because its affinity for term energy store in the body. Greatly increased use
glucose is greater than that of hepatic glucokinase, of fat stores, for example during prolonged fasting,
ensures that glucose enters the metabolic pathways in is associated with ketosis. Adipose tissue cells, acting
these tissues at lower extracellular concentrations than in conjunction with the liver, convert excess glucose
those in the liver. The relatively high insulin activity to triglyceride and store it in this form rather than
after a meal also inhibits the breakdown of triglyceride as glycogen. The components are both derived from
(lipolysis) and protein (proteolysis). If there is relative glucose, fatty acids from the glucose entering hepatic
or absolute insulin deficiency, as in diabetes mellitus, cells and glycerol from that entering adipose tissue cells.
Physiology 181
In the liver, triglycerides are formed from glycerol- Most tissues, other than the brain, can oxidize fatty
3-phosphate (from triose phosphate or glyceraldehyde- acids to acetyl CoA, which can then be used in the TCA
3-phosphate) and fatty acids [from acetyl coenzyme A cycle as an energy source. When the rate of synthesis
(CoA)]. The triglycerides are transported to adipose exceeds its use, the hepatic cells produce acetoacetic acid
tissue cells incorporated in VLDL, where they are by enzymatic condensation of two molecules of acetyl
hydrolysed by lipoprotein lipase. The released fatty CoA; acetoacetic acid can be reduced to b-hydroxybutyric
acids (of hepatic origin) are re-esterified within these acid and decarboxylated to acetone. These ketones can
cells with glycerol-3-phosphate, derived from glucose, be used as an energy source by brain and other tissues at
which has entered this tissue under the influence of a time when glucose is in relatively short supply.
insulin. The resultant triglyceride is stored and is far Ketosis occurs when fat stores are the main energy
more energy dense than glycogen (see Chapter 13). source and may result from fasting or from reduced
During fasting, when exogenous glucose is nutrient absorption, for example due to vomiting. Mild
unavailable and the plasma insulin concentration ketosis may occur after as little as 12 h of fasting. After
is therefore low, endogenous triglycerides are short fasts, metabolic acidosis is not usually detectable,
reconverted to free non-esterified fatty acids (NEFAs) but, after longer periods, more hydrogen ions may
and glycerol by lipolysis (Fig. 12.6). Both are be produced than can be dealt with by homeostatic
transported to the liver in plasma, the NEFA being buffering mechanisms, depleting the plasma bicarbonate
protein bound, predominantly to albumin. Glycerol concentration, which therefore falls (see Chapter 4).
enters the hepatic gluconeogenic pathway at the The plasma glucose concentration is maintained
triose phosphate stage; the glucose synthesized can be principally by hepatic gluconeogenesis, but during
released from these cells, thus minimizing the fall in prolonged starvation, such as that in anorexia nervosa or
glucose concentrations. during childhood, ketotic hypoglycaemia may occur. The
BRAIN
G6P
Acetyl CoA
CO2 + H2O
Glucose KETONES + H+
Triglyceride
G6P
KETONES + H+
+
Figure 12.6 Intermediary metabolism during fasting: ketosis. CoA, coenzyme A; FA, fatty acid; G6P,
glucose-6-phosphate; NEFA, non-esterified fatty acid.
BRAIN
MUSCLE
G6P
Insulin GLYCOGEN
INTESTINE CO2 + H2O
G6P
GLUCOSE
Glucose
Insulin
Insulin
G6P G6P
GLYCOGEN Triose-P
Triose-P
Acetyl CoA
Fatty acid + Glycerol-3-P
Fatty acid + Glycerol-3-P
Glycerol
Triglyceride TRIGLYCERIDE
VLDL
Figure 12.5 Post-prandial metabolism of glucose. CoA, coenzyme A; G6P, glucose-6-phosphate; Glycerol-3-P,
glycerol-3-phosphate; Triose-P, triose phosphate or glyceraldehyde 3-phosphate; VLDL, very low-density lipoprotein.
stimulates the b-cells of the pancreas to secrete insulin. these actions are impaired. Both muscle and adipose
Insulin may further enhance hepatic and muscle tissue store the excess post-prandial glucose, but the
glycogenesis. More importantly, entry of glucose into mode of storage and the function of the two types of
adipose tissue and muscle cells, unlike that into liver and cell are very different, as will be shown later.
brain, is stimulated by insulin and, under physiological
conditions, the plasma glucose concentration falls to Ketosis
near fasting levels. Conversion of intracellular glucose Adipose tissue and the liver
to G6P in adipose and muscle cells is catalysed by the Adipose tissue triglyceride is the most important long-
enzyme hexokinase, which, because its affinity for term energy store in the body. Greatly increased use
glucose is greater than that of hepatic glucokinase, of fat stores, for example during prolonged fasting,
ensures that glucose enters the metabolic pathways in is associated with ketosis. Adipose tissue cells, acting
these tissues at lower extracellular concentrations than in conjunction with the liver, convert excess glucose
those in the liver. The relatively high insulin activity to triglyceride and store it in this form rather than
after a meal also inhibits the breakdown of triglyceride as glycogen. The components are both derived from
(lipolysis) and protein (proteolysis). If there is relative glucose, fatty acids from the glucose entering hepatic
or absolute insulin deficiency, as in diabetes mellitus, cells and glycerol from that entering adipose tissue cells.
Hyperglycaemia and diabetes mellitus 183
During gluconeogenesis, hydrogen ions are reused. ● hepatic and renal gluconeogenesis from lactate
Under aerobic conditions, the liver consumes much cannot occur anaerobically,
more lactate than it produces. ● anaerobic glycolysis is stimulated because the
The physiological accumulation of lactic acid during falling adenosine triphosphate (ATP) levels cannot
muscular contraction is a temporary phenomenon and be regenerated by the TCA cycle under anaerobic
rapidly disappears at rest, when slowing of glycolysis conditions.
allows aerobic processes to ‘catch up’.
The combination of impaired gluconeogenesis and
Pathological lactic acidosis increased anaerobic glycolysis converts the liver from
an organ that consumes lactate and H+ to one that
Lactic acid, produced by anaerobic glycolysis, may either generates large amounts of lactic acid. Severe hypoxia,
be oxidized to CO2 and water in the TCA cycle or be for example following a cardiac arrest, causes marked
reconverted to glucose by gluconeogenesis in the liver. lactic acidosis. If diabetic ketoacidosis is associated with
Both the TCA cycle and gluconeogenesis need oxygen; significant volume depletion, this hypoxic syndrome
anaerobic glycolysis is a non-oxygen-requiring pathway. may aggravate the acidosis. (See Chapter 4 for a further
Pathological accumulation of lactate may occur because: discussion of lactic acidosis.)
● production is increased by an increased rate of The glycolytic pathway as well as the TCA cycle are
anaerobic glycolysis, summarized in Figures 12.1 and 12.2.
● use is decreased by impairment of the TCA cycle or
impairment of gluconeogenesis. HYPERGLYCAEMIA AND DIABETES
Tissue hypoxia (Fig. 12.8) due to the poor tissue MELLITUS
perfusion of the ‘shock’ syndrome is usually the most Hyperglycaemia may be due to:
common cause of lactic acidosis. Hypoxia increases
● intravenous infusion of glucose-containing fluids,
plasma lactate concentrations because:
● severe stress (usually a transient effect) such as
● the TCA cycle cannot function anaerobically and trauma, myocardial infarction or cerebrovascular
oxidation of pyruvate and lactate to CO2 and water accidents,
is impaired, ● diabetes mellitus or impaired glucose regulation.
GLYCOGEN
G6P G6P
GLYCOGEN Pyruvate
LIVER MUSCLE
Genetic syndromes associated with diabetes homeostasis and diabetes mellitus. The definition is
● Down’s syndrome. that the fasting venous plasma glucose is 6.1 mmol/L
● Turner’s syndrome. or more but less than 7.0 mmol/L, and less than
● Klinefelter’s syndrome. 7.8 mmol/L 2 h after an OGTT.
● Myotonic dystrophy.
Subjects at risk of developing diabetes mellitus
Gestational diabetes mellitus
A strong family history of diabetes mellitus may suggest
In the UK, about 4–5 per cent of pregnancies are that an individual is at risk of developing diabetes
complicated by gestational diabetes mellitus (GDM). mellitus (particularly type 2), as may a family history
It is associated with increased fetal abnormalities, of GDM, IGT or IFG. Those with predisposing HLA
for example high birthweight, cardiac defects and types and autoimmune disease may be susceptible
polyhydramnios. In addition, birth complications, to developing type 1 diabetes. Type 2 diabetes is
maternal hypertension and the need for caesarean more common in certain racial groups, such as Afro-
section may occur. If maternal diet/lifestyle factors fail Caribbeans, South Asians and Pacific Islanders. One of
to restore glucose levels, insulin is usually required to the reasons why type 2 diabetes is on the increase is the
try to reduce the risk of these complications. increasing tendency to obesity and central adiposity in
Women at high risk for GDM include those who urbanized and more sedentary populations consuming
have had GDM before, have previously given birth to a high-calorie diets.
high-birthweight baby, are obese, have a family history of The thrifty phenotype (Barker–Hales) hypothesis
diabetes mellitus and/or are from high-risk ethnic groups, proposes that nutritional deficiency in fetal and early
for example black or South Asian. These women should infancy associated with low birthweight increases
be screened at the earliest opportunity and, if normal, the risk of developing type 2 diabetes and insulin
retested at about 24–28 weeks, as glucose tolerance resistance.
progressively deteriorates throughout pregnancy. In some
units 50 g oral glucose is used and the blood glucose is Insulin resistance syndrome or metabolic syndrome
sampled at 1 h – plasma glucose of more than or equal to It has been recognized that certain coronary heart disease
7.8 mmol/L being diagnostic (O’Sullivan’s screening test risk factors occur together. There is an aggregation of
for gestational diabetes). If fasting venous plasma glucose lipid and non-lipid risk factors of metabolic origin.
is 7.0 mmol/L or more and/or the random measurement A particular cluster is known as the metabolic syndrome,
gives a concentration of 11.1 mmol/L or more (some syndrome X or Reaven’s syndrome and is closely linked
doctors prefer to use a lower cut-off of about 9.0 mmol/L to insulin resistance. One definition is the presence of
in pregnancy), the woman has GDM. In equivocal cases, three or more of the following features:
an OGTT is indicated. Six weeks post partum, the woman
should be reclassified with a repeat OGTT. ● Abdominal obesity (waist circumference):
– male more than 102 cm (40 in),
Impaired glucose tolerance – female more than 88 cm (35 in).
The WHO definition of impaired glucose tolerance ● Fasting plasma triglycerides more than 1.7 mmol/L.
(IGT) is a fasting venous plasma glucose concentration ● Fasting plasma high-density lipoprotein (HDL)
of less than 7.0 mmol/L and a plasma glucose cholesterol:
concentration between 7.8 mmol/L and 11.1 mmol/L – male less than 1.0 mmol/L,
2 h after an OGTT. Some patients with IGT develop – female less than 1.3 mmol/L,
diabetes mellitus later and may require an annual OGTT ● Blood pressure more than or equal to 130/85 mmHg.
to monitor for this. However, because of the increased ● Fasting blood glucose more than 5.5 mmol/L.
risk of vascular complications, secondary causes of IGT
should be sought, dietary advice given, if necessary, and Plasma levels of insulin would be expected to be
the patient followed up. In pregnancy IGT is treated as raised, that is, hyperinsulinaemia. Other associated
GDM because of the risks to the fetus. features may include polycystic ovary syndrome, fatty
liver, raised fibrinogen and plasminogen activator
Impaired fasting glucose inhibitor 1 concentrations, renal sodium retention,
Impaired fasting glucose (IFG), like IGT, refers to a hyperuricaemia and dense low-density lipoprotein
metabolic stage intermediate between normal glucose (LDL) particles (see Chapter 13).
186 Carbohydrate metabolism
recommended if further information is required. Insulin proximal tubular cells reabsorb most of the glucose
requirements vary in patients with type 1 diabetes. For in the glomerular filtrate. Glycosuria, as defined
example, the dose may need to be increased during above, occurs only when the plasma, and therefore
any illness or during pregnancy and reduced if there is glomerular filtrate, concentrations exceed the tubular
increased activity or meals are missed. reabsorptive capacity. This may be because the plasma
In patients with type 2 diabetes, plasma glucose and glomerular filtrate concentrations are more than
concentrations may be controlled by diet, associated about 10 mmol/L, and therefore the normal tubular
with weight reduction, and increased physical activity, reabsorptive capacity is significantly exceeded. Very
but insulin may be required during periods of stress rarely, if the glomerular filtration rate is much reduced,
or pregnancy. In this group insulin secretion can be there may be no glycosuria despite plasma glucose
stimulated by the sulphonylurea drugs, such as gliclazide, concentrations more than 10 mmol/L. A diagnosis of
glipizide, glibenclamide or glimepiride. Biguanides, diabetes mellitus should never be made on the basis of
usually metformin, can also be used and are particularly glycosuria.
useful in obese patients. Metformin decreases intestinal Blood glucose
glucose absorption and hepatic gluconeogenesis as well
Blood glucose concentrations may be measured using
as increasing tissue insulin sensitivity. Metformin can
glucose testing reagent strips. The colour change of
inhibit oxidative phosphorylation, which can, under
the strip can be assessed visually or by using a portable
certain circumstances, lead to lactic acid accumulation.
glucose meter and the reaction often involves an enzyme
Acarbose delays post-prandial absorption of glucose by
determination of glucose, for example glucose oxidase.
inhibiting a-glucosidase.
Meters should ideally be overseen by laboratory staff
Other oral agents are the thiazolidinediones or
expert in point of care testing (see Chapter 30). Although
‘glitazones’, for example rosiglitazone and pioglitazone,
the measurement of blood glucose concentrations
which activate g-peroxisome proliferator-activated
involves the discomfort of several skin punctures,
receptors and which can reduce insulin resistance
many motivated patients are able to adjust their insulin
by a number of metabolic pathways, some of which
dose more accurately based on these results than on
involve increasing the transcription of nuclear proteins
those obtained by testing their urine. This method of
that control free fatty acid and tissue glucose uptake.
testing is also useful in the detection of hypoglycaemia.
Repaglinide is a meglitinide that increases insulin release
For patients who do not like blood testing, urinary
from pancreatic b-cells and enhances tissue insulin
glucose testing can be used, but of course cannot detect
sensitivity. The incretins are gastrointestinal hormones
hypoglycaemia and is dependent on the renal glucose
that increase insulin release from the pancreas after
threshold.
eating, for example glucagon-like peptide (GLP-1)
and gastric inhibitory peptide (GIP). They are rapidly Glycated haemoglobin
inactivated by the enzyme dipeptidyl peptidase-4 (DPP- Glycated haemoglobin (HbA1c) is formed by non-
4). Incretin mimetics such as exenatide or liraglutide enzymatic glycation of haemoglobin and is dependent
or DPP-4 inhibitors such as sitagliptin, vildagliptin or on the mean plasma glucose concentrations and on the
saxagliptin are being used in type 2 diabetes mellitus.. lifespan of the red cell; falsely low values may be found
It is now recognized that diabetes mellitus is not in patients with haemolytic disease. Measurement of
just a glucose disorder. It is important also to optimize blood HbA1c may not reveal potentially dangerous
abnormal plasma lipids (see Chapter 13) and correct short-term swings and nor does HbA1c detect
hypertension, particularly if there is microalbuminuria hypoglycaemic episodes and thus plasma glucose
or proteinuria (see Chapter 19). estimations may also be useful.
This was expressed as a percentage of total blood
Monitoring of diabetes mellitus haemoglobin concentration and gives a retrospective
Glycosuria assessment of the mean plasma glucose concentration
Glycosuria can be defined as a concentration of during the preceding 6–8 weeks. The higher the
urinary glucose detectable using relatively insensitive, glycated haemoglobin, the poorer the mean diabetic or
but specific, screening tests. These tests often depend glycaemic control.
on the action of an enzyme, such as glucose oxidase, Glycated haemoglobin used to be expressed in
incorporated into a diagnostic strip. Usually, the percentage units but now is expressed as mmol/mol
188 Carbohydrate metabolism
and conversion between the units is by the following and less than 3.5 g/mol in females. An abnormal result
equation: IFCC-HbA1c (mmol/mol) = [DCCT-HbA1c should be confirmed in two out of three urine samples
(%) – 2.15] ¥ 10.929. HbA1c tests are certified by the in the absence of other causes of proteinuria (see
National Glycohemoglobin Standardization Program Chapter 19). Apart from being predictive of diabetic
(NGSP) to standardize them against the results of renal complications, urinary albumin excretion is also
the 1993 Diabetes Control and Complications Trial associated with increased vascular permeability and
(DCCT) but now are expressed as IFCC (International enhanced risk of cardiovascular disease.
Federation of Clinical Chemistry) units. Intervention Optimization of glycaemic control can slow the
trials for type 1 and type 2 diabetes have shown that progression of microalbuminuria, as can treating
trying to optimize glycaemic control, as judged by HbA1c, hypertension. Some recommend a target blood
to about 7 per cent (or above 53 mmol/mol) reduces pressure lower than 140/80 mmHg in type 2 diabetes, or
the risk of microvascular diabetic complications. 135/75 mmHg or lower if microalbuminuria is present.
The blood pressure targets are usually more aggressive
Fructosamine
in type 1 diabetes, partly as the lifetime risk of overt
The measurement of plasma fructosamine concentrations nephropathy is greater. Angiotensin-converting
may be used to assess glucose control over a shorter enzyme (ACE) inhibitor therapy, such as lisinopril
time course than that of HbA1c (about 2–4 weeks), but in type 1 diabetic patients with microalbuminuria,
the assay has methodological limitations. Fructosamine can result in a decline in the albumin excretion rate;
reflects glucose bound to plasma proteins, predominantly similar findings have been shown with enalapril in
albumin, which has a plasma half-life of about 20 days type 2 diabetes. This action of ACE inhibitors is only
but is problematic in patients with hypoalbuminaemia, partially dependent on their blood pressure-lowering
for example due to severe proteinuria. This assay may ability, and therefore they presumably also have other
sometimes be useful in pregnancy and also if haemoglobin important renal protective actions. The angiotensin II
variants, for example HbS or HbC, exist that may interfere receptor antagonists (ARAs), for example irbesartan
with certain HbA1c assays. and losartan, have also been shown to have renal
Blood ketones protective actions.
Monitoring of blood ketones may have a place
in the home management of type 1 diabetes. A Acute metabolic complications of diabetes mellitus
b-hydroxybutyrate below 0.60 mmol/L is normal, Patients with diabetes mellitus may develop various
whereas values between 0.60 mmol/L and 1.0 mmol/L metabolic complications that require emergency treat-
may necessitate more insulin, and concentrations ment, including coma, and these include the following.
greater than 1.0 mmol/L a warning to seek medical Hypoglycaemia
advice.
This is probably the most common cause of coma seen
Urinary albumin determination and diabetic nephropathy in diabetic patients. Hypoglycaemia is most commonly
One of the earliest signs of diabetic renal dysfunction caused by accidental overadministration of insulin or
is the development of small amounts of albumin in the sulphonylureas or meglitinides. Precipitating causes
urine, called microalbuminuria. Untreated, this can include too high a dose of insulin or hypoglycaemic
progress to overt albuminuria or proteinuria (more drug; conversely, the patient may have missed a meal or
than 300 mg/day), impaired renal function and finally taken excessive exercise after the usual dose of insulin
end-stage renal failure. or oral hypoglycaemic drugs.
Microalbuminuria is defined as a urinary albumin Hypoglycaemia is particularly dangerous, and
excretion of 30–300 mg/day or 20–200 µg/min. An some patients lack awareness of this; that is to say,
albumin concentration less than 30 mg/day or less than they lose warning signs such as sweating, dizziness
20 µg/min is defined as normoalbuminuria. A random and headaches. Driving is a major hazard under such
urine sample or timed overnight collection can be circumstances. Patients should monitor their own
useful to assess urinary albumin excretion, although blood glucose closely, carry glucose preparations
the standard test is the urinary albumin to creatinine to abort severe hypoglycaemia and avoid high-risk
ratio (ACR), which avoids a timed urine collection. activities during which hypoglycaemic attacks could
This should normally be less than 2.5 g/mol in males be dangerous.
Hyperglycaemia and diabetes mellitus 189
CASE 1 CASE 2
A 34-year-old woman with known type 1 diabetes A 24-year-old woman presented to the casualty
mellitus was admitted to hospital following a ‘black department in a coma. The relevant biochemical
out’ while driving. She had recently increased her results were as follows:
insulin dose because she felt unwell with ‘flu’ but Plasma
unwisely had missed two meals during the day. The Sodium 130 mmol/L (135–145)
results of some of her biochemistry tests were as Potassium 5.9 mmol/L (3.5–5.0)
follows: Bicarbonate 10 mmol/L (24–32)
Plasma Chloride 92 mmol/L (95–105)
Sodium 135 mmol/L (135–145) Glucose 35 mmol/L (5.5–11.1)
Potassium 4.0 mmol/L (3.5–5.0) pH 7.10 (7.35–7.45)
Bicarbonate 23 mmol/L (24–32) PaCO2 3.1 kPa (4.6–6.0)
Urea 5.4 mmol/L (2.5–7.0) PaO2 11.1 kPa (9.3–13.3)
Creatinine 100 µmol/L (70–110) Urine was positive for ketones.
Glucose 1.5 mmol/L (5.5–11.1)
DISCUSSION
pH 7.43 (7.35–7.45)
The patient was shown to have type 1 diabetes
PaCO2 5.3 kPa (4.6–6.0)
mellitus and had presented in diabetic ketoacidosis,
PaO2 12.1 kPa (9.3–13.3)
with hyperglycaemia, hyponatraemia, hyperkalaemia
DISCUSSION and a metabolic acidosis.
The blood glucose shows hypoglycaemia, secondary
to the patient having increased her insulin dose
although euglycaemic diabetic ketoacidosis has been
despite having missed meals. Hypoglycaemia can
described when plasma glucose concentrations are only
present with neurological impairment, including
slightly elevated.
impaired memory, loss of consciousness and coma.
Hyperglycaemia causes glycosuria and hence an
This can be treated in the emergency situation by
osmotic diuresis. Water and electrolyte loss due to
giving glucose intravenously to avoid irreversible
vomiting, which is common in this syndrome, increases
neurological damage. It is important for patients on
fluid depletion. There may be haemoconcentration and
insulin to monitor their own blood glucose closely,
reduction of the glomerular filtration rate enough to
particularly if they wish to drive.
cause uraemia due to renal circulatory insufficiency.
The extracellular hyperosmolality causes a shift of water
out of the cellular compartment and severe cellular
Diabetic ketoacidosis dehydration occurs. Loss of water from cerebral cells is
Diabetic ketoacidosis may be precipitated by infection, probably the reason for the confusion and coma. Thus
acute myocardial infarction or vomiting. The patient there is both cellular and extracellular volume depletion.
who reasons ‘no food, therefore no insulin’ could The rate of lipolysis is increased because of decreased
mistakenly withhold insulin. In the absence of insulin, insulin activity; more free fatty acids are produced than
there is increased lipid and protein breakdown, can be metabolized by peripheral tissues. The free fatty
enhanced hepatic gluconeogenesis and impaired acids are either converted to ketones by the liver or,
glucose entry into cells. of less immediate clinical importance, incorporated
The clinical consequences of diabetic ketoacidosis as endogenous triglycerides into VLDL, sometimes
are due to: causing severe hypertriglyceridaemia (see Chapter 13).
Hydrogen ions, produced with ketones other than
● hyperglycaemia causing plasma hyperosmolality,
acetone, are buffered by plasma bicarbonate. However,
● metabolic acidosis,
when their rate of production exceeds the rate of
● glycosuria.
bicarbonate generation, the plasma bicarbonate falls.
Plasma glucose concentrations are usually in the Hydrogen ion secretion causes a fall in urinary pH.
range 20–40 mmol/L, but may be considerably higher, The deep, sighing respiration (Kussmaul’s respiration)
190 Carbohydrate metabolism
and the odour of acetone on the breath are classic Table 12.4 Clinical and biochemical findings in a
features of diabetic ketoacidosis. patient presenting with diabetic ketoacidosis
Plasma potassium concentrations may be raised,
Findings Underlying abnormality
secondarily to the metabolic acidosis, before treatment
Clinical
is started. This is due to failure of glucose entry into
cells in the absence of insulin and because of the low Confusion and later coma Hyperosmolality
glomerular filtration rate. Despite hyperkalaemia, Hyperventilation (Kussmaul’s respiration) Metabolic acidosis
there is a total body deficit due to increased urinary Signs of volume depletion Osmotic diuresis
potassium loss in the presence of an osmotic diuresis. Biochemical
During treatment, plasma potassium concentrations Plasma
may fall as potassium re-enters cells, sometimes causing
Hyperglycaemia Insulin deficiency
severe hypokalaemia unless potassium is prescribed.
Low plasma bicarbonate Metabolic acidosis
Plasma sodium concentrations may be low
(hyponatraemia) or low-normal at presentation, partly Initial hyperkalaemia Intracellular potassium
moves out
because of the osmotic effect of the high extracellular
glucose concentration, which draws water from the cells and Mild uraemia Decreased glomerular
filtration rate
dilutes the sodium. In the presence of a very high plasma
Urine
glucose concentration, a normal or raised plasma sodium
concentration is suggestive of significant water depletion. Glycosuria Insulin deficiency
If there is severe hyperlipidaemia, the possibility of Ketonuria Insulin deficiency
pseudohyponatraemia must be considered (see Chapter
2). When insulin is given, gluconeogenesis is inhibited,
glucose enters cells and sodium-free water follows along of the symptoms, including those of confusion and
the osmotic gradient. If plasma sodium concentrations coma, are related to it. However, the term ‘hyperosmolal’
rise rapidly, the patient may remain confused or even coma or ‘pre-coma’ is usually confined to a condition
comatose as long as the plasma osmolality remains in which there is marked hyperglycaemia but no
significantly raised, despite a satisfactory fall in plasma detectable ketoacidosis. The reason for these different
glucose concentration. This may also occur if isosmolar presentations is not clear. It has been suggested that
or stronger saline solutions are given inappropriately. insulin activity is sufficient to suppress lipolysis but
Hyperphosphataemia followed by hypophosphataemia insufficient to suppress hepatic gluconeogenesis or to
as plasma phosphate concentrations parallel those of facilitate glucose transport into cells.
potassium may persist for several days after recovery from Hyperosmolal non-ketotic (HONK) coma now
diabetic coma. Similarly, hypermagnesaemia can result, may be referred to as hyperosmolar hyperglycaemic
partly because of the acidosis. state (HHS) and may be of sudden onset. It is
Plasma and urinary amylase activities may be more common in older patients. Plasma glucose
markedly elevated and, even in the presence of abdominal concentrations may exceed 50 mmol/L. The effects of
pain mimicking an ‘acute abdomen’, do not necessarily glycosuria are as described above, but hypernatraemia
indicate acute pancreatitis. In some patients the amylase due to predominant water loss is more commonly
is of salivary rather than pancreatic origin. Some plasma found than in ketoacidosis and aggravates the plasma
creatinine assays cross-react with ketones, resulting in a hyperosmolality. Cerebral cellular dehydration, which
spurious plasma creatinine elevation. Sometimes severe contributes to the coma, may also cause hyperventilation,
hypertriglyceridaemia and chylomicronaemia result, and a respiratory alkalosis, although sometimes plasma
due to reduced lipoprotein lipase activity in the face lactic acid may rise, evoking a metabolic acidosis and
of insulin deficiency. A summary of the usual clinical thus a mixed acid–base disturbance may occur. There
and biochemical findings in a patient presenting with may also be an increased risk of thrombosis.
diabetic ketoacidosis is shown in Table 12.4.
Lactic acidosis
Hyperosmolal non-ketotic coma Lactic acidosis can cause a high anion gap metabolic
In diabetic ketoacidosis there is always plasma acidosis and coma. It may be due to the use of
hyperosmolality due to the hyperglycaemia, and many metformin in certain situations, such as high doses in
Hyperglycaemia and diabetes mellitus 191
the very elderly, those with renal, liver or cardiac failure next hour and then 2 h and repeated at 4 h. Monitoring
or those dehydrated or undergoing imaging tests with central venous pressure may be useful to assess fluid
contrast media (see Chapter 4). replacement. Dextrose–saline may be used when the
plasma glucose concentration is less than 15 mmol/L.
Other causes of coma in patients with diabetes mellitus
If the plasma glucose concentration is more than
In addition to the comas described above, a patient 20 mmol/L, 10 U soluble insulin should be given. A
with diabetes mellitus may present with other comas: sliding insulin scale should be instigated. Insulin is
● Cerebrovascular accidents are relatively common in given either by continuous intravenous infusion or
diabetic patients because of the increased incidence by intermittent intramuscular injections, as soon as
of vascular disease. the plasma glucose and potassium concentrations
● Diabetic patients can, of course, have any other are known. Once the patient is eating, subcutaneous
coma, for example drug overdose. insulin can be given instead.
● Diabetic patients are also more at risk of diabetic If the metabolic acidosis is very severe (pH less than
nephropathy and renal failure and thus uraemic coma. 7.0), bicarbonate may be infused, but only until the
blood pH rises to between about 7.15 and 7.20. It is
The assessment of a diabetic patient presenting in
unnecessary and often dangerous to correct the plasma
coma or pre-coma is outlined in Table 12.5.
bicarbonate concentration completely; it rapidly returns
Principles of treatment of diabetic coma
to normal following adequate fluid and insulin therapy.
Remember that 8.4 per cent sodium bicarbonate is
Only the outline of treatment will be discussed. For very hyperosmolar and may cause hypernatraemia and
details of management, the reader should consult a aggravate hyperosmolality. A rapid rise in the blood
textbook of medical emergencies. pH may aggravate the hypokalaemia associated with
Hypoglycaemia treatment.
Hypoglycaemic coma needs prompt glucose replacement The plasma potassium concentration should be
to avoid irreversible brain damage, for example 50 mL measured before insulin is given. It is almost always
of 20 per cent glucose intravenously. If intravenous raised at presentation due to the metabolic acidosis and
access is not an option, glucagon 1 mg can be given reduced glomerular filtration rate, although total body
intramuscularly. Once the patient is awake, glucose- potassium may be decreased. The plasma potassium
containing drinks can be given. concentration may fall rapidly once treatment is
started, and therefore it should be monitored frequently
Diabetic ketoacidosis and potassium given as soon as it starts to fall. Usually
Repletion of fluid and electrolytes should be vigorous. 20 mmol/L potassium is given to each litre bag apart
A 0.9 per cent normal saline solution should be from the first litre and provided there is no oliguria or
administered, usually 1 L initially and then 1 L over the hyperkalaemia. Diabetic ketoacidosis is severe if blood
Table 12.5 Clinical and biochemical features of a diabetic patient presenting in coma
Laboratory findings
Plasma Urine
Diagnosis Clinical features Glucose Bicarbonate Lactate Creatinine Ketones
Hypoglycaemia Sweaty, drowsy Low N N N Neg
Ketoacidosis Volume depletion High Low N N or up Pos
Hyperventilating
Hyperosmolar coma Volume depletion Very high N or slightly low N or up N or up Neg
May be hyperventilating
Lactic acidosis Hyperventilating Variable Low Up N Neg
Uraemia Hyperventilating Variable Low N or up Up Neg
Cerebrovascular accident Neurological May be raised May be low N N Neg
N, normal; Neg, negative; Pos, positive.
192 Carbohydrate metabolism
ketones are greater than 6 mmol/L and the treatment Hyperosmolal non-ketotic coma
aim is for these to be less than 0.30 mmol/L. The treatment of HONK coma is similar to that of
Urinary volume should be monitored; if it fails to ketoacidosis. A sudden reduction of extracellular
rise despite adequate rehydration, further fluid and osmolality may be harmful, and it is important to
potassium should be given only if clinically indicated, give small doses of insulin to reduce plasma glucose
and then with care. The risk of deep vein thrombosis is concentrations slowly, for example 1 U/h. These patients
increased, in part due to dehydration, and thus heparin are often very sensitive to the action of insulin. Hypo-
5000 U every 8 h subcutaneously can be given. osmolal solutions are often used to correct volume
Clinical conditions such as infection that may have depletion, but these too should be given slowly. Heparin
precipitated the coma should be sought and treated. is usually given, as there is an increased risk of venous
Frequent monitoring of plasma glucose, potassium and thrombosis.
sodium concentrations is essential to assess progress and
to detect developing hypoglycaemia, hypokalaemia or
hypernatraemia. Acid–base balance should also be assessed. Initial investigation of a diabetic patient presenting
in coma
A diabetic patient may be in coma due to hyperglycaemia,
CASE 3 hypoglycaemia or any of the causes shown in Tables
12.4 and 12.5. After a thorough clinical assessment,
A 77-year-old man with known type 2 diabetes proceed as follows:
mellitus presented to the casualty department feeling
drowsy. His home blood glucose monitoring had ● Notify the laboratory that specimens are being taken
recently averaged about 25 mmol/L and a recent and ensure that they are delivered promptly. This
glycated haemoglobin (HbA1c) result obtained by his minimizes delays.
general practitioner was 12 per cent (108 mmol/mol). ● Take blood immediately for estimation of:
The following blood results were returned in hospital: – glucose,
Plasma – sodium and potassium,
Sodium 160 mmol/L (135–145) – urea and creatinine,
Potassium 5.0 mmol/L (3.5–5.0) – bicarbonate,
Bicarbonate 21 mmol/L (24–32) – arterial blood gases.
Urea 15 mmol/L (2.5–7.0) ● Do a drug screen for aspirin and paracetamol if
Creatinine 130 µmol/L (70–110) concomitant drug overdose suspected.
Glucose 65 mmol/L (5.5–11.1) ● Determination of plasma lactate will help diagnose a
Osmolality 380 mmol/kg (285–295) lactic acidosis (see Chapter 4).
pH 7.38 (7.35–7.45) ● Test a urine sample or blood for ketones.
PaCO2 5.2 kPa (4.6–6.0) ● A rapid assessment of blood glucose concentration
PaO2 11.8 kPa (9.3–13.3) may be obtained using a point-of-care (POCT)
Urine was negative for ketones. device, but results may be dangerously wrong so
these should always be checked against the results
DISCUSSION
obtained from the laboratory (see Chapter 30).
The patient was found to be in a hyperosmolal non-
● If severe hypoglycaemia is suspected on clinical
ketotic (HONK) diabetic coma. Note the severe
grounds or because of the results obtained using
hyperglycaemia, hypernatraemia and high plasma
reagent strips, glucose should be given immediately
osmolality and presentation in an elderly patient.
while waiting for the laboratory results. It is
HONK coma is associated with type 2 diabetes
less dangerous to give glucose to a patient with
mellitus. Ketoacidosis is usually absent, as there has
hyperglycaemia than to give insulin to a patient with
been no conversion to ketone metabolism. This is
hypoglycaemia.
more common in the elderly, and severe dehydration
● The results of point-of-care testing (see Chapter 30)
is present and there is an increased risk of thrombotic
must be interpreted with caution.
events and focal neurological signs. Treatment is with
● Also look for precipitating causes such as acute
careful intravenous rehydration, insulin and heparin.
myocardial infarction or infection.
Hyperglycaemia and diabetes mellitus 193
In most cases a diagnosis can be established from either Before starting this test, contact your laboratory: local
fasting or random blood glucose determinations. In details may vary.
equivocal cases an OGTT may be required. Procedure
The patient should be resting and should not smoke
Initial investigations during the test.
Blood for plasma glucose estimation should be taken if The patient fasts overnight (for at least 10 h but
a patient presents with symptoms of diabetes mellitus not more than 16 h). Water, but no other beverage, is
or glycosuria or if it is desirable to exclude the diagnosis, allowed.
for example because of a strong family history. A venous sample is withdrawn for plasma glucose
Blood samples may be taken: estimation. If the glucose concentration is measured
in whole blood, the results will be approximately
● at least 10 h after a fast, 1.0 mmol/L lower.
● at random, A solution containing 75 g of anhydrous glucose
● as part of an oral glucose load test. in 300 mL of water is hyperosmolar, and not only
may cause nausea and occasionally vomiting and
Diabetes mellitus is confirmed if one of the following
diarrhoea, but also, because of delayed absorption,
is present:
may affect the results of the test. It is therefore more
● a fasting venous plasma concentration of 7.0 mmol/L usual to give a solution of a mixture of glucose and
or more on two occasions or once with symptoms, its oligosaccharides, because fewer molecules per unit
● a random venous plasma concentration of volume have less osmotic effect than the equivalent
11.1 mmol/L or more on two occasions or once with amount of monosaccharide; the oligosaccharides are
symptoms. all hydrolysed at the brush border, and the glucose
immediately enters the cells.
Diabetes mellitus is unlikely if the fasting venous A solution that contains the equivalent of 75 g of
plasma glucose concentration is less than 5.5 mmol/L anhydrous glucose is: 113 mL of Polycal made up to
on two occasions. Samples taken at random times after approximately 300 mL with water.
meals are less reliable for excluding than for confirming This solution should be drunk slowly over a few
the diagnosis. minutes. Further blood is taken 2 h after the ingestion
The indications for performing an OGTT to diagnose of glucose.
diabetes mellitus may include: Note that in the investigation of acromegaly, sampling
is half-hourly over the 2-h period (see Chapter 7).
● fasting venous plasma glucose concentration
Interpretation of the OGTT is shown in Table
between 5.5 mmol/L and less than 7.0 mmol/L
12.6. There is controversy as to how best to interpret
– this is debatable as the WHO recommends an
the OGTT in pregnancy because of the differences in
OGTT only if fasting plasma glucose is greater than
maternal glucose metabolism, as stated earlier.
6.0 mmol/L,
The following factors may affect the result of the test:
● random venous plasma concentration between
7.0 mmol/L and less than 11.1 mmol/L, ● Previous diet No special restrictions are necessary if
● a high index of clinical suspicion of diabetes mellitus, the patient has been on a normal diet for 3–4 days.
such as a patient at high risk of gestational diabetes However, if the test is performed after a period of
with equivocal blood glucose results. carbohydrate restriction, for example as part of a
weight-reducing diet, this may cause abnormal glucose
The OGTT is sometimes also useful in the diagnosis tolerance, probably because metabolism is adjusted to
of acromegaly (see Chapter 7). the ‘fasted state’ and so favours gluconeogenesis.
It has been suggested that an HbA1c of greater than ● Time of day Most OGTTs are performed in the
6.5 per cent is diagnostic of diabetes mellitus, but morning and the reference values quoted are for this
this is not universally agreed as other factors such as time of day. There is evidence that tests performed
haemoglobin variants and abnormal erythrocyte in the afternoon yield higher plasma glucose
lifespan may affect HbA1c levels. concentrations and that the accepted ‘reference
194 Carbohydrate metabolism
Table 12.6 Interpretation of the oral glucose tolerance test (glucose mmol/L); venous plasma preferred
values’ may not be applicable. This may be due to a Symptoms of hypoglycaemia may develop at higher
circadian variation in islet cell responsiveness. concentrations if there has been a rapid fall from a
● Drug Steroids, oral contraceptives and thiazide previously raised value, when adrenaline secretion is
diuretics may impair glucose tolerance. stimulated and may cause sweating, tachycardia and
agitation. As discussed earlier, cerebral metabolism
HYPOGLYCAEMIA (FIG. 12.9) depends on an adequate supply of glucose from ECF,
By definition, hypoglycaemia is present if the plasma and the symptoms of hypoglycaemia may resemble
glucose concentration is less than 2.5 mmol/L in a those of cerebral hypoxia (neuroglycopenia). Faintness,
specimen collected into a tube containing an inhibitor dizziness or lethargy may progress rapidly to coma and,
of glycolysis, for example fluoride oxalate. Blood if untreated, permanent cerebral damage or death may
cells continue to metabolize glucose in vitro, and low occur. Existing cerebral or cerebrovascular disease may
concentrations found in a specimen collected without aggravate the clinical picture. Whipple’s triad is defined
such an inhibitor can be dangerously misleading as hypoglycaemia, neuroglycopenic symptoms, and
(pseudohypoglycaemia). relief of these symptoms on raising the blood glucose.
Unexplained hypoglycaemia
Related to meals?
No Yes
and high C-peptide can be seen in sulphonylurea or glucose concentration less than 2.5 mmol/L during
meglitinide administration, and a urine or plasma drug an overnight (18-h) fast when assayed on three
screen is thus important. separate occasions.
Autoantibodies positive to the insulin receptor or ● Exercise test Exercise may be used in the induction of
insulin may also evoke hypoglycaemia, such as AIS. If a insulin-induced hypoglycaemia. Blood is collected at
sulphonylurea drug screen and an insulin autoantibody 10-min intervals during 30 min of intense exercise,
screen are negative, raised plasma insulin and C-peptide for example treadmill, and 30 min after stopping.
concentrations are suggestive of an insulinoma. Plasma insulin and C-peptide (and proinsulin, if
Some tumours release proinsulin, which can also be necessary) are assayed and will be inappropriately
assayed. Imaging will also be necessary, such as magnetic high in endogenous hyperinsulinaemia and
resonance imaging or computerized tomography suppressed appropriately in hypoinsulinaemic
scanning, to localize the tumour and to help exclude hypoglycaemia.
MEN syndrome (see Chapter 24). If both the plasma ● Prolonged fast The prolonged fast, of up to 4 h, may
insulin and C-peptide concentrations are suppressed, be reserved for cases where there is a high index
the hypoglycaemia (hypoinsulinaemic hypoglycaemia) of suspicion of hypoglycaemia that has not been
can then be divided into non-ketotic and ketotic forms. provoked spontaneously or by the above tests. It
Hypoglycaemia with low plasma ketone concentrations, should be noted that, during the prolonged fast,
that is, b-hydroxybutyrate less than 600 µmol/L, is the patient must be under close supervision as
suggestive of increased insulin or IGF-1 activity such potentially dangerous.
as may occur in non-islet cell tumour hypoglycaemia
Blood should be taken every 6 h for plasma glucose
(NICTH). In NICTH, there is often increased IGF-2
and insulin estimations and, if symptoms occur, should
secretion. Some spindle cell tumours, for example of
be assayed for glucose immediately. The test can be
the thorax, may release a variant ‘big-IGF-2’ resulting
stopped if hypoglycaemia is demonstrated. The patient
in hypoglycaemia (Doege–Potter syndrome).
should be fed after the test. If prolonged fasting does not
Hypoinsulinaemic hypoglycaemia with
induce hypoglycaemia, endogenous hyperinsulinism
hypoketonaemia can also be seen in hepatic failure or
is unlikely to be the cause of the symptoms. A few
renal disease. Therefore liver and renal function should be
normal individuals may show plasma glucose levels
checked. Conversely, hypoinsulinaemic hypoglycaemia
less than 2.5 mmol/L during the test, but they do not
with high plasma ketones, that is, b-hydroxybutyrate
exhibit neuroglycopenic symptoms or Whipple’s triad.
more than 600 µmol/L, can be found in hypopituitarism
A plasma insulin to C-peptide ratio of less than 1 has
(see Chapter 7) when the plasma GH concentration is
about 89 per cent sensitivity and 100 per cent specificity
usually low. In addition, consider adrenal insufficiency
for insulinoma.
(see Chapter 8) and hypothyroidism (see Chapter 11),
The insulin provocation test is now rarely used (as
in which plasma GH is usually high. High alcohol intake
it can evoke dangerous hypoglycaemia) – in it, insulin
can also present with hypoinsulinaemic hypoglycaemia
administration fails to suppress plasma insulin and
and raised ketone concentrations.
C-peptide in cases of insulinoma. Reactive hypoglycaemia
However, more commonly, the patient has been
should not be diagnosed by a prolonged 75 g OGTT,
referred for investigation of previously documented
as normal individuals may show false-positive results.
hypoglycaemia or with a history strongly suggestive
However, the mixed meal, based on the sort of foods that
of hypoglycaemic attacks. It is also essential to exclude
evoke the attacks, can be used to investigate post-prandial
adrenal insufficiency, especially in diabetes mellitus
neuroglycopenic symptoms. Capillary blood samples are
patients with unexplained hypoglycaemia or reduced
taken prior to and every half-hour for 6 h after mixed-meal
insulin requirements. If no cause is identified at this
ingestion. Reactive hypoglycaemia is a possible diagnosis
point, it may be possible to induce hypoglycaemia by
if the patient develops neuroglycopenic symptoms and
provocation tests, although these are not without the
the capillary plasma glucose concentration is 3.0 mmol/L
risk of severe hypoglycaemic episodes.
or less. Note that venous plasma should ideally not be
Such tests could be as follows:
used, as false-positive results may result because post-
● Overnight fast A majority of patients with prandial glucose concentrations can be 1–2 mmol/L
spontaneous hypoglycaemia manifest one plasma lower than in capillary blood samples.
198 Carbohydrate metabolism
SUMMARY
● Diabetes mellitus is a common medical condition, its complications, such as in the control of blood
and an understanding of its biochemistry aids its glucose, HbA1c, plasma lipids and urinary ACR.
medical management. Type 1 diabetes mellitus is ● Diabetes mellitus can present with various comas,
associated with insulin deficiency and may present including hypoglycaemia, diabetic ketoacidosis
with weight loss and urinary ketones in young (type 1), HONK and lactic acidosis.
individuals. There is a relationship with autoimmune ● Hypoglycaemia can present with neurological
disease. Treatment is with insulin. Conversely, impairment and coma. A useful classification is to
type 2 diabetes mellitus is usually associated with divide hypoglycaemia into that with high plasma
insulin resistance, increased body weight and later insulin and that with low insulin levels. The causes
age presentation. There may be a family history of hyperinsulinaemic hypoglycaemia include
of diabetes mellitus. Treatment involves diet and insulinomas and following insulin administration.
biguanides, sulphonylureas, glitazones or incretins, The causes of hypoinsulinaemic hypoglycaemia
although insulin may sometimes be needed. include severe hepatic disease, adrenal insufficiency,
● Biochemical tests have a major role in the pituitary failure and non-pancreatic tumours
management of diabetes mellitus and in monitoring producing insulin-like substances.
13 Plasma lipids and lipoproteins
CH3(CH2)nCOO–
FATTY ACID
H3C H3C
CH3 CH3
CH3 CH3
HO CH3(CH2)nCOO
O O
1CH 1CH
O 2 O C R1 O 2 O C R1
2CH 2CH
R2 C O O R2 C O O
3CH O C R2 3CH O P O N
2 2
O–
TRIGLYCERIDE PHOSPHOLIPID
Table 13.1 Some of the major fatty acids found in the Carbohydrate Protein
plasma Fat
Two-carbon units rich and, because of their large size, they scatter light,
which can give plasma a turbid appearance (lipaemic)
if present in high concentrations:
Acetoacetyl CoA
● Chylomicrons are the largest and least dense
lipoproteins and transport exogenous lipid from the
3-hydroxy-3-methylglutaryl CoA intestine to all cells.
(HMG-CoA)
● Very low-density lipoproteins (VLDLs) transport
HMG-CoA REDUCTASE
endogenous lipid from the liver to cells.
● Intermediate-density lipoproteins (IDLs), which
are transient and formed during the conversion of
Mevalonic acid
VLDL to low-density lipoprotein (LDL), are not
normally present in plasma.
Isoprenoids
The other two lipoprotein classes contain mainly
cholesterol and are smaller in size:
Squalene
● Low-density lipoproteins are formed from VLDLs
and carry cholesterol to cells.
Lanosterol ● High-density lipoproteins (HDLs) are the most
dense lipoproteins and are involved in the transport
of cholesterol from cells back to the liver (reverse
Cholesterol cholesterol transport). These lipoproteins can be
Figure 13.4 Summary of pathways of cholesterol further divided by density into HDL2 and HDL3.
synthesis. CoA, coenzyme A. Reproduced with kind
If a lipaemic plasma sample, for example after a
permission from Candlish JK and Crook M. Notes on
Clinical Biochemistry. Singapore: World Scientific meal, is left overnight at 4°C, the larger and less dense
Publishing, 1993. chylomicrons form a creamy layer on the surface. The
smaller and denser VLDL and IDL particles do not rise,
and the sample may appear diffusely turbid. The LDL
soluble protein complexes called lipoproteins. Lipids and HDL particles do not contribute to this turbidity
can be derived from food (exogenous) or synthesized because they are small and do not scatter light. Fasting
in the body (endogenous). The water-soluble (polar) plasma from normal individuals contains only VLDL,
groups of proteins, phospholipids and free cholesterol LDL and HDL particles.
face outwards and surround an inner insoluble (non- In some cases of hyperlipidaemia, the lipoprotein
polar) core of triglyceride and cholesterol esters. patterns have been classified (Fredrickson’s
Lipoproteins are classified by their buoyant density, classification) according to their electrophoretic
which inversely reflects their size. The greater the lipid mobility. Four principal bands are formed, based on
to protein ratio, the larger their size and the lower the their relative positions, by protein electrophoresis,
density. Lipoproteins can be classified into five main namely a (HDL), pre-b (VLDL), b (LDL) and
groups (Table 13.2). The first three are triglyceride chylomicrons (Table 13.3).
Table 13.3 Fredrickson’s classification of another lipoprotein called lipoprotein (a), or Lp(a),
hyperlipidaemias has been found. This is similar in lipid composition
to LDL but has a higher protein content. One of its
Type Electrophoretic Increased lipoprotein
proteins, called apolipoprotein (a), shows homology to
I Increased chylomicrons Chylomicrons
plasminogen and may disrupt fibrinolysis, thus evoking
IIa Increased b-lipoproteins LDL a thrombotic tendency. The plasma concentration of
IIb Increased b and pre-b-lipoproteins LDL and VLDL Lp(a) is normally less than 0.30 g/L and it is thought to
III Broad b-lipoproteins IDL be an independent cardiovascular risk factor.
IV Increased pre-b-lipoproteins VLDL The proteins associated with lipoproteins are called
V Increased chylomicrons and pre-b- Chylomicrons and VLDL apolipoproteins (apo). ApoA (mainly apoA1 and apoA2)
lipoproteins is the major group associated with HDL particles. The
IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein;
apoB series (apoB100) is predominantly found with
VLDL, very low-density lipoprotein. LDL particles and is the ligand for the LDL receptor.
Low-density lipoprotein has one molecule of apoB100
per particle. Some reports have suggested that the
Intermediate-density lipoproteins in excess may plasma apoA1 to apoB ratio may be a useful measure of
produce a broad b-band. Some individuals with cardiovascular risk (increased if the ratio is less than 1)
hyperlipidaemia may show varying electrophoretic and it is not significantly influenced by the fasting status
patterns at different times. of the patient. The apoC series is particularly important
Ultracentrifugation (separation based upon particle in triglyceride metabolism and, with the apoE series,
buoyant density) or electrophoretic techniques freely interchanges between various lipoproteins. Some
are rarely used in routine clinical practice as these of the functions of these apolipoproteins are described
may require completed apparatus and experienced in Table 13.4.
operators. Instead, the lipoprotein composition Lipoprotein-associated phospholipase A2 [also
of plasma may be inferred from standard clinical called platelet-activating factor acetylhydrolase (PAF-
laboratory lipid assays. As fasting plasma does not AH)] is present mainly on LDL and to a lesser degree
normally contain chylomicrons, the triglyceride HDL. It is produced by inflammatory cells and is
content reflects VLDL. Furthermore, generally about involved in atherosclerosis formation and levels are
70 per cent of plasma cholesterol is incorporated as associated with increased risk of coronary artery
LDL and 20 per cent as HDL. The latter particles, disease and stroke.
because of their high density, can be quantified
by precipitation techniques that can assay their
cholesterol content by subtraction, although direct
Table 13.4 The main apolipoproteins and their
HDL assays are now often used.
common functions
The Friedewald equation enables plasma LDL
cholesterol concentration to be calculated and is often Apolipoprotein Associated lipoprotein Function
used in clinical laboratories: A1 Chylomicrons and HDL LCAT activator
LDL cholesterol A2 Chylomicrons and HDL LCAT activator
= total cholesterol – HDL cholesterol B48 Chylomicrons and VLDL Secretion of
[triglyceride] chylomicrons/VLDL
–
2.2 (13.1) B100 IDL, VLDL, LDL LDL receptor binding
This equation makes certain assumptions, namely C2 Chylomicrons, HDL, VLDL, IDL Lipoprotein lipase
activator
that the patient is fasting and the plasma triglyceride
C3 Chylomicrons, HDL, VLDL, IDL Lipoprotein lipase
concentration does not exceed 4.5 mmol/L (otherwise
inhibitor
chylomicrons make the equation inaccurate).
E Chylomicrons, HDL, VLDL, IDL IDL and remnant
There has been recent interest in the subdivision particle receptor binding
of LDL particles into small dense LDL2 and LDL3,
HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein;
which appear to be more atherogenic and more easily LCAT, lecithin–cholesterol acyltransferase; LDL, low-density
oxidized than the larger LDL1 particles. Additionally, lipoprotein; VLDL, very low-density lipoprotein.
Two-carbon units rich and, because of their large size, they scatter light,
which can give plasma a turbid appearance (lipaemic)
if present in high concentrations:
Acetoacetyl CoA
● Chylomicrons are the largest and least dense
lipoproteins and transport exogenous lipid from the
3-hydroxy-3-methylglutaryl CoA intestine to all cells.
(HMG-CoA)
● Very low-density lipoproteins (VLDLs) transport
HMG-CoA REDUCTASE
endogenous lipid from the liver to cells.
● Intermediate-density lipoproteins (IDLs), which
are transient and formed during the conversion of
Mevalonic acid
VLDL to low-density lipoprotein (LDL), are not
normally present in plasma.
Isoprenoids
The other two lipoprotein classes contain mainly
cholesterol and are smaller in size:
Squalene
● Low-density lipoproteins are formed from VLDLs
and carry cholesterol to cells.
Lanosterol ● High-density lipoproteins (HDLs) are the most
dense lipoproteins and are involved in the transport
of cholesterol from cells back to the liver (reverse
Cholesterol cholesterol transport). These lipoproteins can be
Figure 13.4 Summary of pathways of cholesterol further divided by density into HDL2 and HDL3.
synthesis. CoA, coenzyme A. Reproduced with kind
If a lipaemic plasma sample, for example after a
permission from Candlish JK and Crook M. Notes on
Clinical Biochemistry. Singapore: World Scientific meal, is left overnight at 4°C, the larger and less dense
Publishing, 1993. chylomicrons form a creamy layer on the surface. The
smaller and denser VLDL and IDL particles do not rise,
and the sample may appear diffusely turbid. The LDL
soluble protein complexes called lipoproteins. Lipids and HDL particles do not contribute to this turbidity
can be derived from food (exogenous) or synthesized because they are small and do not scatter light. Fasting
in the body (endogenous). The water-soluble (polar) plasma from normal individuals contains only VLDL,
groups of proteins, phospholipids and free cholesterol LDL and HDL particles.
face outwards and surround an inner insoluble (non- In some cases of hyperlipidaemia, the lipoprotein
polar) core of triglyceride and cholesterol esters. patterns have been classified (Fredrickson’s
Lipoproteins are classified by their buoyant density, classification) according to their electrophoretic
which inversely reflects their size. The greater the lipid mobility. Four principal bands are formed, based on
to protein ratio, the larger their size and the lower the their relative positions, by protein electrophoresis,
density. Lipoproteins can be classified into five main namely a (HDL), pre-b (VLDL), b (LDL) and
groups (Table 13.2). The first three are triglyceride chylomicrons (Table 13.3).
Triglyceride
LDL
VLDL
A
E B
HDL
E
C
IDL
NEFA
E B E B
VLDL
VLDL Apolipoprotein
Capillary
C C Triglyceride
wall
Cholesterol
Lipoprotein
lipase
Figure 13.6 Endogenous lipid pathways. HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein;
LDL, low-density lipoprotein; NEFA, non-esterified (free) fatty acid; VLDL, very low-density lipoprotein.
from chylomicron remnants via the exogenous pathway binds apoB100. Within the cell, the LDL particles are
or synthesized locally. These lipids are transported from broken down by lysosomes, releasing cholesterol. This
the liver as VLDL. cholesterol can be incorporated into cell membranes or
Very low-density lipoprotein is a large triglyceride- in specific tissues such as the adrenal cortex or gonads
rich particle consisting also of apoB100, apoC and apoE. and utilized in steroid synthesis.
Following hepatic secretion, it incorporates additional Most cells are able to synthesize cholesterol, but, to
apoC from HDL particles within the circulation. Like avoid intracellular accumulation, there is a feedback
chylomicrons, VLDL is hydrolysed by lipoprotein control system reducing the rate of synthesis of the
lipase in the peripheral tissues, albeit more slowly. The LDL receptors. Although most of the plasma LDL is
resulting VLDL remnant or IDL contains cholesterol removed by LDL receptors, if the plasma cholesterol
and triglyceride as well as apoB and apoE and is rapidly concentration is excessive, LDL particles, by virtue
taken up by the liver or converted by the action of of their small size, can infiltrate tissues by passive
hepatic lipase to LDL by losing apoE and triglyceride. diffusion and can even cause damage, as in atheroma
Low-density lipoprotein is a small cholesterol-rich formation within arterial walls. An alternative route of
lipoprotein containing only apoB. It represents about removal of LDL is via the reticuloendothelial system,
70 per cent of the total plasma cholesterol concentration. collectively termed the scavenger cell pathway, which
It can be taken up by most cells, although mainly the recognizes only chemically modified LDL, for example
liver by the LDL or B/E receptor which recognizes and oxidized LDL.
206 Plasma lipids and lipoproteins
The liver has a central role in cholesterol metabolism: saturated fat intake can also suppress LDL receptor
activity and is a bigger driver of cholesterol metabolism
● it contains most of the LDL receptors,
than dietary cholesterol. The richest dietary sources of
● it is responsible for most of the endogenous
cholesterol are egg yolks, dairy products and red meat.
cholesterol synthesis,
Net loss of body cholesterol can occur by bile
● it takes up cholesterol from the diet via lipoproteins,
excretion. Some bile salts are reabsorbed from the
● it can excrete cholesterol from the body in bile.
intestinal lumen and return to the liver via the
Cholesterol is synthesized via a series of enzymatic enterohepatic circulation. Interruption of this process
steps, with HMG-CoA reductase being the rate-limiting results in enhanced conversion of cholesterol to bile
enzyme (Fig. 13.4). Suppression of this enzyme may salts, reduced hepatic cholesterol stores and increased
occur if cholesterol synthesis is excessive. Involved in LDL receptors (Fig. 13.7). The rate of LDL receptor
these processes is a family of transcription-regulating synthesis is also increased by thyroxine and oestrogens
proteins called sterol regulatory element-binding and decreases with age.
proteins. Intracellular cholesterol accumulation also
reduces the number of hepatic LDL receptors, and High-density lipoprotein
therefore LDL entry into cells declines and the plasma The transport of cholesterol from non-hepatic cells
concentration rises. However, if the dietary intake of to the liver involves HDL particles, in a process called
cholesterol is excessive, intracellular accumulation can reverse cholesterol transport (Fig. 13.8). The HDL is
still occur. About 30–60 per cent of the dietary intake synthesized in both hepatic and intestinal cells and
of cholesterol (of 1–2 mmol) is absorbed, this amount secreted from them as small, nascent HDL particles
being increased if the diet is rich in saturated fat. High rich in free cholesterol, phospholipids, apoA and
Acetyl CoA
Nucleus
HMG-CoA
HMG-CoA reductase
Amino acids
Mevalonic acid –
Lysosomal CHOLESTEROL
degradation
+
ACAT
–
CHOLESTEROL
ESTERS
LDL receptor
LDL
Figure 13.7 The low-density lipoprotein (LDL) receptor. ACAT, acyl coenzyme A acyl transferase; CoA, coenzyme
A; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A.
Disorders of lipid metabolism 207
Chylomicron syndrome
This can be due to familial lipoprotein lipase deficiency,
an autosomal recessive disorder affecting about 1 in
1 000 000 people. The gene for lipoprotein lipase is found
on chromosome 8, and genetic studies have shown
insertions or deletions within the gene. Lipoprotein
lipase is involved in the exogenous lipoprotein pathway
by hydrolysing chylomicrons to form chylomicron
remnants, and also in the endogenous pathway by
converting VLDL to IDL particles.
Presentation as a child with abdominal pain (often
with acute pancreatitis) is typical. There is probably no
increased risk of coronary artery disease. Gross elevation
of plasma triglycerides due to the accumulation Figure 13.10 Lipaemia retinalis in a patient with
of uncleared chylomicron particles occurs (Fig. lipoprotein lipase deficiency. Reproduced with kind
13.9). Lipid stigmata include eruptive xanthomata, permission from Nyhan WL and Barshop BA. Atlas of
hepatosplenomegaly and lipaemia retinalis (Fig. 13.10). Inherited Metabolic Diseases, 3rd edition. London:
Other variants of the chylomicron syndrome Hodder Arnold, 2012.
include circulating inhibitors of lipoprotein lipase
and deficiency of its physiological activator apoC2.
Apolipoprotein C2 deficiency is also inherited as an Treatment of the chylomicron syndrome involves
autosomal recessive condition affecting about 1 in a low-fat diet, aiming for less than 20 g of fat a day, if
1 000 000 people. The gene for apoC2 is located on possible, although compliance on such a diet may be
chromosome 19 and mutations resulting in low plasma difficult. Some clinicians supplement the diet with
concentrations have been found. medium-chain triglycerides and also give 1 per cent of
the total calorie intake as linoleic acid.
In cases of apoC2 deficiency, fresh plasma may
temporarily restore plasma apoC2 levels. To confirm
the diagnosis of familial lipoprotein lipase deficiency,
plasma lipoprotein lipase can be assayed after the
intravenous administration of heparin, which releases
the enzyme from endothelial sites. The assay is
complicated in that other plasma lipases (hepatic lipase
and phospholipase, for example) contribute to the
overall plasma lipase activity. Inhibition of lipoprotein
lipase can be performed using protamine, high saline
concentrations or specific antibodies and its overall
activity can be calculated by subtraction.
If apoC2 deficiency is suspected, the plasma
concentrations of this activator can be assayed. Patients
may show a type I or type V Fredrickson’s phenotype.
Family members should be investigated.
Familial hypercholesterolaemia
This condition is usually inherited as an autosomal
Figure 13.9 Whole blood of a patient with lipoprotein
lipase deficiency. Note chylomicron creamy
dominant trait and was described by Goldstein and
appearance. Reproduced with kind permission Brown. The inheritance of one mutant gene that
from Nyhan WL and Barshop BA. Atlas of Inherited encodes for the LDL receptor affects about 1 in every
Metabolic Diseases, 3rd edition. London: Hodder 500 people (more common in certain groups such
Arnold, 2012. as Afrikaners and French Canadians), resulting in
Disorders of lipid metabolism 209
impaired LDL catabolism and hypercholesterolaemia. relative or below the age of 60 years in a second-degree
At least five types of mutation of the LDL receptor relative.
have been described, resulting in reduced synthesis, Typically, patients manifest severe hypercholes-
failure of transport of the synthesized receptor to the terolaemia, with a relatively normal plasma triglyceride
Golgi complex within the cell, defective LDL binding or concentration in conjunction with xanthomata, which
inadequate expression or defective recycling of the LDL can affect the back of the hands, elbows, Achilles tendons
receptor at the cell surface. or the insertion of the patellar tendon into the pretibial
According to the Simon Broome register, definite tuberosity (Fig. 13.11). Premature cardiovascular
familial hypercholesterolaemia (FH) is defined as disease is often observed, along with premature corneal
a plasma cholesterol concentration of more than arci (Fig. 13.12).
7.5 mmol/L in an adult (more than 6.7 mmol/L in Using the Fredrickson’s classification, this
children under 16 years) or a plasma LDL cholesterol condition has also been termed familial type IIa
concentration of more than 4.9 mmol/L in an adult
in the presence of tendon xanthoma. Possible FH is
defined as a plasma cholesterol concentration of more
than 7.5 mmol/L in an adult (more than 6.7 mmol/L in
children under 16 years) or a plasma LDL cholesterol
concentration of more than 4.9 mmol/L in an adult,
plus a family history of either an elevated plasma
cholesterol concentration of more than 7.5 mmol/L in
a first-degree or second-degree relative or myocardial
infarction below the age of 50 years in a first-degree
CASE 1
A 23-year-old woman had her plasma lipids checked
by her general practitioner because her father had
Figure 13.11 Tendinous xanthomas in familial
died of a myocardial infarction aged 44 years. Her
hypercholesterolaemia. Reproduced with kind
24-year-old brother had hyperlipidaemia. Her renal, permission from Nyhan WL and Barshop BA. Atlas of
liver and thyroid function tests were normal, as was Inherited Metabolic Diseases, 3rd edition. London:
her blood glucose. Hodder Arnold, 2012.
Plasma (fasting)
Cholesterol 11.4 mmol/L (3.5–5.0)
Triglyceride 1.1 mmol/L (0.3–1.5)
HDL cholesterol 1.2 mmol/L (1.0–1.8)
On examination, she had tendon xanthomata on her
Achilles tendons and bilateral corneal arci.
DISCUSSION
Note the considerably raised plasma cholesterol
concentration. The absence of an obvious secondary
hyperlipidaemia, in conjunction with the family
history of a first-degree relative with premature
cardiovascular disease and hyperlipidaemia, suggests
a genetic hyperlipidaemia. The presence of tendon
xanthomata and premature corneal arci supports the Figure 13.12 Corneal arcus in familial
diagnosis of familial hypercholesterolaemia. This is hypercholesterolaemia. Reproduced with kind
usually an autosomal dominant disorder and usually permission from Nyhan WL and Barshop BA. Atlas of
Inherited Metabolic Diseases, 3rd edition. London:
a defect of the low-density lipoprotein (LDL) receptor.
Hodder Arnold, 2012.
210 Plasma lipids and lipoproteins
underlying biochemical defect is one of a reduced type III hyperlipoproteinaemia. A concurrent increase
clearance of chylomicron and VLDL remnants. The in plasma VLDL concentration also seems necessary
name broad b-hyperlipidaemia is sometimes used for the condition to be expressed, such as might occur
because of the characteristic plasma lipoprotein in diabetes mellitus, hypothyroidism or obesity. Some
electrophoretic pattern that is often observed (the patients may show either an autosomal recessive or a
broad b-band that is seen being remnant particles). dominant mode of inheritance of the condition.
An association with type III/broad b-hyperlipidaemia The palmar striae (palmar xanthomata) are
and homozygosity for apoE2 or variants of apoE2 has considered pathognomonic for the disorder, but
been described. Apolipoprotein E shows three common tuberoeruptive xanthomata, typically on the elbows and
alleles, E2, E3 and E4, coded for on chromosome 19, knees, xanthelasma and corneal arcus have also been
which are important for the binding of remnant described in this condition. Peripheral vascular disease
particles to the remnant receptor. is a typical feature of this hyperlipidaemic disorder, as
The mechanism for the disorder seems to be that is premature coronary artery disease.
apoE2-bearing particles have poor binding to the Plasma lipid determination frequently reveals
apoB/E (remnant) receptor and thus are not effectively hypercholesterolaemia and hypertriglyceridaemia,
cleared from the circulation. often in similar molar proportions with plasma
It is becoming apparent that it is not just inheriting concentrations of around 9–10 mmol/L. Plasma HDL
the apoE2 genotype that is important in developing cholesterol concentration is usually low. Plasma LDL
broad b-hyperlipidaemia. The prevalence of the apoE2/ concentration may also be low due to the fact that there
E2 genotype is about 1 in 100 in the general population, is reduced conversion from IDL particles, although it
yet only about 1 in 5000–10 000 individuals manifest may also be normal or elevated.
Plasma lipoprotein electrophoresis can show the
classic type III picture with a broad b-band composed
CASE 2 of remnant particles, although this is not always present.
A 43-year-old man attended the vascular surgery out- An association of type III hyperlipoproteinaemia with
patient clinic for peripheral vascular disease. He was homozygosity for apoE2 has been described, and thus
a non-smoker but had undergone a coronary artery apoE phenotyping or genotyping by a specialized
bypass graft the year before. Some of his laboratory laboratory can be useful, although some patients
results were as follows: with broad b-hyperlipidaemia can show other apoE
phenotypes or variants.
Plasma (fasting) Another investigation that can be useful in
Cholesterol 8.7 mmol/L (3.5–5.0) establishing the diagnosis is ultracentrifugation to
Triglyceride 9.1 mmol/L (0.3–1.5) separate the lipoprotein particles. The cholesterol of
HDL cholesterol 0.86 mmol/L (1.0–1.8) the VLDL particles is then quantified and expressed
His apolipoprotein E genotype was E2/E2 as a total of the plasma triglyceride concentration. In
On examination, he had tuberous xanthomata and molar terms, normal individuals show a ratio of less
palmar striae. than 0.30, while ratios more than 0.30 are more likely
in type III hyperlipoproteinaemia, particularly nearer
DISCUSSION
0.60.Treatment consists of dietary measures, correcting
The diagnosis was type III hyperlipoproteinaemia
the precipitating causes and either the statin or fibrate
(familial dysbetalipoproteinaemia or broad
drugs.
b-hyperlipidaemia). Note the mixed hyperlipidaemia
(both cholesterol and triglyceride concentrations Polygenic hypercholesterolaemia
raised) in an approximately 1:1 molar ratio and also This is one of the most common causes of a raised
apoE genotype E2 homozygote (usual is apoE3/E3). plasma cholesterol concentration. This condition is
The type III hyperlipoproteinaemia is associated the result of a complex interaction between multiple
with raised concentration of remnant lipoprotein environmental and genetic factors. In other words, it
particles, which are particularly atherogenic. Note is not due to a single gene abnormality, and it is likely
also the characteristic lipid stigmata and premature that it is the result of more than one metabolic defect.
peripheral vascular and coronary heart disease. There is usually either an increase in LDL production
212 Plasma lipids and lipoproteins
or a decrease in LDL catabolism. The plasma lipid of hyperlipidaemia include obesity, type 2 diabetes
phenotype is usually either IIa or IIb Fredrickson’s mellitus, hypothyroidism, chronic kidney disease,
phenotype. The plasma cholesterol concentration cholestasis and certain drugs. The reader should refer
is usually either mildly or moderately elevated. An to the other chapters in this book for details of the
important negative clinical finding is the absence of relevant diseases.
tendon xanthomata, the presence of which would tend
Other lipid abnormalities
to rule out the diagnosis. Usually less than 10 per cent of
first-degree relations have similar lipid abnormalities, Inherited disorders of low plasma HDL concentration
compared with FH or FCH in which about 50 per cent (hypoalphalipoproteinaemia) occur, and plasma HDL
of first-degree family members are affected. There may cholesterol concentration should ideally be more than
also be a family history of premature coronary artery 1.0 mmol/L. A number of such conditions have been
disease. Individuals may have a high intake of dietary described (such as apoA1 deficiency), many of which
fat and be overweight. Treatment involves dietary are associated with premature cardiovascular disease. In
intervention and sometimes the use of lipid-lowering Tangier’s disease, individuals have very low levels of HDL,
drugs such as the statins. large, yellow tonsils, hepatomegaly and accumulation
of cholesterol esters in the reticuloendothelial system.
Hyperalphalipoproteinaemia There is a defect in the ABC1 gene involved in HDL
Hyperalphalipoproteinaemia results in elevated transport. The causes of a low plasma HDL cholesterol
plasma HDL cholesterol concentration and can be concentration are shown in Box 13.3.
inherited as an autosomal dominant condition or, in Defects of apoB metabolism have also been
some cases, may show polygenic features. The total described. In abetalipoproteinaemia or LDL deficiency
plasma cholesterol concentration can be elevated, there is impaired chylomicron and VLDL synthesis.
with normal LDL cholesterol concentration. There is This results in a failure of lipid transport from the
no increased prevalence of cardiovascular disease in liver and intestine. Transport of fat-soluble vitamins is
this condition; in fact, the contrary probably applies, impaired and steatorrhoea, progressive ataxia, retinitis
with some individuals showing longevity. Plasma HDL
concentration is thought to be cardioprotective, and
individuals displaying this should be reassured. Box Box 13.2 Some important causes of
13.1 gives the causes of raised plasma HDL cholesterol secondary hyperlipidaemia
concentrations.
Predominant hypercholesterolaemia
Secondary hyperlipidaemias
Hypothyroidism
One should not forget that there are many secondary Nephrotic syndrome
causes of hyperlipidaemia. These may present Cholestasis, e.g. primary biliary cirrhosis
alone or sometimes concomitantly with a primary Acute intermittent porphyria
hyperlipidaemia. Some of the causes of secondary Anorexia nervosa/bulimia
hyperlipidaemia are listed in Box 13.2. Secondary causes Certain drugs or toxins, e.g. ciclosporin and
chlorinated hydrocarbons
Predominant hypertriglyceridaemia
Alcohol excess
Box 13.1 Some causes of raised plasma
Obesity
high-density lipoprotein (HDL) cholesterol Diabetes mellitus and metabolic syndrome
Certain drugs, e.g. estrogens, b-blockers (without
Primary intrinsic sympathomimetic activity), thiazide diuretics,
Hyperalphalipoproteinaemia acitretin, protease inhibitors, some neuroleptics and
Cholesterol ester transfer protein deficiency glucocorticoids
Secondary Chronic kidney disease
High ethanol intake Some glycogen storage diseases, e.g. von Gierke’s
Exercise type I
Certain drugs, e.g. estrogens, fibrates, nicotinic acid, Systemic lupus erythematosus
statins, phenytoin, rifampicin Paraproteinaemia
Disorders of lipid metabolism 213
The blood sample should be taken to the laboratory Alternatively, there may be a place for a fibrate drug if
and assayed promptly. The usual fasting lipid profile fasting plasma triglyceride concentrations are raised by
consists of plasma cholesterol, triglyceride and HDL more than 5 mmol/L, particularly if there is also a low
cholesterol concentrations. HDL cholesterol concentration and LDL cholesterol
When faced with a hyperlipidaemia, decide whether it concentration is not much raised. The ideal is to aim
is primary or secondary. A family history, clinical features for fasting plasma cholesterol concentration of about
and appropriate blood tests can be useful to help make 4.0 mmol/L, triglyceride concentration less than
this decision. Lipid stigmata such as tendon xanthomata 1.5 mmol/L and HDL cholesterol concentration more
or premature corneal arci may point to familial than 1.0 mmol/L.
hypercholesterolaemia, and tuberous xanthomata or Severe hypertriglyceridaemia, particularly if the
palmar striae to type III hyperlipoproteinaemia. plasma triglyceride concentration is more than
Blood glucose concentration is useful to help assess 10 mmol/L, is a risk factor for acute pancreatitis.
for diabetes mellitus, liver function tests for liver disease Low-fat diets may help in conjunction with a fibrate
such as cholestasis, urinary protein and plasma albumin (sometimes w-3 fatty acids are used), aiming ideally
concentrations for nephrotic syndrome and thyroid for a fasting plasma triglyceride concentration lower
function tests for hypothyroidism. It is also important than about 1.5 mmol/L. Remember that severe
to determine alcohol consumption and medications hypertriglyceridaemia can cause problems with
from the clinical history. certain assays, for example falsely low plasma amylase
It is generally wise to retest patients’ lipids, a few concentration or pseudohyponatraemia.
months apart, as it is recognized that the within- In terms of primary coronary heart disease
individual variation of lipids can be significant, and prevention, the use of a cardiovascular risk factor
reliance cannot be placed on just one set of readings. assessment may be helpful to determine if a lipid-
It is also useful to assess the patient for other lowering drug is indicated, for example Framingham
cardiovascular risk factors, including smoking habits, Study-based or QRISK cardiovascular risk calculators.
diabetes mellitus, blood pressure, body weight and Close family members should be screened in cases of
family history of cardiovascular disease. These should genetic hyperlipidaemias such as FH.
also be managed in their own right. Assessment should Specialist lipid assays may help define the abnormality.
also be made for possible atherosclerotic disease, for The apoE genotype is useful in the diagnosis of type
example does the patient have evidence of coronary, III hyperlipoproteinaemia, as many of these patients
peripheral or carotid artery disease? are apoE2/E2. Plasma lipoprotein lipase and apoC2
If the patient is known to have coronary artery (its activator) assays may be useful in chylomicron
disease, aim for a plasma LDL cholesterol concentration syndrome, and LDL receptor DNA studies for familial
of about 2.0 mmol/L. Statins are first-line drugs for hypercholesterolaemia. Plasma apoA1 and apoB
patients with raised LDL cholesterol concentration. concentrations and also Lp(a) may help define risk status.
SUMMARY
● Lipids are essential for health, but raised plasma hypothyroidism, chronic kidney disease and
cholesterol and triglyceride concentrations cholestasis.
are associated with an increased incidence of ● The genetic hyperlipidaemias include FH, FCH
cardiovascular disease. and type III hyperlipoproteinaemia. Familial
● Conversely, plasma HDL cholesterol is hypercholesterolaemia usually results from a defect
cardioprotective, partly because of its central role in of the LDL receptor.
reverse cholesterol transport returning cholesterol ● The statins or HMG-CoA reductase inhibitors
from the tissues to the liver. lower plasma cholesterol concentration and are
● There are many cases of secondary hyperlipidaemias, associated with decreased cardiovascular disease.
including obesity, alcohol excess, diabetes mellitus,
14 Nutrition
This chapter gives an outline of certain nutritional been depleted, energy is derived from triglyceride,
abnormalities and how they overlap with aspects of and later from body tissue components such as the
chemical pathology. It is not, however, a substitute for a proteins of cells, including those of muscle. This may
nutrition textbook. The reader may also find Chapters cause severe ketosis from the metabolism of fats and
12 and 13 (on carbohydrate and lipid disorders, ketogenic amino acids and increase nitrogen turnover
respectively), Chapter 15 (on vitamin/trace elements) and loss. The daily energy and nitrogen requirements
and Chapter 16 (on gastrointestinal function) relevant. are not constant, as we will now see.
Nutrition is an important topic, as about 1 billion of the
world’s population are overweight yet, ironically, at the STARVATION
same time approximately 1 billion are undernourished During starvation the body tries to maintain blood
or starving. glucose levels in the acute phase and in the longer term
Adequate nutrition is essential for a variety of to preserve body protein mass.
reasons, including optimal cardiovascular function, Hepatic glycogen stores are largely consumed
muscle strength, respiratory ventilation, protection within 24 h. The obligatory glucose requirements
from infection, wound healing and psychological well- of the brain, erythrocytes and other organs are met
being. by gluconeogenesis. During the first week or so of
The principles of carbohydrate and lipid metabolism acute starvation, up to 150 g of protein is utilized to
and gastrointestinal digestion and absorption all achieve this. Insulin levels decrease, resulting in amino
have important implications in the management of acid (mainly alanine) release from protein, glycogen
nutrition, and of intravenous (parenteral) nutrition conversion to glucose, and adipocyte fatty acid release for
in particular. These principles, including those of fluid energy needs. Plasma insulin concentration is reduced,
and electrolyte homeostasis, must be fully understood but concentrations of glucagon, glucocorticoids,
in order to manage patients receiving parenteral catecholamines and growth hormone are raised. Thus,
nutrition. blood glucose concentrations are generally maintained
Daily energy loss as heat is about 120 kJ (30 kcal) per despite starvation.
kilogram of body weight in a normal adult. In addition, In later starvation, ketone bodies replace glucose as
there is a daily protein turnover of about 3 g/kg body the predominant brain fuel (ketoadaptive phase) and
weight (about 0.5 g of nitrogen), of which about 0.15 g a metabolic acidosis may result. The ketone bodies
of nitrogen/kg body weight is excreted (1 g of nitrogen also inhibit muscle protein degradation and the flow
is derived from about 6.25 g of protein). These losses of alanine into the circulation. There is thus a decrease
are usually balanced by dietary intake of equivalent in urinary nitrogen excretion, a decline in hepatic
amounts of energy, as carbohydrate, fat and protein. gluconeogenesis and increased brain oxidation of
Glucose provides 4 kcal/g, and fat 9 kcal/g. ketone bodies while plasma amino acid concentrations
Excess energy is stored as glycogen and triglyceride. decrease. Plasma insulin is reduced, as are glucagon,
If expenditure exceeds intake, these energy stores are glucocorticoids, catecholamines and growth hormone.
drawn upon. In a well-nourished adult, enough energy Starvation results in initial rapid weight loss,
is stored as hepatic glycogen to last at least a day, and mostly due to protein breakdown and diuresis. The
therefore post-operative patients without complications latter is partly due to increased renal tubule urea load
do not need intravenous feeding. Once this store has and results in renal loss of calcium, phosphate and
Nutritional assessment 217
– Other plasma proteins or peptides that Kwashiorkor occurs in individuals with visceral
have been studied as potential markers of protein loss associated with impaired immunological
nutritional status include retinol-binding function, although other nutritional components
protein, insulin-like growth factor 1 (IGF-1) are satisfactory. Visceral proteins are decreased, but
and fibronectin, but these are rarely used. Pre- body weight, mid-arm circumference and triceps
albumin, now referred to as transthyretin, may skin-fold thickness are relatively normal. Conversely,
be a marker not so much of poor nutrition marasmus is generalized undernutrition with
but of adequate nutritional replacement when reduction in weight, mid-arm circumference and
plasma concentrations rise. skin-fold thickness, although visceral proteins are
– Urinary creatinine-to-height ratio can be a relatively normal.
measure of lean body mass, but this test has the Do not forget that patients can be undernourished
problem of requiring accurate urine collection. while in the hospital ward, particularly the elderly and
– 24-h urinary urea excretion (mmol/L) ¥ 0.034 those who are severely ill, post surgery or in intensive
approximates to urinary nitrogen excretion. care; alas, about 10–40 per cent of all adults in hospitals
This can be used as a guide to nitrogen, and or nursing homes may be undernourished.
thus protein, requirements, that is, catabolic
Nutritional support or artificial nutrition
status.
– Complex laboratory tests are available, such as Nutritional support can take many forms. For example,
the assessment of total body fat by impedance in coeliac disease (Chapter 16), a gluten-free diet is
measurement, or total body nitrogen to assess important, and for lactose intolerance, specific dietary
protein status. However, these are usually used restriction of lactose is required.
only in research settings and not in routine Normally, however, whole nutritional support
clinical practice. should be considered:
– Laboratory tests may also show fatty acid ● when the patient has lost 10 per cent or more of
deficiencies (see Chapter 13) and/or vitamin body weight and continues to lose weight because of
and trace element abnormalities (see Chapter inadequate intake, or
15). Poor nutrition may cause impaired ● if the disease process is thought likely to result in
immunological function with decreased impaired nutritional intake for 10 days or more.
lymphocyte count and abnormalities of
delayed hypersensitivity. Undernutrition is associated with increased
mortality and morbidity and prolonged hospital stay
Prognostic nutritional index and should be remedied promptly. It may be possible
The prognostic nutritional index (PNI) has been devised to encourage eating and, if necessary, enhance it with
as a marker of nutritional status and incorporates a supplements. However, sometimes patients are not
number of the components discussed above including able to eat, and artificial nutrition becomes necessary.
plasma albumin and transferrin concentration, skin- The average daily adult requirements are shown in
fold thickness and lymphocyte function. Table 14.1.
Approximations based on the Harris–Benedict
Subjective global assessment equation give an estimate of the total energy
This looks at a number of subjective weightings on requirements. The daily BMR in kilocalories is:
key variables, including weight loss, gastrointestinal
● for males,
disorders, functional capacity and physical signs of
deficiency. Weighting A is well nourished, B moderately (66.5 + 13.8W + 5.0H – 6.8A)
nourished and C poorly nourished. ¥ activity factor ¥ injury factor (14.2)
● for females,
UNDERNUTRITION
(655.1 + 9.6W + 1.9H – 4.7A)
This starts with reduced intake or nutrient loss.
¥ activity factor ¥ injury factor (14.3)
The stores of nutrients are depleted, which leads
to biochemical and metabolic consequences and, where W = weight (kg), H = height (cm) and A = age
eventually, clinical symptoms and signs. (years). The activity factor, for example, when confined
Undernutrition 219
Water 30–35 mL
If possible, all patients should be fed orally or enterally,
both of which are simpler than parenteral feeding and
Energy 20–35 cal
generally cause fewer complications that might consist
Protein 0.8–1.5 g of infections, metabolic abnormalities and mechanical
Carbohydrate 2–5 g problems for example. The basic rule is that if the gut
Fat 1–3 g works, use it. However, if oral or enteral feeding is
Sodium 1–2 mmol contraindicated, for example when there is intestinal
Potassium 1–2 mmol obstruction or fistula, short bowel syndrome, ileus or
Calcium 0.1–0.3 mmol
persistent vomiting, parenteral feeding may be indicated.
Parenteral nutrition can be given through a
Phosphate 0.2–0.4 mmol
peripheral vein or through a central venous catheter
Magnesium 0.1–0.3 mmol
into a large vein. The amount of energy that can be
Vitamins plus trace elements given into a peripheral vein is limited because glucose
a
All units are expressed as per kilogram of body weight. It is and amino acid solutions are hyperosmolar and cause
important to note that these values are extremely variable irritation to small vessel walls, sometimes causing
depending upon individual circumstances.
thrombophlebitis. Thus, peripheral parenteral feed
osmolarity should usually be less than about 600 mmol/
to bed is 1.2. The injury factor after a minor operation kg. Hyperosmolar solutions infused through a central
is 1.4 and with major sepsis is 1.6. venous catheter minimize the risk of thrombophlebitis,
If the patient cannot take food orally, there are which is more likely via a peripheral line.
two main alternatives depending on the presenting The choice depends partly on the length of time for
indications: enteral or parenteral nutrition. which parenteral feeding is required, as the peripheral
route is used only for short periods, usually 1–2 weeks.
Enteral nutrition Some experts use a glyceryl trinitrate patch at the site of
If the gastrointestinal tract is functioning, enteral insertion of peripheral catheters to facilitate insertion
feeding is usually indicated. This is the most effective and reduce thrombophlebitis. The insertion of central
and natural route, and is preferable because it is venous catheters requires expertise and the involvement
more physiological and has fewer complications. The of a multidisciplinary nutrition team working closely
indications for enteral nutrition may include: with the clinical biochemistry laboratory to ensure
correct positioning of the catheter, and strict attention
● dysphagia,
must be paid to aseptic technique to reduce the risk
● coma or delirious state,
of line infection. The regimen depends on the clinical
● post operative,
condition of the patient and the volume that can safely
● persistent anorexia nervosa.
be infused.
There are various enteral routes available:
Energy source
● nasogastric, The energy source is usually a combination of glucose-
● nasoduodenal, and fat-containing fluids, for example Intralipid. Fat
● nasojejunal. is more dense in energy, has a lower osmolality and
● percutaneous endoscopic gastrostomy (PEG) generates less carbon dioxide. At least 50 per cent of the
● jejunostomy (perioperative placement may energy requirements should be provided as glucose/
be considered if likelihood of ongoing upper lipid. Giving energy as glucose and fat minimizes the
gastrointestinal dysfunction). use of amino acids for gluconeogenesis and reduces
The complications of enteral nutrition include tube urinary nitrogen loss.
complications, poor gut absorption and biochemical Glucose. Severe illness may cause insulin resistance
disturbances (these may be similar to those seen and glucose excess may evoke fatty liver and
in parenteral nutrition – see below). More specific hyperglycaemia.
complications of enteral nutrition include aspiration Fat. The fat particles in emulsion form are similar
pneumonia and diarrhoea. to chylomicrons. Provided that some carbohydrate is
220 Nutrition
given at a constant rate, there is no risk of significant Vitamin, mineral and trace element supplementation
ketoacidosis. Sometimes in sepsis or insulin-resistant or This must be given with all parenteral feeding, in addition
hyperlipidaemic patients intravenous lipid is not fully to meeting the nitrogen and energy requirements.
cleared. Grossly lipaemic plasma may interfere with Urinary trace metal loss is high during the catabolic
some laboratory analyses, particularly that of plasma phase, but falls as anabolism becomes predominant.
sodium. If lipaemia persists, it indicates that the rate If parenteral feeding has been very prolonged,
of administration is faster than the rate at which the fat micronutrient deficiencies may become apparent.
can be metabolized, and the infusion should be slowed. Daily parenteral nutrition solutions are sometimes
Rarely, fat overload can occur. The administration of fat prepared in 3-L bags that contain the appropriate
protects against essential fatty acid deficiency. daily energy, glucose, lipid, nitrogen, vitamin and trace
element requirements in addition to electrolytes, calcium,
Nitrogen supplementation
phosphate and magnesium. Intravenous feeding should
Nitrogen supplements containing amino acids are not be stopped suddenly; the patient should gradually be
needed to replace the daily nitrogen loss and to promote weaned on to oral or enteral feeding.
tissue healing. The assessment of nitrogen requirements
may be difficult. A normal adult patient needs about Monitoring of parenteral feeding
9 g nitrogen/day, but this increases as the catabolic rate Some clinical and metabolic complications associated
increases, for example up to 20 g nitrogen/day in stress with long-term parenteral feeding are shown in Box
and infection. During the catabolic phase, the body 14.1. One of the most important complications is
cannot use administered nitrogen efficiently, and much infection of the central line, and careful nursing
of it is excreted in the urine as urea or free amino acids. attention is therefore essential. It is also important to
About 840 kJ (200 kcal) of energy per gram of nitrogen review the patient’s fluid balance. The biochemical
are needed by a normal adult to synthesize protein investigations that may be used to prevent the onset of
from amino acids. This proportion decreases slightly as these complications are discussed below.
the catabolic rate and nitrogen requirements increase.
Protein intake should be about 10–15 per cent of total
calorie requirement. Box 14.1 Some clinical and metabolic
The essential amino acids are arginine, histidine, complications of long-term parenteral
isoleucine, leucine, threonine, lysine, methionine, nutrition
phenylalanine, tryptophan and valine. These should
constitute about 25 per cent of the total amino acids. Complications of central line
The essential amino acids tend to have more complex Malposition
structures such as aromatic rings or long side-chains; Infection
they can be synthesized by bacteria and plants, but not by Thrombosis
humans. Arginine and histidine may become essential Air embolus
only under certain circumstances. The following is a Hyperglycaemia/hypoglycaemia
useful mnemonic to remember these essential amino Acid–base disturbances
Electrolyte disorders
acids: any help in learning these little molecules proves
Fluid overload
truly valuable. Hypophosphataemia
Glutamine, although not an essential amino acid, is Metabolic bone disease
an important energy source for the cells of the immune Liver disease
system and gut. There are data indicating that the Biliary sludging
addition of glutamine to parenteral feeds may benefit Essential vitamin and mineral deficiencies
certain patients, such as those on intensive care. Aluminium (sometimes by contamination) and
Once tissue repair predominates, the use of amino manganese toxicity
acid increases (anabolic phase) and urinary nitrogen Misinterpretation of laboratory results associated with
loss may fall suddenly; this indicates an increased, not lipid infusion
Gut atrophy
a decreased, need for nitrogen. Conversely, during the
Essential fatty acid deficiency
catabolic phase, increasing nitrogen intake may result Fat overload syndrome
in an increased urinary loss because it cannot be used.
Undernutrition 221
Initially, measurement of the daily plasma It can be associated with significant morbidity
sodium, potassium, bicarbonate and urea/creatinine and mortality. Clinical features include fluid balance
concentrations helps in the assessment of water and abnormalities, abnormal glucose metabolism,
electrolyte needs and renal function. Plasma glucose hypophosphataemia (see Chapter 6), hypomagnesaemia
concentrations must be monitored carefully, as patients and hypokalaemia. In addition, thiamine deficiency
may develop stress-related glucose intolerance with can occur. The conditions associated with refeeding
hyperglycaemia, consequent cell dehydration and syndrome are shown in Box 14.2.
polyuria or rebound hypoglycaemia if glucose infusion Prior to refeeding, electrolyte disorders should be
is stopped suddenly. corrected and the circulatory volume carefully restored.
Plasma albumin, protein, calcium, phosphate and In practice, this may delay the administration of
magnesium concentrations should be measured to nutrition but it can usually be accomplished within the
detect possible metabolic complications. The full blood first 12–24 h.
count, including haemoglobin, white cells and platelets,
as well as clotting, should also be monitored.
Box 14.2 Factors that increase the risk of
A cholestatic type of liver disorder associated with
refeeding syndrome
biliary sludging may develop in some patients receiving
intravenous nutrition; thus, liver function tests also
Kwashiorkor or marasmus
need to be monitored. The plasma alkaline phosphatase Anorexia nervosa
activity increases, with a later rise in plasma transaminase Chronic undernutrition, e.g. associated with carcinoma
activities. Unless significant symptoms occur, such as or old age
severe jaundice, this is not necessarily an indication Chronic alcoholism
to stop parenteral feeding because liver dysfunction Prolonged fasting
usually resolves when the parenteral feeding is stopped. Cancer treatment
Fatty liver may also occur with raised transaminases, Surgery
especially if overinfusion of glucose.
Plasma concentrations of trace elements zinc,
selenium, manganese and copper should be monitored CASE 1
about monthly once the patient is clinically stable,
and similarly for ferritin, vitamin B12 and folate. Some A 64-year-old man with an inoperable oesophageal
patients may not clear the fat in the parenteral feed, carcinoma had been unable to eat solid foods for
and this can be assessed by measuring the plasma about 2 months. A day after he had been commenced
triglyceride concentration. on total parenteral nutrition, the following blood
Estimation of 24-h urinary nitrogen output is very results were obtained.
rarely helpful but has been used to assess nitrogen Plasma
use and the amount that should be replaced. If urea Sodium 136 mmol/L (135–145)
excretion increases quantitatively when nitrogen intake Potassium 2.7 mmol/L (3.5–5.0)
is increased, this indicates that the nitrogen cannot be Urea 2.7 mmol/L (2.5–7.0)
used, and thus should not be increased. If nitrogen loss Creatinine 70 µmol/L (70–110)
falls while supplements are being given, this indicates Albumin-adjusted calcium 2.23 mmol/L (2.15–2.55)
that anabolism is increasing, and supplements should Phosphate 0.21 mmol/L (0.80–1.35)
be increased until urinary excretion increases. Once the Magnesium 0.32 mmol/L (0.70–1.0)
patient has been established on long-term feeding, the DISCUSSION
frequency of monitoring can be reduced. The patient has biochemical features of refeeding
syndrome, with hypokalaemia, hypophosphataemia
Refeeding syndrome
and hypomagnesaemia. Considerable care is needed
This potentially lethal condition can be defined as when feeding patients after prolonged lack of food
severe electrolyte and fluid shifts associated with to avoid these dangerous biochemical features. Other
metabolic abnormalities in undernourished patients associated complications may include thiamine
undergoing refeeding, whether this is oral, enteral or deficiency and fluid balance disturbance.
parenteral.
222 Nutrition
Vitamin and trace element deficiencies should also Table 14.2 Classification of body mass index (BMI)
be corrected and, specifically, thiamine should be
BMI (kg/m2) WHO classification Common description
given before refeeding is instigated. Further thiamine
may be necessary until the patient is stabilized. Some < 18.5 Underweight Thin
preparations containing thiamine have been associated 18.5–24.9 Normal Normal
with anaphylaxis, and therefore facilities for treating 25.0–29.9 Grade 1 Overweight
this should be readily at hand. 30.0–39.9 Grade 2 Obese
The calorie repletion should be slow, at approximately > 40.0 Grade 3 Morbid obesity
20 kcal/kg per day or, on average, 1000 kcal/day initially.
WHO, World Health Organization.
However, this may not meet the patient’s fluid, sodium,
potassium or vitamin requirements unless these are
specifically addressed. The usual protein requirement is adipocytes, is an afferent signal that relays the magnitude
about 1.2–1.5 g/kg per day, or about 0.17 g nitrogen/kg of the fat stores to the central nervous system, primarily
per day. Gradual introduction of calories, particularly the hypothalamus, and plasma levels correlate with
over the first week of refeeding, may be prudent until the adipose tissue mass. In the ob/ob mouse, there is
the patient is metabolically stable. defective leptin production associated with obesity and
insulin resistance. Leptin administration causes weight
ANOREXIA NERVOSA loss in ob/ob mice partly by reducing food intake. In
In this condition, which is more common in females, times of starvation, leptin levels decline. Adiponectin
there are psychological problems with body self- is another adipose-released hormone that is thought to
image and self-inflicted starvation. In some respects sensitize tissues to insulin.
this condition can resemble hypopituitarism with Neuropeptide Y is a potent stimulator of food
amenorrhoea resulting from decreased luteinizing intake, the production of which is inhibited by leptin.
hormone and follicle-stimulating hormone release. Pro-opiomelanocortin is also involved in obesity
However, instead of the loss of axillary and pubic hair, as and is cleaved to form adrenocorticotrophin and
in hypopituitarism, there is additional lanugo hair. There
may be severe weight loss and elevated growth hormone
and cortisol concentrations. Severe hypokalaemia, CASE 2
hypomagnesaemia and hypophosphataemia are
A 49-year-old man presented to his general
also seen, particularly if refeeding syndrome ensues.
practitioner wanting to lose weight. He had
Strangely, hypercholesterolaemia may also occur.
osteoarthritis of his knees and sleep apnoea. His
OBESITY blood pressure was raised, at 168/98 mmHg, and his
BMI was 40.4 kg/m2. His fasting blood glucose was
It may seem strange to talk about obesity in the same
6.0 mmol/L and plasma lipids showed cholesterol
chapter as undernutrition and starvation; however,
6.3 mmol/L, triglyceride 4.8 mmol/L and high-
obesity could be considered a form of malnutrition.
density lipoprotein (HDL) cholesterol 0.67 mmol/L.
About 20 per cent of the European population is obese,
The GP requested an oral glucose tolerance test
with higher prevalence figures in other populations
(OGTT), which had the following results:
such as African Americans and Pacific Islanders.
Although these definitions rely on BMI, an alternative Time ‘zero’ before the OGTT – fasting venous plasma
approach is to measure the waist circumference, with glucose: 5.9 mmol/L.
88 cm for females and 102 cm for males being indicative 2 h after oral 75 g anhydrous glucose load – plasma
of central obesity in some patient groups (Table 14.2). glucose: 9.4 mmol/L.
The reason for this epidemic of obesity is partly the
DISCUSSION
decrease in physical activity seen in sedentary societies
This patient shows grade 3 obesity, which is
combined with the increased intake of calorie-dense
associated with hypertension, mixed hyper-
food such as saturated fat. However, there may also be
lipidaemia, osteoarthritis, sleep apnoea and
underlying genetic or molecular mechanisms, which
impaired glucose tolerance. This is an increasing
are reviewed briefly here.
public health problem.
Leptin, a 16-kDa protein that is expressed in
Obesity 223
a-melanocyte-stimulating hormone (MSH) (see gain are extremely tightly regulated and it only needs
Chapter 7). The latter acts on the melanocorticortin-4 an excess of 100 kcal/day, such as a chocolate biscuit, to
receptor (MC4-R) in the hypothalamus, which in result in a 4-kg gain in a year.
turn increases energy expenditure and reduces food Increased physical activity and reduced calorie
intake. Agouti protein, which is also expressed in hair intake, sometimes combined with psychotherapy or
follicles, antagonizes the actions of MSH by blocking behavioural therapy, are the cornerstone of treatment,
MC4-R. Obese humans paradoxically have high levels although many people fail to achieve satisfactory
of leptin, presumably because of tissue resistance weight loss. Some new drug therapies are available,
to it. A minority of cases of obesity may be due to but these should not be viewed as a universal panacea
mutations in leptin receptors. Also involved in obesity for obesity as there are no short-cut easy answers and
is the b-3-adrenoreceptor, which mediates adipose they may have side effects. One such drug is orlistat,
metabolism by the sympathetic nervous system. a pancreatic lipase inhibitor that causes reduced small
Obesity is associated with numerous medical intestine fat absorption. Other therapeutic possibilities
problems, including type 2 diabetes mellitus, in the future may be neuropeptide Y antagonists, leptin
hyperlipidaemia, hypertension, coronary artery disease analogues and b-3-adrenoreceptor agonists. Surgical
and stroke. There are various treatment strategies for options such as gastric banding or Roux-en-Y gastric
obesity. Remember that energy balance and weight bypass are being used and may be cost-effective.
SUMMARY
● Daily energy loss as heat is about 120 kJ (30 kcal) function, although other nutritional components
per kilogram of body weight in a normal adult. In are satisfactory. Visceral proteins are decreased,
addition, there is a daily protein turnover of about but body weight, mid-arm circumference and
3 g/kg body weight (about 0.5 g of nitrogen), of triceps skin-fold thickness are relatively normal.
which about 0.15 g of nitrogen/kg body weight Conversely, marasmus is generalized undernutrition
is excreted (1 g of nitrogen is derived from about with reduction in weight, mid-arm circumference
6.25 g of protein). These losses are usually balanced and skin-fold thickness, although relatively normal
by dietary intake of equivalent amounts of energy, visceral proteins.
as carbohydrate, fat and protein. Glucose provides ● Enteral or parenteral feeding may be necessary for
4 kcal/g; fat provides 9 kcal/g. patients needing nutritional support.
● During starvation, the body tries to maintain blood ● Although about 1 billion of the world’s population
glucose levels in the acute phase and in the longer are undernourished, approximately 1 billion are
term to preserve body protein mass. overweight. Obesity is increasing, particularly in
● Kwashiorkor occurs in individuals with visceral Western urbanized societies.
protein loss associated with impaired immunological
15 Vitamins, trace elements and metals
This chapter looks at vitamins and trace elements, and ● inadequate absorption, for example malabsorption
might usefully be read in conjunction with Chapter 14 states,
(Nutrition). It also discusses certain elemental metals ● excess loss, for example via gastrointestinal or renal
that are important in disease states. tract,
At one time, vitamins were thought to be amines and ● enhanced utilization, for example sepsis or trauma.
hence the term ‘vitamines’ was coined for substances
that are essential for life but needed in only minute VITAMIN EXCESS
amounts. Vitamins are now known to be organic Some vitamins (notably A and D) are toxic if taken
compounds, not necessarily amines, which are essential in excess, and overdosage has recently become more
for normal growth and development. They must be common, possibly because of the increased availability
included in the diet because the body either cannot of these compounds in over-the-counter preparations.
synthesize them at all or cannot do so in amounts
sufficient for its needs. CLASSIFICATION OF VITAMINS
Trace elements are inorganic compounds that, like
vitamins, are essential for health and needed only Vitamins are classified into two groups on the basis
in small amounts, known as the reference nutrient of their solubilities: fat soluble and water soluble.
intake. A normal mixed diet should provide adequate The distinction is of clinical importance because
amounts of vitamins and trace elements, and thus steatorrhoea may be associated with a deficiency of fat-
supplementation is not usually necessary. soluble vitamins, with relatively little clinical evidence
Testing for vitamin and trace element deficiency of lack of most of the water-soluble vitamins except B12
should be carried out as soon as the diagnosis is and folate.
suspected; the results of laboratory tests usually revert Fat-soluble vitamins
rapidly to normal once the patient has resumed eating The principal fat-soluble vitamins are:
a normal diet, for example after admission to hospital, ● A (retinol),
and it may then be impossible to confirm the original ● D (calciferol),
diagnosis. Where the diagnosis is difficult, a trial of the ● E (a-tocopherol),
micronutrient may be the most reliable and simplest ● K (2-methyl-1,4-naphthoquinone).
method of assessment.
Each of these has more than one active chemical
VITAMIN DEFICIENCIES form, but variations in structure are minimal and
in this chapter each vitamin is considered as a single
These may have the following causes: substance.
● inadequate intake: deficiencies are rarely seen in
affluent populations except in: Vitamin A (retinol)
– individuals with an inadequate dietary intake Sources
or unusual diet, Precursors of vitamin A (the carotenes) are found in
– chronic alcoholism, the yellow and green parts of plants and are especially
– patients with anorexia nervosa, abundant in carrots. The active vitamin is formed by
– patients on parenteral or enteral nutrition, the hydrolysis of b-carotene in the intestinal mucosa;
Classification of vitamins 225
each molecule can produce two molecules of vitamin Causes of vitamin A deficiency
A, which are absorbed as retinol esters and stored in Hepatic stores of vitamin A are large and therefore
the liver. Retinol is transported to tissues bound to the clinical signs develop only after many months, or even
a-globulin retinol-binding protein (RBP). years, of dietary deficiency. Such prolonged deficiency
Vitamin A is stored in animal tissues, particularly the is very rare in affluent communities. In steatorrhoea,
liver, and is also present in milk products and eggs. clinical evidence of vitamin A is rare, although plasma
Functions concentrations may be low. Deficiency is relatively
common in poor countries, especially in children, and
Rhodopsin (visual purple), the retinal pigment
can cause blindness.
that is necessary for vision in poor light (scotopic
vision), consists of a protein (opsin) combined with Diagnosis and treatment of vitamin A deficiency
vitamin A. Rhodopsin decomposes in bright light. It The diagnosis is usually made on the basis of clinical
is partly regenerated in the dark, but, because this is criteria; very low plasma vitamin A concentrations
not quantitatively complete, vitamin A is needed to usually confirm deficiency. In conditions such as non-
maintain retinol levels. Vitamin A is also essential for cirrhotic liver disease, in which plasma concentrations
normal mucopolysaccharide synthesis and mucus of RBP are low, concentrations of vitamin A may be
secretion. decreased despite normal liver stores. In cirrhosis of the
Clinical effects of vitamin A deficiency liver, the stores may be very low. Laboratory tests for
the diagnosis of vitamin A deficiency consist of testing
The clinical effects of vitamin A deficiency include the
for plasma retinol concentration. Retinol-binding
following.
protein is also low in vitamin A deficiency, but this may
● Owing to rhodopsin deficiency: also occur in protein deficiency and the acute-phase
– ‘night blindness’ (nyctalopia): deficiency response.
is associated with poor vision in dim light, Vitamin A deficiency can be treated with retinyl
especially when the eyes have recently been palmitate. High doses of vitamin A should be given to
exposed to bright light. treat xerophthalmia and advanced skin lesions. ‘Night
● Owing to deficient mucus secretion leading to drying blindness’ and early retinal and corneal changes often
and squamous metaplasia of ectodermal tissue: respond rapidly to treatment, although corneal scarring
– Skin secretion is diminished and there may may be irreversible.
be hyperkeratosis of hair follicles. Dry, horny
papules (follicular hyperkeratosis) are found Hypervitaminosis A
mainly on the extensor surfaces of the thighs Vitamin A in large doses is toxic. Acute intoxication
and forearms. Squamous metaplasia of the has been reported in Arctic regions as a result of eating
bronchial epithelium has also been reported polar bear liver, which has very high vitamin A content.
and may be associated with a tendency to More commonly, overdosage is due to the excessive use
develop chest infections. of vitamin preparations.
– The conjunctiva and cornea become dry and In acute poisoning, symptoms include nausea and
wrinkled, with squamous metaplasia of the vomiting, abdominal pain, drowsiness and headache.
epithelium and keratinization of the tissue Pregnant women are usually advised not to eat liver,
(xerosis conjunctivae and xerophthalmia). which is a storage organ for many vitamins, to avoid
Bitot’s spots are elevated white patches, the risk of fetal damage.
composed of keratin debris, found in the Chronic hypervitaminosis A is associated with
conjunctivae. Prolonged deficiency leads to fatigue, insomnia, bone pain, loss of hair, desquamation
keratomalacia, with ulceration and infection and discoloration of the skin, hepatomegaly,
and consequent scarring of the cornea, causing headaches, abdominal pain, bone and joint pain,
blindness. benign intracranial hypertension, osteoporosis and
– Poor bone growth in the skull, leading to weakness.
cranial nerve compression. Additionally, a very high intake of carrots or orange
– Anaemia, which responds to vitamin A but not juice can lead to carotenaemia, which can mimic
to iron therapy. jaundice except that plasma bilirubin is normal.
226 Vitamins, trace elements and metals
The vitamin B complex yeast are particularly rich sources. Adequate amounts
Most of these vitamins act as enzyme cofactors and are present in a normal diet and deficiency is most
many are synthesized by colonic bacteria. As the common in alcoholics and in patients with anorexia
absorption of water-soluble vitamins from the large nervosa.
intestine is poor, probably most of those synthesized
within the colon are unavailable to the body. Functions
Clinical deficiency is rare in affluent communities. Thiamine is a component of thiamine pyrophosphate,
When deficiency does occur, it is usually multiple, which is an essential cofactor for decarboxylation
involving most of the B group, and is associated of 2-oxoacids; one such reaction is the conversion of
with protein undernutrition; for this reason it may pyruvate to acetyl coenzyme A (see Chapter 12). In
be difficult to decide which signs and symptoms are thiamine deficiency, pyruvate cannot be metabolized
specific for an individual vitamin and which are part and accumulates in the blood. Thiamine pyrophosphate
of a general undernutrition syndrome (Fig. 15.1). With is also an essential cofactor for transketolase in the
the exception of vitamin B12, assay of these vitamins pentose–phosphate pathway.
is rarely indicated and treatment is usually given
empirically if a deficiency state is suspected. Clinical effects of thiamine deficiency
Deficiency is usually due to excess ethanol intake with
Thiamine (B1)
high carbohydrate but poor vitamin intake, although
Sources and causes of deficiency
it can also be seen in intensive-care patients with high
Humans cannot synthesize thiamine. It is found in carbohydrate intake. One of the commonest causes
many dietary components; wheat germ, oatmeal and worldwide is a diet high in un-enriched white flour
Role of B vitamins in formation of acetyl CoA
or rice. The level of thiaminase, which breaks down
thiamine, is high in raw fish.
Glucose NICOTINAMIDE PANTOTHENATE
Deficiency of thiamine causes beri beri, in which
anorexia, emaciation, neurological lesions (motor and
sensory polyneuropathy, amnesia and encephalopathy
Pyruvate + NAD+ CoA known as Wernicke–Korsakoff syndrome) and
cardiac arrhythmias may occur. This form is called
TPP THIAMINE ‘dry’ beri beri (shoshin) and is associated with low
cardiac output. In ‘wet’ beri beri there is peripheral
Fats
oedema, sometimes associated with cardiac failure.
Acetyl CoA + CO2 + NADH2
Amino acids Thiamine deficiency can be seen as part of the
Tricarboxylic refeeding syndrome (see Chapter 14). Beri beri may
acid cycle
be aggravated by a high-carbohydrate diet, possibly
because this leads to an increased rate of glycolysis
Role of B vitamins in electron transfer and therefore of pyruvate production.
AH2 NAD+ (NADP+) FpH2 Cytochrome H2O Laboratory diagnosis of thiamine deficiency
One test for thiamine deficiency is the estimation of
A NADH (NADPH) Fp Reduced 1/
2O2
cytochrome erythrocyte transketolase activity, with and without
added thiamine pyrophosphate. Reduced activity, if
due to thiamine deficiency, becomes normal after the
NICOTINAMIDE RIBOFLAVINE
addition of the cofactor. This test is rarely indicated
and of little use once a normal diet, or vitamin
B vitamins in supplementation, has been started because plasma
Figure 15.1 Some biochemical interrelations of the B
concentrations are rapidly corrected. Other tests may
vitamins. A, substrate (e.g. pyruvate); AH2, reduced be useful, including the measurement of both blood
substrate (e.g. lactate); CoA, coenzyme A; Fp, and urinary thiamine concentrations, and a raised
flavoprotein; NAD, nicotinamide adenine dinucleotide; blood pyruvate concentration is suggestive as is a lactic
TPP, thiamine pyrophosphate. acidosis (see Chapter 4).
228 Vitamins, trace elements and metals
states, as may holotranscobalamin, whereas plasma ileal resection or bacterial overgrowth, malabsorption
homocysteine and methylmalonic acid may be elevated. persists. Intrinsic factor and parietal antibodies should
Folate is present in green vegetables and some be tested, as these may be positive in pernicious anaemia
meats. It is easily destroyed during cooking, and dietary (see Chapter 16).
deficiency may occasionally occur. Folate is absorbed Deficiency of vitamin B12, like that of folate, causes
throughout the small intestine and, in contrast to most megaloblastic anaemia. However, unlike that of folate,
of the other B vitamins (except B12), clinical deficiency it can cause subacute combined degeneration of the
is relatively common in intestinal malabsorption spinal cord. Although the megaloblastic anaemia of
syndromes, especially in the ‘contaminated bowel’ vitamin B12 deficiency can be reversed by folate, this
syndrome. In these conditions, and during pregnancy treatment should never be given in pernicious anaemia
and lactation, low red-cell folate concentrations may be because it does not improve, and may even aggravate,
associated with megaloblastic anaemia or macrocytosis. the neurological lesions.
Low maternal folate intake is also associated with neural It has recently become established that homocysteine,
tube defects in the fetus. The active form of the vitamin a sulphur-containing amino acid, is an independent
is tetrahydrofolate, which is essential for the transfer cardiovascular risk factor. Both folate and vitamin
of one-carbon units; it is particularly important in B12 regulate the enzyme methylenetetrahydrofolate
purine and pyrimidine, and therefore deoxyribonucleic reductase and methionine synthase, respectively, while
acid (DNA) and ribonucleic acid (RNA), synthesis. vitamin B6 is a cofactor of the enzyme cystathionine
Methotrexate, a cytotoxic analogue of folate, competes synthase (Fig. 15.2). Supplementation with folate and/
with it for metabolism, and therefore inhibits DNA or vitamin B12 may lower homocysteine plasma levels
synthesis. (see also Chapter 22).
The vitamin B12 group includes several cobalamins, The known biological functions and the clinical
found in animal products but not in green vegetables. syndromes associated with deficiencies of the B vitamins
Dietary deficiency is rare. The cobalamins are are summarized in Table 15.1. Generally, deficiencies of
transported in plasma by a specific carrier protein, this group cause lesions of skin, mucous membranes
transcobalamin II. Deoxyadenosyl-cobalamin and and the nervous system.
methylcobalamin have, like folate, coenzyme activity in
Folate
nucleic acid synthesis. Hydroxycobalamin is the form
most commonly used in treatment, and both it and
cyanocobalamin are converted to cofactor forms in the
body. All forms are absorbed mainly in the terminal Methionine Tetrahydrofolate
ileum, combined with intrinsic factor derived from the
gastric parietal cells. In pernicious anaemia, antibodies
to gastric parietal cells and/or to intrinsic factor cause
S-adenosyl methionine
malabsorption of vitamin B12. The Schilling test has Methylenetetrahydrofolate
been used to diagnose pernicious anaemia. Vit B12 reductase (MTHFR)
labelled vitamin with intrinsic factor restores normal Figure 15.2 Diagram showing simplified pathways of
absorption; if it is due to intestinal disease, for example homocysteine metabolism.
Classification of vitamins 231
Table 15.1 The biochemical functions and clinical deficiency syndromes associated with the B vitamins
the diagnosis, levels probably alter in parallel; plasma Laboratory tests for deficiency include the
assay is technically more satisfactory, but chemical measurement of plasma copper. Copper is carried on
estimations are rarely indicated. There is also an oral the protein caeruloplasmin, the level of which may be
vitamin C loading test that may show a low urinary increased due to an acute-phase response, oestrogens
excretion of vitamin C in deficiency states. Treatment or pregnancy. Copper deficiency can be treated with
of deficiency is with oral vitamin C. certain copper salts, but care is needed in case there is
an effect on zinc and iron absorption.
Ascorbate excess
Copper excess can lead to toxicity, causing renal
Excess vitamin C is rare, but may increase oxalate levels problems, fits, haemolysis and hypotension. Wilson’s
and the likelihood of renal oxalate calculi. disease, in which copper excess and decreased
TRACE METALS caeruloplasmin occur, is an inborn error of metabolism.
Inorganic micronutrients, or trace metals, are essential Wilson’s disease
for normal health and, by definition, make up less than
0.01 per cent of the body’s dry weight. Some plasma copper is loosely bound to albumin, but
most is incorporated in the protein caeruloplasmin.
Zinc Copper is mainly excreted in bile. There are two defects
Zinc is a cofactor for certain enzymes, for example of copper metabolism in Wilson’s disease:
polymerases, carbonic dehydratase and alkaline ● impaired biliary excretion leads to deposition in the
phosphatase. Zinc deficiency may result in a number of liver,
clinical states, including growth retardation, alopecia, ● deficiency of caeruloplasmin results in low plasma
dermatitis, diarrhoea, poor wound healing, infertility copper concentrations. Most is in a loosely bound
and increased risk of infections. form and is therefore deposited in tissues; more
The causes of zinc deficiency are acrodermatitis than normal is filtered at the glomeruli and urinary
enteropathica (a rare autosomal recessive condition copper excretion is increased.
associated with dermatitis, diarrhoea and alopecia
and a defect of the SLC39A4 gene, which encodes for
a membrane zinc-transporting protein) poor intake CASE 2
(e.g. parenteral nutrition or malabsorption), increased
utilization and high levels of dietary phytates. Useful A 21-year-old man was referred to the neurology
laboratory tests for deficiency include the measurement out-patient department because of dysarthria and leg
of plasma or urinary zinc levels. The plasma zinc-carrying weakness. A number of investigations were organized
protein metallothionine may also prove useful. Note that and some of the pertinent results are as follows.
plasma zinc levels may be low during an acute-phase Urine analysis showed amino aciduria.
response, as the zinc is also partly bound to albumin, which Plasma
is a negative acute-phase reactant. Zinc toxicity is rare, Alanine transaminase 98 U/L (< 42)
usually occurring after inappropriate administration, and Bilirubin 12 µmol/L (< 20)
may result in pulmonary oedema, jaundice and oliguria Alkaline phosphatase 367 U/L (< 250)
as well as hypocupraemia (low plasma copper). Albumin 44 g/L (35–45)
g-Glutamyl transferase 234 U/L (< 55)
Copper
Caeruloplasmin 0.08 g/L (0.2–0.6)
Copper functions as an enzyme cofactor, for example Copper 10 µmol/L (11–20)
for cytochromes. Copper deficiency may cause cardiac
arrhythmias, neutropenia, hypochromic anaemia DISCUSSION
and, in children, bony problems such as subperiosteal The patient was confirmed by liver biopsy and
haematomas. genetic studies to have Wilson’s disease presenting
The causes of deficiency include poor intake, for with neurological sequelae, abnormal liver function
example long-term parenteral nutrition. Menke’s tests and renal tubular damage leading to amino
disease is an inborn error of copper transport resulting aciduria. The concentration of copper-binding
in low plasma copper concentrations and abnormal protein caeruloplasmin is low. High urinary free
hair that has a characteristic ‘kink’ appearance. copper concentration would be expected.
Trace metals 233
Excessive deposition of copper in the eyes, basal is tissue damage due, for example, to inflammation
ganglia of the brain, liver and renal tubules produces: or neoplasia. These may account for the rare finding
of ‘normal’ plasma caeruloplasmin concentrations in
● Kayser–Fleischer rings at the edges of the cornea due
patients with Wilson’s disease.
to deposition of copper in Desçemet’s membranes
Histological examination, with the demonstration of
(Fig 15.3),
an increased copper content, of a liver biopsy specimen
● neurological symptoms due to degeneration of the
is often necessary to confirm the diagnosis.
basal ganglia,
Wilson’s disease has a recessive mode of inheritance,
● liver damage leading to cirrhosis,
but reduced plasma caeruloplasmin concentrations
● renal tubular damage with any or all of the associated
may be demonstrable in heterozygotes. The
biochemical features, including amino aciduria
distinction between presymptomatic homozygotes
(Fanconi’s syndrome).
and heterozygotes is important because the former
Diagnosis can be treated. Treatment with copper-chelating agents
such as D-penicillamine may reduce tissue copper
Most patients have low plasma caeruloplasmin (less
concentrations.
than 0.2 g/L) and copper concentrations (less than
12 µmol/L) and high urinary copper excretion (more Selenium
than 1.2 µmol/day). The main function of selenium is to mediate the activity
The penicillamine test is sometimes used in the of the enzyme glutathione peroxidase, which acts as an
diagnosis of Wilson’s disease and is based on the antioxidant.
principle that penicillamine solubilizes copper and Deficiency can be caused by poor intake and
enhances copper urinary excretion. Following a dose is most commonly seen in certain areas of China.
of oral penicillamine, urinary copper excretion is more Patients on artificial nutrition, for example parenteral
than 25 µmol/day, indicative of Wilson’s disease. support, are also more susceptible. Selenium deficiency
Low plasma caeruloplasmin concentrations may may also cause cardiomyopathy (Keshan’s disease),
also occur during the first few months of life due osteoarthropathy (Kaschin–Beck disease), myopathy,
to undernutrition, and in the nephrotic syndrome macrocytosis and anaemia.
due to urinary loss. Raised plasma caeruloplasmin Selenium may slow the progression of mild Graves’
concentrations are found in active liver disease, in orbitopathy (see Chapter 11).
women taking oral contraceptives, during the last Diagnostic laboratory tests for selenium deficiency
trimester of pregnancy and non-specifically when there include the measurement of urinary or blood selenium
or finding decreased erythrocyte glutathione peroxidase
activity.
Manganese
This is an enzyme cofactor, for example of superoxide
dismutase. Deficiency is associated with vitamin K
deficiency and may be seen in patients who are being
fed artificially.
Manganese excess can occur, for example in miners
of manganese ores, and can result in Parkinson-like
disease and defects of the basal ganglia. Sometimes
parenteral nutrition regimens may cause raised plasma
manganese concentration. Manganese is mainly biliary
excreted, and therefore toxicity may occur with hepatic
dysfunction or cholestasis.
Figure 15.3 Patient with Wilson’s disease manifesting Chromium
the Kayser–Fleischer ring. Reproduced with kind
permission from Nyhan WL, Barshop BA and Ozand Chromium is an insulin cofactor. Chromium deficiency
PT. Atlas of Metabolic Diseases, 2nd edition. London: can occur on long-term parenteral nutrition, leading to
Hodder Arnold, 2005. glucose intolerance and neuropathy.
234 Vitamins, trace elements and metals
Toxicity is associated with gastrointestinal problems, The diagnosis of mercury poisoning can be
lung cancer and hepatitis. established from urinary or blood mercury levels.
Sometimes hair mercury levels can be used to detect
Molybdenum
long-term exposure.
Molybdenum is a cofactor of xanthine oxidase and
other enzymes. Aluminium
Deficiency can occur on long-term parenteral Aluminium toxicity, although rare, is well described in
nutrition and may lead to tachycardia, central scotomas, patients with renal impairment. Contamination of the
‘night blindness’ and coma. water used in dialysis fluid has been implicated in renal
Cobalt osteodystrophy and dialysis dementia. Dialysis water
is now usually treated to decontaminate aluminium.
This is important for vitamin B12 metabolism. Toxicity
Toxicity has also been seen in individuals with excess
is rare and has been described in dialysis patients and
oral intake, such as of aluminium-containing antacids
heavy drinkers of beer that is contaminated with cobalt,
for dyspepsia. Contamination from plastic tubing
leading to cardiomyopathy.
and glass vials may occur in some long-term total
METAL POISONING parenteral nutrition patients. Aluminium exposure
It would be highly unusual to have a deficiency of the is diagnosed by plasma aluminium measurements.
following metals, although, for completeness, metal Desferrioxamine has been used to chelate aluminium
poisoning is included in this chapter. in toxicity.
Mercury Cadmium
Mercury poisoning can occur from organic or inorganic Cadmium toxicity can occur in industrial workers
salts or elemental mercury vapour. Acute toxicity exposed to cadmium fumes. Clinical features include
may result in a metallic taste and respiratory distress, nephrotoxicity, hepatotoxicity and bone disease.
nausea and vomiting. More chronic features include Cadmium exposure can be assessed from the levels in
neuropathy and renal dysfunction. blood and urine. Renal tubular damage can be monitored
Acute toxicity can be treated with dimercaprol- by raised urinary b2-microglobulin concentration.
chelating agents, which increase excretion via urine and
bile. In chronic exposure, N-acetyl penicillamine has Lead
been used to chelate mercury. For a discussion of lead poisoning, see Chapter 21.
SUMMARY
● Vitamins are essential for biochemical processes ● Vitamin B12 deficiency can be seen in pernicious
and can be divided into those that are fat soluble anaemia.
and those that are water soluble. The former include ● Vitamin C deficiency causes scurvy.
vitamin A, D, E and K; the latter are vitamins B ● Trace elements are also essential, such as zinc,
and C. copper, manganese and selenium. Deficiencies
● Vitamin A and E deficiencies are more common can occur, usually if the patient has poor dietary
in developing countries. Vitamin D deficiency intake.
can result in rickets or osteomalacia (discussed in ● Trace element excess can occur, for example in
Chapter 6). Vitamin K deficiency can cause bleeding Wilson’s disease, in which plasma caeruloplasmin
problems with prolonged prothrombin time. concentration is low, resulting in excess unbound
● Vitamin B1 (thiamine) deficiency is associated with copper.
beri beri and Wernicke–Korsakoff syndrome.
16 The gastrointestinal tract
Gastrointestinal disorders present relatively commonly such a way that the conditions for enzyme activity
to doctors, and biochemical laboratory tests have are near optimal for digestion. Adjustment of pH
important roles in their investigation. This chapter and electrolyte concentrations occurs in the distal
looks at gastrointestinal pathophysiology and how ileum and colon; sodium reabsorption (and therefore
chemical pathology tests may be useful in diagnosis and water reabsorption) is enhanced by aldosterone in
treatment. the colon. The colon can metabolize undigested
carbohydrates into short-chain fatty acids such
PHYSIOLOGY AND BIOCHEMISTRY OF as butyrate, propionate and acetate. These are an
NORMAL DIGESTION important energy source for the colon.
The major functions of the gastrointestinal tract are Disturbances of water, electrolyte and hydrogen ion
the digestion and absorption of nutrients. Additionally, homeostasis may occur in small intestinal or colonic
it synthesizes certain hormones along its length that disease. If there is loss of fluid and electrolytes from the
act locally (paracrine hormones), so controlling gut upper intestinal tract because of vomiting or because
motility and the release of digestive enzymes and the function of intestinal cells is so perturbed that the
secretions. The gastrointestinal tract handles about amount of fluid and electrolytes entering the distal
8–9 L of fluid per day, with the majority of this derived parts exceeds the reabsorptive capacity, similar changes
from endogenous secretions. Fluid reabsorption by may occur (see also Chapter 2).
the gastrointestinal tract (mainly the small intestine) Digestive enzymes usually act on complex molecules
is highly efficient (98 per cent), with only 100–200 mL such as protein, polysaccharides and fat. This process
lost daily in the stools. starts in the mouth, where food is broken down
Digestion and absorption depend on food and the by mastication and is mixed with saliva containing
products of its digestion being diluted with a large a-amylase. In the stomach, acid fluid is added and
volume of fluid, mostly an ultrafiltrate of extracellular the low pH initiates protein digestion by pepsin.
fluid filtered through the ‘tight junctions’ between The stomach also secretes intrinsic factor, essential
epithelial cells, mainly in the duodenum. Some is for vitamin B12 absorption from the terminal ileum.
reabsorbed in the proximal jejunum, along the osmotic However, most digestion takes place in the duodenum
gradient created by reabsorption of the products of and upper jejunum, where alkaline fluid is added to
digestion; a large amount remains in the lumen to be the now liquid intestinal contents. Pancreatic enzymes
reclaimed more distally. digest proteins to small peptides and amino acids,
A small amount of the nutrients from desquamated polysaccharides to monosaccharides, disaccharides and
intestinal cells also enters the lumen; most is oligosaccharides, and fat to monoglycerides and fatty
reabsorbed. Almost all the fat in normal faeces acids.
is of endogenous origin, which is relevant when
interpreting the results of a faecal fat estimation. GASTRIC FUNCTION
A much smaller volume of fluid enters the lumen The main components of gastric secretion are
by active secretion. As a result, the pH and the hydrochloric acid, pepsin and intrinsic factor, all
electrolyte and enzyme concentrations change of which are important for normal digestion and
during the passage of fluid through the tract in absorption. Loss of hydrochloric acid by vomiting, as
236 The gastrointestinal tract
in pyloric stenosis, may cause a metabolic alkalosis and by histological examination of the biopsy material
(Chapter 4). Gastric secretion may be stimulated by the obtained.
following:
Post-gastrectomy syndromes
● The vagus nerve, which in turn responds to stimuli There may be some degree of malabsorption after
from the cerebral cortex, normally resulting from gastrectomy. Rapid passage of the contents of the small
the sight, smell and taste of food. Hypoglycaemia gastric remnant into the duodenum may be associated
can stimulate gastric secretion and so can be used to with the following:
assess the completeness of a vagotomy.
● Gastrin, which is carried by the bloodstream to the ● The early dumping syndrome Soon after a meal, the
parietal cells of the stomach; its action is mediated patient may experience abdominal discomfort and
by histamine. Gastrin secretion is inhibited (by feel faint and nauseated. These symptoms may be
negative feedback) by acid in the pylorus. Calcium caused by the rapid passage of hypertonic fluid into
also stimulates gastrin secretion and this may explain the duodenum. Before this abnormally large load
the relatively high incidence of peptic ulceration in can be absorbed, water passes along the osmotic
patients with chronic hypercalcaemia. gradient from the extracellular space into the lumen.
The reduced plasma volume causes faintness and the
Histamine stimulates gastric secretion after binding large volume of fluid causes abdominal discomfort.
to specific cell surface receptors, of which there are at ● The late dumping syndrome (or post-gastrectomy
least two types: hypoglycaemia) If a meal containing a high glucose
● those for which antihistamines compete with concentration passes quickly into the duodenum,
histamine (H1 receptors): these are found on smooth glucose absorption is very rapid, stimulating a
muscle cells, surge in insulin secretion. The resultant ‘overswing’
● those on which antihistamines have no effect: these of plasma glucose concentration may cause
H2 receptors are found on gastric parietal cells. hypoglycaemic symptoms, typically occurring
about 2 h after a meal. This is a form of reactive
Hypersecretion of gastric juice may cause duodenal hypoglycaemia (see Chapter 12).
ulceration. However, there is overlap between the
amount of acid secreted in normal subjects and in those Both these dumping syndromes can be minimized if
with duodenal ulceration. The estimation of gastric frequent, small, low-carbohydrate meals are taken.
acidity is of very limited diagnostic value. In the very
rare Zollinger–Ellison syndrome, acid secretion is very NORMAL INTESTINAL ABSORPTION
high due to excessive gastrin produced by a gastrinoma There are three phases of digestion and absorption:
– a tumour more usually involving the pancreas or luminal phase, mucosal phase and post-absorptive
duodenum (see later). phase.
Drugs such as ranitidine compete with histamine Absorption depends on:
for the H2 receptors, so directly reducing acid secretion.
● the presence of nutrient in an absorbable form (and
Proton pump inhibitors such as omeprazole and
therefore on normal digestion),
lansoprazole have to some degree surpassed these
● the integrity and large surface area of absorptive
agents.
cells,
Hyposecretion of gastric juice occurs most
● a normal ratio of the rate of absorption to the rate
commonly in association with pernicious anaemia,
of passage of contents through the intestinal tract.
due to the formation of antibodies to the parietal cells
of the gastric mucosa. Extensive carcinoma of the The absorptive area of the small intestine is very
stomach and chronic gastritis may also cause gastric large. The mucosa forms macroscopically visible folds,
hyposecretion. Plasma gastrin concentrations are increasing the area considerably. Microscopically, these
raised, because reduced acid secretion causes the loss of folds are covered with villi lined with absorptive cells
negative feedback inhibition. (enterocytes), which increase the area about eight-fold.
Tests of gastric function involving the measurement A large number of minute projections (microvilli)
of acid secretion have largely been superseded by cover the surface of each enterocyte, separated by
endoscopic examination of the stomach and duodenum microvillous spaces. These increase the absorptive
Normal intestinal absorption 237
area about a further 20-fold. The absorptive area is disaccharidases, b-galactosidases (lactase) and
significantly reduced if the villi are flattened, as they a-glucosidases (maltase, sucrase), located on the
are, for example, in gluten-sensitive enteropathy. surface of enterocytes (‘brush border’), especially in the
Absorption also depends on whether a molecule is: proximal jejunum. Sucrase also hydrolyses the 1:4, and
isomaltase the 1:6, linkages of limit dextrans.
● water soluble, so that it can be absorbed either by
Monosaccharides are actively absorbed in the
active transport against a physicochemical gradient
duodenum and proximal jejunum. Unlike for fructose,
or by passive diffusion along gradients,
a common active process absorbs glucose and galactose.
● lipid soluble, enabling it to diffuse across the lipid
Carbohydrate absorption depends on:
membranes of the enterocytes.
● the presence of amylase, and therefore on normal
The release of intestinal secretions and the control
pancreatic function (polysaccharides only),
of gut motility are, in part, controlled by a series of
● the presence of disaccharidases on the luminal
peptide hormones produced in the mucosa of the
membrane of intestinal cells (disaccharides),
gastrointestinal tract. Like other hormones, their release
● normal intestinal mucosal cells with normal active
is under feedback control by either the product or the
transport mechanisms (monosaccharides).
physiological response of the target tissue.
Cells that synthesize gut peptides are scattered Polysaccharides can be absorbed only if all three
throughout the length of the gastrointestinal tract, mechanisms are functioning.
although the secretory cells are often concentrated in
one segment of the tract. Many of these peptides are Xylose absorption test
present in other organs, particularly the brain, where
they probably act as neurotransmitters. This can be used to test the integrity of the intestinal
mucosa. Xylose is a pentose that, like glucose, can be
● Gastrin is released from the gastrin-secreting G absorbed without digestion (by facilitated diffusion),
cells in the gastric antrum in response to distension but the metabolism of which is not hormone controlled;
and to protein. It stimulates the contraction of the therefore, unlike glucose, it is not affected by insulin
stomach muscles and the secretion of gastric acid. secretion.
Acid inhibits gastrin release by negative feedback. An oral dose of xylose is absorbed by normally
● Cholecystokinin stimulates contraction of the gall functioning upper small intestinal cells. Metabolism
bladder and the release of pancreatic digestive of the absorbed pentose is very slow and, because it
enzymes. is freely filtered by the glomeruli, most of it appears
● Secretin stimulates the release of pancreatic fluid rich in the urine. In the xylose absorption test, either the
in bicarbonate. amount excreted in the urine during a fixed period or,
● Pancreatic polypeptide inhibits pancreatic secretion. in children, the plasma concentration at a defined time
● Vasoactive intestinal polypeptide (VIP) and motilin after the dose is measured.
regulate gut motility. Intestinal disease (for example blind loops) or
impaired mucosal surface area (as in coeliac disease) may
Carbohydrate absorption impair xylose absorption and therefore reduces excretion.
Pancreatic disease does not affect either process. Therefore
Polysaccharides, such as starch, consist either of straight
this test can distinguish between pancreatic insufficiency
chains of glucose molecules joined by 1:4 linkages,
and conditions affecting the intestinal mucosa.
called amylose, or of branch chains, in which the
Unfortunately, there are several sources of error in
branches are joined by 1:6 linkages, called amylopectin.
the xylose test:
Salivary and pancreatic a-amylases hydrolyse starch to
1:4 disaccharides such as maltose (glucose + glucose). ● Xylose is absorbed mostly from the upper small
Pancreatic amylase is quantitatively the more important. intestine and absorption may therefore be normal
A few larger branch-chain saccharides (limit dextrans) despite significant ileal dysfunction, for example in
remain undigested. Crohn’s disease.
Disaccharides [maltose, sucrose (glucose + fructose) ● The urine test depends on accurate collections
and lactose (glucose + galactose)] are hydrolysed to over a short time interval and therefore even more
their constituent monosaccharides by the appropriate significant errors may occur than in 24-h collections.
238 The gastrointestinal tract
● Mildly impaired renal function decreases glomerular by enterokinase (enteropeptidase) located on the brush
filtration of xylose and may give falsely low results in border. The products of digestion are small peptides
the urine tests, or a falsely high plasma concentration. and amino acids. Many peptides are further hydrolysed
This is a common cause of misleading results in by peptidases on the brush border.
the elderly. The xylose absorption test should not Amino acids are actively absorbed in the small
be performed if glomerular function is even only intestine. Small peptides are absorbed by an active
marginally impaired. transport mechanism, independently of amino acids
● Both plasma concentrations and urine excretion and, with a few exceptions, are hydrolysed intracellularly.
depend partly on the volume of distribution of Protein absorption depends on the presence of
the absorbed xylose. In oedematous or very obese pancreatic proteolytic enzymes (and therefore on
patients, results may be falsely low. normal pancreatic function) and an intestinal mucosa
with normal active transport mechanisms.
A possible scheme for carrying out the xylose test is
now discussed. Lipid absorption
Procedure Digestion of fats
A dose of 5 g of xylose is preferable to one of 25 g because Triglycerides are the main form of dietary fat and are
a high xylose concentration within the intestinal lumen esters of glycerol with three, usually different, fatty
may, like a large dose of glucose, have an osmotic effect acids. They are insoluble in water. Cholesterol, in the
that causes symptoms and affects the results. intestinal lumen, is derived from bile salts and from the
diet; only about 30 per cent of the dietary cholesterol is
● The patient fasts overnight. absorbed (see Chapter 13).
● 08.00 h The bladder is emptied and the specimen Primary bile acids are synthesized in the liver from
discarded. Then 5 g of xylose dissolved in 200 mL of cholesterol, conjugated with the amino acid glycine or
water is given orally. All specimens passed between taurine in the liver, and then enter the intestinal lumen
08.00 h and 10.00 h are put into a bottle labelled in the bile. In the alkaline duodenal fluid their sodium
number 1. salts act as detergents, emulsifying and so facilitating the
● 10.00 h The bladder is emptied and the specimen is digestion and absorption of fats. Most of the bile salts are
put into bottle number 1, which is now complete. All actively reabsorbed in the distal ileum, although some
specimens passed between 10.00 h and 13.00 h are enter the colon where they are converted by bacteria
put into a bottle labelled number 2. to secondary bile salts, some of which are absorbed.
● 13.00 h The bladder is emptied and the specimen is The absorbed bile salts are recirculated to the liver and
put into bottle number 2, which is now complete. resecreted into the bile (the enterohepatic circulation).
Both bottles are sent to the laboratory for analysis. Some bacteria within the gut lumen contain enzymes
that catalyse the deconjugation of bile salts; unconjugated
Interpretation
bile salts emulsify fat less effectively than conjugated
In the normal subject, more than 1.5 g xylose should be ones and this may contribute to fat malabsorption.
excreted during the 5 h. About 50 per cent or more of Triglycerides are emulsified by bile salts within the
the total excretion should occur during the first 2 h. In duodenum. They are hydrolysed by pancreatic lipase at
mild intestinal malabsorption, the total 5-h excretion the glycerol/fatty acid bond, primarily in positions 1 and
may be normal, but delayed absorption is reflected 3. The end products are mainly 2-monoglycerides, some
in a 2:5-h ratio of less than 40 per cent. In pancreatic diglycerides and free fatty acids. Colipase, a peptide
malabsorption, the result should be normal. coenzyme secreted by the pancreas, is essential for lipase
activity. It anchors lipase at the fat/water interface,
Protein absorption prevents the inhibition of the enzyme by bile salts and
Protein in the intestinal lumen is derived from the reduces its pH optimum from about 8.5 to about 6.5;
diet, intestinal secretions and desquamated mucosal the latter is the pH in the upper intestinal lumen.
cells. Dietary protein is broken down by gastric
pepsin and in the duodenum by trypsin and other Micelle formation
proteolytic enzymes secreted in pancreatic juice; Monoglycerides and free fatty acids aggregate with
pancreatic trypsinogen is converted to active trypsin bile salts to form water-miscible micelles. The micelles
Normal intestinal absorption 239
also contain free cholesterol liberated by the hydrolysis almost all the fat is of endogenous origin. However, if
of cholesterol esters in the lumen and phospholipids, the patient is on a very low-fat diet, mild steatorrhoea
as well as fat-soluble vitamins (A, D, E and K). The may be undetected.
diameter of the negatively charged micelles is between
The triolein breath test
100 and 1000 times smaller than that of the emulsion
particles. Both their small size and their charge allow The triolein breath test has also been used to assess fat
them to pass through the narrow microvillous spaces. absorption. Absorbed fat is ultimately metabolized to
Within enterocytes, triglycerides are resynthesized CO2 and water. If 14C-labelled triglyceride (triolein) is
from monoglycerides and fatty acids, and cholesterol given by mouth, the ratio of 14CO2 to unlabelled CO2 can
is re-esterified. Triglycerides, cholesterol esters and be measured in expired air. A low ratio usually indicates
phospholipids, together with fat-soluble vitamins, impaired fat absorption. The assay uses expensive
combine with apolipoproteins manufactured in isotopes and requires special expertise. Although the
enterocytes to form chylomicrons. These are suspended results correlate with faecal fat measurements, these
in water and pass into the lymphatic circulation. limitations have prevented its widespread adoption.
Some short-chain and medium-chain free fatty acids Vitamin B12 absorption
pass directly through the intestinal cells into the portal
Vitamin B12 can be absorbed only when it has formed a
bloodstream.
complex with intrinsic factor, a glycoprotein secreted by
The absorption of lipids and fat-soluble vitamins
the parietal cells of the stomach (see Chapter 15). This
depends on:
complex is resistant to proteolytic digestion and binds
● an adequate absorptive area of the intestinal mucosa to specific cell receptors in the distal ileum, from where
for chylomicron formation, it is absorbed. Some intestinal bacteria need vitamin
● the presence of bile salts, B12 for growth and may prevent its absorption by
● the digestion of triglycerides by lipase, and therefore competing for it with intestinal cells. Intestinal bacterial
on normal pancreatic function. overgrowth is associated with intestinal strictures,
diverticula and ‘blind loops’. Vitamin B12 absorption
Faecal fat estimation test therefore depends on normal gastric secretion of
Faecal fat excretion should represent the difference intrinsic factor, intestinal mucosa in the distal ileum
between the fat absorbed and that entering the and intestinal bacterial flora.
gastrointestinal tract from the diet and from the
body. The absorption of fat and, more importantly, Absorption of other water-soluble vitamins
the addition of fat to the intestinal contents occur Most of the other water-soluble vitamins (C and the
throughout the small intestine. A single 24-h collection B group, except B12) are absorbed in the upper small
of faeces gives very inaccurate results for two reasons: intestine, probably by specific transport mechanisms.
the transit time from the duodenum to the rectum is Clinical deficiencies of all but folate are relatively
variable, and rectal emptying is variable and may not uncommon in the malabsorption syndromes, probably
be complete. because their absorption does not depend on that
It has been suggested by some experts that the faecal of fat. Folate deficiency may be caused by intestinal
fat test is too inaccurate for clinical use and should be malabsorption, inadequate intake or increased use by
abandoned. Indeed visual inspection looking for fat intestinal bacteria (see Chapter 15).
globules in stools may be as sensitive and specific. Calcium and magnesium absorption
Consecutive 24-h collections give faecal fat results
that may vary by several hundred per cent. The Calcium is absorbed mainly from the duodenum
longer the period of collection, the more accurate the and upper jejunum in the free ionized form;
calculated daily mean result; ideally, stools should be this is enhanced by the vitamin D metabolite
collected for a minimum of 3 days. 1,25-dihydroxycholecalciferol, which influences
both active transport and passive diffusion. Calcium
Interpretation absorption is impaired by the formation of insoluble
A mean daily fat excretion of more than 18 mmol (5 g) salts with free fatty acids and with phosphate. Much
indicates steatorrhoea. The result is not affected by diet of the faecal calcium is endogenous in origin, being
within very wide limits, because in the normal subject derived from intestinal secretions (see Chapter 6).
240 The gastrointestinal tract
An active process that may be shared with calcium activity increases about five-fold in acute pancreatitis,
also absorbs magnesium. Calcium and magnesium but plasma enzyme activities of up to, and even above,
absorption depends on: this value may be reached in a number of other disorders,
in particular after intestinal perforation and renal failure.
● a low concentration of fatty acids and phosphate in
Occasionally, the plasma enzyme activity in acute
the intestinal lumen,
pancreatitis may not be very high and usually falls rapidly
● the absorption and metabolism of vitamin D, and
as the enzyme is excreted in the urine. Consequently, a
therefore on normal fat absorption,
high plasma amylase activity is only a rough guide to
● normal intestinal mucosal cells.
the presence of acute pancreatitis, and normal or only
slightly raised values do not exclude the diagnosis.
Iron absorption
Plasma amylase concentrations can be spuriously
Iron is absorbed in the duodenum and upper jejunum low or normal in acute pancreatitis evoked by severe
in the ferrous form (Fe2+). Absorption is increased by hypertriglyceridaemia. In such circumstances a raised
anaemia of any kind and probably also by vitamin C. urinary amylase concentration may facilitate diagnosis
Some of the iron absorbed into intestinal cells enters or clearing the plasma lipaemia by centrifugation prior to
the plasma, but some is lost into the lumen with assay. Haemorrhagic pancreatitis or chronic pancreatitis
desquamated cells (see Chapter 21). can also show a normal plasma amylase concentration.
NORMAL PANCREATIC FUNCTION Lipase
Pancreatic secretions can be divided into endocrine This enzyme is more specific for the pancreas and can
and exocrine components. The endocrine function be useful to measure if the source of a raised plasma
and insulin and glucagon that control the plasma amylase concentration is not obvious. Lipase has a
glucose concentration are discussed in Chapter 12. The longer half-life than amylase and thus may be useful in
exocrine secretions are made up of two components, the the late diagnosis of acute pancreatitis, when amylase
alkaline pancreatic fluid and the digestive enzymes. The activity can fall within the reference range.
alkaline fluid is primarily responsible for neutralizing
gastric acid secretions, thus providing an optimal Trypsin
environment for duodenal digestive enzyme activity. This may be used to screen for cystic fibrosis (see
These enzymes include the proteases trypsin, elastase later and Chapter 27) during the first 6 weeks of life.
and chymotrypsin, and amylase and lipase. Some of the Blockage of pancreatic ductules by sticky mucous
proteases are secreted as precursors and are converted secretion causes high plasma trypsin concentrations.
to the active form within the intestinal lumen. After about 6 weeks, plasma concentrations may fall as
Gut peptides control pancreatic secretion and are pancreatic insufficiency develops; normal levels do not
released from the duodenum in response to a rise in the exclude the diagnosis.
hydrogen ion concentration or to the presence of food.
They include secretin (which stimulates the release of Duodenal enzymes
a high volume of alkaline fluid) and cholecystokinin Measurement of pancreatic enzymes and the
(which stimulates the release of a fluid rich in enzymes). bicarbonate concentration in duodenal aspirates
before and after stimulation with cholecystokinin and
Tests of exocrine pancreatic function
secretin is not very suitable for routine use because of
Plasma enzymes the difficulty in positioning the duodenal tube and in
Plasma enzyme measurements are of limited value in quantitative sampling of the secretions.
assessing exocrine function. They include the following. ‘Tubeless tests’ have been developed that avoid the
Amylase
need for intubation and that overcome the difficulties
of invasive sample collection.
This enzyme consists of two forms, one of salivary gland
origin and one of pancreatic origin. In acute pancreatitis, The PABA test
total plasma amylase activity is usually significantly A synthetic peptide, labelled with para-aminobenzoic
increased due to release from damaged cells. acid (PABA), is taken orally. After PABA has been
Plasma amylase is discussed more fully in Chapter 18 split from the peptide by chymotrypsin, it is absorbed
in the context of enzymology. Typically, plasma amylase and excreted in the urine. Urinary excretion of PABA
Pancreatic disorders 241
Mechanisms of malabsorption
Reduction of absorptive areas or generalized impairment of
transport mechanisms
Villous atrophy
Malabsorption may be caused by a reduction of the
absorptive area because of flattened intestinal villi,
demonstrated by microscopic examination of a mucosal
biopsy specimen, and by the following:
● Coeliac disease (gluten-sensitive enteropathy)
e is caused by sensitivity to the protein a-gliadin,
present in gluten in wheat and rye. The condition
Figure 16.1 Obstructive jaundice due to carcinoma of
is characterized by villous atrophy, mainly in the
head of pancreas. Reproduced with kind permission
from Kinirons M and Ellis H. French’s Index of
proximal small intestine, which is usually reversible
Differential Diagnosis, 15th edition. London: Hodder following withdrawal of gluten from the diet.
Arnold, 2011. Clinical symptoms usually become evident at about
1 year of age with failure to thrive and diarrhoea,
but may present at any age, including in adulthood.
MALABSORPTION SYNDROMES
Clinical features may include abdominal pain and
Generalized malabsorption may result from either
intestinal or pancreatic disease:
CASE 2
● In intestinal malabsorption, fat digestion is usually
normal, but absorption of the products of digestion A 31-year-old woman presented to the gastro-
is impaired. enterologists with weight loss of about 15 kg in 1 year,
● In pancreatic malabsorption, the absorptive capacity frequent diarrhoea and abdominal distension. There
is normal, but fat cannot be digested because there is was a family history of coeliac disease. On examination,
deficiency of digestive enzymes. she was found to have abdominal tenderness and
looked pale. Her blood count showed a haemoglobin
level of 8.9 g/dL and she had a low plasma folate
Steatorrhoea
concentration. Her anti-transglutaminase antibodies
Malabsorption is usually, but not always, associated with were strongly positive. A jejunal biopsy was reported
impaired fat absorption (steatorrhoea). No biochemical as showing villous atrophy.
test is sensitive enough for the early detection, or precise
DISCUSSION
enough for the differential diagnosis, of steatorrhoea;
The clinical history and clinical features are typical
endoscopy and ultrasound have reduced the need
of coeliac disease. Note also the folate deficiency. The
for laboratory tests. However, biochemical tests are
patient’s symptoms improved on a gluten-free diet,
important in detecting and monitoring the treatment
with gain in body weight and normalization of her
of the metabolic effects of prolonged and severe
haemoglobin.
malabsorption.
Malabsorption syndromes 243
distension, diarrhoea, tiredness, mouth ulcers, Increased rate of transit through the small intestine
anaemia, malabsorption and increased risk of Food passes through the intestine more rapidly than
intestinal adenocarcinoma and lymphoma. It can usual in the following circumstances.
be associated also with hyposplenism, infertility,
abnormal hepatic function, dermatitis herpetiformis ● After gastrectomy, when normal mixing of food with
and other autoimmune disorders such as type 1 fluid, acid and pepsin does not occur in the stomach,
diabetes mellitus, autoimmune thyroiditis and resulting in impaired enzyme activity within the
primary biliary cirrhosis. small intestine. An increased rate of transit through
Antibodies to a-gliadin or endomysium or the duodenum may contribute to the malabsorption.
tissue transglutaminase (tTG) also may be detected However, post-gastrectomy malabsorption is rarely
in plasma and their measurement can be used severe, although iron deficiency is a relatively
in diagnosis and to monitor compliance with a common complication.
gluten-free diet. Antibodies to tTG have about ● In carcinoid syndrome, which is a rare condition
95 per cent sensitivity for coeliac disease. Exclude associated with excessive production of
immunoglobulin A (IgA) deficiency, which is more 5-hydroxytryptamine (serotonin) by tumours of
common in patients with coeliac disease, as some argentaffin cells usually arising in the small intestine,
assays for coeliac screening look at IgA antibodies, or by their metastases, for example in the liver. It
which may be falsely negative in IgA deficiency may occasionally present with malabsorption, which
(see Chapter 19). Indeed, IgA anti-tTG assay is a is probably the result of increased intestinal motility
screening test, but, in IgA deficiency, IgG anti-tTG due to the concomitant release of the gut peptide
assay is preferable to help ascertain the need for substance P (see Chapter 24).
intestinal biopsy. However, IgG anti-tTG is not as Impaired digestive enzyme activity
sensitive or specific as IgA antibodies, and biopsy Failure of digestive enzyme secretion
may still be clinically indicated if this test is negative.
Pancreatic dysfunction may cause malabsorption
It is important to also note that false-positive tests
by reducing the secretion of digestive enzymes. The
can occur in other bowel diseases such as Crohn’s
following are some important examples:
disease.
● Tropical sprue, in which villous atrophy does not ● Chronic pancreatitis: commonly due to alcohol
respond to a gluten-free diet. There may be a bacterial excess, this is not always associated with steatorrhoea
cause because the condition sometimes responds to or malabsorption. It rarely follows a severe attack of
broad-spectrum antibiotics and folate supplements. acute pancreatitis, but is more likely after repeated
● Extensive surgical resection or fistulae of the small acute or subacute attacks. Diabetes mellitus requiring
intestine may so reduce the absorptive area as to cause insulin may occur due to islet cell damage. There
malabsorption. Normally the small bowel spans from may be steatorrhoea due to the impairment of lipase
260 cm to 800 cm. Any disease, trauma, radiation or activity because of bile salt deficiency. Pancreatic
vascular event that leaves less than 200 cm of viable lipase activity can be reduced by up to 75 per cent
small bowel remaining puts the patient at risk of before chemical steatorrhoea occurs.
developing short-gut syndrome. The short-gut Low plasma trypsin levels are relatively specific
syndrome can present with malabsorption, diarrhoea for chronic pancreatitis, but malabsorption does
and undernutrition. Electrolyte disturbances may not tend to occur until more than 90 per cent of
include severe hypokalaemia and hypomagnesaemia the pancreas has been destroyed. Similarly, there
as well as fluid depletion. Intestinal failure can be may also be low faecal elastase or chymotrypsin
defined as a condition in which there is inability of the concentrations. Direct pancreatic enzyme tests are
intestine to absorb nutrients and water sufficiently. sometimes needed to detect the disease in its early
● Infiltration or inflammation of the small intestinal stages.
wall, for example caused by Crohn’s or Whipple’s Direct invasive tests require specialist expertise
disease or small intestinal lymphoma, may impair and involve collecting duodenal aspirates by a tube
absorption. Malabsorption may be aggravated by in a cannulated pancreatic duct, sometimes in
alteration of the bacterial flora due to reduced conjunction with endoscopic retrograde cholangio-
intestinal motility. pancreatography (ERCP) after stimulation by
244 The gastrointestinal tract
– A dimorphic (mixed iron deficiency and intestinal histological picture is normal, the most
macrocytic) picture is very suggestive of likely possibilities are:
intestinal malabsorption. – localized intestinal disease, such as Crohn’s
– Low red cell folate and/or plasma vitamin B12 disease,
concentrations suggest intestinal malabsorption. – contaminated small bowel (‘blind loop’)
● If doubt remains, faecal fat estimation may help. syndrome,
However, because of the difficulty of obtaining an – pancreatic disease such as chronic pancreatitis.
accurately timed faecal collection, only very high ● Intestinal malabsorption may need further
results are of unequivocal significance; in such cases anatomical delineation. Ileal disease may require a
steatorrhoea will probably be obvious visually. For Schilling test or 14C-radiolabelled glycocholic acid
this reason many hospital laboratories no longer test. Bacterial intestinal overgrowth may require one
perform this test. In any case, normal faecal fat of the breath tests.
does not exclude malabsorption, as carbohydrate Figure 16.2 gives a summary algorithm for the
absorption may be impaired. investigation of steatorrhoea (see also Box 16.1).
● Slow stool elastase levels are indicative of
malabsorption of pancreatic origin. Laboratory tests to identify some metabolic
● The xylose absorption test is sometimes helpful complications of malabsorption
in distinguishing pancreatic malabsorption from Laboratory investigations of plasma can be carried out
intestinal problems. If it is unequivocally normal, an to identify some of the complications of generalized
upper intestinal lesion is unlikely. Such a result does malabsorption:
not, however, exclude ileal or pancreatic disease,
contaminated bowel syndrome, or even some cases
of coeliac disease. Steatorrhoea?
● A pancreolauryl test or PABA test if available
may also help confirm the presence of pancreatic Faecal fat
insufficiency. Some authorities suggest the gold
standard test for assessing pancreatic function is the
secretin– pancreozymin test, but this is invasive and Normal Abnormal
requires specialist skill.
● If any or all of the above investigations suggest Probably not Xylose
steatorrhoea absorption
steatorrhoea, a cause should be sought. The following
if no hepatobiliary test
are the most definitive tests: disease present
– Endoscopy, with histological examination of
an intestinal biopsy specimen. Flattened villi
Normal Abnormal
suggest: coeliac disease or tropical sprue.
– Giardia lamblia may be detected
PABA or Upper small
microscopically, and stool culture is useful in
pancreolauryl intestine disease
case of other organisms. test or faecal likely. Confirm
– Radiological examination may detect abnormal elastase with biopsy
intestinal mucosa.
● The diagnosis of chronic pancreatitis and of causes
of pancreatic malabsorption may also need imaging Normal Abnormal
techniques such as ERCP or MRCP. Tests such as
abdominal ultrasound or CT scanning may help, Bacterial Pancreatic
especially if pancreatic carcinoma is suspected. bowel disease?
overgrowth?
Plain abdominal radiographs may show pancreatic
calcification. Figure 16.2 Algorithm for the biochemical investigation
● If steatorrhoea is obvious on clinical grounds of steatorrhoea (imaging and endoscopy investigations
and after visual inspection of the stool, and if the may also be required). PABA, para-amino benzoic acid.
Malabsorption syndromes 249
● Plasma electrolytes may show hypernatraemia and Other gastrointestinal conditions in which biochemical
impaired renal function if there is significant water tests may be useful
loss. Purgative or laxative abuse
● Plasma potassium and magnesium may reveal the Some individuals abuse purgatives and laxatives,
presence of hypokalaemia and hypomagnesaemia. and may have psychiatric problems, including
● There may sometimes be a metabolic acidosis, Munchausen’s syndrome. Stool samples can be screened
which may be due to D-lactic acidosis (if bacterial for purgatives such as senna or phenolphthalein by thin
overgrowth), or a hyperchloraemic acidosis (see layer chromatography. Also look out for associated
Chapter 4). hypokalaemia due to gastrointestinal potassium loss
Investigations may also reveal the following: resulting from this (see Chapter 5).
Diarrhoea fluid can be classified into two types:
● Anaemia: haematological investigations such as osmotic and secretory. The former could be due to
ferritin, folate and vitamin B12. lactose intolerance, or osmotic laxatives containing
● Rickets/osteomalacia: plasma calcium with albumin, magnesium or lactulose. The latter could be the result
phosphate and alkaline phosphatase activity. of infections, inflammatory bowel disease, carcinoid
● Abnormal bleeding (vitamin K deficiency): clotting syndrome or VIPoma, to name but a few. The osmolality
studies such as prolonged prothrombin time. of faecal water can be calculated from:
● Low plasma immunoglobulin (hypogammaglobul-
inaemia) or low plasma cholesterol (hypocholest- 2 [sodium + potassium] (16.1)
erolaemia) concentrations. If the measured osmolality is greater than the
calculated osmolality, an osmotically active constituent
Box 16.1 Some causes of malabsorption is likely to be present, suggestive of osmotic diarrhoea
(see Chapter 2).
Luminal phase abnormalities
Helicobacter pylori infection
Abnormal hydrolysis of nutrients
Pancreatic insufficiency Helicobacter pylori is found in up to 50 per cent of
Rapid gut transit European adults and is associated with peptic ulcer,
Enzyme inactivation by gastric hypersecretion gastric adenocarcinoma and non-Hodgkin’s lymphoma
Enzyme deficiencies, e.g. enterokinase or trypsin of the stomach. There are many laboratory tests for this
Decreased bile acid secretion, e.g. hepatic organism, including microscopy, bacterial culture and
disease, biliary obstruction the direct urease test using gastric mucosa biopsies. Non-
Intestinal stasis, e.g. scleroderma, obstruction,
invasive tests include blood, stool or saliva IgG antibody
blind loops
Abnormal luminal processing
studies to detect Helicobacter pylori infection, or breath
Bacterial overgrowth tests that look at labelled carbon, which is released by
Lack of intrinsic factor, e.g. vitamin B12 deficiency this urea-splitting organism. The latter test is useful in
Mucosal phase abnormalities determining whether the organism has been eradicated.
Brush-border abnormality, e.g. lactase or sucrase–
Inflammatory bowel disease
isomaltase deficiency
Glucose–galactose malabsorption, Hartnup’s disease In inflammatory bowel disease such as Crohn’s disease
Acquired defects elevated faecal calprotectin may occur and may be
Gut resection a marker of disease activity. Calprotectin is a 36-kDa
Poor absorbing surface, e.g. coeliac disease, calcium-binding protein secreted by neutrophils and
sprue, radiation damage, gut infiltrations such as concentrations are usually normal in irritable bowel
lymphoma syndrome.
Infection such as acquired immunodeficiency
syndrome (AIDS), giardiasis, Whipple’s disease Colorectal carcinoma
Post-absorptive phase abnormalities
Colorectal carcinoma is one of the most prevalent
Lymphatic obstruction, e.g. intestinal lymphangiectasia
Lymphoma
cancers. Screening has been shown to reduce mortality,
Protein-losing enteropathy but direct visualization by colonscopy is invasive, costly
and requires skilled personnel.
250 The gastrointestinal tract
At present, faecal occult blood tests are being used for ● In cases in which basal plasma gastrin concentrations
screening purposes, as the tumour often releases blood are equivocal, the secretin test may be useful.
into the gastrointestinal tract. This can be observed by Intravenous secretin (2 units/kg body weight) raises
point-of-care testing or laboratory tests that analyse the plasma gastrin concentration to more than 200 µg/L
blood in small faecal samples. However, false-positives within 10 min if a gastrinoma is present.
can occur in people with a high meat intake. ● Imaging techniques such as CT, MRI and endoscopic
Future screening tests may look at altered faecal ultrasound may be useful, and also somatostatin
deoxyribonucleic acid (DNA) derived from the tumour. receptor scintigraphy to localize tumours and
Plasma carcinoembryonic antigen (CEA) can be used metastases.
to monitor the treatment of colorectal carcinoma. (See
Chapter 24 for tumour markers.) Glucagonomas
Glucagonomas usually arise from the a-cells of the
Tumours of the enteropancreatic system secreting gut
hormones pancreatic islets. The presenting clinical feature is a
bullous rash, known as necrolytic migratory erythema,
(It is suggested that this section is read in conjunction
sometimes accompanied by psychiatric disturbances,
with Chapter 24.) These tumours are very rare. They
thromboembolism, glossitis, weight loss, impaired
are usually found in the pancreatic islets and cause
glucose tolerance, anaemia and a raised erythrocyte
well-recognized clinical and pathological syndromes.
sedimentation rate. Diagnosis may be confirmed by
Gastrinomas
finding a very high plasma glucagon concentration.
Gastrinomas, usually in the pancreatic islets, secrete VIPomas
large quantities of gastrin, despite high gastric VIPomas are extremely rare tumours, usually of the
hydrogen ion levels, and may cause the Zollinger– pancreatic islet cells, that secrete VIP (vasoactive
Ellison syndrome. About 60 per cent of gastrinomas intestinal polypeptide), a hormone that increases
are malignant. Of the remaining 40 per cent, only intestinal motility. They cause a syndrome in which
about one-third (13 per cent of the total) are single, there is very profuse, watery diarrhoea – the Verner–
resectable adenomas, the rest being multiple. The very Morrison or watery diarrhoea, hypokalaemia and
high rate of gastric acid secretion causes ulceration in achlorhydria syndrome. Plasma concentrations of VIP
the stomach and proximal small intestine with severe are high.
diarrhoea; inhibition of pancreatic lipase activity by the
low pH may cause steatorrhoea. This syndrome may be LABORATORY INVESTIGATION OF
associated with benign, and usually non-functioning, ABDOMINAL PAIN
adenomas elsewhere, for example in the anterior In some cases, a clinical history and examination with
pituitary, parathyroid and thyroid glands and in the imaging techniques such as abdominal radiograph,
adrenal cortex [multiple endocrine neoplasia (MEN1) ultrasound, CT or even laparotomy may reveal a
syndrome]. The diagnosis depends on the following: diagnosis. However, sometimes biochemical tests may
help, particularly in the following situations:
● Fasting plasma gastrin concentrations are up to
30 times the upper reference limit in Zollinger– ● Plasma amylase or lipase concentration may be
Ellison syndrome. Plasma concentrations more than raised in acute pancreatitis (see Chapter 18).
1000 µg/L with acid hypersecretion strongly support ● Gallstones may present with abnormal liver function
this diagnosis. tests, in particular plasma bilirubin and alkaline
● However, high plasma gastrin concentrations may phosphatase. They may sometimes be associated
also be caused by hypochlorhydria, for example with jaundice (see Chapter 17).
atrophic gastritis, pernicious anaemia or post ● Diabetes ketoacidosis can present with an acute
vagotomy. H2 blockers and proton pump inhibitors abdomen, and therefore hyperglycaemia and urinary
are usually stopped prior to sampling. ketones are present (see Chapter 12).
● The Zollinger–Ellison syndrome basal gastric acid ● Hypercalcaemia is another cause of abdominal pain
output may be more than 15 mmol/h, with large (see Chapter 6).
gastric secretory volumes and gastric pH of less ● Severe hypokalaemia can evoke a paralytic ileus and
than 2.0. intestinal obstruction (see Chapter 5).
Laboratory investigation of abdominal pain 251
● Sickle cell disease or crisis can be investigated by by a raised concentration of plasma b-human
haemoglobin studies for HbS. chorionic gonadotrophin and pelvic ultrasound (see
● Acute porphyria can present as an acute abdomen Chapter 10).
(see Chapter 21).
● Addison’s disease can present as an adrenal crisis (see Clinical biochemistry laboratory tests can also
Chapter 8). be used in the management of abdominal pain, for
● Heavy metal poisoning, for example lead, can cause example post-operatively. Plasma urea and electrolytes
abdominal pain (see Chapter 21). and creatinine as well as chloride and bicarbonate
● Ectopic pregnancy should be considered in concentrations are useful, particularly if intravenous
women of reproductive age and can be detected fluids are given or an acid–base disturbance is suspected.
SUMMARY
● The routine hospital chemical pathology laboratory Vitamin deficiencies may result, as can
has a role in the investigation of gastrointestinal hypoalbuminaemia and hypogammaglobulinaemia.
disorders, but the tests carried out in the laboratory ● Acute pancreatitis is a potentially lethal condition;
do not replace more specialized invasive tests such its diagnosis is supported by the presence of elevated
as endoscopy or radiology. plasma amylase or lipase.
● Normal intestinal, hepatic and pancreatic function ● Intestinal failure, for example gastrointestinal
is essential for digestion and absorption. fistula or short gut, can result in severe fluid loss
● The gut is not merely an absorbing surface, but and electrolyte disturbance.
also secretes hormones and is controlled by both ● Abdominal pain is common and some of its causes
humoral and neural pathways. are biochemical.
● Malabsorption can be due to various pathologies, ● The gastrointestinal tract is the source of various
which should be sought and which can present as tumours, the diagnosis and treatment of which can
abdominal discomfort, weight loss and steatorrhoea. be assisted by biochemical tests.
17 Liver disorders and gallstones
This chapter looks at the chemical pathology of liver lobule are the portal tracts that contain branches of
and gall bladder disorders. These are common in the hepatic artery, the portal vein and bile ducts. Blood
clinical practice, and liver function tests constitute one flows from the portal tracts towards the central hepatic
of the most frequently requested clinical biochemistry vein. Therefore:
laboratory profiles.
● Hypoxia and toxins that are metabolized in the liver
FUNCTIONS OF THE LIVER cause damage to the centrilobular area first.
The liver has essential synthetic and excretory functions ● Toxins that do not depend on hepatic metabolism
and can be thought of as a large ‘metabolic factory’. It also primarily affect the periphery of the lobule.
detoxifies and, like the kidneys, excretes the end products Almost all nutrients from the gastrointestinal tract
of metabolism. The main blood supply to the liver is via pass through the sinusoidal spaces prior to entering the
the portal vein. The liver is made up of hexagonal lobules systemic circulation. The hepatic architecture may be
of cells (Fig. 17.1). Rows of hepatocytes radiate from disturbed in cirrhosis (fibrosis).
the central hepatic vein and are separated by sinusoidal
spaces, along the walls of which are interspersed hepatic General metabolic functions
macrophages, the Kupffer cells. These phagocytic cells When the glucose concentration is high in the portal
are part of the reticuloendothelial system and have an vein, it is converted to glycogen and the carbon skeletons
important detoxifying function. At the corners of each of fatty acids, which are transported to adipose tissue as
very low-density lipoprotein (VLDL). During fasting,
the systemic plasma glucose concentration is maintained
by the breakdown of glycogen (glycogenolysis) or by the
Hepatic artery
Portal vein
Bile duct synthesis of glucose from substrates such as glycerol,
lactate and amino acids (gluconeogenesis). Fatty acids
reaching the liver from fat stores may be metabolized
in the tricarboxylic acid cycle, converted to ketones or
incorporated into triglycerides (see Chapter 13).
Central
hepatic Synthetic functions
vein
Hepatocytes synthesize:
● plasma proteins, excluding immunoglobulins and
complement,
● most coagulation factors, including fibrinogen and
Portal tract factors II (prothrombin), V, VII, IX, X, XI, XII and
XIII – of these, prothrombin (II) and factors VII, IX
Figure 17.1 Diagrammatic representation of a cross- and X cannot be synthesized without vitamin K,
section of a hepatic lobule showing the relation between ● primary bile acids,
the central hepatic vein and the portal tracts. Blood flows ● the lipoproteins, such as VLDL and high-density
towards the central vein, as indicated by the arrows. lipoprotein (HDL) (see Chapter 13).
Functions of the liver 253
The liver has a very large functional reserve. ● Many drugs – which are metabolized and inactivated
Deficiencies in synthetic function can be detected only if by enzymes of the endoplasmic reticulum system;
liver disease is extensive. Before a fall in plasma albumin some are excreted in the bile.
concentration is attributed to advanced liver disease, ● Toxins – the reticuloendothelial Kupffer cells in
extrahepatic causes must be excluded, such as the loss the hepatic sinusoids are well placed to extract
of protein through the kidney, gut or skin, or across toxic substances that have been absorbed from the
capillary membranes into the interstitial space, as in gastrointestinal tract.
even mild inflammation or infection (see Chapter 19).
Efficient excretion of the end products of metabolism
Prothrombin levels, assessed by measuring the
and of bilirubin depends on:
prothrombin time, may be reduced because of impaired
hepatic synthesis, whether due to failure to absorb ● normally functioning liver cells,
vitamin K or to hepatocellular damage. If hepatocellular ● normal blood flow through the liver,
function is adequate, parenteral administration of ● patent biliary ducts.
vitamin K may reverse the abnormality.
Formation and excretion of bilirubin (Fig. 17.2)
Excretion and detoxification
At the end of their lifespan, red blood cells are broken
The excretion of bilirubin is considered in more detail
down by the reticuloendothelial system, mainly in the
below. Other substances that are inactivated and
spleen. The released haemoglobin is split into globin,
excreted by the liver include the following:
which enters the general protein pool, and haem, which
● Cholesterol – excreted in the bile either unchanged or is converted to bilirubin after the removal of iron,
after conversion to bile acids. which is reused (see Chapter 21).
● Amino acids – which are deaminated in the liver. About 80 per cent of bilirubin is derived from haem
Amino groups, and the ammonia produced by within the reticuloendothelial system. Other sources
intestinal bacterial action and absorbed into the include the breakdown of immature red cells in the
portal vein, are converted to urea. bone marrow and of compounds chemically related to
● Steroid hormones – which are metabolized and haemoglobin, such as myoglobin and the cytochromes.
inactivated by conjugation with glucuronate and Less than 300 µmol of bilirubin is produced daily from
sulphate and excreted in the urine in these water- the breakdown of erythrocytes, while the normal
soluble forms. liver is able to conjugate up to about 1 mmol/day, and
SITE METABOLISM EXCRETION
Reticuloendothelial Haem
system
Uptake by
ligandin
Liver Conjugation
Intestinal tract
Faeces 170–300 µmol/day
Bacterial action
secretory) function are relatively insensitive indicators Patients with prolonged and more widespread
of liver disease. There are a number of new tests that are cholestasis may present with severe jaundice and
being devised to improve the accuracy of the diagnosis pruritus due to the deposition of retained bile salts in
of hepatic disorders. Tests of hepatocellular activity have the skin; the plasma bilirubin concentration may be
been proposed, such as galactose elimination capacity, more than 800 µmol/L. More rarely, there is bleeding
the aminopyrine breath test, indocyanine green due to malabsorption of vitamin K, with consequent
clearance, and monoethylglycinexylidide (MEGX) prothrombin deficiency. Cholesterol retention may
production. All these tests are indirect measures of cause hypercholesterolaemia. Dark urine and pale
hepatic activity that rely on measuring compounds or stools suggest biliary retention of conjugated bilirubin.
their metabolites after they have been acted on by the The jaundice caused by extrahepatic obstruction due
liver. As yet, they do not have a place in routine clinical to malignant tissue is typically painless and progressive,
diagnosis. but there may be a history of vague persistent back pain
and weight loss. By contrast, intraluminal obstruction
DISEASES OF THE LIVER by a gallstone may cause severe pain, which, like the
Cholestasis jaundice, is often intermittent. Gallstones may not
Cholestasis may be either: always cause such symptoms. If a large stone lodges in
the lower end of the common bile duct, the picture may
● intrahepatic, in which bile secretion from the
be indistinguishable from that of malignant obstruction.
hepatocytes into the canaliculi is impaired, due to:
Although most of the findings are directly
– viral hepatitis,
attributable to cholestasis, biliary back pressure may
– drugs such as chlorpromazine or toxins such as
damage hepatocytes, and plasma aminotransferase
alcohol,
activities may increase. Unless the cause is clinically
– inflammation of the biliary tract (cholangitis),
obvious, evidence of dilated ducts due to extrahepatic
– autoimmune disease (primary biliary
obstruction should be sought using tests such as
cirrhosis),
ultrasound, computerized tomography (CT) scanning
– cystic fibrosis,
or cholangiography.
● extrahepatic, due to obstruction to the flow of bile
CASE 1 CASE 2
A 52-year-old woman was referred to the hepatology A 22-year-old woman who was an intravenous drug
clinic because of jaundice, pruritus, hepatomegaly, addict was referred to the hepatology clinic because
xanthelasma and the following abnormal liver test of the following abnormal liver test results:
results: Plasma
Plasma Bilirubin 93 µmol/L (< 20)
Bilirubin 93 µmol/L (< 20) Alanine aminotransferase 761 U/L (< 42)
Alanine aminotransferase 111 U/L (< 42) Alkaline phosphatase 306 U/L (< 250)
Alkaline phosphatase (ALP) 826 U/L (< 250) Albumin 44 g/L (35–45)
Albumin 34 g/L (35–45) g-Glutamyl transferase 324 U/L (< 55)
g-Glutamyl transferase (GGT) 764 U/L (< 55) Urinary bilirubin positive.
She had a positive test result for anti-mitochondrial Further investigations, including hepatitis screen,
antibodies. showed her to be hepatitis B positive.
DISCUSSION DISCUSSION
Subsequent studies, including liver biopsy, showed Note the grossly elevated plasma aminotransferase
the patient to have primary biliary cirrhosis. Note activities, indicative of extensive hepatocyte damage.
the predominant cholestatic biochemical picture Intravenous drug addicts are at increased risk
with raised plasma ALP and GGT activities. This of hepatitis B infection. The urinary bilirubin is
condition is associated with hyperlipidaemia, hence positive, as the hyperbilirubinaemia is predominantly
the xanthelasma, and is more common in middle- conjugated, that is, water soluble.
aged women with other autoimmune disorders.
There may also be raised plasma IgM concentration
more sporadically than hepatitis A. It has a longer
and osteoporosis and osteomalacia.
incubation period, of between 40 and 180 days.
Some patients may be anicteric; some may develop
Acute hepatitis fulminant hepatitis or chronic active hepatitis and
The biochemical findings in acute hepatitis are later cirrhosis and hepatocarcinoma. They may
predominantly those of cell membrane damage with an become asymptomatic carriers of the disease.
increase in plasma ALT activity greater than that of AST. ● Hepatitis C (non-A, non-B hepatitis), which may be
There may be a superimposed cholestatic picture and, the result of sexual transmission or the transfusion
in very severe cases, impaired prothrombin synthesis. of blood products, has an incubation period of
between 15 and 50 days. It may progress to cirrhosis.
Viral hepatitis
In all types there may be a 3- to 4-day history of
Viral hepatitis may be associated with many viral anorexia, nausea and tenderness or discomfort over the
infections, such as infectious mononucleosis (Epstein– liver before the onset of jaundice. Some patients remain
Barr virus), rubella and cytomegalovirus. However, the anicteric. Plasma aminotransferase activities are very
term is most commonly used to describe three principal high from the onset of symptoms; they peak about
types of viral infection in which the clinical features of 4 days later, when jaundice becomes detectable, but
the acute illness are very similar, although they have a may remain elevated for several months. Once jaundice
different incubation period: appears, some of the initial symptoms improve.
● Hepatitis A (‘infectious hepatitis’), transmitted by In the early stages there is often a cholestatic
the faecal–oral route as a food-borne infection, is element, with pale stools due to reduced intestinal
relatively common in schools and other institutions bilirubin, and dark urine due to a rise in plasma
and has an incubation period of between 15 and 45 conjugated bilirubin concentration; unconjugated
days. Relapses may occur, but it rarely progresses to bilirubin concentrations also increase due to impaired
chronic hepatitis. hepatocellular conjugation.
● Hepatitis B (‘serum hepatitis’) is transmitted by Plasma bilirubin concentrations rarely exceed
blood products and other body fluids; it occurs 350 µmol/L, and the plasma ALP activity is only
Diseases of the liver 259
moderately raised, or even normal. If hepatocellular after exposure to the virus in about 50 per cent of
damage is severe and extensive, the prothrombin time patients.
may be increased and, in patients with cholestasis, It should be noted that there are other possible
malabsorption of vitamin K may be a contributory viruses that can cause liver dysfunction such as hepatitis
factor. D and E. Hepatitis D is a ribonucleic acid (RNA)
subviral satellite and needs hepatitis B to propagate,
Serological findings
while hepatitis E is a RNA virus spread more commonly
Testing for viral antigens, or for antibodies synthesized by the oral–faecal route.
in response to the virus, can be used to diagnose viral
hepatitis. Alcoholic hepatitis
Hepatitis A viral (HAV) antibodies of the IgM class Alcoholic hepatitis occurs in heavy drinkers, often
are detectable in the plasma of patients at the onset of after a period of increased alcohol intake. Although
symptoms. The presence of an IgG anti-HAV antibody the clinical features may mimic acute viral hepatitis,
is suggestive of previous infection. Most cases of the plasma aminotransferase activities and bilirubin
hepatitis A recover completely. concentration are not usually as markedly elevated,
Hepatitis B viral (HBV) infection, during the although GGT may be.
prodromal illness, can be diagnosed by the presence in There is no perfect laboratory marker of alcohol
the plasma of a viral surface antigen (HBsAg) and a core abuse. A raised mean corpuscular red cell volume
antigen (HBe), an internal component of the virus. These (MCV), hypertriglyceridaemia, hyperuricaemia and
antigens are short lived. During the next few weeks, elevated plasma GGT are clues, but are not specific.
an antibody response occurs, with the appearance of Increased plasma desialylated or carbohydrate-
plasma antibodies to the viral core (anti-HBc), to HBe deficient transferrin of greater than 2 per cent or
and finally to the surface antibody (anti-HBs), which raised plasma sialic acid levels have been proposed as
may be used to document previous infection (Fig. 17.3). markers of alcohol abuse, as sialic acid metabolism may
The presence of the HBe antigen correlates with be perturbed in the presence of high concentrations
infectivity, and its disappearance is a good prognostic of alcohol. Note that the consumption of more than
sign; HBsAg may persist, especially in patients with an 80 g a day of alcohol may raise GGT concentrations,
impaired immune response, and indicates chronicity not necessarily by hepatic damage, but by enzyme
associated with raised plasma aminotransferase activities. induction.
Hepatitis C viral (HCV; non-A, non-B hepatitis)
infection is often diagnosed by exclusion. Anti-HCV Drugs and other toxins
anti-bodies may be detected in plasma about 12 weeks
Various drugs and other toxins are hepatotoxic,
sometimes directly and sometimes due to a
hypersensitivity reaction; in the latter case, the damage
is not dose related. The clinical picture may resemble
Plasma ALT
that of acute viral hepatitis or cholestasis. A drug
U/L
Erythromycin +
Ferrous sulphate +a
Halothane + + +
Indometacin +
Isoniazid + +
Methotrexate +
Methyldopa + + + +
Nitrofurantoin + + +
Paracetamol + a
+
Phenothiazines +
Phenylbutazone + +
Phenytoin +
Salicylate (aspirin) + a
signs or symptoms and without a significant change muscle and antinuclear antibodies. As the disease
in activity over many years. The activities rarely exceed progresses, more cells are destroyed and the plasma AST
three times the upper reference limits. Jaundice is activity may rise to or exceed that of ALT; slight jaundice
unusual. may develop. If there is significant hepatocellular
destruction, the plasma albumin concentration falls.
Chronic active hepatitis
This is caused by active hepatocellular destruction with Cirrhosis
episodes of relapses and remissions. It may progress to Cirrhosis is the end result of many inflammatory
cirrhosis. It occurs at any age, but is most common in and metabolic diseases involving the liver, including
women. It may: prolonged toxic damage, usually due to alcohol. In
‘cryptogenic cirrhosis’, the cause is unknown. The
● be associated with, or a consequence of, viral
fibrous scar tissue distorts the hepatic architecture,
infections such as HBV or HCV, or may be drug
and regenerating nodules of hepatocytes disrupt the
induced,
blood supply, sometimes increasing the pressure in the
● be part of an autoimmune process that sometimes
portal vein, causing portal hypertension. Blood may be
involves more than one organ,
shunted from the portal into the hepatic vein, bypassing
● have no obvious cause.
the liver.
The earliest findings that differentiate it from In the early stages there may be no abnormal
chronic persistent hepatitis are an increasing plasma biochemical findings. During phases of active cellular
IgG concentration, perhaps detected by a rising plasma destruction, the plasma AST, and sometimes ALT,
g-globulin concentration, and the presence of smooth activities rise. In advanced cases, the biochemical
Diseases of the liver 261
JAUNDICE
Haemolytic jaundice
There are many causes of haemolysis, including sickle-
cell anaemia, thalassaemia and spherocytosis, and it
can also be drug or autoimmune induced. In adults,
unconjugated hyperbilirubinaemia is usually mild
because of the large reserve of hepatic secretory capacity.
The plasma bilirubin concentration is usually less than
70 µmol/L. Erythrocytes contain high amounts of AST
and lactate dehydrogenase (LDH1 and LDH2) (see
Chapter 18). Blood reticulocytes may be raised, with
Figure 17.4 Patient with severe a1-antitrypsin
abnormal blood film and a low plasma haptoglobin
deficiency, showing hepatosplenomegaly, who
developed cirrhosis and oesophageal varices. concentration. A haemolytic component may be seen
Reproduced with kind permission from Nyhan WL and in alcoholic hepatitis known as Zieve’s syndrome.
Barshop BA. Atlas of Inherited Metabolic Diseases, Urinary bilirubin concentration is usually not raised in
3rd edition. London: Hodder Arnold, 2012. haemolysis (acholuric jaundice).
264 Liver disorders and gallstones
Conjugated hyperbilirubinaemia
Drugs, e.g. see Table 17.2
Infections, e.g. hepatitis, cytomegalovirus, Epstein– Primary bile acids Cholate Chenodeoxycholate
Barr virus, sepsis
Damage to bile ducts, e.g. primary biliary cirrhosis, Taurine/glycine
sclerosing cholangitis conjugates
Alcohol, haemochromatosis, Wilson’s disease
Gallstones, cholangiocarcinoma, bile duct strictures,
pancreatic tumours Deconjugation by
intestinal bacteria
Metabolic disorders, e.g. a1-antitrypsin, Reye’s syndrome,
fatty liver, intrahepatic cholestasis of pregnancy
Diffusion infiltration, e.g. sarcoid, lymphoma, amyloid Secondary bile acids Deoxycholate Lithocholate
Inborn errors, e.g. Dubin–Johnson syndrome, Rotor’s
Figure 17.5 Synthesis of bile acids in the liver and
syndrome
their conversion to secondary bile salts in the intestine.
Unconjugated hyperbilirubinaemia
Physiological jaundice of the newborn
Gilbert’s syndrome
Crigler–Najjar syndrome isosmotic amount of water. Consequently, gall bladder
Haemolysis bile is 10 times more concentrated than hepatic bile;
Rarely thyrotoxicosis sodium is the major cation and bile salts the major
anions. The concentrations of other non-absorbable
molecules, such as conjugated bilirubin, cholesterol
in the liver from cholesterol and are called primary and phospholipids, also increase.
bile acids. They are secreted in bile as sodium salts, Gallstones
conjugated with the amino acid glycine or taurine
(primary bile salts). These are converted by bacteria Although most gallstones contain all biliary
within the intestinal lumen to the secondary bile salts, constituents, they consist predominantly of one.
deoxycholate and lithocholate, respectively (Fig. 17.5). Only about 10 per cent contain enough calcium to be
Secondary bile salts are partly absorbed from the radio-opaque and in this way they differ from renal
terminal ileum and colon and are re-excreted by the liver calculi. They can be shown on gall bladder ultrasound
(enterohepatic circulation of bile salts). Therefore, bile (Fig. 17.6).
contains a mixture of primary and secondary bile salts.
Deficiency of bile salts in the intestinal lumen leads
to impaired micelle formation and malabsorption of
fat (see Chapter 13). Such deficiency may be caused by
cholestatic liver disease (failure of bile salts to reach the
intestinal lumen) or by ileal resection or disease (failure
of reabsorption causing a reduced bile salt pool). Bile
salts are also important biochemical modulators and
activate various nuclear receptors.
Formation of bile
Between 1 L and 2 L of bile is produced daily by the
liver. This hepatic bile contains bilirubin, bile salts,
phospholipids and cholesterol, as well as electrolytes Figure 17.6 Ultrasound of the gall bladder
in concentrations similar to those in plasma. Small demonstrating a cluster of gallstones (arrowed).
amounts of protein are also present. Reproduced with kind permission from Kinirons M and
In the gall bladder there is active reabsorption of Ellis H. French’s Index of Differential Diagnosis, 15th
sodium, chloride and bicarbonate, together with an edition. London: Hodder Arnold, 2011.
266 Liver disorders and gallstones
Hyperbilirubinaemia
Exclude drug causes (see Table 17.2) Exclude drug causes (see Table 17.2)
Yes No Yes No
Yes No
Yes No
Consider
Gilbert’s syndrome Evidence of
or rare causes in intrahepatic cholestasis?
Box 17.1
Yes No
(see Table 17.4)
Evidence of
extrahepatic cholestasis?
Yes No
(see Table 17.4)
Consider rare causes
(see Box 17.1)
Figure 17.7 Algorithm for the investigation of jaundice in an adult.
well patient is usually between acute hepatocellular – signs of liver decompensation such as liver flap,
damage and cholestasis. A suggested scheme is as follows ‘liver palms’, spider naevi, ascites etc.
(for neonatal jaundice, see Chapter 26): ● Measure plasma bilirubin and unconjugated/
conjugated bilirubin fractions:
● When taking the history pay special attention to: – Predominantly unconjugated hyperbilirubi-
– recent exposure to hepatitis or infectious naemia with plasma conjugated bilirubin
mononucleosis, including a sexual and levels less than about 10 per cent of the total,
occupational history, and with little or no bilirubinuria, may suggest
– recent administration of blood or blood haemolysis as a cause. Haemolysis is supported
products, by a raised reticulocyte count and diagnostic
– medication and alcohol intake (see Table 17.2), blood film, reduced plasma haptoglobin
– intravenous drug abuse or tattoos, and raised plasma lactate dehydrogenase
– associated symptoms such as abdominal pain, concentrations.
pruritus, weight loss or anorexia and nausea, – If haemolysis is excluded, consider Gilbert’s
– recent changes in the colour of the urine or syndrome provided other liver tests are normal
stools, and other hepatic disorders have been excluded.
– recent foreign travel. ● A fresh urine sample should be examined. This test
● On clinical examination, look for: may show the presence of bilirubin if conjugated
– severity of jaundice, hyperbilirubinaemia is present. Dark-yellow or
– hepatomegaly and splenomegaly, brown urine suggests biliary obstruction. An absence
268 Liver disorders and gallstones
of urinary urobilinogen is seen in biliary obstruction. – alcohol intake and medication history, for
Reagent strips are available for testing for bilirubin example paracetamol,
and urobilinogen in urine. – the presence, or history, of other autoimmune
● Pale stools suggest biliary obstruction as a cause of disorders,
jaundice. – jaundice, pruritus or features of malabsorption,
● Whatever the results of the urine and stool inspection – family history of liver disease.
and testing, request plasma aminotransferase, ALP ● Request tests for plasma bilirubin, aminotransferases
and GGT assays: (ALT and AST), albumin, GGT and ALP:
– In hepatitis, there is a predominant increase in – A raised plasma ALT activity higher than that
the concentrations of plasma aminotransferases; of the AST may be due to reversible alcoholic
usually the plasma ALT activity is higher than hepatitis, to chronic persistent hepatitis, or to
the AST activity. If hepatitis or infectious early chronic active hepatitis.
mononucleosis is suggested by the history, – A raised plasma AST activity higher than
request serological tests. that of ALT may be due to cirrhosis or severe
– In cholestasis, there is predominant elevation chronic active hepatitis.
of the plasma ALP and GGT activities. Bile duct – A high plasma ALP activity with raised GGT
dilatation should be sought using ultrasound concentration suggests cholestasis.
or other radiological tests: – Aminotransferase levels of more than 10 times
• if the bile ducts are dilated, there is normal suggest primary hepatocyte damage, as
obstruction that may require surgery, in viral hepatitis or caused by drugs/toxins.
• if the plasma ALP activity is high but ● An alcohol-related aetiology is supported by a
dilated ducts are not demonstrated, there is macrocytosis and plasma GGT concentration, but
probably intrahepatic cholestasis. neither test is fully diagnostic. See above.
● Other tests, such as ferritin and iron saturation ● Check hepatitis serology, for example A, B and C.
(haemochromatosis), and autoantibody (such as ● Detectable plasma mitochondrial or smooth-muscle
smooth muscle, p-anti-neutrophil cytoplasmic antibodies are suggestive of primary biliary cirrhosis
antigen, mitochondrial, nuclear antibodies) and or chronic active hepatitis, respectively.
immunoglobulin levels, may also be indicated. ● A raised plasma ferritin concentration with high
● If acute alcoholic hepatitis is suspected, contributory iron saturation (see Chapter 21) may reveal
evidence may be the finding of a disproportionately haemochromatosis.
high plasma GGT activity compared with those of the ● Plasma protein electrophoresis and immunoglobulin
aminotransferases. There may also be macrocytosis, assay may help in the diagnosis of:
hypertriglyceridaemia and hyperuricaemia. – cirrhosis – high plasma IgG and IgA
● In obstructive jaundice (biliary obstruction), the plasma concentrations causing b–g fusion on the
ALP is usually more than four to five times and GGT electrophoretic strip,
more than 10 times normal. Liver/biliary ultrasound is – alcoholic cirrhosis – may present with raised
useful to distinguish between obstructive jaundice with IgA concentration,
dilated biliary ducts or undilated ducts. Endoscopic – chronic active hepatitis – a high plasma IgG
retrograde cholangiopancreatography (ERCP) or concentration and normal IgA,
percutaneous cholangiography may be indicated. – primary biliary cirrhosis – a high plasma IgM
● If the diagnosis is in doubt, a liver biopsy may be concentration.
indicated, although there may be a risk of bleeding A low plasma albumin concentration (hypo-
and therefore the prothrombin time should be albuminaemia) in the face of abnormal liver function
measured beforehand. tests can be seen in cirrhosis implying chronicity.
● A prolonged prothrombin time implies poor hepatic
Suspected liver disease showing abnormal synthetic capacity, for example clotting factors, and
plasma hepatic enzymes is prognostic in paracetamol overdose. It is also
● Relevant points in the clinical evaluation are: important if a liver biopsy is considered.
– a previous history of hepatitis, intravenous ● Significant infiltration of the liver by tumour
drug use, occupational and sexual history, cells, or by granulomas such as sarcoidosis
Investigation of suspected liver disease 269
(possibly with raised plasma angiotensin- and type 2 diabetes mellitus or impaired glucose
converting enzyme activity), may occur. In this regulation.
situation raised plasma AST activity may be the ● If primary hepatocellular carcinoma is suspected,
most sensitive test, despite a normal plasma ALT the plasma a-fetoprotein level may also be high.
activity. Hepatic space-occupying lesions may ● Low plasma copper and caeruloplasmin
additionally present with raised GGT and ALP concentrations may suggest Wilson’s disease (see
concentrations, and a liver ultrasound is useful to Chapter 15), and plasma a1-antitrypsin deficiency
detect these. can result in cirrhosis (see Chapter 19).
● A fatty liver may be revealed by liver ultrasound ● Radionuclide scans or other imaging procedures
as this may show increased echogenicity. This may (CT or MRI) may be useful. A liver biopsy may be
be associated with hypertriglyceridaemia, obesity indicated to clarify a histological diagnosis.
SUMMARY
● The liver has an enormous synthetic capacity and is ● Raised plasma aminotransferase activities suggest
involved in numerous metabolic pathways, including hepatocyte damage and raised hepatic ALP
vitamin storage, amino acid deamination, bile salt and concentration is associated with cholestasis.
cholesterol synthesis, and the production of various ● Plasma GGT is a sensitive marker of hepatic
proteins such as clotting factors and hormones. damage but its activity can also be raised as a result
● Compounds ‘released’ from the liver into the plasma of drug enzyme induction, hypertriglyceridaemia
can be used as markers of liver damage, including and increased alcohol intake.
bilirubin, ALT, AST, GGT and ALP. ● A prolonged prothrombin time and low plasma
● Hyperbilirubinaemia can be due to raised albumin concentration may both reflect reduced
unconjugated or conjugated bilirubin concentration. hepatic synthetic capacity.
The former may be due to haemolysis and the latter
to hepatic or extrahepatic causes.
18 Plasma enzymes in diagnosis
(clinical enzymology)
Assessment of cell damage and proliferation 270 Normal plasma enzyme activities 272
Factors affecting results of plasma enzyme assays 271 Plasma enzyme patterns in disease 280
This chapter discusses the principles of clinical will now see, changes in plasma enzyme activities may
enzymology, which have already been encountered in be useful to detect and localize tissue cell damage or
some of the preceding chapters. Enzymology can be proliferation, or to monitor the treatment and progress
defined as the assay of an enzyme(s) in body fluids, of disease.
usually blood, that can be used diagnostically or to
monitor a clinical condition. ASSESSMENT OF CELL DAMAGE AND
An enzyme is a protein that catalyses one or more PROLIFERATION
specific biochemical reactions. It is usually easier to Plasma enzyme levels depend on the extent of cell
measure enzyme activity in body fluids, by monitoring damage and the rate of release from damaged cells,
changes in either substrate or product concentrations, which, in turn, depends on the rate at which damage
than to measure enzyme protein concentration is occurring.
directly, although this is sometimes done. However, In the absence of cell damage, the rate of release
measurement of the enzyme protein concentration is depends on the degree of induction of enzyme synthesis
more specific and less prone to analytical variation. and the rate of cell proliferation.
Generally, enzymes are present in cells at much These factors are balanced by the rate of enzyme
higher concentrations than in plasma. Some occur clearance from the circulation.
predominantly in cells of certain tissues, where they Acute cell damage, for example in viral hepatitis,
may be located in different cellular compartments such may cause very high plasma aminotransferase activities
as the cytoplasm or the mitochondria. ‘Normal’ plasma that reduce as the condition resolves. By contrast,
enzyme concentrations reflect the balance between the the liver may be much more extensively involved in
rate of synthesis and release into plasma during cell advanced cirrhosis but the rate of cell damage is often
turnover, and the rate of clearance from the circulation. low, and consequently plasma enzyme activities may
The enzyme activity in plasma may be: be only slightly raised or within the reference range. In
very severe liver disease, plasma enzyme activities may
● higher than normal, due to the proliferation of cells,
even fall terminally when the number of hepatocytes is
an increase in the rate of cell turnover or damage
grossly reduced (see Chapter 17).
or in enzyme synthesis (induction), or to reduced
Relatively small enzymes, such as amylase, can be
clearance from plasma,
cleared by the kidneys. Thus, plasma amylase activity
● lower than normal, due to reduced synthesis,
may be high as a result of renal glomerular impairment
congenital deficiency or the presence of inherited
rather than pancreatic damage. However, most
variants of relatively low biological activity –
enzymes are large proteins and may be catabolized
examples of the latter are the cholinesterase variants.
by plasma proteases before being taken up by the
Sometimes macroenzymes are found, that is to say, reticuloendothelial system.
a high-molecular-weight form of a native enzyme. In healthy individuals, each enzyme has a fairly
Often these are enzymes [such as lactate dehydrogenase constant and characteristic biological half-life, a fact
(LDH), creatine kinase (CK) and alkaline phosphatase that may be used to assess the time since the onset of an
(ALP)] complexed with immunoglobulins and are acute illness. After a myocardial infarction, for example,
more common in individuals with autoimmune plasma levels of CK and aspartate aminotransferase
disease. It is important to recognize macroenzymes as (AST) fall to normal before those of LDH, which has a
they can sometimes cause diagnostic confusion. As we longer half-life (see Chapter 22).
Factors affecting results of plasma enzyme assays 271
Localization of damage Slight rises in plasma ALT and AST activities are
Most of the enzymes commonly measured to assess tissue common, non-specific findings in many illnesses.
damage are present in nearly all body cells, although Moderate exercise, or a large intramuscular injection,
their relative concentrations in certain tissues may differ. may lead to a rise in plasma CK activity; isoenzyme
Measurement of the plasma activity of an enzyme known determination may identify skeletal muscle as the tissue
to be in high concentration within cells of a particular of origin.
tissue may indicate an abnormality of those cells, but the Some drugs, such as the anticonvulsants phenytoin
results will rarely enable a specific diagnosis to be made. and phenobarbital, may induce the synthesis of the
For example, if there is circulatory failure after a cardiac microsomal enzyme g-glutamyl transferase (GGT),
arrest, very high plasma concentrations of enzymes and so increase its plasma activity in the absence of
originating from many tissues may occur because of disease.
hypoxic damage to cells and reduced rates of clearance. Plasma enzyme activities may be raised if the rate
The distribution of enzymes within cells may of clearance from the circulation is reduced. In the
differ. Alanine aminotransferase (ALT) and LDH are absence of hepatic or renal disease, this may occur if,
predominantly located in cytoplasm, and glutamate for example, the plasma enzyme forms complexes with
dehydrogenase (although this is not usually measured immunoglobulins, known as a macroenzyme.
clinically) in mitochondria, whereas AST occurs in both Various enzymes can form clinically significant
these cellular compartments. Different disease processes macroenzymes including amylase, LDH, ALP and
in the same tissue may affect the cell in different ways, CK.
causing alteration in the relative plasma enzyme activities.
The diagnostic precision of plasma enzyme analysis FACTORS AFFECTING RESULTS OF
may be improved by the following: PLASMA ENZYME ASSAYS
Analytical factors
● Serial enzyme estimations The rate of change of
plasma enzyme activity is related to a balance The total concentration of all plasma enzyme proteins
between the rate of entry and the rate of removal is less than 1 g/L. The results of enzyme assays are not
from the circulation. A persistently raised plasma usually expressed as concentrations, but as activities.
enzyme activity is suggestive of a chronic disorder Changes in concentration may give rise to proportional
or, occasionally, impaired clearance. changes in catalytic activity, but the results of such
● Isoenzyme determination Some enzymes exist in measurements depend on many analytical factors,
more than one form; these isoenzymes may be including:
separated by their different physical or chemical ● substrate concentration,
properties. If they originate in different tissues, ● product concentration,
such identification will give more information than ● enzyme concentration,
the measurement of plasma total enzyme activity; ● reaction temperature,
for example, CK may be derived from skeletal or ● reaction pH,
cardiac muscle, but one of its isoenzymes is found ● presence of activators or inhibitors.
predominantly in the myocardium.
The definition of ‘international units’ does not take
● Estimation of more than one enzyme Many
these factors into account, and the results from different
enzymes are widely distributed, but their relative
laboratories, which are apparently expressed in the
concentrations may vary in different tissues. For
same units, may not be directly comparable. Therefore,
example, although both ALT and AST are abundant
plasma enzyme activities must be interpreted in relation
in the liver, the concentration of AST is much greater
to the reference ranges from the issuing laboratory.
than that of ALT in heart muscle.
In some countries such as the UK there are plans to
harmonize or converge laboratory reference ranges for
Non-specific causes of raised plasma
certain analytes.
enzyme activities
Before attributing a change in plasma enzyme activity Non-disease factors
to a specific disease process, it is important to exclude Examples of non-disease factors affecting enzyme
the presence of factitious or non-specific causes. activities include the following.
272 Plasma enzymes in diagnosis (clinical enzymology)
Figure 18.2 Algorithm for the investigation of a raised plasma alkaline phosphatase. GGT, g-glutamyl transferase.
Normal plasma enzyme activities 277
● Bone disease:
– rickets and osteomalacia (see Chapter 6), CASE 3
– Paget’s disease of bone (may be very high), A 64-year-old man with lung carcinoma attended
– secondary malignant deposits in bone, the oncology clinic. Some of his blood results were
– osteogenic sarcoma (only if very extensive), as follows:
– primary hyperparathyroidism with extensive
Plasma
bone disease (usually normal but may be
Bilirubin 10 µmol/L (< 20)
slightly elevated),
Alanine aminotransferase 23 U/L (< 42)
– secondary hyperparathyroidism.
Alkaline phosphatase (ALP) 426 U/L (< 250)
● Liver disease:
g-Glutamyl transferase (GGT) 50 U/L (< 55)
– intrahepatic or extrahepatic cholestasis (see
Albumin 36 g/L (35–45)
Chapter 17),
Albumin-adjusted calcium 2.22 mmol/L (2.15–2.55)
– space-occupying lesions, tumours, granulomas
Phosphate 1.11 mmol/L (0.80–1.35)
and other causes of hepatic infiltration.
● Inflammatory bowel disease: the gut ALP isoenzyme A liver ultrasound and bone scan were normal.
can be increased in ulcerative colitis.
DISCUSSION
● Malignancy: bone or liver involvement or direct
The question here is what is the source of the raised
tumour production.
ALP activity? The normal GGT activity makes
A placental-like, so-called ‘Regan’ isoenzyme a hepatic source unlikely. Similarly, the normal
may occasionally be identified in plasma in patients plasma calcium concentration and bone scan do
not make a bone source likely either. However,
ALP isoenzymes showed the presence of a Regan
CASE 2 isoenzyme, thought to be ectopically released from
his lung carcinoma.
A 23-year-old man was investigated in the
gastroenterology clinic because his plasma amylase
was 445 U/L (< 200) and some of his blood results
were as follows: with malignant disease, especially carcinoma of the
bronchus. There is also a Nago isoenzyme released by
Plasma
certain tumours.
Sodium 139 mmol/L (135–145)
Transient very high levels of ALP (up to 30 times
Potassium 4.0 mmol/L (3.5–5.0)
URL) have been recorded in children, but the clinical
Bicarbonate 26 mmol/L (24–32)
significance of this finding is unknown. This has
Urea 4.5 mmol/L (2.5–7.0)
been called transient hyperphosphatasaemia and may
Creatinine 100 µmol/L (70–110)
be either the bone or the liver isoenzyme. It may be
Glucose 4.5 mmol/L (3.5–5.5)
associated with abdominal symptoms and usually
Albumin-adjusted calcium 2.39 mmol/L (2.15–2.55)
resolves within a few months.
No other investigations revealed any abnormality, Plasma total ALP activity is not usually increased in
including upper gastrointestinal endoscopy and myelomatosis, despite the radiographic appearance of
abdominal ultrasound. A urine analysis did not find multiple ‘punched-out’ osteolytic lesions. The lesions
any amylase activity. are in the marrow cavity, not the bone substance, and
osteoblastic activity is not stimulated. However, ALP
DISCUSSION
activity may be raised if there is liver involvement
The question here is what is the explanation for the
or, more rarely, if there is healing of very extensive
raised plasma amylase activity? Amylase is normally
pathological fractures.
present in the urine because of its relatively small
molecular size. Its absence in urine and serum amylase Possible causes of low plasma alkaline phosphatase
electrophoretic studies confirmed macroamylasaemia. activity
This is a large form of amylase, usually immunoglobulin A low plasma ALP concentration is less usual, but may
A bound, that is not renally cleared. be caused by the following:
278 Plasma enzymes in diagnosis (clinical enzymology)
● Arrested bone growth: The placental isoenzyme does not cross the placenta
– achondroplasia, and is therefore not detectable in the plasma of the
– hypothyroidism, newborn infant (Fig. 18.3).
– severe vitamin C and vitamin B12 deficiency.
Acid phosphatase
● Magnesium and zinc deficiency.
● Hypophosphatasia, an autosomal recessive disorder, Acid phosphatase (ACP) is found in cells of the prostate,
associated with rickets or osteomalacia. liver, erythrocytes, platelets and bone. The main
● Treatment of hyperlipidaemia with a fibrate drug, for indication for estimation was to help diagnose prostatic
example bezafibrate. This observation can be useful carcinoma and to monitor its treatment. Estimation has
to determine drug compliance. been largely replaced by the measurement of plasma
prostate-specific antigen (PSA), a protein derived from
the prostate (see Chapter 24). This test is more specific
Isoenzymes of alkaline phosphatase and sensitive for diagnosis and monitoring treatment
Bone disease with increased osteoblastic activity and and has essentially rendered the plasma ACP assay
liver disease with involvement of the biliary tracts are obsolete in the diagnosis and management of prostatic
the most common causes of an increased total ALP carcinoma.
activity. Haemolysed blood samples should also be avoided,
Rarely, the cause is not apparent and further tests as ACP is found in erythrocytes. Normally, ACP drains
may be helpful. The isoenzymes originating from from the prostate, through the prostatic ducts and into
the cells of bone, liver, intestine and placenta may be the urethra, and very little can be detected in plasma.
separated by electrophoresis, but interpretation may be In extensive prostatic carcinoma, particularly if it has
difficult if the total activity is only marginally raised. spread extensively or has metastasized, plasma ACP
The placental and ‘Regan’ isoenzymes are more stable activity rises, probably because of the increased number
at 65°C than the bone, liver and intestinal isoenzymes, of prostatic ACP-containing cells. Another problem
and heat inactivation may help to differentiate the heat- with plasma ACP is that its concentration can increase
stable from the heat-labile fraction. after rectal examination.
Figure 18.3 Electrophoresis separation of alkaline phosphatase (ALP) isoenzymes. Liver ALP runs more anodal
than bone and heat and inhibition studies can be used to determine the various isoenzyme forms. Reproduced
with kind permission of Sebia.
Normal plasma enzyme activities 279
Causes of decreased plasma cholinesterase activity angiotensin I and bradykinin. One of its actions is
● Hepatic parenchymal disease (reduced synthesis). releasing angiotensin II by cleaving the dipeptide
● Ingestion, or absorption through the skin, of such histidyl-leucine from angiotensin I. The major site of
anticholinesterases as organophosphates. ACE production is the pulmonary endothelium.
● Inherited abnormal cholinesterase variants, with This enzyme can be measured in plasma as a marker
low biological activity. of disease activity in sarcoidosis. It can be elevated in
● Pregnancy. other lung conditions and therefore is not specific for
sarcoidosis and has limited diagnostic value. However,
Causes of increased plasma cholinesterase activity high plasma ACE activity may decline on treatment of
● Recovery from liver damage (actively growing sarcoidosis with steroids.
hepatocytes). Tryptase
● Nephrotic syndrome.
Classic allergy such as bronchospasm and urticaria
Suxamethonium sensitivity
generally produces allergen-specific IgE to certain
agents, with IgE-producing mast-cell degranulation.
The muscle relaxants suxamethonium (succinyl Tryptase is a serine protease found in mast cells. In
choline, ‘scoline’) and mivacurium are usually broken cases of mast-cell degranulation, as occurs in systemic
down by plasma cholinesterase, and this limits the anaphylaxis, tryptase levels rise within 1 h and remain
duration of their action. Giving these agents to patients elevated for 4–6 h. In patients with life-threatening
with a low cholinesterase activity (usually due to an systemic reactions to diverse allergens (such as
enzyme variant) may result in a prolonged period of venoms, latex and drugs), the finding of elevated
apnoea (‘scoline apnoea’); such patients may need plasma tryptase levels (more than 1 µg/L) between 1
prolonged ventilatory support after an operation. and 6 h after the event is highly sensitive and specific
The abnormal cholinesterase variants may be for confirming mast-cell degranulation as the cause of
classified by measuring the percentage inhibition of the the event.
enzyme activity by dibucaine (dibucaine number) or by
fluoride (fluoride number). A low dibucaine number
and/or fluoride number with or without low plasma PLASMA ENZYME PATTERNS IN
activity is suggestive of a cholinesterase variant, for DISEASE
example A, K, J, F types. There is also a silent gene type. Liver disease
The identification of patients susceptible to Plasma enzyme changes in liver disease are discussed
suxamethonium, and of their affected relatives, is in Chapter 17 and are important in the diagnosis of
important. Close blood relatives should be traced and various liver disorders.
investigated to identify their genotype, and so to predict
the chance of future anaesthetic risk. All affected Muscle disease
individuals should carry a warning card or a ‘Medic In the muscular dystrophies, for example Duchenne’s,
Alert’ bracelet and should notify the anaesthetist if they plasma levels of the muscle enzyme CK (isoenzyme
require an anaesthetic. CK-MM) and also of LDH and the aminotransferases
(mainly AST) are increased as a result of leakage from
Acetylcholinesterase the diseased cells. Results of plasma CK estimation
Reduced red cell acetylcholinesterase activity can be are more specific than LDH and AST for muscle
useful as a measure of exposure to organophosphates, disease.
for example certain pesticides, and this activity can be Points to consider in the interpretation of CK levels
used to monitor people at risk of organophosphate in Duchenne’s muscular dystrophy are:
exposure. ● Activities are highest (may be greater than 5–10
times the URL) in the early stages of the disease;
Angiotensin-converting enzyme later, when much of the muscle has wasted, they are
Angiotensin-converting enzyme (ACE) is a dipeptidyl lower and may even be normal.
carboxypeptidase and cleaves dipeptides from the ● Activities are higher following muscular activity
free carboxy end of various polypeptides, including immediately after rest (because of the release of CK
Plasma enzyme patterns in disease 281
built up in muscle during rest) than after prolonged (Regan or Nago isoenzymes), LDH1 (HBD) or
activity. CK-BB, assays of which may be used as an aid to
diagnosis or for monitoring treatment.
The clinical diagnosis of the Duchenne type of
● Although plasma prostatic (tartrate-labile) ACP
muscular dystrophy can be confirmed by measuring
activity rises in some cases of malignancy of the
the plasma CK activity, examining a muscle biopsy
prostate gland, plasma PSA determination has now
specimen and by deoxyribonucleic acid (DNA) analysis
essentially replaced its clinical use.
in the majority of cases (see Chapter 30).
Carriers of the disorder can often be detected by Haematological disorders
DNA analysis of the dystrophin gene and by finding Very high activities of LDH may be found in
raised plasma CK activities. The rise is only moderate, megaloblastic anaemias and leukaemias and in other
and non-specific causes of raised enzyme activities conditions in which bone marrow activity is abnormal.
must be excluded. Typically there is much less change in the plasma AST
Less marked changes in plasma CK, LDH and AST than in the LDH activities. Severe in vivo haemolysis
are found in some patients with the other muscle produces changes in both AST and LDH activities,
disorders described above. which mimic those of myocardial infarction.
Myocardial infarction
Enzymes in malignancy
The diagnosis of a myocardial infarction is usually made
Plasma total enzyme activities may be raised, or an
on the clinical presentation and electrocardiographic
abnormal isoenzyme detected, in several neoplastic
findings and confirmed by the characteristic changes
disorders:
in plasma enzyme activities or troponins. The latter
● Malignancies may be associated with a non- now have superseded enzyme analysis in this context
specific increase in plasma LDH and, occasionally, and enzymes are rarely measured (see Chapter 22).
aminotransferase activity. It should be evident that enzymology is important
● Plasma aminotransferase and ALP estimations may in diagnostic processes. (The reader is also referred to
be of value to monitor the treatment of malignant Chapter 16 on gastrointestinal and pancreatic disorders,
disease. Raised ALP levels may indicate secondary Chapter 17 concerning liver function tests, Chapter 22
deposits in liver or in bone. Liver deposits may on cardiovascular chemical pathology and Chapter 24
also cause an increase in plasma LDH or GGT on tumour markers.) Enzymes can also be measured
concentration. in other body fluids, such as faeces, that is, elastase (see
● Tumours occasionally produce a number of enzymes, Chapter 16), and pleural fluid, for example adenosine
some of which are placental or placental-like ALP deaminase (see Chapter 23).
SUMMARY
● Enzymes can be measured in body fluids, ● Certain aminotransferases (AST and ALT) and also
mainly plasma, to aid the clinical diagnosis and GGT can be used in the investigation of hepatic
management of certain conditions. This is called disease.
clinical enzymology. ● Clinical enzymology has limitations, as generally
● Enzymes may exist as isoenzymes (molecular variants plasma enzyme activity lacks specificity. Isoenzyme
of enzymes), which may be present in different determination or measuring a number of enzymes
tissues. Examples are ALP (hepatic, bone, intestinal may increase diagnostic accuracy.
and placental isoenzymes) and CK (striated muscle, ● Some enzymes may complex with larger molecules,
MM; brain, BB; and cardiac muscle, MB). such as immunoglobulins, forming macroenzymes.
19 Proteins in plasma and urine
constituents, such as albumin and immunoglobulins, are protein concentrations may be due to dilution, for
likely to alter total protein concentrations significantly. example if blood is taken near the site of an intravenous
The acute-phase response can result in rapid albumin saline infusion, hypoalbuminaemia or severe
extravasation and thus hypoalbuminaemia. immunoglobulin deficiency.
Plasma total protein concentrations may be
misleading for other reasons. They may be normal in Qualitative methods for studying plasma
the presence of quite marked changes in the constituent and urinary proteins
proteins. For example, a fall in plasma albumin Electrophoresis
concentration may be approximately balanced by a Electrophoresis is a technique that separates
rise in immunoglobulin concentrations. Also, most compounds such as proteins according to their
individual proteins apart from albumin contribute different electrical charges. It is usually performed by
little to the total protein concentration; therefore, quite applying a small amount of serum to a strip of cellulose
a large percentage change in the concentration of one acetate or agarose and passing a current across it for a
may not cause a detectable change in the total protein standard time. In this way, five main groups of proteins
concentration. – namely albumin and the a1, a2, b and g globulins –
Raised plasma total protein concentrations may may be distinguished after protein staining and may
be due to loss of protein-free fluid, or excessive stasis be visually compared with those in a normal control
during venepuncture, or a major increase in the serum. Each of the globulin fractions contains several
concentration of one or more of the immunoglobulins, proteins (Fig. 19.1).
including paraproteins. The difference between serum The following description applies to the normal
total protein and albumin concentration is termed appearance, in adults, of the principal bands seen after
the serum globulin concentration. Low plasma total electrophoresis on cellulose acetate:
Prealbumin ANODE
Albumin
-Lipoprotein
1-Acid–glycoprotein
1
1-Antitrypsin
1-Antichymotrypsin
2-Macroglobulin 2
Haptoglobin
Lipoprotein
1
Transferrin
2
IgA
Origin
C3
IgG
IgM
CATHODE
Figure 19.1 The normal serum electrophoretic pattern. In this example the globulin has separated into b1 and b2
fractions. This finding is not uncommon, especially in stored specimens.
284 Proteins in plasma and urine
● Albumin, usually a single protein, makes up the most Parallel changes in all protein fractions
obvious band. Reduction may occur in severe undernutrition,
● a1-globulins consist almost entirely of a1-antitrypsin sometimes due to malabsorption, unless accompanied
and a1-antichymotrypsin. by infection and haemodilution. An increase may occur
● a2-globulins consist mainly of a2-macroglobulin and in haemoconcentration.
haptoglobin.
● b-globulins often separate into two; b1 consists The acute-phase pattern
mainly of transferrin with a contribution from low- Tissue damage usually triggers the sequence of
density lipoprotein (LDL), and b2 consists of the C3 biochemical and cellular events associated with
component of complement. inflammation. The biochemical changes include the
● g-globulins are immunoglobulins; some immuno- stimulation of synthesis of the so-called acute-phase
globulins are also found in the a2 and b regions. proteins, with a rise in the a1-globulin and a2-globulin
fractions. The plasma concentrations of these proteins
If plasma rather than serum is used, fibrinogen
reflect the activity of the inflammatory response,
appears as a distinct band in the b–g region. This
and their presence is responsible for the rise in the
may make interpretation difficult. Blood should be
erythrocyte sedimentation rate (ESR) and increased
allowed to clot and serum used, rather than plasma, if
plasma viscosity characteristic of such a response.
electrophoresis is to be performed.
Chronic inflammatory states
Electrophoretic patterns in disease (Fig. 19.2) In chronic inflammation, the usual increase in
Some abnormal electrophoretic patterns are immunoglobulin synthesis may be visible as a diffuse
characteristic of a particular disorder or group of rise in g-globulin. If there is an active inflammatory
related disorders, while others indicate non-specific reaction, the increased density in the g-globulin region
pathological processes. For example, the a2 band, which is associated with an increase in the a1 and a2 fractions
contains haptoglobin, may be reduced if there is in vivo of the acute-phase response.
haemolysis and may split into two if in vitro haemolysis
has occurred. Cirrhosis of the liver
The changes in the concentrations of plasma proteins
in liver disease are discussed in Chapter 17. They are
Normal control usually ‘non-specific’, but in cirrhosis a characteristic
pattern is sometimes seen. Albumin and often a1-
globulin concentrations are reduced and the g-globulin
Acute-phase reaction
concentration is markedly raised, with apparent fusion
or ‘bridging’ of the b and g bands because of an increase
Chronic inflammation
in plasma IgA concentrations.
Figure 19.2 Serum protein electrophoretic patterns in The a1 band consists almost entirely of a1-antitrypsin
disease. and its absence or an obvious reduction in its density
Plasma proteins 285
suggests a1-antitrypsin deficiency. Occasionally, a1- ● Recumbency: plasma albumin concentrations may be
antitrypsin variants may present with a split a1 band. 5–10 g/L lower in the recumbent than in the upright
position because of the redistribution of fluid.
Other protein abnormalities
● Increased capillary membrane permeability: this is the
Hypogammaglobulinaemia shows reduced plasma usual cause of the rapid fall in plasma concentration
g-globulins and is discussed in the sections on found in many circumstances, for example post-
immunoglobulins. operatively and in most illnesses.
Multiple myeloma and paraproteinaemia may show
a band or bands on the electrophoretic strip and are The slight fall in plasma albumin concentration
also discussed later. found in mild illness may be due to a combination of
the above two factors.
Albumin
Decreased synthesis of albumin
Albumin, with a molecular weight of about 65 kDa, is Normally, about 4 per cent of the body albumin is
synthesized by the liver. It has a normal plasma biological replaced each day. Hepatic impairment, such as cirrhosis,
half-life of about 20 days. About 60 per cent in the causes hypoalbuminaemia if the rate of synthesis of new
extracellular fluid is in the interstitial compartment. amino acids is inadequate to replace those deaminated
However, the concentration of albumin in the smaller during metabolism; most of the amino nitrogen is then
intravascular compartment is much higher because of lost as urinary urea. Hypoalbuminaemia may therefore
the relative impermeability of the blood vessel wall. This be due to:
concentration gradient across the capillary membrane is
important in maintaining plasma volume (see Chapter 2). ● undernutrition, resulting in an inadequate supply of
There are several inherited abnormalities of albumin dietary nitrogen,
synthesis: ● malabsorption, resulting in impaired absorption of
dietary peptides and amino acids,
● the bisalbuminaemias, in which two forms of ● impairment of synthesis, due to chronic liver
albumin are present; there are usually no clinical dysfunction, for example cirrhosis.
consequences,
● analbuminaemia, in which there is deficient synthesis Increased loss of albumin from the body
of the protein; clinical consequences are slight, and Because of its relatively low molecular weight, significant
oedema, although present, is surprisingly mild. amounts of albumin and other low-molecular-weight
An abnormally high plasma albumin concentration proteins are lost in conditions associated with increased
is found only artefactually in a sample taken with membrane permeability. The plasma concentration of
prolonged venous stasis or after loss of protein-free fluid. albumin is higher than that of the other proteins and its
loss is therefore more obvious. Albumin loss may occur
Causes of hypoalbuminaemia through:
A low plasma albumin concentration may be due to ● the skin, because of extensive burns or skin diseases
dilution or to redistribution. True albumin deficiency such as psoriasis; a large part of the interstitial fluid
may be caused by a decreased rate of synthesis, or by an is subcutaneous,
increased rate of catabolism or loss from the body. ● the intestinal wall, in protein-losing enteropathy,
● the glomeruli, in the nephrotic syndrome.
Dilutional hypoalbuminaemia
Dilutional hypoalbuminaemia may, as in the case of Increased catabolism of albumin
total protein, result from artefactual changes due to Catabolism (and therefore nitrogen loss) is increased in
taking blood from the arm into which an infusion is many illnesses, including sepsis and hyperthyroidism. This
flowing, administration of an excess of protein-free may worsen the hypoalbuminaemia due to other causes.
fluid or fluid retention, usually in oedematous states or
during late pregnancy. Consequences of hypoalbuminaemia
Redistribution of albumin The following may occur.
Redistribution of albumin from plasma into the ● Fluid distribution Albumin is quantitatively the
interstitial fluid space results from: most important protein contributing to the plasma
286 Proteins in plasma and urine
colloid osmotic pressure. Oedema may occur in subdivided using antibodies against the cell
severe hypoalbuminaemia (see Chapter 2). surface markers; this is known as cluster
● Binding functions Albumin binds calcium, bilirubin differentiation (Fig. 19.3).
and free fatty acids. (See Chapter 6 for a discussion The inflammatory response and the ability to kill
of albumin-adjusted calcium.) foreign organisms may be impaired if there is deficiency
of either cellular or humoral components.
A number of drugs, such as salicylates, penicillin
and sulphonamides, may become albumin bound. Acute-phase proteins
The albumin-bound fractions are physiologically and
pharmacologically inactive. A marked reduction in The innate or natural immune system, which is
plasma albumin, by reducing the binding capacity, phylogenetically older than so-called acquired or adaptive
may increase the plasma free concentration of those immunity, is a rapid first-line defence mechanism based
substances leading to toxic effects. on non-lymphoid tissue components. The acute-phase
Albumin-bound drugs may, if administered response is part of this system and results in pronounced
together, compete for binding sites, thus increasing free changes in the concentration of plasma proteins in
concentrations; an example of this is the simultaneous response to a variety of stresses, including infection,
administration of salicylates and the anticoagulant tissue injury and inflammation. The concentration of
warfarin, thus potentiating the effect of the latter (see some of these proteins increases (positive acute-phase
Chapter 25). proteins), such as CRP, fibrinogen and plasma amyloid
A, whereas that of others decreases (negative acute-phase
The acute-phase response proteins), such as albumin and transferrin (Fig. 19.4.)
The body responds to tissue damage and to the presence
of infecting organisms or other foreign substances by
a complex, inter-related series of cellular and humoral Macrophage
responses. These act together to initiate and control
the inflammatory reaction and so to remove damaged
tissues and foreign substances. Defence mechanisms Virus
TNF FEVER
can be separated into two main groups.
● The inflammatory response: this is non-specific,
Macrophage
requires changes in the permeability of membranes IL-1
and depends on humoral factors such as acute- IFN-
of biological variation at these low concentrations inflammation (chemotaxis), which, by increasing the
repeat determinations are useful. permeability of the capillary wall to both cellular and
chemical components, allows them to reach affected
Procalcitonin is a 14-kDa protein encoded by the
cells. Activation of the complete complement pathway
Calc1 gene. Blood levels of procalcitonin are increased
on foreign cell surfaces results in their lysis and, together
in systemic inflammation and procalcitonin has been
with immunoglobulins, some of the complement
proposed as a possibly better marker of bacterial sepsis
components act as opsonins, which enhance
than CRP.
phagocytosis. Because of inhibitors, complement
usually circulates in an inactive form.
Complement
Clinically, the most important of the complement
Macrophages and hepatocytes synthesize a group of proteins is C3. Two main pathways are concerned with
proteins called the complement system, which is part its activation (Fig. 19.5). Activation of either results in
of the innate immune system. Sequential activation low plasma C3 concentrations.
reduces complement plasma concentrations, but in
many instances this is compensated for by increased Classic pathway
synthesis of acute-phase reactants. The products In the classic pathway, C3 is activated by the products
of activation attract phagocytes to the area of of a pathway initiated by the activation of C1, usually
ACTIVATOR
Ag–Ab complex
C1 C1 inhibitor
C4
CLASSIC
PATHWAY
C2
Opsonization
C3 C3b C5–9
ALTERNATIVE
PATHWAY INHIBITORS
ACTIVATORS
IgA complexes
Organisms
Chemotaxis
Figure 19.5 The complement pathway, much simplified. Ag, antigen; Ab, antibody; IgA, immunoglobulin A.
Plasma proteins 289
by antigen-bound IgG or IgM (immune complexes), They migrate to sites of chronic inflammation, where
by protein within the cell walls of many strains of they develop into macrophages. Under T-cell control,
Staphylococcus aureus, or by CRP. C4 (and C2) are used macrophages are activated and develop a greatly
during the resultant sequence of events, which also enhanced ability to phagocytose and digest pathogenic
activates the alternative pathway via C3; plasma C3 and organisms.
C4 concentrations fall. Once the formation of immune The most important functions of T cells in the
complexes stops, plasma C3 and C4 concentrations are inflammatory response are the initiation and mainten-
restored. The enzyme C1q esterase inhibits the serine ance of chronic cellular inflammatory responses
protease C1 (which is composed of C1q, C1r and C1s). to invading organisms and the control of immune
inflammation by providing helper and suppressor
Alternative pathway influences on other cells. B cells need the co-operation
In the alternative pathway, which is the more important of T cells (helper cells) to make antibodies to complex
of the two, C3 is activated by IgA immune complexes, antigens. T-cell modulation of inflammatory and
and substances such as lipopolysaccharides and immune responses is mediated by chemical messengers
peptidoglycans in the walls of certain bacteria and called lymphokines. T cells also have a cytotoxic
viruses, factor B and properdin are involved. C3b function directed towards destroying the body’s own
is formed, which in turn activates more C3. Other cells infected by intracellular pathogens such as viruses.
products of C3b cause vasodilatation and cell lysis.
The cyclical process is self-perpetuating and plasma C3 Immune response
concentrations fall. If the initial stimulus is removed, Immunoglobulins
inhibitors such as those formed in the acute-phase Immunoglobulins, synthesized by B lymphocytes, are
reaction control the reaction and C3 concentrations incorporated into cell membranes, where they act as
return to normal; factors H and I inhibit the enzyme C3 antigen receptors. On exposure to a specific antigen
convertase. The alternative pathway is most important in the presence of T-helper cells, these B lymphocytes
in the absence of preformed antibodies. proliferate and differentiate into plasma cells
Recently, a third pathway of C3 activation has been synthesizing and secreting immunoglobulins.
described involving mannose-binding lectin, one of
the colectin proteins. After C3 activation, the terminal Structure
complement system consists of other activated proteins The basic immunoglobulin is a Y-shaped molecule
such as C5–9, collectively known as the membrane (depicted schematically in Fig. 19.6).
attack complex. This is involved in puncturing cell Usually four polypeptide chains are linked by
membranes. disulphide bonds. There are two heavy (H) and two light
In immune-complex disease, such as SLE, circulating (L) chains in each unit. The H chains in a single unit are
immune complexes persist. The products of C3, released identical and determine the immunoglobulin class of
by continued activation of the classic pathway, may the protein. Heavy chains g, a, µ, d and e occur in IgG,
damage blood vessels, joints and the kidneys. Plasma IgA, IgM, IgD and IgE respectively. The L chains are of
concentrations of both C3 and C4 are low. Persistently two types, k and l. In a single molecule, the L chains are
low plasma C3 concentrations may help to distinguish
chronic mesangiocapillary glomerulonephritis, with a Chains
poor prognosis, from the less serious and self-limiting
Fc L
acute post-streptococcal glomerulonephritis, in which
plasma C3 concentrations return to normal within a H
F(ab)2 piece
few months. H
Fc
L
Normal cellular response to infection
Variable region
Two types of phagocytic cells help to combat infection. Hinge Constant region
Polymorphonuclear neutrophil leucocytes migrate into region
Disulphide bonds
sites of acute inflammation and represent a non-
specific first line of defence against pyogenic bacteria. Figure 19.6 Diagram of an immunoglobulin monomer.
Mononuclear phagocytes circulate in blood as monocytes. L, light; H, heavy.
290 Proteins in plasma and urine
of the same type, although the immunoglobulin class as Normal immunoglobulin response to infection
a whole contains both types. In response to infection, each plasma cell produces an
There are two antigen-combining sites per immunoglobulin of a single class.
monomer, together known as the F(ab)2 piece. These
lie at the ends of the arms of the Y; both H and L chains Immunoglobulin G
are necessary for full antibody activity. The amino acid Immunoglobulin G concentrations rise slightly later
composition of this part of the chain varies in different in response to soluble antigens such as bacterial toxins.
units (variable region). When the F(ab)2 site combines Owing to their relatively low molecular weight, they can
with antigen, conformational changes are transmitted diffuse into the interstitial fluid to fight against tissue
through the hinge region of the heavy chain, and the Fc infection. Within a few weeks of the initial infection,
segment of the molecule becomes activated and reacts raised plasma concentrations of all immunoglobulins
with Fc receptors on a number of immune cell types may be demonstrated and may be detectable as a
resulting in immune activation. diffusely increased g zone on the electrophoretic strip.
The rest of the H and L chains is less variable There are four subclasses (IgG 1–4) in humans.
(constant region, which includes the Fc segment). The
activated Fc segments of the H chains are responsible Immunoglobulin M
for such properties as the ability to bind complement or Immunoglobulin M is synthesized first in response to
actively to cross the placental membrane. The H chains particulate antigens such as blood-borne organisms.
are associated with a variable amount of carbohydrate; Because of the relatively large size of IgM, it mostly
IgM has the highest content. remains in the vascular compartment. This, together
Some immunoglobulin molecules contain more than with the speed of synthetic response, makes it the
one basic unit bound together by ‘J’ chains; for example, first line of defence among immunoglobulins against
the IgM molecule consists of five units. Because of the invading organisms. The fetus can synthesize IgM, and
variation in size and therefore in density, the classes can high plasma concentrations at birth usually indicate
be separated by ultracentrifugation. They are classified that there has been an intrauterine infection.
by their sedimentation coefficient (Svedberg units, S),
the S value of a protein increasing with increasing size. Immunoglobulin A
The S values of immunoglobulins, together with their Immunoglobulin A is synthesized predominantly
most important functions and properties, are shown submucosally and is present in intestinal and
in Table 19.1. However, now with the development respiratory secretions, sweat, tears and colostrum. It is
of reliable immunoassays, ultracentrifugation is affected more than other immunoglobulins in diseases
not used in routine clinical practice to quantify of the gastrointestinal and respiratory tracts. Secretory
immunoglobulins. IgA is a dimer, in which two subunits are joined by a
Table 19.1 The properties and functions of the plasma immunoglobulins (Ig)
peptide ‘J’ chain, and has a ‘secretory piece’ synthesized Table 19.3 Diseases associated with increased serum
by epithelial cells. immunoglobulin E
Infections, particularly if chronic, activate B
Allergic disease (type 1 ‘Extrinsic’ asthma
lymphocytes and cause a polyclonal immunoglobulin hypersensitivity) Hay fever
response. However, immunoglobulin estimation adds
little to the observation of increased g-globulin visible Atopic eczema
in the serum electrophoretic pattern. Urticaria–angioedema
In certain conditions (some of which are listed Anaphylaxis
in Table 19.2), one or more immunoglobulin class Allergic disease arising Some genetic defects
predominates. Although there is considerable secondary to immunodeficiency Other primary T-cell deficiencies and
overlap, individual immunoglobulin estimation may or immunosuppression acquired immunodeficiency syndrome
occasionally help in the diagnosis of some of these (AIDS)
conditions. Direct response to infections Mainly parasitic helminths (e.g.
echinococcosis, ascariasis and
Immunoglobulin response to allergy toxocariasis)
Immunoglobulin E
Immunoglobulin E is synthesized by plasma cells The diagnosis of an allergic disorder is usually made
beneath the mucosae of the gastrointestinal and from the history, but skin testing and the measurement
respiratory tracts and in the lymphoid tissue of the of plasma specific IgE concentrations may be needed
nasopharynx. Circulating IgE is rapidly bound to cell as total serum IgE concentrations are relatively
surfaces, particularly those of mast cells and basophils; insensitive for allergy diagnosis. In skin testing, a small
plasma concentrations are therefore very low. drop of dilute allergen is injected subcutaneously;
The combination of antigen with this cell-bound a positive ‘wheal and flare’ develops within a few
antibody stimulates cells to release biologically minutes (immediate hypersensitivity). This test is
active mediators and accounts for such immediate contraindicated in patients with a history of eczema or
hypersensitivity reactions as occur in hay fever, the most anaphylaxis, and plasma IgE may be assessed to detect
severe form of hypersensitivity being anaphylactic shock. specific allergens.
Raised plasma IgE concentrations are found in
several diseases with an allergic (atopic) component, Deficiencies of components of the
inflammatory response and of proteins
such as some cases of eczema, asthma due to allergens
of the immune system
(‘extrinsic’) and parasitic infestation (Table 19.3).
Deficiencies of the inflammatory or immune response
of the host defence system may result in an increased
Table 19.2 Some abnormalities of plasma risk of infection and lack of immune regulation. Plasma
immunoglobulins in disease immunoglobulins reflect only the humoral phase of
Predominant class Examples of clinical conditions the immune system; their measurement does not assess
of immunoglobulin cellular immunity. Normal plasma immunoglobulin
(Ig) increased concentrations do not exclude an immune deficiency
IgG Autoimmune diseases, e.g. systemic lupus state; they may, in fact, be raised, as in the acquired
erythematosus and chronic active hepatitis immunodeficiency syndrome (AIDS). The human
IgA Diseases of the intestinal tract, e.g. Crohn’s immunodeficiency virus (HIV) binds to CD4 cells and
disease becomes internalized.
Diseases of the respiratory tract, e.g. tuberculosis, As a general rule, any patient with a severe, protracted,
bronchiectasis unusual or recurrent infection should be investigated
IgM Primary biliary cirrhosis for an underlying deficiency in ‘defence’ mechanisms.
Haemoprotozoan infections, e.g. malaria
B-cell and immunoglobulin deficiency
At birth, indicating intrauterine infections Dysgammaglobulinaemia
Viral hepatitis and some acute viral infections Dysgammaglobulinaemia is defined as an abnormality
IgG, IgA, IgM Chronic bacterial infections, sarcoidosis, acquired in the structure or distribution in the class(es) of
immunodeficiency syndrome (AIDS) immunoglobulins.
292 Proteins in plasma and urine
The plasma globulin concentration may be reduced Several rare syndromes, usually familial,
or normal. have been described. In infantile sex-linked
Immunoglobulin subclass determination is rarely agammaglobulinaemia (Bruton’s disease), which
needed clinically. However, IgG subclass deficiency may occurs only in males, there is almost complete
be associated with certain recurrent respiratory tract absence of B- cells and circulating immunoglobulins,
infections. while cellular (T-cell) immunity is normal. Clinical
consequences are increased susceptibility to various
Hypogammaglobulinaemia
infections, commonly of the respiratory tract. Other
Hypogammaglobulinaemia can be defined as a decrease syndromes have varying degrees of immunoglobulin
in plasma immunoglobulins. Total plasma protein deficiency and impaired cellular immunity and can
concentration is usually low. A marked reduction occur in either sex.
in plasma immunoglobulin concentrations may be
detectable as obvious hypogammaglobulinaemia in the Secondary immunoglobulin deficiency
serum electrophoretic pattern, but usually measurement Low plasma immunoglobulin concentrations are
of individual proteins is needed to make a diagnosis. The most common in patients with malignant disease,
effects of deficiencies of individual immunoglobulins particularly of the haemopoietic and immune
are related to their functions and distribution: systems, and often precipitated by chemotherapy or
radiotherapy; they are an almost invariable finding
● IgA deficiency may be symptomless, or may be
in patients with myelomatosis (immune paresis). In
associated with recurrent, mild respiratory tract
severe protein-losing states, such as the nephrotic
infections or intestinal diseases.
syndrome or protein-losing enteropathy, low plasma
● IgG deficiency may result in recurrent pyogenic
immunoglobulin concentrations (especially of IgG) are
infections of tissue spaces, especially in the lungs
due partly to the loss of relatively low-molecular-weight
and skin, by toxin-producing organisms such as
immunoglobulins and partly to increased catabolism.
staphylococci and streptococci.
Other causes are shown in Box 19.1.
● IgM deficiency frequently predisposes to septicaemia.
Primary immunoglobulin deficiency is less common T-cell deficiency
than that which is secondary to other disorders. Various Patients with T-cell deficiency present with severe,
classifications have been proposed for these deficiencies, protracted infections caused by viruses, fungi or
some of which are discussed below. rarely detected organisms such as Pneumocystis carinii
Transient immunoglobulin deficiency
and Cryptococcus. It is important to identify T-cell
deficiencies, because the administration of live vaccines
In the newborn infant, circulating IgG is derived
from the mother by active placental transfer. Plasma
concentrations decrease over the first 3–6 months of Box 19.1 Some causes of
life and then gradually rise as endogenous IgG synthesis hypogammaglobulinaemia
increases. In some infants, onset of synthesis is delayed
and ‘physiological hypogammaglobulinaemia’ may Primary
persist for several months. Bruton’s or Swiss-type agammaglobulinaemia
During pregnancy, most of the IgG transfer across Secondary
the placenta takes place in the last 3 months. Severe Protein-losing states, e.g. nephrotic syndrome, major
deficiency may therefore develop in very premature burns and protein-losing enteropathy
babies because the concentration of IgG derived from Undernutrition
Diabetes mellitus
the mother falls before the endogenous concentration
Cushing’s syndrome
rises.
Malignancy, including chronic lymphocytic leukaemia,
Primary immunoglobulin deficiency lymphomas, paraproteinaemia
Chronic kidney disease
Primary IgA deficiency in plasma, saliva and other
Chronic infections
secretions is relatively common, with an incidence Drugs such as steroids, azathioprine, cyclophosphamide
of about 1 in 500 of the population. Most cases are Irradiation
symptom free.
Plasma proteins 293
and blood products is then contraindicated. T-cell In hereditary angioneurotic oedema, C1 esterase
deficiency may be classified as: inhibitor is deficient or of low activity. The condition
is characterized by episodes of increased capillary
● primary (congenital) deficiencies, which are rare,
permeability and consequent oedema of the
often severe and usually present soon after birth;
subcutaneous tissues and mucous membranes of the
they range from total or partial T-cell depletion to
upper respiratory or gastrointestinal tract. Laryngeal
loss of function,
oedema may be fatal. Plasma C1 esterase inhibitor
● secondary (acquired) deficiencies, associated with
concentration or activity can be measured.
metabolic disorders such as diabetes mellitus,
Complement deficiences can be assessed by
undernutrition, drug abuse, auto-immunity,
measuring individual complement components and
malignancy and immunosuppressive treatment or
also CH50 (a measure of the complement activity).
radiation; secondary T-cell deficiency is relatively
common, often mild and presents at any age.
a1-Antitrypsin deficiency
In severe combined immunodeficiency (SCID)
syndrome, T-cell function is affected, often also with B-cell a1-Antitrypsin normally controls the proteolytic action
abnormalities. This syndrome can be caused by a number of lysosomal enzymes from phagocytes (protease
of genetic conditions that interfere with lymphocyte inhibitors, PIs), and is an acute-phase reactant. Plasma
development and function, including SCID-X1, a defect concentrations rise 2–3 days after trauma or acute
of the g-chain of interleukin receptors, and adenosine infection.
deaminase deficiency, which allows a lymphocyte There are more than 50 genetic variants of a1-
toxic build-up of adenosine diphosphate, guanosine antitrypsin, inherited as autosomal codominant
triphosphate and deoxyadenosine triphosphate. alleles. The most common allele is PIM with a genotype
T cells may be decreased in number or may function MM. At least seven of the phenotypes associated with
abnormally. The total lymphocyte count may be normal low functional plasma a1-antitrypsin concentrations,
despite severe T lymphocytopenia, such as occurs in the most important of which is PI null, are associated
acquired deficiencies, or malignancy, as in chronic with complete deficiency of the protein. Others
lymphocytic leukaemia. T-cell function may be assessed include PIS and PIZ, in which protein accumulates in
by determining cell-surface markers and therefore the liver because it cannot be secreted after synthesis,
changes in the different T-cell subsets. Changes in and PIM Duarte, in which plasma concentrations
the ratio of helper to suppressor cells occur in many of the protein may be normal but there is deficient
conditions, but are profound in AIDS, in which the functional activity.
virus specifically affects the CD4 cell population and a1-Antitrypsin deficiency may be suspected after
numbers can go below 200 cells/mm3. serum electrophoresis if the a1 band is much reduced,
absent or split; the condition may present clinically
Deficiencies of other proteins of the with the following:
inflammatory response ● Hepatic cirrhosis, occurring in 10–20 per cent of
Other proteins involved in complement activation or subjects, such as PIZ homozygotes, in whom the
as acute-phase reactants may be deficient in addition protein cannot be secreted by hepatocytes. The
to immunoglobulins. The most clinically significant are condition may present as hepatitis in the neonatal
discussed below. period or as cirrhosis in children or young adults
(see Chapter 17).
Complement deficiency ● Pulmonary disease: the unopposed action of
Complement deficiencies, for example of C5–9, are proteases from phagocytes in the lung may, by
very rare, although they may predispose individuals destroying elastic tissue, cause basal panlobular
to infections, for example meningococcal. C1, C2 emphysema in adults, aged between 30 and 40 years,
and C4 deficiency are associated with SLE. Mannose- who are homozygous for the abnormal alleles. The
binding lectin deficiency increases the risk of Neisseria condition may be exacerbated by cigarette smoking,
meningitidis and Streptococcus pneumoniae infection. air pollution or infection.
C5 deficiency may lead to Leiner’s disease, associated The diagnosis of a1-antitrypsin deficiency can
with diarrhoea, seborrhoeic dermatitis and wasting. usually be confirmed by demonstrating that the
294 Proteins in plasma and urine
CASE 1
Normal control A 72-year-old man presented to his general
practitioner with back pain and weakness. The
following are the results of some of his laboratory
Figure 19.7 Serum and urinary protein electrophoretic
tests:
patterns in myelomatosis. Patient A with paraprotein Plasma
and immune paresis in serum and Bence Jones protein Sodium 136 mmol/L (135–145)
(BJP) band in urine. Patient B with paraprotein and Potassium 4.9 mmol/L (3.5–5.0)
immune paresis in serum and with heavy BJP on the Urea 13.7 mmol/L (2.5–7.0)
right and leakage of albumin and other low-molecular-
Creatinine 160 µmol/L (70–110)
weight proteins (glomerular permeability) in urine.
Estimated glomerular filtration rate (eGFR)
39 mL/min per 1.73 m2
Other findings in patients with malignant B-cell Albumin-adjusted calcium 3.20 mmol/L (2.15–2.55)
tumours are: Total protein 98 g/L (60–75)
● normochromic normocytic anaemia, a common Albumin 34 g/L (35–45)
presenting feature of any malignant disease, Globulins 64 g/L (15–30)
● haemorrhages, perhaps due to complexing of DISCUSSION
coagulation factors by the paraprotein, Note the impaired renal function, hypercalcaemia,
● Raynaud’s phenomenon, which may occur if the hypoalbuminaemia and raised plasma globulins
paraprotein is a cryoglobulin, and raised total plasma protein concentration.
● hypercalcaemia may also be present. The patient was also found to be anaemic, with a
haemoglobin concentration of 9.3 g/dL. Dipstick
b2-Microglobulin is a low-molecular-weight protein
urine testing showed proteinuria. Serum and urinary
(11.8 kDa) that forms part of the human lymphocyte
protein electrophoresis showed an IgG k-paraprotein
antigen on the surface of all nucleated cells. The
with immune paresis and Bence Jones proteinuria.
protein is readily filtered by glomeruli, and plasma
Skeletal bone survey showed lytic bone lesions, and
concentrations are normally low. In myelomatosis the
bone marrow biopsy supported the diagnosis of
plasma b2-microglobulin concentration is an index
multiple myeloma.
of the extent of the disease and of the prognosis.
296 Proteins in plasma and urine
in the skull, vertebrae, ribs and pelvis. There may be the cells resemble lymphocytes rather than plasma
generalized osteoporosis. Pathological fractures may cells. There is generalized lymphadenopathy. Like
occur. Histologically there is little osteoblastic activity myelomatosis, macroglobulinaemia usually occurs in
around the lesion, which arises in the marrow rather older patients, over the age of 60, but, unlike myelomatosis,
than in the bone itself; consequently, the plasma alkaline it is more common in men than in women. Symptoms
phosphatase activity is normal unless there is liver of the ‘hyperviscosity syndrome’ are commoner than in
involvement, in which case the raised level is of hepatic, myelomatosis, probably because of the large size of the
not bony, origin. A normal plasma alkaline phosphatase IgM molecule; skeletal manifestations are rarer.
activity, in cases with bone lesions, suggests myelomatosis The laboratory findings and diagnosis include the
rather than bony metastases. Hypercalcaemia may occur following:
and also renal failure. The latter may occur as a result of
● IgM monoclonal paraprotein in the g-region on
Bence Jones proteinuria as well as the hypercalcaemia.
protein electrophoresis. The plasma IgA concentration
Amyloidosis is also associated.
is usually reduced, but that of IgG may be raised.
Disordered immunoglobulin synthesis and immune paresis ● The bone marrow aspirate or lymph node biopsy
The immunoglobulin most frequently increased is IgG, contains atypical lymphocytoid cells.
and, less commonly, IgA (about 2.5:1). Occasionally,
IgM, IgD or IgE is found, the last two being very rare. Heavy-chain disease
Bence Jones protein may sometimes be present in The heavy-chain diseases are a rare group of disorders
plasma if renal failure is present. In about 20 per cent characterized by the presence of an abnormal protein
of cases, no paraprotein is detected in the plasma but in plasma or urine, identifiable as part of the H chain
BJP is detected in urine. Rarely, neither a paraprotein (a, g or µ). The clinical picture is that of:
nor BJP can be found. In either case there is usually
immune paresis. In IgD myelomatosis an increase in ● intestinal lymphomatous lesions, with severe
g-globulin concentration may not be detectable by malabsorption (a-chain disease); the condition
routine electrophoresis. The paraprotein should be usually affects young adults of Mediterranean
typed and the plasma concentration monitored to origin;
follow progress. The serum free light chain ratio (k to ● generalized lymphadenopathy with recurrent
l) may also be abnormal. This ratio is normally about infections in the elderly (g-chain disease);
0.26–1.65 and can be useful in monitoring patients with ● chronic lymphatic leukaemia (µ-chain disease).
low levels of paraprotein (monoclonal or ‘M’ protein) In some of these conditions a paraprotein may be
and of early treatment response and disease relapse. detectable.
Bone marrow appearance
Cryoglobulinaemia
The proportion of plasma cells in the bone marrow is
increased and many of these cells are atypical (‘myeloma Cryoglobulins are proteins that precipitate when cooled
cells’). Examination of a bone marrow aspirate must be below body temperature. They may be associated with
carried out before myelomatosis is diagnosed or excluded. diseases known to produce paraproteins. About half
of them can be shown to consist of a monoclonal
Soft-tissue plasmacytoma immunoglobulin (usually IgM or IgG). The patient
Rarely, myeloma involves soft tissues, without marrow usually presents with other symptoms of the underlying
changes (extramedullary plasmacytoma). Although the disease and the cryoglobulin is found during
protein abnormalities of myelomatosis are often found, investigation. Occasionally, intravascular precipitation
the behaviour and prognosis of the two conditions are may occur at temperatures above 22°C and, if the
different. Spread of soft-tissue plasmacytoma is slow concentration of protein is high, presenting symptoms
and tends to be local. Local excision of the solitary and signs may be skin lesions such as purpura and
tumour may be effective. Raynaud’s phenomenon. In some cases the protein
is polyclonal and includes complement; these cases
Waldenström’s macroglobulinaemia may be associated with immune-complex disease.
Waldenström’s macroglobulinaemia is caused by a Occasionally no underlying abnormality can be found
malignancy of B cells, with increased synthesis of IgM; (essential cryoglobulinaemia).
Proteins in urine 297
To determine cryoglobulinaemia, the blood should be light-chain fragment (abbreviated L); thus, patients
collected in prewarmed tubes at 37°C and transported with these amyloidoses are now said to have light-
quickly to the laboratory. Aliquots can be taken, one at chain amyloidosis (AL). In instances previously termed
room temperature (22°C) and the other at 37°C. The senile cardiac amyloidosis and in cases formerly called
cryoglobulin protein can be quantified by subtracting familial amyloid polyneuropathy, the fibrils consist
the difference between the 37°C sample and the of the transport protein transthyretin (TTR); these
supernatant of the 22°C sample, as at 22°C the protein/ diseases are now termed ATTR. (See Chapter 11.)
gel would be expected to precipitate, as lower than body
temperature. PROTEINS IN URINE
The loss of most plasma proteins through the
Monoclonal gammopathy of undetermined significance
glomeruli is restricted by the size of the pores in, and
If a paraprotein is found on electrophoresis, investigation by a negative charge on, the basement membrane that
for one of the diseases discussed above should be initiated. repel negatively charged protein molecules. Alteration
In up to 30 per cent of hospital cases, and probably of either of these factors by glomerular disease may
more in the ‘well population’, no cause can be found. allow albumin and larger proteins to enter the filtrate.
The condition, which may be transient, has been called Low-molecular-weight proteins are filtered even under
‘benign’ or ‘essential’ paraproteinaemia or monoclonal normal conditions; most are absorbed and metabolized
gammopathy of undetermined significance (MGUS). The by tubular cells. Normal subjects excrete up to 0.08 g
diagnosis should be made provisionally and the patients of protein a day in the urine, amounts undetectable by
followed up; they may later develop myelomatosis or usual screening tests. Proteinuria of more than 0.15 g a
macroglobulinaemia. This condition is more common day almost always indicates disease.
in the elderly; BJP is not usually present and serum Significant proteinuria may be due to renal disease
paraprotein (or M protein) is often less than 30 g/L with or, more rarely, may occur because large amounts of
no immune paresis or amyloidosis and no evidence of low-molecular-weight proteins are circulating and
myeloma organ or tissue impairment and with the bone therefore being filtered. Blood and pus in the urine and
marrow less than 10 per cent of clonal plasma cells. also urinary infection give positive tests for protein (see
also Chapter 3).
Amyloid
Amyloid diseases are secondary protein structure Renal proteinuria
diseases in which insoluble protein fibrils accumulate Glomerular proteinuria
extracellularly. At least 20 different types of fibrils
have been described in human amyloidosis, each with Glomerular proteinuria is due to increased glomerular
a different clinical picture. All types of tissue amyloid permeability, as in nephrotic syndrome. Albumin is
consist of a major fibrillar protein that defines their usually the predominant protein in the urine.
type. All amyloid types show certain features: Transient proteinuria
● soluble in water and in buffers of low ionic strength, Transient proteinuria may be associated with physical
● amorphous eosinophilic appearance on light exertion, trauma, cardiac failure, fever and other acute
microscopy after haematoxylin and eosin staining, illness.
● green fluorescence seen under polarized light after ‘Orthostatic’ (‘postural’) proteinuria
Congo red staining,
Proteinuria is usually more severe in the upright
● regular fibrillar structure as observed by electron
than in the prone position. The term ‘orthostatic’ or
microscopy,
‘postural’ has been applied to proteinuria, often severe,
● X-ray diffraction shows b-pleated sheet structure.
which disappears at night. Overnight urine collection
Amyloidosis is usually classified biochemically. shows normal albumin excretion (i.e. less than 50 mg
The amyloidoses are referred to with a capital A (for during the 8-h period). It appears to be glomerular
amyloid) followed by an abbreviation for the fibril in origin and is most common in adolescents and
protein. For example, in cases formerly termed primary young adults, typically those who are tall and thin. It
amyloidosis and in myeloma-associated amyloidosis, may be associated with severe lordosis. Renal function
the fibril protein is an immunoglobulin light chain or is normal and proteinuria is usually less than 1 g/day.
298 Proteins in plasma and urine
Although it is often harmless, evidence of renal disease a-globulins and b-globulins are sensitive markers
may occur after some years. of renal tubular damage. Tubular proteinuria can be
Microalbuminuria
diagnosed by measuring certain low-molecular-weight
proteins in urine, such as retinol-binding protein (RBP),
Sensitive immunological assays have shown the normal N-acetyl-b-D-glucosaminidase or a1-microglobulin.
daily excretion of albumin to be less than 0.05 g. However, because the b2-microglobulins in urine
Patients with diabetes mellitus who excrete more are unstable, other proteins, such as RBP, may be better
than this, but whose total urinary protein excretion is indicators of tubular damage. Tubular proteinuria
‘normal’, are said to have microalbuminuria and to be is associated with Fanconi’s syndrome and with
at greater risk of developing progressive renal disease glycosuria, amino aciduria, hyperphosphaturia
than those whose albumin excretion is normal. This (resulting in hypophosphataemia) and renal tubular
can be assessed from the urinary albumin to creatinine acidosis. This can be primary or secondary, for example
ratio (ACR) The incidence of this complication may be to heavy metals and drugs such as cisplatin.
reduced by optimization of glycaemic control and also
blood pressure using angiotensin-converting enzyme Overflow proteinuria
(ACE) inhibitors (see Chapter 12). Microalbuminuria This occurs when proteins of low molecular weight are
can also occur in other diseases, such as inflammatory filtered normally by the glomerulus and reabsorbed at
bowel disease or rheumatoid arthritis. the proximal tubule but are produced in amounts greater
Tubular proteinuria
than the reabsorptive capacity of the proximal tubule.
Overflow proteinuria can be due to the production
Tubular proteinuria may be due to renal tubular damage of BJP, to severe haemolysis with haemoglobinuria,
from any cause, especially pyelonephritis. If glomerular or to severe muscle damage (rhabdomyolysis) with
permeability is normal, proteinuria is usually less than myoglobinuria. In the last two cases, the urine may be
1 g/day and consists mainly of low-molecular-weight red or brown in colour.
globulins and not albumin. Low-molecular-weight Bence Jones proteinuria can be inferred by comparison
of urinary and serum electrophoretic patterns, by
immunochemical assays and confirmation of free light
CASE 2 chains by immunofixation. Bence Jones protein is the
A 65-year-old man with type 2 diabetes mellitus only protein of a molecular weight lower than albumin
had glycated haemoglobin (HbA1c) of 10.0 per cent that is likely to be found in significant amounts in
(86 mmol/mol) and random blood glucose unconcentrated urine in the absence of haemoglobinuria
concentration of 23.8 mmol/L in the diabetes clinic. or myoglobinuria. The presence of a band in urine that is
He was taking metformin 500 mg twice a day. His denser than that of albumin, especially if not present in
blood pressure was 180/100 mmHg. The urinary the serum, is suggestive of BJP.
albumin to creatinine ratio (ACR) was 9.9 g/mol. A
year prior to this, his urinary ACR was normal, at less Apparent proteinuria
than 2.5 g/mol. Apparent proteinuria has occasionally been found
because the patient, or someone else, has added egg
DISCUSSION
white or animal blood to their urine for example.
The patient has microalbuminuria, which was
confirmed on repeat urine sampling. This can Nephrotic syndrome
progress to proteinuria and eventually chronic kidney
An established case of the nephrotic syndrome is
disease. The aim of the clinician should be to try to
characterized by proteinuria, hypoalbuminaemia,
optimize glycaemic control. In this case, the patient’s
oedema and hyperlipidaemia. The clinical condition is
HbA1c was high, reflecting poor control, and was
caused by increased glomerular permeability, resulting
improved by increasing his metformin and adding
in a daily urinary protein loss of, by definition, more
the sulphonylurea gliclazide, which brought his HbA1c
than 3 g (although some definitions state different
to 8 per cent (64 mmol/mol). He is hypertensive,
values). There may be hypertension and evidence of
which also needs treating, and he was started on an
other impaired renal function. Some of the causes are
angiotensin-converting enzyme inhibitor.
shown in Box 19.2.
Proteins in urine 299
DISCUSSION
The patient had nephrotic syndrome presenting Normal control
with peripheral oedema, hypoproteinaemia,
hypoalbuminaemia and severe hyperlipidaemia. Note
that the plasma creatinine and urea concentrations
may not be too abnormal unless chronic kidney
Patient A
disease ensues. A renal biopsy showed that the patient Serum
had focal segmental glomerulonephritis.
Urine
Laboratory findings of nephrotic syndrome
Protein abnormalities
Proteinuria in the nephrotic syndrome usually ranges
Patient B
from 3 g or more a day (or a urine protein to creatinine Serum
ratio of > 300 mg/mmol; see Chapter 3). The relative
proportion of different proteins lost is an index of the
severity of the glomerular lesion. In mild cases, albumin Urine
(molecular weight 65 kDa) and transferrin (molecular
weight 80 kDa) are the predominant urinary proteins,
but a1-antitrypsin (molecular weight 50 kDa) is also
present. With increasing glomerular permeability, Figure 19.8 Serum and urinary protein electrophoretic
larger proteins such as IgG (molecular weight 160 kDa) patterns from patients with the nephrotic syndrome.
appear. Patient A has selective glomerular proteinuria, and
patient B has non-selective proteinuria.
The serum electrophoretic picture depends on the
severity of the renal lesion. In early cases a low serum
albumin concentration may be the only abnormality, globulin because of an increase in the high-molecular-
but the typical pattern includes reduced albumin, weight a2-macroglobulin (Fig. 19.8). The g-globulin
a1- and g-globulin bands, with an increase in a2- concentration may be normal or raised. Urinary
300 Proteins in plasma and urine
electrophoresis gives some idea of the severity of the pH 3. At this pH it is yellow in the absence of protein.
glomerular lesion. Protein forms a complex with the dye, stabilizing it in
The differential protein clearance is a more precise the blue form; it is green or bluish-green if protein is
measure of the selectivity of the lesion. The clearance present. The colour after testing is compared with
of a low-molecular-weight protein, such as transferrin the colours on the chart provided, which indicate the
or albumin, is compared with that of a larger one, approximate protein concentration. The strips should
such as IgG. The result is usually expressed as a ratio, be kept in the screwtop bottles, in a cool place and the
obviating the need for timed urine collections. A ratio instructions should be followed carefully. However, due
of IgG to transferrin or albumin clearance of less than to some of the problems associated with such methods
0.2 indicates high selectivity (predominant loss of small in some countries there has been a move away from
molecules) and has a more favourable prognosis than a such testing, preferring instead the spot urine ACR.
higher ratio; such cases usually respond well to steroid False-positive results occur:
or cyclophosphamide therapy.
● if the specimen is contaminated with vaginal or
The consequences of the protein abnormalities are
urethral secretions, including haematuria, semen or
oedema caused by a fall in the intravascular colloid
menstrual fluid,
osmotic pressure, due to hypoalbuminaemia (see
● in strongly alkaline (infected or stale) urine, when
Chapter 2), and reduction in the concentration of
buffering capacity is exceeded; a green colour in this
protein-bound substances due to loss of carrier pro-
case is a reflection of the alkaline pH,
teins; it is important not to misinterpret low plasma
● if the urine container is contaminated with
total concentrations of calcium, thyroxine, cortisol and
disinfectants such as chlorhexidine.
iron.
In mild cases, plasma LDL concentrations increase, False-negative results occur if acid has been added
with consequent hypercholesterolaemia probably due to the urine as a preservative (for example for the
to the increased rate of protein synthesis, including estimation of urinary calcium).
that of apolipoprotein B (apoB) lipoprotein, in the liver
(see Chapter 17). In more severe cases, a rise in plasma Investigation of proteinuria (Fig. 19.9)
very low-density lipoprotein (VLDL) (triglycerides) ● Normally urinary total protein is about 50–150 mg/
concentration may cause plasma turbidity. Fatty casts day. Rather than a 24-h urinary protein collection,
may be detectable in the urine. it may be more convenient to use a spot urine ACR
In the early stages, glomerular permeability is high or in pregnancy a spot urinary protein to creatinine
and the plasma urea and creatinine concentrations are (P:C) ratio. Normally the latter is less than 15 mg/
normal, although uraemia may develop. At this stage, mmol. Remember that urine dipstick tests can give
protein loss is reduced and plasma concentrations of false-positive results (see above).
protein and lipid may revert to normal, but this does ● Exclude urinary tract infection; a midstream urine
not indicate recovery. Secondary hyperaldosteronism test may be useful.
may also occur (see Chapters 2 and 3). ● Is the proteinuria transient? Confirm on at least two
or three occasions.
Testing for urinary protein ● Renal function tests, i.e. plasma urea and creatinine,
It is essential that the sample is fresh. There are should be performed and it is also important to check
limitations of screening tests, such as the following. plasma albumin and protein for hypoalbuminaemia
and hypoproteinaemia.
● The tests were mainly developed to detect albumin
● Take a drug history for renal toxic agents, for example
and may be negative in the presence of other
gold or penicillamine.
proteins, such as BJP.
● Clinical history and examination may reveal systemic
● Because the tests depend on protein concentrations,
illnesses that can cause proteinuria (see above).
very dilute urine may give negative results despite
Check plasma prostate-specific antigen in men to
significant proteinuria.
help assess for prostatic disease.
One example of a urinary protein screening test is ● Consider orthostatic or postural proteinuria when
Albustix. The test area of the reagent strip is impregnated an early morning urine sample is negative for protein
with an indicator (tetrabromophenol blue) buffered to but pre-bed samples show proteinuria.
Blood sampling for protein estimations 301
in hydration, or to changes in capillary permeability inspection of a bone marrow aspirate or biopsy and
(see Chapter 2). Dehydration may evoke raised usually also a skeletal survey. Plasma immunoglobulin
plasma albumin and protein concentrations, whereas concentrations should be measured to assess the degree
the converse is true in overhydration. of immune paresis.
● To help assess nutritional status Plasma albumin
concentrations are sometimes used to help ● To evaluate apparently abnormal concentrations
assess nutritional status. However, low albumin or changes in concentrations of a protein-bound
concentrations, although associated with substance Estimation of plasma albumin
undernutrition, are not specific as they can be low concentration should always accompany that of total
for other reasons (see Chapter 14). plasma calcium. If changes in this or other protein-
● To investigate suspected myelomatosis or bound substances parallel those of albumin, they are
macroglobulinaemia The difference between the probably due to changes in protein concentrations.
total plasma protein and albumin concentration is ● As part of the investigation of hypogamma-
called the ‘globulin’ concentration. This is raised in globulinaemia A low total plasma protein
clinical situations when the globulin concentration concentration, if not due to hypoalbuminaemia,
is raised, such as in myelomatosis. may be due to hypogammaglobulinaemia. Plasma
● Raised plasma globulin also occurs in liver disease, immunoglobulin concentrations can be measured
for example autoimmune hepatitis. as part of the assessment of the adequacy of the
immune system.
Protein electrophoresis should be carried out on
serum to detect a paraprotein or M protein and, Note that these tests assess only humoral immunity:
importantly, also on urine to detect BJP. The diagnosis T-cell abnormalities and their investigations are outside
of myelomatosis should be confirmed by microscopic the scope of this book.
SUMMARY
● Abnormalities of plasma and urinary proteins are synthesis and humoral immunity, whereas T cells
relatively common in clinical practice, and therefore are involved in cellular immunity.
knowledge of protein biochemistry is necessary for ● Multiple myeloma is a plasma cell malignancy
a full understanding of these conditions. associated with paraproteinaemia, Bence
● Diagnostically, certain proteins can be specifically Jones proteinuria, osteolytic bone lesions and
assayed or protein electrophoresis of serum and/or hypercalcaemia.
urine can be performed to show pattern changes. ● Proteinuria can be due to either glomerular or tubular
● The acute-phase response and inflammation are disorders. The nephrotic syndrome is associated
essential mechanisms to protect the body. B cells with peripheral oedema, hypoalbuminaemia and
are concerned primarily with immunoglobulin hypoproteinaemia.
20 Purine and urate metabolism
Abnormalities of purine metabolism are often found when its products increase. The control of this rate-
in clinical practice, notably hyperuricaemia and limiting step may be impaired in primary gout.
gout. After exploring purine metabolic pathways, Glycine is added to phosphoribosylamine. The rate
this chapter discusses the various disorders of purine of purine synthesis is increased in primary gout. In
metabolism, including their clinical features, diagnosis Figure 20.1, the atoms in the glycine molecule have
and treatment. been numbered to correspond with those in the purine
and urate molecules, and the solid lines further indicate
PURINES the final position of the amino acid in these molecules.
Normal purine metabolism Via a series of metabolic steps, purine ribonucleotides
Urate is the end product of purine metabolism in humans. (purine ribose phosphates) are formed, which control
In most other mammals, it is further metabolized to the the second step in the synthetic pathway – the formation
more water-soluble allantoin. Humans lack uricase and of phosphoribosylamine. Ribose phosphate is cleaved
thus their uric acid levels are higher. It is because of off, thereby releasing the purines. Some cytotoxic drugs
the poor solubility of urate that humans are prone to inhibit various stages of the pathway, thus preventing
the clinical effects of hyperuricaemia, such as gout and DNA formation and cell growth.
renal damage. The purines adenine and guanine are
constituents of nucleic acid from deoxyribonucleic acid Fate of purines
(DNA) and ribonucleic acid (RNA). The purines used Purines synthesized in the body, those derived from
by the body for nucleic acid synthesis may be derived the diet and those liberated by endogenous catabolism
from the breakdown of ingested nucleic acid, mostly of nucleic acids may be oxidized to urate or reused for
from cell-rich meat, or they may be synthesized de novo nucleic acid synthesis.
from small molecules. About two-thirds of the body’s Purines oxidized to urate Some adenine is oxidized
urate (3–4 mmol/day) is produced endogenously, with to hypoxanthine, which is further oxidized to xanthine.
one-third coming from exogenous dietary purines Guanine can also form xanthine. Xanthine, in turn,
(1–2 mmol/day). is oxidized to form urate. The oxidation of both
hypoxanthine and xanthine is catalysed by xanthine
Synthesis of purines oxidase in the liver. Thus the formation of urate from
The upper part of Figure 20.1 summarizes some of the purines depends on xanthine oxidase activity: gout may
more important synthetic steps. be treated using an inhibitor of this enzyme (allopurinol).
The first step in purine synthesis is condensation Purines reused for nucleic acid synthesis Some xanthine,
of pyrophosphate with phosphoribose to form hypoxanthine and guanine can be resynthesized to purine
phosphoribose diphosphate (phosphoribosyl nucleotides by pathways involving, among other enzymes,
pyrophosphate, PRPP). hypoxanthine–guanine phosphoribosyl transferase
The amino group of glutamine is incorporated into (HGPRT) and adenine phosphoribosyl transferase (APRT).
the ribose phosphate molecule and pyrophosphate is
released. Amidophosphoribosyl transferase catalyses this Excretion of urate
rate-limiting or controlling step. The enzyme is subject Urate is filtered through the glomeruli and most
to feedback inhibition by increasing concentrations of is reabsorbed in the proximal tubules. More than
purine nucleotides; thus the rate of synthesis is slowed 80 per cent of that formed in urine is derived from more
304 Purine and urate metabolism
Purine synthesis
NH3+
H2C NH3+
7
–OOC H2C 5
Glycine
GluNH3+ Glu– + PP + O C
4
9
NH3+ NH
Ne
g at
ive
Ribose P Pyrophosphate
fe
ed
(PP)
ba
ck 6 N
N1 5 7
8
2 4 9
3
N N
Ribose P
Purine nucleotide
Guanine Adenine
Urate formation
O
6 N
HN 1 5 7 xanthine xanthine Hypoxanthine
8 O– oxidase
Xanthine
oxidase
2
3 4 9
O N N
H H
Several steps
URATE
Negative feedback
Figure 20.1 Summary of purine synthesis and breakdown, showing the steps of clinical importance. APRT,
adenine phosphoribosyl transferase; GluNH3+, glutamine; Glu–, glutamate; HGPRT, hypoxanthine–guanine
phosphoribosyl transferase.
distal tubular secretion. Urinary excretion is slightly in urine. The remaining 25 per cent passes into the
lower in males than in females, which may contribute intestinal lumen, where it is broken down by intestinal
to the higher incidence of hyperuricaemia in men. bacteria, the process being known as uricolysis.
Renal secretion may be enhanced by uricosuric
HYPERURICAEMIA
drugs (e.g. probenecid or sulfinpyrazone), which
block tubular urate reabsorption. Tubular secretion Causes of hyperuricaemia
of urates is inhibited by organic acids, such as lactic Generally speaking, hyperuricaemia can occur by two
and oxoacids, and by ketones and thiazide diuretics. main mechanisms: increased production (overproducers)
Seventy-five per cent of urate leaving the body is and/or decreased excretion (underexcretors).
Hyperuricaemia 305
The factors that may contribute to hyperuricaemia converts urate to uric acid, which is less soluble than
(summarized in Fig. 20.2) are: urate, and a vicious circle is set up in which further
precipitation, and therefore further inflammation,
● increased synthesis of purines (step 1),
occurs. In acute attacks of gouty arthritis, local factors are
● increased intake of purines (step 2),
more important than the plasma urate concentration,
● increased turnover of nucleic acids (step 3),
which is usually normal during the attack.
● increased rate of urate formation (step 4),
Precipitation may occur in subcutaneous tissues,
● reduced rate of excretion (step 5).
especially of the ears, and in the olecranon and
Increased synthesis, due to impaired feedback control, patellar bursae and tendons. Such deposits are called
is probably the most important mechanism causing gouty tophi (Fig. 20.3). A potentially serious effect of
primary hyperuricaemia, whereas abnormalities of the hyperuricaemia is precipitation of urate in the kidneys
other steps are causes of secondary hyperuricaemia. and renal calculi, causing progressive renal damage.
For this reason it has been recommended that even
Consequences of hyperuricaemia
asymptomatic cases should probably be treated if the
Urate is poorly soluble in plasma. At plasma pH, most plasma urate concentration is consistently higher
urate is ionized at position 8 of the purine ring (see than 600 µmol/L. Hyperuricaemia can be primary or
Fig. 20.1). This anionic group is associated with the secondary.
predominant extracellular cation, sodium. Ionization
of uric acid decreases as the pH falls, and it therefore Primary hyperuricaemia and gout
becomes less soluble; at a urinary pH below about 6, Familial incidence
uric acid may form renal calculi. In some cases a hereditary component is present,
Crystallization in joints, especially those of the feet, probably involving abnormalities of urate metabolic
produces the classic picture of gout. Urate precipitation pathways.
at these sites causes an inflammatory response with
leucocytic infiltration, and it is thought that lactic acid Sex and age incidence
production by these cells causes a local fall in pH: this Primary hyperuricaemia and gout are very rare in
PURINE 1
children and unusual in women of child-bearing age.
SYNTHESIS Plasma urate concentrations are low in children and
BODY PURINE TISSUE rise in both sexes at puberty, more so in males than in
NUCLEOTIDES NUCLEIC ACIDS
females; concentrations are higher in males. Women
DIETARY
3 become more prone to hyperuricaemia and gout in the
PURINES 2
post-menopausal period.
PURINES
URIC ACID
Intestinal uricolysis
by bacteria (25%)
5
gout is a rare complication. It may, however, be ● Colchicine, which has an anti-inflammatory effect
precipitated in those patients with a gouty tendency. and inhibits neutrophil activation, can be used
● Other drugs, including low doses of salicylates, in acute gouty arthritis but does not affect urate
ciclosporin, nicotinic acid, methoxyflurane, metabolism. It is sometimes used if NSAIDs are
levodopa, ethambutol and pyrazinamide. contraindicated.
● Renal glomerular dysfunction: before the diagnosis ● Reducing urate production by using drugs that
of primary hyperuricaemia is made, the plasma inhibit xanthine oxidase activity, such as allopurinol
concentration of creatinine should be assayed (hydroxypyrazolopyrimidine), which is structurally
on the same specimen as urate, to exclude renal similar to hypoxanthine, so acting as a competitive
dysfunction as a cause. It may be difficult to decide inhibitor of the enzyme. De novo synthesis may
which abnormality is the primary one because also be decreased by this drug. However, allopurinol
hyperuricaemia can cause renal dysfunction. The may worsen an acute attack of gout. Initiation of
urate to creatinine concentration ratio in a spot urine allopurinol should be made a few weeks after the
sample may help distinguish hyperuricaemia due acute attack under the cover of an NSAID, assuming
to reduced renal excretion from renal impairment that it is not contraindicated. Febuxostat is a non-
secondary to hyperuricaemia. A ratio of less than competitive inhibitor of xanthine oxidase and may
0.7 implies that renal impairment is responsible have a place in those intolerant to allopurinol.
for the hyperuricaemia. A ratio of greater than 0.7 ● Increasing the renal excretion of urate with uricosuric
suggests urate nephropathy. Clinical gout is rare in drugs, such as probenecid or sulfinpyrazone. These
the secondary hyperuricaemia of renal disease. drugs may be effective if renal function is normal, but
● Prolonged metabolic acidosis (see Chapters 3 and 4). are less so if there is renal glomerular dysfunction.
● Chronic lead intoxication (see Chapter 21). Fluid intake is usually kept high. Low doses of most
uricosuric drugs reduce urate secretion; they are
Combined (both increased production and reduced rarely used unless allopurinol is contraindicated.
excretion) causes
● High alcohol intake.
● Prolonged and severe exercise. Investigation of hyperuricaemia (Fig. 20.4)
● Dyslipidaemia and impaired glucose tolerance or type ● A careful drug history is important, as various drugs
2 diabetes mellitus. Hyperuricaemia is associated with (Box 20.1), such as thiazide diuretics, can evoke
insulin resistance and the metabolic syndrome. hyperuricaemia, as well as a high alcohol intake.
● Glucose-6-phosphatase deficiency or von Gierke’s ● A family history may reveal a primary hereditary
disease (see Chapter 27). The tendency to form of gout or hyperuricaemia.
hyperuricaemia in these patients may be directly ● Check renal function with plasma urea and
related to the inability to convert glucose-6- creatinine, as renal impairment is a common cause
phosphate (G6P) to glucose. More G6P is available of hyperuricaemia. A spot urinary urate to creatinine
for metabolism through intracellular pathways, concentration ratio may help to distinguish
including: hyperuricaemia due to reduced renal excretion from
– the pentose phosphate pathway, thus increasing renal impairment secondary to hyperuricaemia.
ribose phosphate (phosphoribose) synthesis; this ● Consider secondary conditions with high
may accelerate the first step in purine synthesis, cell turnover, such as tumours, psoriasis and
with consequent urate overproduction, polycythaemia (see Box 20.1).
– glycolysis, thus increasing lactic acid production; ● Hyperuricaemia can also be seen in a metabolic
lactic acid may reduce renal urate excretion. acidosis due to reduced renal urate excretion (see
Chapter 4).
Principles of treatment of hyperuricaemia ● There is an association between hyperuricaemia and
● Reducing dietary purine intake, for example red the metabolic syndrome with hypertriglyceridaemia,
meats. This treatment is rarely effective by itself. obesity and insulin resistance.
● An acute attack of gout can be treated with a non- ● A 24-h urine analysis for urate excretion on and off a
steroidal anti-inflammatory drug (NSAID) or low-purine diet may give an indication of whether the
sometimes steroids. patient is an overproducer or underexcretor of urate.
308 Purine and urate metabolism
Hyperuricaemia
No Yes
Measure
urine urate excretion
Low Normal/high
No Yes
No Yes
Evidence of
metabolic acidosis Consider rare enzyme
defects of purine metabolism
(see Box 20.1)
PSEUDOGOUT
Box 20.1 Some causes of hyperuricaemia
Pseudogout is not a disorder of purine metabolism
Drugs: e.g. thiazides, nicotinic acid, warfarin, although the clinical presentation is similar to that
ciclosporin, pyrazinamide, didanosine and ethambutol of gout. Calcium pyrophosphate precipitates in joint
Poisoning by other agents, such as alcohol and lead cavities, and calcification of cartilages is demonstrable
Metabolic syndrome associated with insulin radiologically (chondrocalcinosis). The plasma urate
resistance and hypertriglyceridaemia, obesity and concentration is usually normal. The crystals of
hypertension calcium pyrophosphate may be identified in joint
Impaired urate excretion fluid using a polarizing microscope, when positive
Acute kidney injury or chronic kidney disease bifringence is observed. Patients may present with acute
Metabolic acidosis pseudogout arthopathy or chronic chondrocalcinosis.
High cell turnover
Pseudogout and chondrocalcinosis are associated
Haemolytic anaemia
Myeloproliferative disease with hyperparathyroidism, hypophosphataemia,
Malignancies hypothyroidism, haemochromatosis, renal
Psoriasis osteodystrophy, Wilson’s disease and acromegaly.
Tumour lysis syndrome
Hereditary or genetic conditions CAUSES OF LOW PLASMA URATE
Glucose-6-phosphatase deficiency (von Gierke’s CONCENTRATIONS (HYPOURICAEMIA)
disease) This is rare unless it is the result of the treatment of
Lesch–Nyman syndrome
hyperuricaemia with, for example, allopurinol or
Down’s syndrome
probenecid. It is associated with proximal renal tubular
damage, in which the reabsorption of urate is reduced,
● Do not forget the rare causes of hyperuricaemia, for and can be seen in Fanconi’s syndrome. Hypouricaemia
example Lesch–Nyman syndrome or von Gierke’s is also a finding in some patients receiving parenteral
disease. nutrition and in pregnancy, the syndrome of
Causes of low plasma urate concentrations (hypouricaemia) 309
inappropriate antidiuretic hormone (SIADH, see Purine breakdown stops at the xanthine–hypoxanthine
Chapter 2) and type 1 diabetes mellitus. stage, and plasma and urinary urate concentrations
are very low. Increased xanthine excretion may lead to
Xanthinuria the formation of xanthine stones; this does not occur
This is a very rare inborn error of purine metabolism, during the treatment of gout with xanthine oxidase
inherited as an autosomal recessive disorder, in which inhibitors, perhaps because the drugs also inhibit
there is a deficiency of xanthine oxidase in the liver. purine synthesis.
SUMMARY
● Urate is formed as a product of purine metabolism ● Allopurinol, a xanthine oxidase inhibitor, can
and is renally excreted. be used clinically to lower plasma uric acid
● Raised plasma urate concentrations (hyperuricaemia) concentrations.
are associated with gout. ● Hypouricaemia is usually not of major clinical
● Gout can be primary or secondary, and the significance in itself, although factors associated
clinical features include acute arthritis leading to with it include SIADH, pregnancy, Fanconi’s
chronic arthropathy, gouty tophi, renal calculi and syndrome and xanthinuria.
obstructive uropathy.
21 Disorders of haem metabolism:
iron and the porphyrias
Red blood cells are broken down by the Figure 21.1 Biosynthesis pathways of haem. CoA,
reticuloendothelial system, predominantly in the coenzyme A.
spleen. Released haemoglobin is split into the
peptide chain, globin, which enters the general
protein pool, and haem. The haem ring is split, a synthetic pathway and is regulated by feedback
process catalysed by haem oxidase, to form a linear inhibition by haem.
molecule, biliverdin. Released iron is reused and ● Two molecules of ALA condense to form a
biliverdin is reduced to lipid-soluble bilirubin. The monopyrrole, porphobilinogen (PBG).
metabolism of bilirubin is discussed in Chapter 17 ● Four molecules of PBG combine to form a
in the context of jaundice. tetrapyrrole ring, uroporphyrinogen (Fig. 21.2). Two
Biosynthesis of haem and haemoglobin isomers are formed, I and III. The major pathway
involves the III isomer.
The main steps in the synthesis of haem are outlined
● Haem is formed by the successive production of
below and in Figure 21.1.
coproporphyrinogen and protoporphyrin, followed
● 5-Aminolaevulinic acid (ALA) is formed by by incorporation of Fe2+ into the centre of the ring.
condensation of glycine and succinate. The reaction ● Haemoglobin consists of four haem molecules,
requires pyridoxal phosphate and is catalysed by covalently linked to four (two pairs of) polypeptide
ALA synthase. This is the rate-limiting step in the chains.
Haem metabolism 311
A P A P M V
+H N–CH
3 2
H2C CH2 HC CH
N N A M N M
H A H
NH HN N Fe N
H P P V
P N N
H2C CH2 HC CH
P A P M
Figure 21.2 Porphobilinogen, the tetrapyrrole uroporphyrinogen III, which incorporates four porphobilinogen units
and haemoglobin. Uroporphyrinogen I differs only in the order of the side chains on one of the rings. Side chains:
A, acetate; M, methyl; P, propionate; V, vinyl.
Excretion of haem precursors Consciousness is not lost until the carboxy form has
Excess intermediates on the haem pathway are excreted replaced about half the oxyhaemoglobin. (See oximetry
in either urine or faeces. and blood gases in Chapter 4.)
The porphyrin precursors ALA, PBG and
Methaemoglobin
uroporphyrinogen are water soluble and appear in the
urine. They are colourless, but PBG may spontaneously Methaemoglobin is haemoglobin in which iron is in the
oxidize to form uroporphyrin when exposed to air and ferric (Fe3+) form (haemin); therefore, it cannot carry
light. oxygen. It is brown and is normally present in very low
Porphyrinogens also oxidize spontaneously to plasma concentrations; drugs such as sulphonamides
the corresponding porphyrins, which are dark red or nitrites/nitrates may increase methaemoglobin. The
and fluoresce in ultraviolet light. A urine specimen symptoms of methaemoglobinaemia are due to hypoxia,
containing large amounts of porphyrinogens or their which causes cyanosis and an increased respiratory rate
precursors will gradually darken if left standing. and, if methaemoglobin is greater than 70 per cent of
Protoporphyrin is excreted in bile and appears in the the total haemoglobin, can be fatal. Methemoglobin
faeces, whereas coproporphyrin(ogen) may be excreted may cause a pulse oximeter to read about 85 per cent
by either route. regardless of the actual amount of oxygen saturation.
Methaemoglobinaemia is associated with glucose-
Haemoglobin and related compounds 6-phosphate dehydrogenase (G6PD) deficiency (see
When oxygen is incorporated into haemoglobin to Chapter 27).
form oxyhaemoglobin, the spatial arrangement of the
haem complexes is altered in such a way as to facilitate Methaemalbumin
further oxygen uptake. Other compounds related to Methaemalbumin is also brown. It is formed when haem
haemoglobin may sometimes be formed and some of combines with plasma albumin in conditions such as
these may hinder the oxygen-carrying capacity. severe intravascular haemolysis or acute haemorrhagic
pancreatitis when haemoglobin has been converted
Carboxyhaemoglobin to haemin in the abdominal cavity and absorbed.
Carboxyhaemoglobin is cherry red in colour and is Methaemalbumin occurs when the haemoglobin-
formed when carbon monoxide binds to haemoglobin or binding capacity of haptoglobin has been exceeded.
displaces oxygen from oxyhaemoglobin; haemoglobin
has a greater affinity for carbon monoxide than for Sulphaemoglobin
oxygen. This occurs in carbon monoxide poisoning, Sulphaemoglobin is similar to methaemoglobin but
which can be lethal. Once carbon monoxide is removed contains sulphur; unlike methaemoglobin, it cannot be
from inspired air, oxyhaemoglobin is reformed. reconverted to haemoglobin in vivo. It remains in intact
312 Disorders of haem metabolism: iron and the porphyrias
Stores
18 000 µmol
(1000 mg)
Marrow
2000 µmol
(115 mg) Plasma 70 µmol (4 mg)
Erythrocytes
45 000 µmol
(2500 mg)
Normal iron loss is so small, and normal iron stores iron. The following factors are known to affect plasma
are so large, that it would take about 3 years to become concentrations within a population:
iron deficient on a completely iron-free diet. Of course,
● Sex and age differences Plasma iron concentrations,
this period is much shorter if there is any blood loss,
like those of haemoglobin and the erythrocyte
such as menstruation.
count, are higher in men than in women, probably
Iron transport in plasma for hormonal reasons. The difference is first evident
Iron is transported in the plasma in the ferric form, at puberty, before significant menstrual loss has
attached to the specific binding protein transferrin occurred, and disappears at the menopause.
at a concentration of about 18 µmol/L. Transferrin is Androgens tend to increase the plasma iron
normally capable of binding about 54 µmol/L of iron concentration and oestrogens to lower it.
and is therefore about one-third saturated. Transferrin- ● Pregnancy and oral contraceptives In the first few
bound iron is carried to stores and to bone marrow weeks of pregnancy, the plasma iron may rise to
cells and in the latter some iron passes directly into concentrations similar to those found in men. A similar
developing erythrocytes to form haemoglobin. rise occurs in women taking some oral contraceptives.
Factors affecting plasma iron concentration Iron concentrations can vary within an individual
by up to 100 per cent or more. This can be due to the
The plasma iron concentration is likely to be a poor
following:
index of the total body content because only a very
small proportion is in this compartment; it has no ● Random variations Day-to-day variations may be as
function, except as a protein-bound transport fraction. much as three-fold and usually overshadow cyclical
Plasma iron concentration is very variable, even under changes. They may be associated with physical or
physiological conditions. mental stress or diet, but usually no cause can be
found.
Physiological factors ● Circadian (diurnal) rhythm The plasma iron
The causes of physiological changes in plasma iron concentration is higher in the morning than in the
concentrations are not well understood, but alterations evening. If subjects are kept awake at night, this
can be very rapid and almost certainly represent shifts difference is less marked; it is reversed in night
between plasma and stores, not changes in total body workers.
314 Disorders of haem metabolism: iron and the porphyrias
● Monthly variations in women The plasma iron may system. Marrow iron stores and plasma ferritin
reach very low concentrations just before or during concentrations are usually increased in chronic
the menstrual period. The reduction is probably due haemolytic conditions.
to hormonal factors rather than blood loss. ● Acute liver disease Disruption of hepatocytes may
release ferritin iron into the bloodstream and cause
Pathological and clinical factors a transient rise in the plasma iron concentration.
Cirrhosis may be associated with a similar finding,
Iron deficiency and iron overload usually cause low
perhaps due to increased iron absorption and intake.
and high plasma iron concentrations respectively. Iron
deficiency is associated with a hypochromic, microcytic
Transferrin and total iron-binding capacity
anaemia and with reduced amounts of stainable
bone marrow iron. Plasma ferritin concentrations are Plasma iron concentrations alone rarely give
usually, but not always, low. Iron overload is associated information about the state of iron stores. In rare
with increased amounts of stainable iron in liver biopsy situations in which doubt remains after haematological
specimens, and plasma ferritin concentrations are high. investigation, diagnostic precision may sometimes be
Plasma iron is of little value in the diagnosis of iron improved by measuring both the plasma transferrin
deficiency not least because other pathological factors and the iron concentrations.
may affect plasma iron concentrations including the Plasma transferrin can be directly assayed or
following: measured indirectly by adding an excess of inorganic
iron to the plasma, any not bound to protein being
● Any acute or chronic illness (even a bad cold) causing a removed, usually with an exchange resin. The
fall in plasma iron concentration Chronic conditions concentration of iron remaining is assayed and the result
such as malignancy, renal disease, rheumatoid expressed as the total iron-binding capacity (TIBC).
arthritis and chronic infections are often associated Unsaturated iron-binding capacity (UIBC) is the TIBC
with normochromic, normocytic anaemia. Iron minus plasma iron concentration. This is usually a
stores and plasma ferritin concentrations are normal valid approximation of the transferrin concentration.
or even increased; the anaemia does not respond to In rare circumstances, of which the most common is
iron therapy. Iron deficiency may be superimposed severe liver disease, plasma ferritin concentrations are
on the anaemia of chronic illness, especially if drugs high enough to bind significant amounts of iron, and
are being taken that cause gastrointestinal bleeding; the results of iron-binding capacity measurements are
plasma ferritin concentrations are then variable. The then misleading as an assessment of the transferrin
finding of hypochromic erythrocytes is the most concentration.
sensitive index of this complication. Low plasma
iron concentrations occur whether or not there is Physiological changes in the plasma
any iron deficiency. transferrin concentration
● Disorders in which the marrow cannot use iron, either The plasma transferrin concentration is less labile than
because it is hypoplastic or because some other that of iron. However, it rises:
essential erythropoietic factor, such as vitamin B12 ● after about the 28th week of pregnancy even if iron
or folate, is deficient; plasma iron concentrations stores are normal,
are often high. Blood and marrow films may show ● in women taking some oral contraceptive
a typical picture, but, for example in pyridoxine- preparations,
responsive anaemia and in thalassaemia, the findings ● in any patient treated with estrogens.
in the blood film may resemble those of iron
deficiency; in the last two conditions the presence of Pathological changes in the plasma
stainable marrow iron stores excludes the diagnosis transferrin concentration
of iron deficiency.
Plasma transferrin concentration and TIBC:
● Haemolytic anaemia The plasma iron concentration
may be high during a haemolytic episode, as iron, ● rise in iron deficiency and fall in iron overload,
liberated from the destroyed erythrocytes, enters the ● fall in those chronic illnesses associated with low
plasma; it is usually normal during the quiescent plasma iron concentrations,
periods when the iron enters the reticuloendothelial ● may be unchanged in acute illness,
The soluble transferrin receptor 315
Plasma concentrations
Iron Transferrin Ferritin TfR Marrow iron stores
Low iron concentration
Iron deficiency Ø ≠ Usually Ø ≠ Ø
Acute illness Ø – – or ≠ – –
Chronic illness Ø Ø – or ≠ – Usually ≠
High iron concentration
Early pregnancy ≠ – – – –
Late pregnancy or oral contraceptives Variable ≠ – ≠ –
Iron overload ≠ Ø ≠ Ø ≠
Liver disease ≠ Ø ≠ – or ≠ May be ≠
Haemolysis ≠ – or Ø ≠ ≠ ≠
TfR, transferrin receptor; ≠, up; –, normal; Ø, down.
316 Disorders of haem metabolism: iron and the porphyrias
Causes of iron overload The effect of the accumulated iron depends on its
distribution in the body. This, in turn, is influenced
● Increased intestinal absorption:
partly by the route of entry. Two main patterns are seen
– hereditary or primary haemochromatosis,
at postmortem or in biopsy specimens.
– anaemia with increased, but ineffective
Parenchymal iron overload occurs in
erythropoiesis, for example sideroblastic
haemochromatosis and in patients with ineffective
anaemia, thalassaemia, myelodysplasia,
erythropoiesis. Iron accumulates in the parenchymal
– liver disease (rare cause),
cells of the liver, pancreas, heart and other organs.
– dietary excess or iron poisoning,
There is usually associated functional disturbance or
– inappropriate oral iron therapy.
tissue damage.
● Parenteral administration:
Reticuloendothelial iron overload is seen after
– multiple blood transfusions,
excessive parenteral administration of iron or multiple
– inappropriate parenteral iron therapy.
blood transfusions. The iron accumulates initially in
A very rare cause of iron overload is an inherited the reticuloendothelial cells of the liver, spleen and
deficiency of transferrin. bone marrow.
Iron overload 317
In dietary iron overload, both hepatic The relatives of patients with proven hereditary
reticuloendothelial and parenchymal overload may haemochromatosis should be investigated. Genetic
occur, associated with scurvy and osteoporosis. tests are now available, with a majority of cases of
Whatever the cause of massive iron overload, there may haemochromatosis usually being due to the C282Y
be parenchymal accumulation and tissue damage. or H63D HFE gene mutations. There are rarer non-
Haemosiderosis is a histological definition. An HFE forms of haemochromatosis associated with gene
increase in iron stores as haemosiderin can be seen. It defects of hepcidin, ferroportin, the transferrin receptor
does not necessarily mean that there is an increase in and DMT-1.
total body iron; for example, in many types of anaemia Treatment is often by venesection, aiming to reduce
there is reduced haemoglobin iron but increased storage the plasma ferritin to less than 100 µg/L. Each 500 mL
iron. Haemochromatosis describes the clinical disorder unit of blood removes about 250 mg of iron from the
due to parenchymal iron-induced damage. body.
Syndromes of iron overload
Secondary iron overload
Hereditary or primary haemochromatosis Anaemia and iron overload
Hereditary haemochromatosis usually has an Several types of anaemia may be associated with iron
autosomal recessive mode of inheritance and a defect overload. In some, such as hypoplastic anaemia and the
of the haemochromatosis gene (HFE). Approximately anaemia of chronic kidney disease, the cause is multiple
1 in 10 people in Western societies are carriers and blood transfusions; the iron initially accumulates in the
about 1 in 1000 are homozygous. The gene for this reticuloendothelial system. If overload is massive (often
disorder is closely associated with the human leucocyte over 100 units of blood), deposition may occur in
antigen (HLA) gene locus on chromosome 6. Some parenchymal cells, with the development of secondary
forms of haemochromatosis result in low hepcidin haemochromatosis.
concentrations. Factors such as alcohol abuse may hasten In anaemias characterized by erythroid marrow
the accumulation of iron and the development of liver hyperplasia but with ineffective erythropoiesis, such
damage. Males are more likely to manifest the condition, as thalassaemia major and sideroblastic anaemia, there
as menstruation in females lowers tissue iron stores. is, in addition, increased absorption of iron. Secondary
There is increased intestinal absorption of iron haemochromatosis develops at a lower transfusion load
over many years, which produces large iron stores in than in hypoplastic anaemia.
the tissues, such as the liver, pancreas, joints, heart and
gonads. It presents, usually in middle age, as cirrhosis Treatment of iron overload of anaemia
of the liver, sometimes with diabetes mellitus, joint This can obviously not be treated by venesection. The
pains, cardiomyopathy, hypogonadism and greyish tendency for transfusion to aggravate iron overload
skin pigmentation due to melanin, not iron. It has been further can be minimized by giving the iron-chelating
referred to as ‘bronzed diabetes’, although such skin agent desferrioxamine each time; this can be excreted in
features tend to occur late. the urine with any non-haemoglobin iron.
Examination of liver biopsy specimens may be useful,
giving an idea of disease severity and showing increased Dietary iron overload
iron content when stained by Perl’s blue staining, Increased iron absorption due to excessive intake is rare.
although magnetic resonance imaging (MRI) is also One well-described form is, however, relatively common
able to determine iron content non-invasively. There in the rural black population of southern Africa. The
is an association with hepatocellular carcinoma, which source is beer brewed in iron containers. Usually, the
can be screened for by assay of plasma a-fetoprotein excess is confined to the reticuloendothelial system and
(AFP). the liver (both portal tracts and parenchymal cells), and
The diagnosis may be made on the basis of the there is no tissue damage. In a small number of cases,
measurement of plasma ferritin, which is often greater deposition in the parenchymal cells of other organs
than 1000 µg/L. Also plasma iron concentration occurs and the clinical picture may resemble that of
and TIBC, with a saturation of greater than about primary haemochromatosis; it may be distinguished by
45 per cent, are indicative of haemochromatosis and the high concentration of iron in the reticuloendothelial
may increase before plasma ferritin concentration does. system seen in the bone marrow.
318 Disorders of haem metabolism: iron and the porphyrias
Scurvy and osteoporosis may occur in this form low plasma ferritin concentration due to iron deficiency.
of iron overload. The ascorbate deficiency may be A plasma ferritin concentration more than 200 µg/L
due to its irreversible oxidation in the presence of under these circumstances probably rules out iron
excessive amounts of iron, and osteoporosis sometimes deficiency anaemia, but values more than 10 µg/L but
accompanies scurvy. Ascorbate deficiency also less than 200 µg/L are equivocal and do not distinguish
interferes with normal mobilization of iron from the between iron deficiency anaemia and anaemia of
reticuloendothelial cells; plasma iron concentrations chronic disorders. Under these circumstances, plasma-
may be low and the response to chelating agents poor, soluble TfR assay may be useful; this should be raised in
despite iron overload. iron deficiency but not in anaemia of chronic disorders.
An unequivocally low plasma ferritin concentration
Investigation of disorders of iron confirms iron deficiency, but a normal or high one
metabolism should not be assumed to exclude it. A bone marrow
Investigation of iron deficiency of anaemia film for iron staining may be needed to confirm the
There are many causes of anaemia, which may be diagnosis of iron deficiency in ambiguous cases. If a
due either to iron deficiency or to a variety of other diagnosis of iron deficiency is made, it is essential to rule
conditions, sometimes associated with high iron out blood loss and its source. Particularly important
stores. The subject of the diagnosis of anaemia, such is to exclude gastrointestinal malignancy and also
as haemolytic or pernicious anaemia, is covered more gynaecological pathology. Faecal occult blood samples
fully in textbooks of haematology. Blood haemoglobin may show the presence of gastrointestinal blood loss,
reflects the major metabolic pool of iron, ferritin the which may necessitate prompt endoscopic investigation
storage pool and transferrin the transit pool. The (see Chapter 16). A patient with proven iron deficiency
clinical impression of anaemia should be confirmed who shows no response to oral iron treatment within
by blood haemoglobin estimation. Iron deficiency can, a few weeks may not be taking the tablets. If iron has
however, exist with haemoglobin concentrations within been taken, malabsorption (usually as part of a general
the reference range. absorption defect) is a possible explanation of a poor
The mean corpuscular volume (MCV) and mean response.
corpuscular haemoglobin (MCH) should be checked
and often a blood film examined. Iron deficiency Investigation of suspected iron overload
anaemia is microcytic and hypochromic in type, and Initial tests
these findings may be evident before the haemoglobin Plasma iron, percentage saturation of transferrin, plasma
concentration has fallen below the reference range. ferritin
Normochromic, normocytic anaemia is a non-specific All of these should be measured. The plasma iron
finding, usually associated with other chronic disease; it concentration is almost invariably high in primary
is associated with iron deficiency only if there has been haemochromatosis, often more than 36 µmol/L.
very recent blood loss. This is associated with a reduced plasma transferrin
In most cases of anaemia, consideration of these concentration and TIBC, and the percentage
findings together with the clinical picture may elucidate saturation is usually greater than about 45 per cent,
the cause. Anaemias such as those due to sickle cell and may be nearer 90–100 per cent. In the presence
disease, thalassaemia or of the sideroblastic type, of infection or malignancy, however, the plasma iron
although rarer, are most likely to confuse the picture, concentration and percentage saturation may be lower
since they too are hypochromic but are not due to iron than expected; the plasma transferrin concentration
deficiency. A low plasma ferritin concentration (less remains low.
than 10 µg/L) is indicative of iron deficiency due to low Plasma ferritin concentrations are high in
iron stores. most patients with iron overload (whether
One major diagnostic dilemma is to separate iron reticuloendothelial or parenchymal). It is rare in
deficiency anaemia from anaemia of chronic disorders, hereditary haemochromatosis to have a normal plasma
for example carcinoma or inflammatory disease. In ferritin concentration, although this may occur in the
the latter there is often an acute-phase response, which early stages, when the iron saturation may be more
can raise plasma ferritin concentrations, which are useful diagnostically. Remember that plasma ferritin
influenced by inflammation. This can, therefore, ‘mask’ a can be elevated in hepatic disease and high alcohol
Disorders of haem synthesis: the porphyrias 319
The diagnosis of iron overload usually needs ALA Æ PBG Æ URO Æ COPRO Æ PROTO Æ HAEM
demonstration of increased iron stores.
Excreted in urine faeces
Liver biopsy
Figure 21.4 Sites of enzyme deficiencies in (1) acute
Liver biopsy specimens contain large amounts of intermittent porphyria, (2) hereditary coproporphyria,
stainable iron, which may be mainly in parenchymal or (3) variegate porphyria. ALA, 5-aminolaevulinic acid;
mainly in reticuloendothelial cells. If haemochromatosis COPRO, coproporphyrinogen; PBG, porphobilinogen;
is confirmed, liver function tests are necessary, as is URO, uroporphyrinogen; PROTO, protoporphyrinogen.
320 Disorders of haem metabolism: iron and the porphyrias
deficiency, include alcohol abuse, iron overload or excretion of porphyrins. Carotene treatment may be
high-dose estrogen therapy. Symptoms improve when useful in erythropoietic porphyria.
the offending substance is withdrawn. Some liver
Other causes of excessive porphyrin
toxins such as hexachlorobenzene directly inhibit the
excretion
activity of the enzyme. There is an association with
hepatitis C. Iron overload can also be present and Porphyria is not the only cause of disordered porphyrin
haemochromatosis needs to be excluded. metabolism, and positive screening tests must be
confirmed by quantitative analysis, with identification
The impaired conversion leads to an accumulation of the porphyrin. Other causes must be considered.
of uroporphyrinogen and porphyrins intermediate
between it and coproporphyrinogen. These deposit Lead poisoning
in the skin and are excreted in the urine in increased Lead poisoning inhibits several of the enzymes
amounts. Faecal porphyrins are not increased but the involved in haem synthesis, including PBG synthase
abnormal pattern of intermediate porphyrins may and ferrochelatase, and eventually causes anaemia. The
be detected by chromatography; this finding is of urine contains increased amounts of ALA (an early
diagnostic value. It is important not to confuse this and sensitive test), and coproporphyrin. Some of the
disorder with the coproporphyrinuria of liver disease symptoms of lead poisoning, such as abdominal pain
(see below). and peripheral neuropathy, are similar to those of the
acute porphyric attack, and may cause difficulty in
Erythropoietic porphyrias
diagnosis. However, the excretion of PBG is not usually
Two rare inherited disorders are associated with the increased. Zinc protoporphyrin concentration rises
accumulation of porphyrins in erythrocytes. Acute with increased lead exposure, although the method of
porphyric attacks do not occur and ALA and PBG choice for assessing exposure to inorganic lead is blood
excretion are normal. lead concentration. Other features of lead poisoning
Congenital erythropoietic porphyria
include lead staining of the teeth and basophilic
stippling of red blood cells.
Congenital erythropoietic porphyria is inherited
as an autosomal recessive characteristic due to Liver disease
uroporphyrinogen III synthase deficiency. Usually, blood
This may increase urinary coproporphyrin levels,
erythrocyte and plasma uroporphyrin I concentrations
possibly because of decreased biliary excretion. It is
are very high from infancy onwards and there is severe
probably the most common cause of porphyrinuria.
photosensitivity. Porphyrins are also deposited in bones
Occasionally there is mild photosensitivity (in
and teeth, which fluoresce in ultraviolet light; the teeth
porphyria cutanea tarda, the more severe skin lesions
may be brownish-pink in colour. Hirsutism, especially
are due to uroporphyrin excess).
of the face, also occurs and there is haemolytic anaemia.
Urinary porphyrin concentrations are grossly increased, Bleeding lesions of upper gastrointestinal tract
although faecal porphyrin levels are less so.
These lesions may produce raised levels of faecal
Protoporphyria porphyrin by degradation of haemoglobin. If there is
This is an autosomal dominantly inherited disorder due bleeding from the lower part of the tract, the blood
to ferrochelatase deficiency in which protoporphyrin reaches the rectum before there is time for conversion;
concentrations are increased in erythrocytes and faeces. this may be of help in locating the approximate site of
There is mild photosensitivity, and hepatocellular bleeding.
damage may lead to liver failure.
Iron deficiency and sideroblastic anaemia
These two conditions tend to give rise to
photosensitivity, whereas there is increased skin fragility This may also result in increased concentrations of
in the other porphyrias that involve the skin. The erythrocyte porphyrins.
erythropoietic porphyrias can be treated by sunlight
avoidance, for example by using skin sunblocks. In Investigation of suspected porphyria
porphyria cutanea tarda, venesection may be used to The laboratory should be notified and a check made
reduce iron stores and oral chloroquine to increase the of which type of samples is required. Porphyrins
Disorders of haem synthesis: the porphyrias 323
are relatively stable, but samples must be protected abnormal faecal porphyrins. The following should be
from light. Porphobilinogen rapidly polymerizes to remembered.
uroporphyrins and consequently a random fresh Patients with acute intermittent porphyria who
urine sample is more suitable than a 24-h collection. have once had an acute attack may continue to excrete
All samples should be analysed as soon as possible increased amounts of PBG for many months. This
after collection. Samples should not be sent simply condition does not show skin lesions, unlike the other
requesting a ‘porphyrin screen’; an indication should acute porphyrias. Plasma fluorescence may reveal
be given of which type of porphyria is suspected, giving different emission peaks when stimulated at 405 nm,
the relevant clinical details, so that the laboratory can for example variegate porphyria may show a peak at
select the appropriate tests. 625 nm when stimulated at 405 nm, whereas the other
Generally, urine, faeces and blood (both plasma and acute porphyrias show a peak at 615 nm.
erythrocytes) are needed for complete analysis. The It is possible to assay enzymes of haem metabolism in
technique of plasma fluorescence has recently proved red blood cells. For example, decreased PBG deaminase
useful to distinguish some of the porphyrias (Table is found in patients with acute intermittent porphyria.
21.3). Specialist laboratories can carry out enzyme There is often overlap in levels between affected and
assays and genetic tests. unaffected people but, within a family, carriers can be
shown to have levels about half those of unaffected
Suspected acute porphyria members. This test may also detect affected children.
Fresh urine should be tested immediately for PBG However, the enzyme activity is related to the mean
using Ehrlich’s reagent. If PBG is not present, it is highly age of the red blood cells; this assay is not suitable
unlikely that the patient is suffering from an acute for children under the age of about 9 months or for
porphyric attack unless he or she has the rare PBG individuals with haemolytic disorders. Negative urine
synthase deficiency, in which case ALA concentration and faeces porphyrin tests do not exclude the diagnosis
would be expected to be raised. A patient with a history of latent porphyria, and in these cases enzyme and
of repeated attacks of abdominal pain or neurological genetic tests may be useful.
symptoms may have acute intermittent porphyria,
variegate porphyria or hereditary coproporphyria. Suspected porphyria with skin lesions
If an acute porphyria is confirmed, the concentration Skin lesions may occur in any type of porphyria
of porphyrins in urine and in a random sample of faeces other than acute intermittent porphyria. Blood,
should be measured. Raised values suggest variegate urine and faeces should be sent for testing. Increased
porphyria (protoporphyrin and coproporphyrin) or concentrations of erythrocyte porphyrins suggest
hereditary coproporphyria (coproporphyrin). Acute protoporphyria or congenital erythropoietic porphyria
intermittent porphyria does not normally show
Urine
Porphyria PBG POR Faeces Red blood cells Plasma fluorescence (nm)a
AIP + Copro III – – 615
HC + Copro III Copro III – 615
VP + Copro III Proto IX – 624–626
PCT – Urohepta Isocopro – 615
CEP – Uro I Copro I Zn-Proto 615
Copro I
PP – – Proto Proto 632
+, present; –, absent; AIP, acute intermittent porphyria; CEP, congenital erythropoietic porphyria; Copro, coproporphyrinogen I or
III; HC, hereditary coproporphyria; Isocopro, isocoproporphyrinogen; PBG, porphobilinogen; PCT, porphyria cutanea tarda; POR,
porphyrin; PP, protoporphyria; Proto, protoporphyrinogen; Urohepta, uroheptaporphyrinogen; Uro, uroporphyrinogen; VP, variegate
porphyria; Zn, zinc.
a
Plasma fluorescence emission wavelength peak when stimulated at 405 nm.
324 Disorders of haem metabolism: iron and the porphyrias
(very rare). High concentrations may also occur in iron cell PBG deaminase activity. Genetic studies are now
deficiency anaemia and lead poisoning. possible in specialist laboratories to investigate the
Increased concentrations of urinary porphyrins porphyrias.
suggest porphyria cutanea tarda or congenital
erythropoietic porphyria. The increased uroporphyrin
excretion in these conditions must be distinguished
from the coproporphyrinuria of liver disease.
Increased concentrations of faecal porphyrins occur
in protoporphyria, variegate porphyria and hereditary
coproporphyria. These may be distinguished by
chromatographic separation of porphyrins; this will
also demonstrate the abnormal pattern of porphyria
cutanea tarda.
Protoporphyria shows a unique plasma fluorescence
emission peak of 632 nm when stimulated at 405 nm.
Enzyme and genetic tests may be useful.
SUMMARY
● Haemoglobin contains most (70 per cent) of diabetes mellitus, skin pigmentation, and cardiac
the body’s iron. Iron is mandatory for normal and endocrine problems.
haemopoiesis. ● The haem moiety of haemoglobin contains
● Ferritin is an intracellular iron-binding protein. tetrapyrrole rings synthesized from porphyrinogen
Iron is also absorbed in the small intestine and precursors. Disorders of the haem synthetic
bound to transferrin within the circulation. It is pathways can lead to porphyria. Most of the
stored bound to ferritin. porphyrias are autosomal dominant.
● Iron deficiency is common and poor iron intake ● The porphyrias can be divided into (a) the acute
or blood loss needs to be excluded. A low plasma group, which can present with acute abdomen,
ferritin concentration suggests iron deficiency. neuropsychiatric symptoms and hypertension, and
● Unbound iron is toxic to the body. Haemochroma- (b) cutaneous (non-acute) forms that may present
tosis is an autosomal recessive condition of chronic with skin symptoms.
iron overload. This is associated with liver disease,
22 Cardiovascular disease
The clinical biochemistry laboratory often plays a findings and confirmed by the characteristic changes in
central role in the diagnosis and management of acute plasma enzyme activities or troponin levels. Myocardial
myocardial infarction. A number of cardiovascular infarction can be defined as a typical rise and/or fall
risk factors have been described, some of which can be of cardiac biomarkers (preferably troponin) with at
assayed in the biochemistry laboratory, and these are least one value above the 99th percentile of the upper
discussed in this chapter. reference limit together with evidence of ischaemia
with at least one of the following: symptoms of
MYOCARDIAL INFARCTION ischaemia, electrocardiogram (ECG) changes (new ST
One of the largest killers in Western urbanized societies is changes or new left bundle branch block), development
acute myocardial infarction. Its diagnosis is usually made of ECG pathological Q waves or imaging evidence of
on the clinical presentation and electrocardiographic myocardial ischaemia. (Fig. 22.1). This classification
CASE 1 CASE 2
A 46-year-old man was admitted late at night A 66-year-old man was sent by his general
(23.30 h) to casualty by ambulance because of practitioner to casualty because of tight chest pain
a tight central chest pain that had started at that had occurred 3 days previously. The pain had
03.00 h that morning. He had delayed calling for largely resolved after 6 h, but he was left feeling weak
medical assistance because he thought it was only and breathless, which worsened over a few days,
indigestion. In the ambulance, the paramedics gave causing him eventually to seek medical attention.
him an intramuscular injection of diamorphine for The following laboratory test results were found:
pain relief. The ECG was normal. Plasma
Plasma Creatine kinase (CK) 235 U/L (< 250)
Creatine kinase (CK) 565 U/L (< 250) Troponin T 13.0 ng/L (< 10)
Troponin T 1.0 ng/L (< 10) An electrocardiogram showed changes suggestive of
DISCUSSION a myocardial infarction in the lateral leads V4–V6.
The normal plasma troponin T makes a diagnosis of
acute myocardial infarction unlikely, as this would DISCUSSION
be expected to be elevated in view of when the chest The plasma CK activity has returned to normal
pain started. The elevated plasma CK concentration because of the time delay since myocardial infarction,
is not specific for cardiac damage and may have been while the plasma troponin T concentration still
raised as a result of muscle damage secondary to remains elevated. Plasma CK usually starts to rise
the intramuscular injection: beware of interpreting 4–6 h after a myocardial infarction and to normalize
elevated plasma CK concentrations in the presence after a couple of days. Conversely, plasma troponin
of intramuscular injections. A CK-MB concentration T starts to rise at 4–6 h post infarct and remains
would be less likely than total CK concentration to elevated for as long as about 10 days. Troponins are
be elevated in skeletal muscle damage, as CK-MB is thus useful cardiac markers in both the early hours
predominantly cardiac in origin. and a few days later.
326 Cardiovascular
ST SEGMENT disease
ABNORMALITIES
DEPRESSION ELEVATION
angina infarct
horizontal pericarditis
upward angina
sloping variant
(A)
Figure 22.2 Coronary arteriogram showing critical right
also recognizes that patients previously classified as coronary artery stenosis (arrowed). Reproduced with
having unstable angina or minor myocardial injury are kind permission from Browse NL, Black J, Burnand
now reclassified as having non-ST segment elevation KG, Corbett SA and Thomas WEG (eds). Browse’s
myocardial infarction (NSTEMI). Introduction to the Investigation and Management of
Surgical Disease. London: Hodder Arnold, 2010.
It is useful to note that acute myocardial infarction
and NSTEMI have a common pathophysiological
pathway, namely acute coronary artery plaque rupture,
followed by a series of events resulting in thrombosis differences between cardiac and skeletal muscle TnC.
formation. The outcome is dependent on the state However, the cardiac and skeletal forms of TnI and TnT
of the collateral circulation and the severity of artery are structurally different and can be distinguished by
occlusion (Fig. 22.2). In other words, clinically we no immunological assays.
longer think in terms of the presence or absence of an Troponin I and TnT appear in the plasma 4–8 h
acute myocardial infarction, but instead of a spectrum or earlier with high-sensitivity troponin assays after
of disease ranging from angina pectoris through to symptoms of acute myocardial infarction, and are
acute myocardial infarction; this stratification is based best measured 12 h after the start of chest pain. They
upon cardiac markers reflecting ischaemic damage. are therefore not early markers of acute myocardial
Acute coronary syndrome is the term used to embrace infarction, but they do stay elevated for about 7–10
ST segment elevation myocardial infarction (STEMI), days in plasma, which makes them useful in the late
as well as NSTEMI and unstable angina pectoris presentation of chest pain.
It is also particularly important to diagnose acute Troponin T may be elevated in patients with
myocardial infarction promptly, as thrombolytic chronic kidney disease and thus may not be so cardio-
therapy may be indicated to try to dissolve the thrombus specific. However, TnI can be extensively modified by
in situ or angioplasty. The earlier this therapy is given, proteolysis after release into the plasma. This means
the better the prognosis, as ‘minutes are myocardium’. that measurements may give varying results as different
Thus, biochemical markers need to be specific, sensitive antigenic epitopes are revealed, depending upon the
and change rapidly to optimize the diagnosis and commercial supplier of the assay. There is no universal
treatment of acute myocardial infarction. agreement as to which troponin assay, i.e. TnI or TnT,
is best.
Troponins An increased TnI or TnT concentration is a sensitive
Troponins are muscle-regulatory proteins present in marker of occult myocardial damage even in non-
skeletal and cardiac muscle. Three troponins have ischaemic conditions. Plasma troponin concentrations
been reported, namely troponin C (TnC), troponin I are increased in subarachnoid haemorrhage (due to
(TnI) and troponin T (TnT). There are no structural vasoactive peptide release affecting the myocardium),
Myocardial infarction 327
hypertension, tachyarrhythmias, cardiac surgery, sepsis, in the diagnosis of acute myocardial infarction unless
congestive cardiac failure, pulmonary embolism and used in conjunction with other markers.
hypothyroidism. It should be remembered that the
Cardiac enzymes
universal definition of an acute myocardial infarction
requires not only a change in troponin but also clinical The plasma enzyme estimation of greatest value
or ECG evidence. is that of CK (see also enzymology, Chapter 18),
Research has shown that troponins allow a subset although this has been largely superseded by the
of patients with rest-unstable angina to be classified assay of cardiac troponins. At one time, the enzymes
despite normal plasma CK-MB concentrations. Those lactate dehydrogenase (LDH) or hydroxybutyrate
with raised plasma cardiac troponin concentrations dehydrogenase (HBD) and aspartate aminotransferase
were found to be more likely to have an acute (AST) were used diagnostically, although they are now
myocardial infarction or die within 6 months of rarely assayed in the diagnosis of acute myocardial
the event. In other words, troponins allow risk infarction or NSTEMI.
stratification of patients with acute coronary An approximate guide to the sequence of changes
syndrome. In another study it was found that, unlike after acute myocardial infarction is given in Table 22.1
CK-MB, cardiac troponins have prognostic value (see also Fig. 22.3).
after coronary thrombolysis therapy, with troponin All plasma enzyme activities (including that of CK-
concentrations on admission correlating with 6-week MB) may be normal until at least 4 h after the onset of
mortality. Low-molecular-weight heparin or platelet- chest pain due to a myocardial infarction; blood should
receptor blockers (glycoprotein GIIB/IIIA inhibitors) not be taken for enzyme assay until this time has elapsed. If
reduce cardiac events in non-Q-wave coronary the initial plasma CK activity is normal, a second sample
syndromes with raised concentrations of cardiac should be taken about 4–6 h later. A rise in the plasma
troponins compared with those without troponin CK activity supports the diagnosis of an infarction. The
concentration elevation. There are also new sensitivity simultaneous measurement of plasma CK-MB activity,
troponin assays which may allow earlier diagnosis and which is shown to exceed about 5 per cent of the total
better risk stratification. CK activity, may occasionally help in early diagnosis; a
In view of these findings, cardiac troponins have raised plasma CK-MB activity or concentration alone is
been recommended as the biochemical cardiac marker not diagnostic of an infarction.
of choice. However, sometimes a panel of cardiac Most of the CK released after a myocardial infarction
markers may be useful in making the diagnosis of is, in fact, the MM isoenzyme (CK-MM), which is
acute myocardial infarction, for example myoglobin found in both skeletal and myocardial muscle and has a
rises early, thus alerting the clinician to possible cardiac longer half-life than the MB fraction. After about 24 h,
problems, while the later rise of troponin allows the finding of a high MM and undetectable MB does
greater specificity. Other cardiac markers that are not exclude myocardial damage as a cause of high total
being studied include ischaemia-modified albumin, CK activities; by this time the plasma HBD activity is
pro-brain natriuretic peptide (pro-BNP) and glycogen usually raised.
phosphorylase isoenzymes BB.
Table 22.1 The time sequence of changes in plasma
Myoglobin cardiac markers after acute myocardial infarction
As mentioned in Chapter 21, myoglobin is a low-
molecular-weight haem-containing protein found Cardiac marker Starts to rise Time after Duration of
(hours) infarction for rise (days)
in both skeletal and cardiac muscle. Because of its peak rise (hours)
low molecular weight, it is rapidly released from the
CK (total) 4–6 24–48 2–4
myocardium upon damage, and a typical rise occurs
AST 6–8 24–48 4–6
within 2–4 h after the onset of acute myocardial
infarction. This is useful for the early diagnosis of acute LDH/HBD 12–24 48–72 7–12
myocardial infarction, as this rise is generally earlier Myoglobin 2–4 12–24 2–4
than that of the other currently used cardiac markers. Troponin 4–6 12–24 7–10
Unfortunately, myoglobin is not cardiac specific, being AST, aspartate transaminase; CK, creatine kinase; HBD,
also found in skeletal muscle, and thus is less useful hydroxybutyrate dehydrogenase; LDH, lactate dehydrogenase.
328 Cardiovascular disease
studies have not shown convincingly a reduction in for coronary heart disease and stroke. In most cases the
cardiovascular risk. cause is unknown and it is called essential hypertension.
There are many causes of hypertension, including
C-reactive protein obesity, steroid use, insulin resistance and high alcohol
Research has shown that atherosclerosis involves intake. Rarer secondary causes include renal disease
inflammatory processes. Macrophages, monocytes and such as polycystic disease, scleroderma, pyelonephritis
T lymphocytes are found in the atheromatous plaques, and renal artery stenosis (Table 22.3). Endocrine causes
as are raised concentrations of cytokines with acute- include phaeochromocytoma (Chapter 24), Cushing’s
phase reactants. Cytokines such as interleukin 6 (IL-6) syndrome (Chapter 8), Conn’s syndrome (Chapter
increase the hepatic synthesis of acute-phase proteins, 8), acromegaly (Chapter 7) and hyperparathyroidism
including C-reactive protein (CRP, see Chapter 19). (Chapter 6). Also consider aortic coarctation and, if the
Plasma CRP concentration may reflect the likelihood patient is pregnant, pre-eclampsia (Chapter 10).
of the atheromatous plaque rupturing. High-sensitivity Clinical biochemistry tests have an important part to
CRP assays (hs-CRP) have recently been developed, play in the management of hypertension. First, they have
and a positive association between future coronary a role in diagnosis. The biochemical investigation of
events and plasma hs-CRP concentration has been hyperparathyroidism, acromegaly, Cushing’s syndrome,
found. Plasma hs-CRP may also have prognostic value primary hyperaldosteronism (Conn’s syndrome) and
in patients with acute coronary syndrome, with those phaeochromocytoma, and of other rare conditions such
with an hs-CRP of more than 3 mg/L at onset being as Liddle’s syndrome and Gordon’s syndrome, which
more likely to suffer acute myocardial infarction or may present with hypokalaemia or hyperkalaemia
cardiovascular death. Desirable values are less than respectively, are covered in other chapters of this book.
1 mg/L, with those at intermediate cardiovascular risk Biochemical testing also has a role in assessing
having values 1–3 mg/L. In terms of cardiovascular renal function (see Chapter 3) and in monitoring
risk event prediction, plasma hs-CRP may be stronger antihypertensive therapy. Hypokalaemia can develop in
than the plasma total cholesterol to HDL ratio (see patients treated with thiazide diuretics (non-potassium
Chapter 13). However, currently in the UK hs-CRP sparing), as can hyperuricaemia, hypercalcaemia,
is not recommended for patient risk stratification or hyperglycaemia and hyponatraemia.
treatment decisions. Angiotensin-converting enzyme (ACE) inhibitors
and angiotensin II receptor antagonists (ARAs) or
Hypertension blockers (ARBs) are also used to treat hypertension
Hypertension can be defined as sustained systolic blood and may cause hyperkalaemia and a rise in plasma
pressure of more than 140 mmHg and/or diastolic blood creatinine, particularly in patients with renal artery
pressure more than 90 mmHg. It is a major risk factor stenosis. In renal artery stenosis, glomerular filtration
Table 22.3 Some secondary causes of hypertension and initial biochemical investigations
that may be useful
is maintained by angiotensin II and these drugs can is for this reason that renal function should be closely
thus reduce the glomerular filtration rate, with a monitored within days of initiating an ACE inhibitor
concomitant impairment in renal function, for example or ARA and after an increase in dose. Plasma renin
a 10–20 per cent rise in plasma creatinine may occur. It concentration is usually raised in renal artery stenosis.
SUMMARY
● Cardiovascular disease is one of the world’s biggest ● The troponins are more specific cardiac markers
killers. It has many risk factors, which can be than CK. Also, plasma troponin stays elevated for
divided into those that are modifiable and those longer than CK after an infarct. Troponins are
that are not. Of the former, the major contributors therefore useful for the early and late diagnosis of
are smoking, diabetes mellitus, abnormal lipids, myocardial infarction.
hypertension and obesity. ● Plasma myoglobin becomes elevated within about
● The diagnosis of acute myocardial infarction can 2–4 h of myocardial infarction but is not specific
be difficult, but it is important to make a prompt for cardiac damage, as it is also found in skeletal
diagnosis, as intervention needs to be given early. muscle.
Plasma CK can be used, and exists as three major ● Plasma natriuretic peptides may be elevated in
isoenzymes (BB, MB and MM). Total plasma CK lacks ventricular dysfunction and are therefore useful in
cardiac specificity, and the CK-MB isoenzyme is more the diagnosis of cardiac failure.
specific and either its activity or its mass amount can ● Biochemistry investigations may be useful in the
be assayed. However, the troponins have essentially management and investigation of hypertension.
taken over from CK for diagnostic purposes.
23 Cerebrospinal, pleural and ascitic fluids
necessary, any remaining specimen can then be used CSF bilirubin can, however, also be raised when serum
for biochemical analysis. If a CSF glucose concentration bilirubin is elevated.
is indicated, 0.5 mL should be collected into a fluoride
tube and promptly sent to the laboratory with a blood Turbidity
sample taken at the same time. The CSF is potentially Turbidity is usually due to infection or high CSF protein
highly infectious and must be handled and transported content. It may also occur after haemorrhage.
with care.
Biochemical estimations
Appearance The following are the most commonly requested CSF
Normal CSF is completely clear and colourless; slight biochemical tests.
turbidity is most easily detected by visual comparison
with water. Glucose
The CSF glucose concentration is slightly lower than
Spontaneous clotting that in plasma and, under normal circumstances, is
Clotting occurs when there is excess fibrinogen in the rarely less than 50 per cent of the plasma concentration.
specimen, usually associated with a very high protein Provided that CSF for glucose assay has been preserved
concentration. This finding occurs with tuberculous with fluoride, an abnormally low glucose concentration
meningitis or with tumours of the CNS. occurs in the following:
● Hypoglycaemia The CSF glucose concentration
Colour parallels that of plasma, although there is a delay
A bright red colour may result from damage to a blood before changes in plasma glucose concentrations are
vessel during lumbar puncture (traumatic tap) or a reflected in the CSF. Hypoglycaemia may cause coma
recent haemorrhage into the subarachnoid space. and low CSF glucose concentrations in the absence
If CSF is collected as three separate aliquots, blood of any primary cerebral abnormality (see Chapter
staining will be progressively less in the aliquots if 12). Both plasma and CSF concentrations must be
bleeding is due to the lumbar puncture itself, whereas measured.
all aliquots would be expected to be bloody if there was ● Infection If there is an increased polymorphonuclear
a subarachnoid bleed. leucocyte count or a bacterial infection, the CSF
Xanthochromia is defined as a yellow coloration glucose concentration may be very low because of
of the CSF and results from altered haemoglobin, the increased metabolism of glucose. The CSF glucose
colour appearing several days after a subarachnoid concentration may be particularly low in pyogenic
haemorrhage and, depending on the extent of the meningitis and tuberculous meningitis; in viral
bleeding, lasting for up to a week or more or jaundice meningitis, it is often normal. The estimation of
(which will be clinically obvious and may impart a CSF glucose does not reliably distinguish between
yellow colour to the CSF). different forms of infective meningitis because the
Visual appraisal of CSF is not sufficiently sensitive result may be normal in any form.
to detect subtle degrees of xanthochromia for the ● Widespread malignant infiltration of the meninges
diagnosis of subarachnoid haemorrhage. In such may also be associated with low CSF glucose
cases spectrophotometric examination of CSF is concentrations.
important. Spectrophotometric analysis may reveal
an oxyhaemoglobin absorption peak of 413–415 nm; Protein
bilirubin shows an additional peak at 450–460 nm. This The CSF protein concentration in the lumbar spine
test is particularly useful in patients with subarachnoid is up to three times higher than that in the ventricles;
haemorrhage who have a negative brain CT scan. the normal lumbar concentration is below 0.4 g/L. In
The presence of methaemoglobin or bilirubin is newborn infants, because of the relatively high vascular
strongly suggestive of a subarachnoid haemorrhage, as permeability, the CSF protein concentration is about
oxyhaemoglobin alone is not necessarily confirmatory three times that of the adult.
because it may simply reflect a ‘bloody’ CSF tap. The test Changes in concentration of, for example,
should be done at least 12 h after initiation of headache. immunoglobulin G (IgG) do not necessarily indicate
334 Cerebrospinal, pleural and ascitic fluids
cerebral disease, but may simply reflect changes in may, however, detect abnormalities when the total protein
plasma (normally 80 per cent of total CSF protein has concentration is equivocally raised or normal, and may
transferred across from the plasma). Therefore the help to elucidate the cause of a high concentration.
results of assays can only be interpreted if those of the Increased capillary permeability, with a similar
two fluids are compared. increase in the permeability of the blood–brain
Cerebral disease may change the total concentration barrier, may be demonstrated by finding relatively
of CSF protein and the proportions of its constituents, high-molecular-weight proteins not normally present
for two reasons: in CSF. This non-specific pattern is associated with a
large number of inflammatory conditions, but may
● The vascular and meningeal permeabilities can
sometimes facilitate diagnosis.
increase, allowing more protein to enter the CSF.
● Proteins (immunoglobulins) may be synthesized Abnormal cerebrospinal fluid protein synthesis
within the cerebrospinal canal by inflammatory or The identification of immunoglobulins synthesized
other invading cells. within the CSF, particularly IgG and IgA, may help
Measurement of total cerebrospinal fluid protein concentration with the diagnosis of multiple sclerosis or other
demyelinating disorders. Abnormal immunoglobulin
Measurement of CSF total protein is a relatively
synthesis may be detected by the following.
insensitive test for the diagnosis of cerebral disease,
because early changes in the concentration of a specific ● The finding of a characteristic electrophoretic
protein do not always cause a detectable rise in the total pattern with multiple (‘oligoclonal’) bands in the
protein concentration. g-globulin region. Oligoclonal bands signify cerebral
The CSF total protein concentration may be disease only if they are found in the CSF and not
increased in the following situations. in the serum. Although such bands can be detected
in more than 90 per cent of patients with multiple
● In the presence of blood, due to haemoglobin and
sclerosis, the finding is not specific for this condition.
plasma proteins.
Occasionally, intrathecal malignant B lymphocytes
● In the presence of pus, due to cell protein and to
produce a local monoclonal band.
exudation from inflamed surfaces.
● Comparison of the IgG and albumin CSF to plasma
● In non-purulent inflammation of cerebral tissue,
ratio. Albumin has a lower molecular weight than IgG
when there may be a definite rise in total protein
and its concentration increases disproportionately
concentration despite the absence of detectable cells
in CSF if increased vascular permeability due to
in the CSF. Cells may also be undetectable in some
cases of bacterial meningitis, particularly in children,
in immunocompromised patients, or if antibiotics CASE 2
have been given before lumbar puncture.
● If there is blockage of the spinal canal which, by A 43-year-old man attended the ear, nose and throat
impairing the flow of CSF distal to the block, allows (ENT) department because of a nasal discharge. He
longer for equilibrium with the circulation and so had had brain surgery 3 years previously following a
brings the composition of CSF slightly nearer to that head injury. Although he had initially been treated
of plasma (Froin’s syndrome). Increased pressure in as having allergic rhinitis, the ENT consultant sent
the CSF may also increase protein. Such blockage some of the nasal fluid to the clinical biochemistry
may be caused by: laboratory for tau protein analysis. The results
– spinal tumours, returned showed the presence of tau protein in his
– vertebral fractures, nasal fluid.
– spinal tuberculosis. DISCUSSION
● Where there is local synthesis of immunoglobulins by Nasal fluid should not normally contain tau
plasma cells within the CSF. (asialotransferrin) protein, as this is usually found
Measurement of individual cerebrospinal fluid protein
in cerebrospinal fluid (CSF). Therefore, the findings
concentrations suggest that the nasal discharge contained CSF. The
previous head injury had resulted in a dura tear,
In the presence of blood or pus, tests for individual CSF
allowing CSF to leak from the nose.
proteins are not useful and may even be misleading. They
Pleural fluid 335
inflammation is the cause of the high protein However, these tests are not specific, as both can be
concentration. In this case the CSF to plasma ratio elevated in other conditions. Microbiological tests are
of IgG to albumin will be low or, if permeability is so often more useful if infection is suspected.
increased that the two proteins diffuse at almost the
same rate, normal. In conditions such as multiple Procedure for examination of the cerebrospinal fluid
sclerosis in which IgG has been synthesized within the Consider the following:
CSF, the ratio is high. This method is less sensitive than
● Heavily bloodstained (assuming a non-traumatic
the detection of oligoclonal bands (see Chapter 19).
lumbar puncture) in three consecutive specimens:
Increased CSF immunoglobulin synthesis, with there has probably been a cerebral haemorrhage.
oligoclonal bands, may be found in: ● Send for microbiological examination and for
● multiple sclerosis (the most important indication for estimation for glucose and protein concentrations.
the test), ● Xanthochromic: in addition to the above tests, send
● viral infections: the specimen for spectrophotometric examination.
– meningitis, ● If indicated, intrathecal immunoglobulin synthesis
– encephalitis, may be confirmed, either using the CSF to plasma
– subacute sclerosing panencephalitis, ratio of IgG to albumin or by the detection of
● prion disease, for example Creutzfeldt–Jakob disease, multiple (oligoclonal) bands in the CSF.
● bacterial meningitis,
PLEURAL FLUID
● tuberculosis,
● neurosyphilis, This is a plasma ultrafiltrate; there is usually less than
● acute idiopathic polyneuropathy (Guillain–Barré 10 mL of this fluid in each pleural cavity. If the rate of
syndrome), removal is less than the rate of formation, pleural fluid
● systemic lupus erythematosus, will accumulate. Decreased removal may be due to
● cerebral sarcoidosis, decreased pleural space pressure, for example bronchial
● cerebral tumours (rarely). obstruction, or impaired lymphatic drainage, for
example neoplasms (Fig 23.1). Pleural fluid production
Tau protein
As in any ultrafiltrate, the concentration of high-
molecular-weight CSF proteins is very much lower CASE 3
than in plasma. Also, electrophoresis shows that the
proportions of individual proteins are slightly different A 69-year-old male smoker presented to the chest
in CSF from those in serum. For example, in the CSF, the clinic with shortness of breath, haemoptysis, cough
concentration of pre-albumin is higher and of g-globulin and weight loss. A chest radiograph revealed a left-
lower relative to other proteins than in plasma. The tau sided pleural effusion and upper zone ‘shadow’. The
protein, detectable in the b–g region in CSF, is a variant effusion was ‘tapped’ and the following results were
of transferrin (asialotransferrin), which cannot be obtained:
absorbed as the ultrafiltrate passes through the choroid Pleural fluid lactate dehydrogenase (LDH) 566 U/L
plexus; this modified form cannot be reabsorbed into the (< 200)
circulation and therefore is not found in plasma. Pleural fluid protein 114 g/L, with concomitant
In plasma, the asialotransferrin concentration is very plasma protein 60 g/L
low, as desialylated glycoproteins are rapidly removed
from the circulation by the hepatic asialoglycoprotein DISCUSSION
receptor. A nasal secretion (rhinorrhoea) containing These biochemical tests suggest a pleural exudate.
tau protein is suggestive of the source being CSF, as Note the raised pleural fluid LDH and considerably
normal nasal secretions do not contain tau protein. raised pleural fluid protein (more than 30 g/L)
concentrations. The cytology showed malignant
Other cerebrospinal fluid analytes cells. A lung carcinoma was found on bronchoscopy.
C-reactive protein and lactate have also been used in Exudate pleural fluid is associated with malignant
certain circumstances to indicate bacterial infection. lung disease.
336 Cerebrospinal, pleural and ascitic fluids
abdominal distension and breathing difficulties. concentration). A high gradient of > 11 g/L suggests
Ascitic fluid drainage (paracentesis) may be necessary portal hypertension, e.g. cirrhosis, Budd–Chiari
to facilitate diagnosis and relieve symptoms. syndrome, constrictive pericarditis, kwashiorkor or
● Like pleural fluid, ascites can be divided into cardiac failure, and a gradient < 11 g/L is indicative
exudates and transudates. Similar tests to those of a non-portal hypertensive aetiology, e.g.
done for pleural fluid can be requested. However, carcinoma, infection such as spontaneous bacterial
it has been suggested that, rather than classifying peritonitis, nephrotic syndrome or pancreatitis
into transudate or exudate, it may be more (see Chapter 17).
useful to classify the ascitic fluid upon the basis ● Meig’s syndrome is the triad of pleural effusion,
of the serum ascites albumin gradient (serum ascites and ovarian tumour (fibroma).
albumin concentration minus ascites albumin
SUMMARY
● In adults, the total volume of CSF is about ● Pleural fluid can also be analysed in the laboratory
135 mL, produced at a rate of 500 mL/day. This is and divided into transudates (protein less than
predominantly formed by plasma ultrafiltration 30 g/L) and exudates (protein more than 30 g/L).
through the capillary walls of the choroid plexuses This may be useful when searching for the cause of
in the brain’s lateral ventricles. the effusion.
● Laboratory analysis of CSF can be useful in the ● Laboratory tests, including the serum to ascites
diagnosis of various diseases, including meningitis albumin gradient, can also help in the diagnosis of
and subarachnoid haemorrhage. ascitic fluid accumulation.
24 Metabolic effects of tumours
This chapter looks at how clinical biochemistry tests embryonic neural crest and is composed of two types of
can be used to diagnose and monitor patients with cells,the chromaffin cells and the nerve cells,both of which
malignant disease. Malignant disease is one of the can synthesize active catecholamines (dihydroxylated
world’s major ‘killers’, and it is therefore important to phenolic amines). Adrenaline (epinephrine) is almost
know the basic principles of biochemical diagnosis and exclusively a product of the adrenal medulla, whereas
monitoring in such patients. noradrenaline (norepinephrine) is predominantly
Neoplastic cells of differentiated tissues sometimes formed at sympathetic nerve endings.
synthesize enough compounds not normally thought
of as coming from that tissue to be detectable in body
fluids. These substances fall into two principal groups: CASE 1
● those that alter metabolism and thus may produce A 34-year-old man was referred to the hypertension
clinical effects, some of which are hormonal clinic because of uncontrolled hypertension. His
syndromes, blood pressure in the clinic was 186/104 mmHg,
● those that, although biologically inactive, may be despite treatment with bendroflumethiazide,
analytically detectable in body fluids; these are enalapril and felodipine. Some biochemistry tests
sometimes used as tumour markers. were requested, which gave the following results:
Plasma
THE DIFFUSE ENDOCRINE SYSTEM Sodium 138 mmol/L (135–145)
Certain rare syndromes with endocrine or Potassium 4.2 mmol/L (3.5–5.0)
neurotransmitter properties are associated with Urea 5.7 mmol/L (2.5–7.0)
neoplasia. These cells may share common cytological Creatinine 90 µmol/L (70–110)
characteristics, originating in the embryonic ectoblast.
Urine
Staining techniques have shown their ability for amine
Normetanephrine 23.1 mmol/24 h (normally
precursor uptake and decarboxylation (APUD) with
< 2.0 µmol/24 h)
the production of amines. Tumours of these cells
Metanephrine 17.3 mmol/24 h (normally
have therefore been called APUDomas (although
< 1.5 µmol/24 h)
this older classification has been challenged). Some
secrete physiologically active amines, whereas others, DISCUSSION
such as the pituitary and parathyroid glands and the Phaeochromocytoma is the likely diagnosis, given
calcitonin-producing C cells of the thyroid, secrete the raised urinary metanephrine concentrations.
peptide hormones. Many occur in the gastrointestinal This was confirmed by further tests, including a
tract and pancreas. Secreting tumours of tissues of the radioisotope metaiodobenzylguanidine (MIBG)
sympathetic nervous system and the amine-secreting scan and an adrenal MRI scan. The MEN syndrome
carcinoid tumours are discussed in this chapter. needs to be excluded, given the known association
with phaeochromocytoma. It is often wise to seek a
CATECHOLAMINES secondary cause of hypertension in young individuals
with hypertension, particularly those refractory to
The sympathetic nervous tissue comprising the adrenal
treatment.
medulla and sympathetic ganglia is derived from the
Catecholamines 339
Metabolism of catecholamines although many are isolated tumours. The symptoms and
Adrenaline and noradrenaline are formed from the signs can be related to very high plasma concentrations
amine precursor tyrosine via dihydroxyphenylalanine of catecholamines.
and dihydroxyphenylethylamine (dopamine) and bear The hypertension associated with phaeochromocy-
the catechol (3,4-dihydroxyphenyl)-moiety. Adrenaline toma is potentially curable by surgery (Fig. 24.2).
and noradrenaline are both metabolized to the inactive Its presence should be excluded, particularly if a
4-hydroxy-3-methoxymandelic acid (HMMA), young adult presents with paroxysmal or persistent
incorrectly called vanillyl mandelic acid (VMA), hypertension for no apparent reason or a patient
by similar pathways in which metadrenaline and presents with refractory hypertension or shows the
normetadrenaline, respectively, are intermediates (Fig. classic triad of sweating, headaches and tachycardia
24.1). Adrenaline, noradrenaline and their metabolites associated with hypertension. Hyperglycaemia and,
can be measured in urine. if the plasma glucose concentration is high enough,
glycosuria may occur during the attack.
Action of catecholamines
Diagnosis of phaechromocytomas
Noradrenaline causes generalized vasoconstriction,
with hypertension and pallor. Adrenaline dilates blood The biochemical diagnosis of these tumours can
vessels in muscles, with variable effects on blood be made by measuring the daily urinary excretion
pressure and pulse rate. Adrenaline may also cause of catecholamines (adrenaline, noradrenaline and
hyperglycaemia due to stimulation of glycogenolysis dopamine) or HMMA, the major catabolic product
and other anti-insulin effects. of catecholamines. Extra-adrenal tumours may
only secrete dopamine. Metanephrines (which are
Catecholamine-secreting tumours also metabolites of catecholamines and include
Phaeochromocytomas metadrenaline, normetadrenaline and methoxy-
Phaeochromocytomas present mainly in adults and tyramine) are preferable because they are more
occur in chromaffin tissue. About 10 per cent are sensitive than catecholamines and can also be measured
outside the adrenal medulla, 10 per cent are bilateral in urine and their concentration may be raised in some
and 10 per cent are malignant. They are associated with phaeochromocytomas when urinary catecholamines
the multiple endocrine neoplasia (MEN) syndrome, are normal. A slightly increased excretion of urinary
neurofibromatosis and Von Hippel–Lindau disease catecholamines can be found in cases of essential
OH
DOPA DOPAMINE
CH2CHNH3 COO OH OH
Synthesis Tyrosine
OH OH
OH OH
CHOH CHOH
O CH3 CH2NH2 CH3 CH2NH3 O CH3
Adrenaline Noradrenaline
CH2NH2CH3 CH2NH3
Metadrenaline Normetadrenaline
Figure 24.1 Synthesis and metabolism of catecholamines. DOPA, dihydroxyphenylalanine; HMMA, 4-hydroxy-3-
methoxymandelic acid.
340 Metabolic effects of tumours
100
Neuroblastomas
Neuroblastomas are very malignant tumours of
Tumour out the sympathetic nervous tissue and usually occur
Nitroprusside in children. About 40 per cent occur in the adrenal
medulla, and about 60 per cent are extra-adrenal.
0 1/2 1 11/2 The plasma catecholamine concentrations may
Hours
be as high as, or higher than, those in patients with
phaeochromocytomas. Some neuroblastomas secrete
Figure 24.2 Showing large fluctuations in systolic blood dopamine and its metabolite homovanillic acid (HVA)
pressure during manipulation of a phaeochromocytoma
whose concentration is raised in the urine and may be
during its surgical removal. The blood pressure was
used as a tumour marker.
treated with nitroprusside infusion. Adapted with kind
permission from Kinirons M and Ellis H. French’s THE CARCINOID SYNDROME
Index of Differential Diagnosis, 15th edition. London:
Normal metabolism of 5-hydroxytryptamine
Hodder Arnold, 2011.
Some cells are called argentaffin because they reduce,
and therefore stain with, silver salts. These are normally
hypertension. Various dietary components and drugs found in tissues derived from the embryonic gut and
affect some of the analytical methods, and therefore it
is important to consult your laboratory before starting a
urine collection. Labetalol, paracetamol, a-methyldopa CASE 2
and Sinemet (combined levodopa and carbidopa) may A 56-year-old man attended the gastroenterology
interfere with some of these assays. department because of profuse diarrhoea. He also
In some cases, particularly if the tumours are small mentioned that he had felt increasingly breathless, and
or hypertension is paroxysmal, metabolite excretion in he looked flushed. A number of investigations were
a 24-h urine collection may be misleading. If there is a requested, but it was a 24-h urinary 5-hydroxyindole
high index of suspicion, it may be useful to repeat this acetic acid (5-HIAA) concentration of 544 µmol/day
estimation on three consecutive days. Raised plasma (less than 25) that suggested the diagnosis.
metanephrine concentrations have nearly 100 per cent
DISCUSSION
sensitivity for the diagnosis of phaeochromocytoma
The patient had considerably raised urinary 5-HIAA
and may be particularly helpful if the patient is
concentration and symptoms suggestive of carcinoid
unable to give a reliable urine sample. However, the
syndrome. Note the flushing and diarrhoea as well
concentration of plasma metanephrines may also be
as the breathlessness, probably due to bronchospasm
raised in renal failure.
induced by vasoactive and bronchoactive amines.
The clonidine or pentolinium suppression tests may
The patient was subsequently found to have
have a place in the investigation if urinary catecholamines
carcinoid syndrome involving a tumour of the ileum
are positive or equivocal and imaging studies are
that had metastasized to the liver. Urine 5-HIAA can
negative. In normal individuals, concentration of
be used to monitor disease assuming that a change in
urinary and plasma catecholamines should decrease
management would improve clinical prognosis.
after the administration of the suppressing agent, unlike
Multiple endocrine neoplasia 341
are most abundant in the ileum and appendix, but has also been suggested as a cause of the flushing. A
they are also found in the pancreas, stomach and pellagra-type syndrome may occasionally develop,
rectum. They synthesize the biologically active amine because tryptophan is diverted from nicotinamide
5-hydroxytryptamine (5-HT; serotonin) from the amine to 5-HT synthesis. A carcinoid crisis occurs when the
precursor tryptophan, the intermediate product being tumour releases large amounts of vasoactive substances
5-hydroxytryptophan (5-HTP). 5-Hydroxytryptophan into the circulation.
is inactivated by deamination and oxidation by
Diagnosis of the carcinoid syndrome
monoamine oxidases to 5-hydroxyindole acetic acid
(5-HIAA) (Fig. 24.3). Urinary 5-HIAA secretion is usually very high in the
The oxidases and aromatic amino acid decarboxylases carcinoid syndrome. A daily excretion of more than
are present in other tissues as well as in argentaffin cells; 25 µmol is elevated, and greater than 100 µmol indicates
5-HIAA is usually the main urinary excretion product carcinoid syndrome if plums, pineapples, kiwi fruit,
of argentaffin cells. Argentaffin cells may also excrete the avocados, tomatoes, walnuts and bananas (which are
peptide substance P, an excess of which causes flushing, high in hydroxyindoles) have been excluded from
tachycardia, increased bowel motility and hypotension. the diet for about 24 h before the urinary collection
is started. Elevated urinary 5-HIAA has also been
Causes of the carcinoid syndrome reported in association with small bowel disease and
The carcinoid syndrome is usually associated with intestinal obstruction, for example coeliac disease and
abnormally high concentrations of plasma 5-HT. Ileal sprue, but usually concentrations are not as high as in
and appendiceal tumours only produce the typical clinical carcinoid syndrome.
syndrome when they have metastasized, usually to the In very rare cases, usually of bronchial or gastric
liver. The products of intestinal tumours are inactivated in tumours, the argentaffin cells lack aromatic amino
the liver, but those from other sites, such as the bronchus, acid decarboxylase. In such cases, despite increased
are released directly into the systemic circulation in an secretion of 5-HTP, urinary 5-HIAA excretion may
active form and may therefore cause symptoms. not be increased to diagnostic levels. If there is a strong
Only one enzyme, tryptophan-5-hydroxylase, is clinical suspicion of the carcinoid syndrome, despite
needed to convert tryptophan to 5-HTP (see Fig. 24.3), the finding of normal 5-HIAA excretion, estimation
and its derepression in malignant syndromes leads of total 5-hydroxyindole (5-HTP, 5-HT and 5-HIAA)
to excessive synthesis of 5-HTP. The decarboxylase excretion may be indicated. In some cases, plasma 5-HT
and monoamine oxidases are present in normal non- or its metabolites may be measured.
argentaffin tissues, so 5-HTP accumulates. Carcinoid tumours may also secrete
The symptoms and signs of the carcinoid syndrome adrenocorticotrophic hormone (ACTH), which can
include: result in Cushing’s syndrome as well as being associated
with MEN 1 syndrome. Octreotide, a somatostatin
● diarrhoea, which may be severe enough to cause the
analogue, can be used to antagonize some of the effects
malabsorption syndrome,
of the released mediators in carcinoid syndrome.
● flushing,
● bronchospasm and right-sided fibrotic lesions MULTIPLE ENDOCRINE NEOPLASIA
of the heart, such as tricuspid incompetence and
In the rare syndromes of MEN (pluriglandular
pulmonary stenosis; the heart lesions do not occur if
syndrome), two or more endocrine glands secrete
the primary tumour is in the bronchus.
inappropriately high amounts of hormones, usually
The symptoms and signs are more likely to be due from adenomas. There are two main groups of
to the presence of substance P than of 5-HT. Histamine syndromes.
MEN 1 may involve two or more of the following with neoplasia, which are sometimes associated with
endocrine tissues; the glands involved are listed in order the synthesis of significant amounts of a peptide not
of decreasing incidence of involvement: normally detectable in the fully differentiated tissue.
There are two possible hypotheses, both of which
● parathyroid gland (hyperplasia or adenoma),
include derepression:
● pancreatic islet cells:
– gastrinomas, ● of part of the genome of the tissue cell that codes for
– insulinomas, the peptides, which are then synthesized in excess,
– glucagonomas, ● of specialized neuroendocrine cells, which are
– vasoactive intestinal polypeptide-producing scattered through many tissues.
tumour (VIPoma),
Some substances are secreted more often by one
– pancreatic polypeptide-producing tumour
type of tumour than by others, whereas some types of
(PPoma),
tumour secrete more than one ‘foreign’ substance.
● anterior pituitary gland,
● adrenal cortex. Hormonal syndromes
Remember the three Ps: parathyroids, pancreas and Hormones produced at ectopic sites, such as those
pituitary. MEN 2 includes: secreted by pathological overactivity of endocrine
● medullary carcinoma of the thyroid gland, glands, are not under normal feedback control, and
● phaeochromocytoma, secretion continues under conditions in which it
● adenoma or carcinoma of the parathyroid gland. should be suppressed; such secretion is therefore
inappropriate. Ectopic production is most clinically
MEN 2 can be subdivided into 2A (Sipple’s obvious if the peptide has hormone-like effects. Many
syndrome) and 2B, the latter also being associated with of the syndromes associated with such hormonal
marfanoid habitus, mucosal neuromas and abnormal syndromes are discussed in the relevant chapters of this
corneal nerve fibres. book.
Both types of MEN are usually familial, inherited as The following may be due to hormone secretion by
a Mendelian dominant trait with variable penetrance. the tumour.
MEN 1 is due to a mutation in the MENIN gene, a
tumour-suppressor gene; MEN 2 is the result of a Hyponatraemia due to antidiuretic hormone secretion
proto-oncogene mutation known as RET. Genetic tests Although ectopic antidiuretic hormone (ADH)
can be used to screen families. production is commonly associated with the relatively
Calcitonin (see Chapter 6) is secreted by the thyroid rare small cell carcinoma of the bronchus, the
parafollicular ‘C’ cells. Plasma calcitonin concentration syndrome of inappropriate ADH secretion has been
can be raised in medullary carcinoma of the thyroid reported in a wide variety of tumours. This syndrome,
associated with MEN 2 syndrome (rarely in other which is not confined to malignant disease, is discussed
tumours). It can thus be used as a tumour marker in Chapter 2.
for medullary thyroid carcinoma. Plasma calcitonin
concentration can be used for screening for familial Hypokalaemia due to adrenocorticotrophic hormone
thyroid medullary carcinoma. In some cases, basal secretion
plasma calcitonin levels are not significantly raised Ectopic ACTH secretion stimulates the secretion of
but may become so after intravenous pentagastrin all adrenocortical hormones except aldosterone; the
stimulation. clinical presentation often resembles that of primary
hyperaldosteronism (glucocorticoid hormones have
ECTOPIC HORMONE PRODUCTION some mineralocorticoid action). The condition is
Mechanism of production usually associated with a small cell carcinoma of
All cells have the potential to produce any peptide or the bronchus but has occasionally been described in
protein coded for in the fertilized ovum. Malignant cells association with a variety of other malignant lesions,
may produce ectopic hormones or other peptides if they especially pulmonary carcinoid tumours. The patient
partly revert to their early embryonic, pluripotential may present with severe hypokalaemic alkalosis (see
state. This may cause the histological changes associated Chapters 4 and 5).
Tumour markers 343
Tumour markers may be used for the following: Some examples of tumour markers
● To screen for disease Very few markers are sufficiently Prostate-specific antigen
sensitive or specific to be used to screen for the Prostate-specific antigen (PSA) is a marker for prostatic
presence of a tumour (see Chapter 1). carcinoma, a common male tumour, and is a 33-kDa
● To diagnose a tumour If a patient presents with protein and is homologous with the protease kallikrein
clinical signs or symptoms, the measurement of a family; it has a plasma half-life of about 3 days. One
marker in plasma or urine may very occasionally be of its probable functions is to help liquidize semen. Its
used to confirm a diagnosis. level is raised in benign prostatic hyperplasia (BPH)
● To determine the prognosis In some cases the and prostatic carcinoma but also in prostate infection,
concentration of a specific marker is related to the for example prostatitis, and after rectal examination.
mass or spread of the tumour. Levels of PSA increase with age, which is mainly
● To monitor the response to treatment If a tumour due to the increase in the volume of the prostate that
marker is present, the rate of its decrease in occurs. Therefore age-adjusted reference ranges should
concentration may be used to assess the response be used. There may also be a place for expressing
to treatment such as surgery, chemotherapy or plasma PSA in terms of prostate volume as found on
radiotherapy. ultrasound examination.
● To identify the recurrence of a tumour If the One diagnostic limitation is that the values of PSA
concentration of the marker was previously raised, overlap in BPH and prostatic carcinoma. After a radical
intermittent measurement during remission prostatectomy, plasma PSA levels become undetectable
may sometimes be used to identify recurrence. at 2–3 weeks. Finasteride, a 5-a-reductase inhibitor
Occasionally, however, tumours may dedifferentiate that is sometimes used to treat BPH, decreases plasma
and fail to express the marker despite continued PSA by up to 50 per cent.
growth and spread. The PSA is bound in the plasma to either a1-
antichymotrypsin or a2-macroglobulin. The
concentration of bound or complexed PSA is higher
in prostate carcinoma, whereas that of free PSA is
CASE 4 higher in BPH. The ratio of free to total PSA is lower
in men with prostatic carcinoma. The PSA index
A 67-year-old man attended the urology out-patient is expressed as the percentage of the total plasma
clinic because of urinary hesitancy, lower back PSA that is free; an index above about 17 per cent is
pain and nocturia. Rectal examination revealed a suggestive of BPH and one of less than 17 per cent of
large, firm and craggy prostate gland. Some of his prostate carcinoma.
biochemistry results were as follows: Plasma PSA concentrations greater than 10 µg/L are
Plasma strongly suggestive of carcinoma, although carcinoma
Prostate-specific antigen (PSA) 28 µg/L (< 4) may be present even if values fall within the reference
Albumin-adjusted calcium 2.98 mmol/L (2.15–2.55) range. A PSA above 20 µg/L is suggestive of prostatic
Phosphate 0.92 mmol/L (0.80–1.35) carcinoma that has spread beyond the prostate gland.
Alkaline phosphatase (ALP) 654 U/L (< 250) Plasma PSA assays in conjunction with digital
rectal examination may be used as part of a screening
DISCUSSION
programme for prostatic carcinoma in at-risk males.
The grossly elevated plasma PSA concentration is
There is, however, no universally agreed screening
suggestive of malignant prostate disease. A bone
protocol for prostatic carcinoma in the general
scan showed osteosclerotic bony secondaries
population. Prostate cancer antigen 3 (PCA3) and
in his lumbar spine. Further investigations,
specific PSA isoforms may also prove useful markers in
including prostatic biopsy, confirmed metastatic
conjunction with PSA.
prostatic carcinoma. This would also be a likely
Prostate biopsy is usually necessary if the PSA
explanation for his hypercalcaemia. The raised
concentration is above 10 µg/L; however, the decision
plasma ALP suggests a bone source due to the
regarding biopsy is more difficult if PSA levels are
bony secondaries.
4–10 µg/L, although the PSA index may help.
Tumour markers 345
with AFP and hCG, it is useful in the diagnosis and ● Chromogranin A is released from neuroendocrine
monitoring of extragonadal and gonadal germ cell cells such as in phaeochromocytoma and carcinoid
tumours. However, plasma levels are also elevated in tumours.
smokers (see Chapter 18). ● Protein S100B is a calcium-binding protein. It is
● Thyroglobulin: this high-molecular-weight protein expressed in brain astrocytes and glial cells and also
is produced in the follicular cells of the thyroid. in melanocytes and may be useful in monitoring
Its concentration is raised in follicular or papillary therapy in malignant melanoma.
carcinoma of the thyroid. Spuriously low levels may ● Human epididymis protein (HE4) is being used as a
be found in the presence of thyroglobulin antibodies, tumour marker for ovarian carcinoma.
which interfere with the assay.
New tumour markers are likely to be revealed in
● Neuronal-specific enolase: plasma levels may be raised
the future and this chapter is not all-inclusive. Genetic
in small cell lung carcinoma and neuroblastoma; it is
tests are also being developed that may be useful to
derived from neurodectal tissue.
predict those individuals at risk of developing various
● Inhibin: this is secreted by the granulosa cells of
carcinomas, for example BRCA gene mutations for breast
the ovary and by the Sertoli cells of the testis. It
carcinoma. Tumour-modified deoxyribonucleic acid
can be used as a plasma tumour marker of ovarian
(DNA), ribonucleic acid (RNA) and nucleic acids also
granulosa cell tumours and testicular Sertoli cell
circulate in the blood and may be useful in the diagnosis
tumours.
of certain cancers.
● Squamous cell carcinoma antigen: this is a plasma
tumour marker of potential use in squamous cell
carcinoma of the cervix.
SUMMARY
● Laboratory biochemical tests can be useful in the ● Similarly, raised urinary 5-HIAA concentration may
investigation and management of certain malignant be useful in the diagnosis of carcinoid syndrome.
disorders. ● For the measurement of a tumour marker to be
● Tumours alter metabolism and thus may produce clinically useful, the result should clearly separate
clinical effects, some of which are hormonal those patients with a tumour from those without.
syndromes. In addition, tumours may release Therefore a tumour marker should ideally be
compounds that, although biologically inactive, sensitive (concentrations should be raised if the
may be analytically detectable in body fluids. These tumour is present) and specific (levels should not
are sometimes used as tumour markers. be raised if the tumour is not present).
● In the rare syndromes of MEN (pluriglandular ● Tumour markers are generally best used for
syndrome), two or more endocrine glands secrete monitoring the progression of tumours with
inappropriately high amounts of hormones, usually therapy (remission or relapse). Many are of little
from adenomas. value in diagnosis or screening of disease.
● The diagnosis of phaeochromocytomas may be
facilitated by the assay of urinary catecholamines
or metanephrines.
25 Therapeutic drug monitoring and
poisoning
Drug overdose and the consequences of drug side effects ● plasma potassium concentrations during potassium
are common causes of hospital medical admissions. supplementation (Chapter 5),
This chapter also looks at therapeutic monitoring of ● thyroid-stimulating hormone concentrations while
particular drugs and how the clinical biochemistry on thyroxine therapy (Chapter 11),
laboratory can be involved in the management of ● plasma calcium concentration and alkaline
various drug overdoses. phosphatase activity during vitamin D treatment for
The blood (usually plasma or serum) concentrations hypocalcaemia or osteomalacia (Chapter 6),
of many drugs can be measured or, more rarely, those ● plasma cholesterol after hydroxy-malonyl-glutaryl
of other body fluids, although in only a few situations is coenzyme A reductase inhibitor (statin) therapy
this of proven benefit. (Chapter 13),
Pharmacokinetics is the study of the fate of drugs after ● clotting status as judged by prothrombin time
administration and is concerned with their absorption, and international normalized ratio (INR) for
distribution in body compartments, metabolism and determining warfarin dosage.
excretion. Absorption depends on whether the drug is
The drug or metabolite concentrations can also be
taken orally, by intravenous or intramuscular injection,
measured in biological fluids, although these may not
sublingually or rectally.
parallel cellular effects. However, the measurement
Possible indications for measuring drug
of plasma concentrations may be indicated in the
concentrations in various body fluids are to:
following situations:
● check that the patient is taking the drug as prescribed
● If the desired result cannot be measured precisely:
(compliance),
for example, the incidence of epileptic fits is a poor
● ensure that the dose is sufficient to produce the
indicator of the optimal dosage of anticonvulsants.
required effect but not so high as to be likely to cause
● If the range of plasma levels that is most effective
toxic effects,
in producing the desired result without toxic side
● help diagnose drug side effects and drug interactions,
effects (the therapeutic range) has been defined:
● determine the type of drug or drugs taken in cases
this is particularly true if there is a narrow margin
of suspected overdose and to assess the need for
between therapeutic and toxic drug concentrations,
treatment.
such as in the case of digoxin or lithium. This is
of particular value when the relation between
MONITORING DRUG TREATMENT dose and plasma concentrations of a drug is
One can assess whether the dose of a drug is optimal unpredictable.
either clinically or by laboratory assays. An example ● If the prescribed drug is the main active compound
of the former would be monitoring the action of and is not metabolized significantly to an active
antihypertensive drugs by measuring the blood pressure. metabolite: otherwise the active metabolite may be
Examples of laboratory biochemical effects, which are measured, for example phenobarbital in primidone
discussed in the relevant chapters, include the following: treatment.
348 Therapeutic drug monitoring and poisoning
FACTORS AFFECTING DRUG PLASMA at all, take more than the prescribed dose, take the drug
CONCENTRATIONS intermittently or become confused about the timing
The total amount of drug in the extracellular fluid (ECF) and dose, particularly if taking more than one drug.
depends on the balance between that entering and that Clinicians should realize that poor compliance might
leaving the compartment; the plasma concentration cause a poor clinical response or toxicity. A regular
depends on the volume of fluid through which the review of therapy and careful explanation to the patient
retained drug is distributed. are important. Assay of plasma concentrations is only
a crude method of assessing compliance and only tests
Timing of the sample the situation at the time when blood was taken.
Blood samples must be taken at a standard time after
Entry of the drug into, and distribution
ingestion of the drug, the exact time varying with the
through, the extracellular fluid
known differences in the rate of absorption, metabolism
and excretion of different drugs (Table 25.1). Absorption
The following factors may affect the blood Lipid-soluble drugs can pass through cell membranes
concentration of some drugs. more readily, and are therefore absorbed more rapidly,
than water-soluble ones; they reach the highest plasma
Patient compliance concentrations at between 30 and 60 min after ingestion.
It has been shown that not all patients take a drug The rate of absorption in an individual patient may be
exactly as prescribed. The patient may not take the drug affected by:
● the timing of ingestion in relation to meals, found in hepatic cirrhosis or the nephrotic syndrome,
● the rate of gastric emptying: this must be allowed for, may reduce the proportion of protein-bound drugs.
for example during treatment with drugs affecting ● Competition for binding sites on protein Many drugs,
gut motility or after gastric surgery, unconjugated bilirubin fatty acids and hydrogen
● vomiting, diarrhoea or malabsorption syndromes, ions compete with each other for binding sites.
● the administration of other compounds, such as the
Metabolism and excretion of drugs
bile acid sequestrants which may also bind certain
drugs within the intestinal lumen. The blood concentrations of a drug depend on normal
hepatic and renal function as well as on acid–base and
Volume of distribution electrolyte balance. The time taken for the plasma drug
concentration to fall to half its original concentration
The final plasma concentration of a drug reached after
is called its effective half-life. To maintain a reasonably
a standard amount has been absorbed depends on the
steady plasma concentration, drugs with a short half-
volume through which it has been distributed. For
life should be taken more frequently than those with
example, it may be difficult to predict the appropriate
a long one. After starting treatment, a steady state is
dose in oedematous or obese patients who, because
usually reached after about five times its half-life has
they have a larger than normal volume of distribution,
elapsed; the first specimen of blood for monitoring
may have unexpectedly low plasma concentrations.
should not be taken earlier than this.
By contrast, in small children, with a low volume of
The rate at which the plasma drug concentration
distribution, there is a danger of overdosage. The
falls after it has reached peak concentration depends on
patient’s weight or surface area may need to be allowed
the rate of distribution through the ECF (see above),
for when the dose is calculated.
the rate of entry into cells and the rate at which it is
metabolized and excreted, whether in urine or bile.
Binding to plasma albumin
The rate of elimination of most drugs depends on
Many drugs, like many endogenous substances, are their plasma concentration. A small increase in the dose
partly inactivated by protein binding, usually albumin. of some, such as phenytoin, may exceed the capacity
Most drug assays estimate the total concentration of the of the metabolic or excretory pathways and so cause a
free plus the protein-bound drug. Biological feedback disproportionate increase in plasma levels; the plasma
mechanisms do not control free drug concentrations as concentrations of these drugs should be monitored
they do those of plasma calcium and hormones; therefore, carefully. The rate at which this occurs is termed
the method of interpretation to allow for altered protein saturation kinetics.
binding is different. Measured plasma concentrations
fall little due to a reduction in protein binding; a larger Metabolic conversion to active or inactive metabolites
proportion of the measured drug will be in the unbound, Some drugs are active only after metabolic conversion;
free, active form, so that metabolism and excretion will others are inactivated, usually by conjugation in the
increase. Unless this is realized, dangerously high plasma liver. Ideally, a drug assay should measure all the active
free concentrations may be interpreted as being within, forms, whether the parent compound or its active
or even below, the therapeutic range if the plasma metabolite, and none of the inactive forms.
albumin concentration is very low.
The bound proportion varies with differing plasma Drug–drug interactions
albumin concentrations, and there is no valid correction If a number of different drugs are being prescribed,
factor that allows for protein abnormalities. About one may affect the plasma concentration of another
90 per cent of phenytoin, 70 per cent of salicylic acid, by altering its binding to plasma proteins, rate of
50 per cent of phenobarbital and 20 per cent of digoxin metabolism or excretion. For example, sodium
is protein bound. However, binding may be affected by valproate displaces phenytoin from its protein-binding
the following: sites and reduces its rate of metabolism.
● Abnormalities in plasma albumin concentration Blood
should be taken without stasis to minimize a possible Tolerance
rise in plasma albumin, and albumin-bound drug, Some drugs (for example phenytoin) may induce
concentrations. Low concentrations, such as those often the synthesis of enzymes that inactivate them, and
350 Therapeutic drug monitoring and poisoning
cross-react with endogenous compounds – so-called may result in a large increase in plasma concentration
digoxin-like immunoreactive substances. The latter and be associated with toxicity. Sodium valproate
can occur in pregnancy. Sometimes digoxin overdose/ displaces phenytoin from its binding sites and reduces
toxicity can be treated by the administration of its rate of metabolism; free plasma phenytoin may
inactivating digoxin antibodies (Digibind). It should then cause toxicity despite apparently appropriate total
be also remembered that Digibind interferes with some plasma concentrations. Phenytoin is a potent enzyme-
immunoassays used to measure digoxin. inducing drug (as, indeed, are phenobarbital and
carbamazepine) and may enhance the metabolism of
Amiodarone other drugs as well as causing an elevation in plasma
This is used to treat various cardiac arrhythmias, g-glutamyl transferase, which does not necessarily
including ventricular tachycardias and atrial mean abnormal hepatic toxicity (see Chapter 17).
fibrillation. The drug is highly protein bound and has
a long half-life, up to 10–40 days after a single dose and Carbamazepine
up to 100 days on chronic therapy. It is not excreted Like phenytoin, carbamazepine can be used in the
in the urine, but is predominantly metabolized in treatment of partial and generalized tonic–clonic
the liver. The metabolite desethylamiodarone is also seizures by inhibiting voltage-gated sodium channels.
active. Amiodarone is potentially very toxic, sometimes Additionally, it can be used in the treatment of
causing photosensitivity reactions, pulmonary fibrosis trigeminal neuralgia and diabetic neuropathy and also
and corneal microdeposits. Additionally, it blocks free for prophylaxis of bipolar disorders. It has an active
thyroxine to free tri-iodothyronine conversion and can metabolite formed in the liver called carbamazepine
evoke hypothyroidism. Conversely, as it contains iodine, 10,11-epoxide. Carbamazepine induces its own
it can also cause thyrotoxicosis in some circumstances metabolism and thus plasma concentrations may fall at
(see Chapter 11). the end of the first month of therapy, which results in
the need for a dose increase.
Flecainide
This is another antiarrhythmic drug for which Valproate
therapeutic drug monitoring may be beneficial. This is used to treat myoclonic seizures, generalized
absence seizures and partial and generalized tonic–
Anticonvulsants clonic seizures. It is also used in the treatment of bipolar
Once the dose that gives stable plasma concentrations affective disorders.
within the therapeutic range has been determined, Plasma concentrations of sodium valproate correlate
monitoring is probably necessary only in the following poorly with the dose prescribed, the clinical response
cases: and toxicity, probably because of the presence of active
metabolites. Consequently, the main justification
● to assess compliance,
for measuring plasma concentrations is to assess
● if the frequency of fits increases in a previously well-
compliance. However, valproate is hepatotoxic and
controlled patient,
it is therefore important to check liver function tests.
● if the clinical picture suggests toxicity,
Phenobarbital, primidone and ethosuximide are less
● if another drug is prescribed that may affect the
used today in the treatment of epilepsy, although
plasma concentrations.
therapeutic drug monitoring may be useful.
Plasma concentrations in children and in pregnant There are several new antiepileptic drugs now
women should be monitored more frequently, as these available, for which the need to monitor plasma
groups are more likely to develop toxicity. concentrations is under evaluation. Vigabatrin, an
inhibitor of g-aminobutyric acid metabolism, has
Phenytoin a long action because it binds avidly to receptors;
Phenytoin is used in the treatment of partial and therefore, plasma concentrations are unlikely to
generalized tonic–clonic seizures by inhibiting voltage- predict therapeutic response or toxicity. Other newer
gated sodium channels. Measurement of plasma antiepileptic drugs include lamotrigine, which
phenytoin may be useful, because it exhibits saturation inhibits excitatory neurotransmitter release possibly
kinetics. Thus a small increase in dose of phenytoin by action upon voltage-sensitive sodium channels,
352 Therapeutic drug monitoring and poisoning
and gabapentin, but it remains to be seen how useful reduced salt intake or diuretic usage, can reduce
therapeutic drug monitoring will be in patient lithium clearance. It is important, therefore, to monitor
management. Lamotrigine also has found use in the thyroid and renal function tests in such patients.
treatment of bipolar disorders as well as partial and Plasma lithium levels above 2.5 mmol/L are associated
tonic–clonic seizures. with significant mortality and may necessitate dialysis.
Rarely hypercalcaemia may result possibly mediated by
Lithium
hyperparathyroidism.
The margin between therapeutic and toxic
concentrations of lithium at high dosage is narrow, and Theophylline
therefore plasma concentrations should be monitored Theophylline assays may be useful, especially in acute
regularly. Lithium may be given to psychiatric patients asthmatic attacks that are not responding clinically; the
for bipolar affective disorders. It is not protein results may help to ensure that the poor response is not
bound, and interpretation of the results of assays is due to underdosage and, if it is not, that increasing the
not complicated by protein abnormalities. Plasma dosage will not lead to toxic plasma levels.
concentrations should be measured on a specimen Theophylline and its active metabolite caffeine are
taken 12 h after the evening dose. used in the management of recurrent apnoea in the
Lithium usage is associated with hypothyroidism newborn infant; at this age, their clearance is slow and
(see Chapter 11), and plasma concentrations above theophylline is partially metabolized to caffeine. Both
about 1.4 mmol/L can be nephrotoxic. Oliguria and plasma theophylline and caffeine concentrations may
acute kidney injury, which can be precipitated by need to be assayed if theophylline has been given, in
order to assess therapeutic or toxic effects.
CASE 2 Samples for theophylline assays should be taken at
between 2 and 4 h after the last dose. The half-life of
A 40-year-old woman was admitted to casualty theophylline is shortened in smokers.
because of confusion. She was known to be on
lithium therapy for a bipolar disorder. The following Antibiotics
blood results were obtained: Aminoglycosides, such as gentamicin, are given
Plasma intramuscularly or intravenously and, in contrast
Sodium 150 mmol/L (135–145) to many other antibiotics, the margin between the
Potassium 5.0 mmol/L (3.5–5.0) therapeutic range and toxic levels is narrow. Toxic levels
Urea 7.8 mmol/L (2.5–7.5) may cause ototoxicity and nephrotoxicity. Measurement
Creatinine 134 µmol/L (70–110) is particularly indicated if there is serious infection or
Estimated glomerular filtration rate 40 mL/min per the treatment is so prolonged that the risk of toxicity is
1.73 m2 increased or renal function is impaired.
Thyroid-stimulating hormone 18.7 mU/L (0.20–5.0) Aminoglycoside concentrations are often measured
Free thyroxine 6.8 pmol/L (12–25) twice. The first specimen should be taken immediately
Lithium 2.8 mmol/L (therapeutic range 0.5–1.0) before injection, when the ‘trough level’ is expected, to
ensure that concentrations are not already high and that
DISCUSSION
further administration is not likely to cause toxicity,
The elevated plasma lithium concentration indicates
or that they are not so low as to be ineffective. The
a considerable risk of toxicity. The level should
second should be taken about an hour after injection,
be checked to ensure correct sample timing, as
at the time of the anticipated ‘peak level’, to ensure
incorrect timing, i.e. not trough levels, is a common
that an adequate concentration has been achieved
cause of elevated drug levels.The hypernatraemia
for antibacterial action. Two glycopeptide antibiotics,
can be explained by the polyuria and nephrogenic
namely vancomycin and teicoplanin, are used to
diabetes insipidus secondary to lithium toxicity
treat aerobic and anaerobic gram-positive bacteria.
upon the kidney. Primary hypothyroidism can
Like gentamicin, they may cause nephrotoxicity
also be secondary to lithium toxicity. Note also
and ototoxicty, a risk increased by concomitant
the abnormal renal function, which is essential to
aminoglycoside usage. Therapeutic drug monitoring
monitor in patients on lithium.
also has a role in the use of these drugs.
Biochemical monitoring of possible side effects of drug treatment 353
Methotrexate Tacrolimus
Methotrexate is used as a cytotoxic agent and, to achieve This is a macrolide lactone and, like ciclosporin,
this, plasma concentrations should exceed 1 µmol/L inhibits calcineurin phosphatase, thereby inhibiting
for at least 36 h. It is a folate antagonist, which inhibits T-lymphocyte activation.
deoxyribonucleic acid (DNA) synthesis at lower Although useful as an immunosuppressant, it is
concentrations. It is also used as an immunosuppressant nephrotoxic and cardiomyopathy may occur. It has an
in psoriasis and rheumatoid arthritis. advantage over ciclosporin in having a greater potency,
When used as chemotherapy, plasma levels above and impaired biliary function is less likely to alter its
5–10 µmol/L at 24 h, 0.5–1.0 µmol/L at 48 h and absorption. Thereapeutic drug monitoring seems to
0.1 µmol/L at 72 h post dose are associated with an be of value, and levels over 25 µg/L are associated with
increased risk of marrow suppression. Therapeutic drug increased toxicity.
monitoring is less useful when lower immunosuppressant Atypical antipsychotic drugs
doses are used unless toxicity is suspected.
These drugs, such as clozapine, may benefit from
Methotrexate excretion is predominantly renal and
therapeutic drug monitoring to monitor compliance
is facilitated by a good urine flow and an alkaline pH
and help detect toxicity. There is a risk of agranulocytosis
greater than 6.5. After high-dose methotrexate, ‘rescue
with some agents and full blood counts should be
therapy’ with folinic acid is given to reduce toxicity by
monitored. Some of these agents may cause weight gain
providing cells with folate co-factors for DNA synthesis.
and risk of diabetes mellitus, as well as cardiac toxicity.
For liver monitoring and methotrexate use, see Chapter
17. PHARMACOGENETICS
Immunosuppressants Various enzymes are responsible for the metabolism
of drugs. For example, the inactivation of isoniazid by
Ciclosporin
acetylation depends on whether the patient is genetically
This is an immunosuppressant that inhibits capable of carrying out this process at a normal rate;
T-lymphocyte activation by binding to calcineurin toxicity is likely at lower plasma concentrations in ‘slow
phosphatase and is used to prevent the rejection of acetylators’ than in ‘fast acetylators’.
transplanted organs, for example kidney. Ciclosporin is Thiopurine methyltransferase (TPMT) is involved
a cyclic peptide derived from the fungus Tolypocladium in the metabolism of azathioprine, which is used as a
inflatum (Gams). It is also used (usually at lower cytotoxic and also as an immunosuppressant agent. The
dose) in the treatment of inflammatory bowel disease, enzyme TPMT shows pharmocogenetic variation and
rheumatoid arthritis and psoriasis. about 90 per cent of the UK population have high enzyme
The dosage may be difficult to assess because there levels. However, about 10 per cent are heterozygotic for
is considerable variation among individuals in the rate TPMT deficiency and about 0.3 per cent are homozygotic
of absorption and clearance; there is also a relatively for no functional activity. The latter two groups are more
narrow margin between therapeutic and toxic drug susceptible to azathioprine toxicity. Determination of red
concentrations. The main toxic action is on the kidney, cell TPMT activity prior to azathioprine administration
although hepatic dysfunction can also occur. During the may give a guide as to in whom the drug should be
first 6 months, when the risks of rejection are highest, avoided or given at a lower dose.
plasma concentrations should be measured regularly. Similarly, debrisoquine hydroxylase activity can
Peak ciclosporin concentrations occur 1–4 h after oral be used as a guide to cytochrome P450 phenotype.
dosage and require adequate bile flow. Over half of the The cytochrome family is involved in the metabolism
drug binds to red blood cells and then redistributes in of various drugs. Debrisoquine hydroxylase activity
plasma, where a majority is found bound to lipoproteins, reflects cytochrome (CY) 2D6 status, which is involved
as ciclosporin is strongly lipophilic. Ciclosporin levels in the metabolism of a number of drugs, including
should be determined on trough ethylenediamine certain antiarrhythmics and antidepressants.
tetra-acetic acid (EDTA) anticoagulated samples, that
is, 12 h after the previous dose on a twice-daily regimen. BIOCHEMICAL MONITORING OF POSSIBLE
Daily monitoring is usual after transplantation; in the SIDE EFFECTS OF DRUG TREATMENT
first few months, levels of 100–400 µg/L are usual, Some drugs have harmful side effects. For example,
decreasing to about 100–200 µg/L afterwards. the plasma urea, creatinine, sodium and potassium
354 Therapeutic drug monitoring and poisoning
concentrations should be checked for patients on diuretic Drugs or poisons for which there is a
therapy or taking angiotensin-converting enzyme (ACE) specific antidote
inhibitors. The assessment of liver damage (for example Paracetamol (acetaminophen)
plasma transaminase activities) or of renal function It is essential to measure plasma drug concentrations
may be indicated during treatment with potentially in suspected paracetamol poisoning, for the following
hepatotoxic (for example valproate) or nephrotoxic (for reasons:
example ciclosporin) drugs, respectively. The statins may
sometimes cause muscle damage or rhabdomyolysis, ● A metabolite of paracetamol is hepatotoxic:
which can be detected by measuring the plasma its metabolism is saturable and is detoxified
concentration of creatine kinase (CK). by conjugation with glutathione. Depletion of
glutathione is implicated in the liver damage. The
DIAGNOSIS OF DRUG OR SUBSTANCE patient may die of liver failure despite recovery from
OVERDOSE OF UNKNOWN CAUSE the immediate effects.
Drug overdosage must always be excluded as a cause ● A specific antidote to the hepatotoxic effect is available.
of coma of unknown origin. Unexplained clinical or ● The antidote is only useful if given within a defined
laboratory findings may suggest effects due to drug period of time and if defined plasma concentrations
or alcohol ingestion despite denial by the patient. are reached.
Hypokalaemia, for example, may be due to purgative ● The likelihood of hepatotoxicity cannot be predicted
abuse, and hypoglycaemia may be a presenting finding from the clinical picture at presentation (Fig. 25.1).
associated with alcohol ingestion. Raised plasma
transaminase activities, may be due to alcoholic liver CASE 3
disease,. A raised plasma CK concentration may be
indicative of ‘Ecstasy’ or cocaine abuse. Measurement A 23-year-old woman took a paracetamol overdose
of blood gases is useful to determine whether there is an after an argument with her boyfriend. She had a
acid–base disturbance, for example a metabolic acidosis history of anorexia nervosa. The following results
may be seen in paracetamol overdose. As discussed were obtained 2 days after treatment with intravenous
above, some drugs, such as digoxin and lithium, may N-acetylcysteine.
cause renal impairment. Plasma
About half the patients attempting suicide take Bilirubin 33 µmol/L (< 20)
several drugs, sometimes with alcohol. Qualitative Alanine aminotransferase (ALT) 881 U/L (< 42)
screening of plasma, urine or gastric aspirate may be Alkaline phosphatase 127 U/L (< 250)
needed to help identify the drugs. The measurement Albumin 42 g/L (35–45)
of plasma drug concentrations (and also sometimes g-Glutamyl transferase 266 U/L (<55)
of other biological fluids such as vomit or gastric International normalized ratio (INR) 3.1 (2–3)
contents) may be supplemented by screening the
DISCUSSION
urine for drugs or their metabolites. Screening is rarely
advised and it is best to discuss cases with a toxicologist The results show severely abnormal liver function
to decide whether this is necessary. The careful labelling tests secondary to paracetamol overdose. The raised
and identification of samples that may have associated ALT activity suggests hepatocyte damage evoked by
medicolegal issues is essential. the paracetamol. Paracetamol overdose can induce
It is often unnecessary to measure the plasma a number of biochemical disturbances, including a
concentrations of drugs that the patient is known to metabolic acidosis, raised plasma anion and osmolar
have taken in overdose. The need for gastric lavage gap. The raised INR is concerning and suggests severe
and measures to increase the urinary excretion of paracetamol toxicity and the risk of life-threatening
the drug and to maintain adequate respiration and hepatic damage, in some cases necessitating liver
circulation are not usually affected by knowledge of transplant. It should be remembered that those
drug concentrations. The treatment of many drug who are undernourished or have alcoholism or are
overdoses is often supportive, with close monitoring on certain medications, such as enzyme-inducing
of the patient and clinical intervention when necessary. drugs, may be more at risk of paracetamol toxicity;
However, in some cases specific antidotes are indicated. see Fig. 25.1
Diagnosis of drug or substance overdose of unknown cause 355
200
1.3
190
180 1.2
170
1.1
Figure 25.1 Patients whose plasma paracetamol concentrations are above the normal treatment line should
be treated with acetylcysteine by intravenous infusion (or, if acetylcysteine cannot be used, with methionine
by mouth provided the overdose has been taken within 10–12 h and the patient is not vomiting). Patients on
enzyme-inducing drugs (e.g. carbamazepine, phenobarbital, phenytoin, primidone, rifampicin, alcohol and
St John’s wort) or who are undernourished (e.g. in anorexia nervosa, in alcoholism, or those who are human
immunodeficiency virus-positive) should be treated if their plasma paracetamol concentrations are above the
high-risk treatment line. The prognostic accuracy after 15 h is uncertain, but a plasma paracetamol concentration
above the relevant treatment line should be regarded as carrying a serious risk of liver damage. Reproduced
courtesy of University of Wales College of Medicine Therapeutics and Toxicology Centre.
N-acetylcysteine and methionine are both relatively ingestion. Concentrations should be measured on
effective antidotes, because they enable paracetamol to specimens taken as early as possible, ideally between 4
be converted to non-toxic metabolites. Methionine is and 20 h after ingestion (see Fig. 25.1).
given orally but may cause vomiting. N-acetylcysteine Patients taking enzyme-inducing agents such as
is most often used and is given intravenously. It is only phenytoin, rifampicin, carbamazepine or alcohol or
effective in clearly defined circumstances and should those who are undernourished, for example because of
only be given after the following factors have been anorexia or infection with human immunodeficiency
taken into account. virus (HIV), are more at risk of paracetamol toxicity.
Plasma concentrations of paracetamol cannot Monitor plasma transaminase activities (which may
be interpreted and should not be measured until rise to 10 000 U/L or more), renal function (as renal
absorption and distribution are nearly complete, at failure can develop) and prothrombin time or INR.
about 4 h after ingestion. If treatment is to be effective, Prothrombin time or INR is probably the best marker
it should be started as soon as possible following of the severity of liver failure.
356 Therapeutic drug monitoring and poisoning
of alkalosis often further increases the excretion rate of uncoupling of oxidative phosphorylation). Children
acidic drugs), but neither forced diuresis nor induction seem less likely to develop the respiratory alkalosis.
of alkalosis is without risk. If an alkaline diuresis is Hypokalaemia and either hyperglycaemia or
induced, plasma potassium concentrations, as well as hypoglycaemia may also occur.
blood pH, must be monitored because of the danger of Where there is no specific drug antidote, treatment is
acute hypokalaemia. supportive, although biochemical tests for electrolytes,
In adults, a respiratory alkalosis occurs initially, acid–base balance, renal and hepatic function and
followed by a metabolic acidosis (possibly due to blood gases are often useful.
SUMMARY
● The biochemical laboratory plays an important role changes in drug levels if used in combination
in the monitoring of the concentrations of some with other drugs to help avoid drug interaction.
drugs, usually those for which there is a narrow Therapeutic drug monitoring can be used to ‘tailor’
therapeutic window. The common drugs for which drug dosage to patient needs.
this may be useful are the antiepileptic drugs and ● Drug or substance overdoses are a common cause
some of the cardiac antiarrhythmic agents, as well as of hospital admissions, and measuring drug
certain antibiotics, lithium and chemotherapy drugs. concentrations and biochemical monitoring, for
● Therapeutic drug monitoring may be useful for example of renal, liver and acid–base status, can be
detecting patient drug compliance and for observing useful.
26 Clinical biochemistry at the extremes
of age
This chapter looks at clinical biochemistry at the is not fully developed until the age of about 2 years.
extremes of age, that is, in neonates and the elderly. Glomerular function develops more rapidly than that
of the tubules. Fetal urine is produced from about the
NEONATES ninth week.
Some preliminary definitions are useful. A premature The glomerular filtration rate (GFR) doubles during
or preterm baby is one born before 37 completed weeks’ the first 2 weeks of life because of an increase in renal
gestation. A neonate is a live baby within 1 month of blood flow. The GFR (which is corrected for surface
birth. A low-birthweight baby is one less than 2.5 kg area) reaches adult values by about 6 months of age.
and a very low-birthweight baby is one less than 1.5 kg. The plasma urea concentration is low in newborn
One of the most significant advances in recent medical infants compared with that in adults, despite the
care has been the survival rate of very small preterm relatively low GFR; the high anabolic rate results in more
infants, which has increased because of improved nitrogen being incorporated into protein rather than
specialized medical and nursing techniques for treating into urea than in adults. Plasma urea concentrations
the neonate. However, these infants can experience a fluctuate markedly with varying nutritional states,
number of abnormal biochemical conditions, which metabolic rates and states of hydration.
are briefly summarized in this chapter. The plasma creatinine concentration (which is
Diseases occurring during the neonatal period can inversely related to GFR) at birth is similar to the
be divided into two main groups: those of infants born mother’s. It decreases rapidly at first, averaging about
before term, in whom immaturity contributes to the 35 µmol/L, the fall being slower in preterm infants, and
severity of the disease, and those of full-term infants. then rises before reaching adult values (corrected for
The most common disorders in both groups are the body surface area) by about 6 months.
perinatal asphyxia, the respiratory distress syndrome Renal function in newborn infants can maintain
(RDS), infection and inborn errors. basic homeostasis but may not be able to respond
Compared with about 5 L in a 70-kg adult, the adequately to illness or other stresses. It is often
blood volume of a premature infant weighing 1 kg is difficult to determine if there is renal impairment
only about 90 mL. Therefore only a small amount of because of the unsatisfactory nature of renal function
blood can be taken without causing volume depletion tests at this age. A plasma urea concentration above
or anaemia. Sometimes capillary samples are used as about 8 mmol/L suggests retention due to glomerular
an alternative to venous (or arterial) samples, although impairment, whether parenchymal or pre-renal in
the former may be more prone to contamination from origin, especially if the urinary output can be shown
the interstitial and cellular fluids. Prior to requesting to be low. Management depends to a large extent on
tests on small samples, the clinician should contact the clinical criteria. In the neonate, neither the plasma
laboratory to discuss the best strategy for obtaining urea nor the creatinine concentration is a very sensitive
test samples and prioritizing of investigations. The indicator of renal function.
interpretation of test results may be influenced by the Renal dysfunction in neonates can be due to factors
different reference ranges at different ages, which can such as hypotension, birth asphyxia, renal toxic drugs,
be difficult to define. renal agenesis, septicaemia or dehydration or can occur
post surgery.
Renal function The daily excretion of urine is about 15–60 mL in
The kidneys usually have a full complement of nephrons full-term neonates, increasing to about 500–600 mL/
by about the 36th week of gestation, but renal function day at about 1 year (see Chapter 3).
Neonates 359
considered as a possible cause of hyponatraemia at this for example pyloric stenosis. In this age group also
age. consider renal tubular acidosis (type I or II). Iatrogenic
Clinical signs of hyponatraemia may be associated causes include diuretic use.
with hypotension, drowsiness and convulsions.
Hyperkalaemia
Hypernatraemia Iatrogenic causes include excess potassium treatment.
Hypernatraemia develops if water loss exceeds that of Other common causes include acute kidney injury,
sodium, or if an excess of sodium relative to water is exchange transfusion and tissue damage resulting
infused or fed. from hypoxia or birth trauma, for example severe
If replacement is inadequate, the high ‘insensible’ bruising or haematoma. Consider also congenital
water loss, aggravated by impaired urinary water adrenal hyperplasia or adrenal insufficiency (see
retention, may cause rapid ECF depletion with Chapter 5).
hypotension and, if water depletion is predominant,
hypernatraemia. This is particularly likely if ‘insensible’ Pulmonary function
loss is increased through the: Perinatal asphyxia
● skin – for example due to sweating caused by pyrexia Renal complications and disturbances of electrolyte
or overhead heaters or to phototherapy for jaundice, balance are more likely to develop in infants with
● lungs – if the infant is hyperventilating, for example perinatal asphyxia. Cerebral oedema or haemorrhage
because of pneumonia, may stimulate ADH secretion, causing oliguria and
● intestine – if there is vomiting or diarrhoea. a dilutional hyponatraemia with hypo-osmolality,
accompanied by a high urinary sodium concentration
Iatrogenic causes in infants include giving sodium due to plasma volume expansion.
bicarbonate or excess salt. Neonates are more The hypotension occurring during asphyxia may
susceptible than adults to developing hypernatraemia reduce renal blood flow enough to cause acute oliguric
because the addition of a given amount of extra sodium renal failure (acute kidney injury). In addition to the
increases the very low extracellular sodium content oliguria and hyponatraemia, there may be uraemia and
proportionally more than if it were diluted in a larger hyperkalaemia, with proteinuria. Hypoglycaemia and
pool, as it is in adults; this tendency is aggravated hypocalcaemia may also occur.
by impaired renal excretion. Nephrogenic diabetes Fetal pH can be measured during delivery, usually
insipidus is a rare cause of neonatal hypernatraemia. from a capillary sample from the baby’s scalp. This
may be indicated if changes in cardiotocograph or fetal
Potassium heart rate indicate fetal distress. A pH value below 7.2
The total body potassium of a newborn infant of 1 kg is indicates the need for urgent delivery.
less than 100 mmol, compared with about 3000 mmol
in an adult. A normal full-term infant requires about Hypoxaemia
2–4 mmol/kg of potassium a day (compared with about Fetal lung fluid production ceases at birth, probably as
1 mmol/kg in an adult) to replace losses. a result of increases in adrenaline concentration during
As in the adult, artefactual causes of abnormal delivery. Labour squeezes liquid from the mouth, and
plasma potassium concentrations must be excluded. absorption takes place into the pulmonary lymphatics
Pseudohyperkalaemia is especially likely if capillary and capillaries. Type 2 pneumocytes produce surfactant,
samples are used, because tissue cells may be damaged which serves to reduce lung surface tension, thus
if the skin is squeezed as in a heel prick. It may also be facilitating lung expansion. Deficiency of surfactant as
due to in vitro haemolysis, or to withdrawal of blood in prematurity can cause RDS.
from a cannula through which a potassium solution is Normal term neonatal arterial PO2 breathing air is
being infused. Conversely, pseudohypokalaemia may about 8–11 kPa. Oxygen therapy can cause toxicity,
result if the infused fluid is potassium free. including bronchopulmonary dysplasia and stiff lungs,
and should be closely monitored, for example by
Hypokalaemia transcutaneous gas analysis. Retinopathy of prematurity
Hypokalaemia may be caused by increased (previously known as retrolental fibroplasia) may lead to
gastrointestinal loss due to diarrhoea or an alkalosis, blindness.
Neonates 361
such as plasma a1-antitrypsin deficiency, plasma pregnancy. Very premature infants therefore have little
and urinary amino acids. Exclude galactosaemia (see liver glycogen and adipose tissue and are especially
Chapter 27). Liver ultrasound may also be useful. prone to hypoglycaemia. Full-term infants may
● It is important that biliary atresia is excluded, for become hypoglycaemic if initially adequate stores
example with imaging techniques. are drawn on more rapidly than normal, for example
● In the presence of conjugated hyperbilirubinaemia during perinatal asphyxia. Infants at greater risk of
and normal liver function tests, Dubin–Johnson or hypoglycaemia include those with infections, asphyxia,
Rotor’s syndrome should be considered. rhesus haemolytic disease and exchange transfusion,
those who are small for dates and those born to
Glucose metabolism and hypoglycaemia
diabetic mothers.
The newborn infant Plasma glucose concentrations as low as 1.7 mmol/L
Causes of hypoglycaemia in the newborn period during the first 72 h of life in the preterm infant, or
(and, for completeness, also in childhood) are 2.0 mmol/L during the later neonatal period, may not
shown in Box 26.1. Hepatic glycogen stores increase be associated with any clinical signs. When clinical
about three-fold and adipose tissue (another source features do occur they include tremors, apnoeic attacks
of energy) is laid down during the last 10 weeks of and convulsions. However, impaired neurological
development has been reported in full-term infants
Box 26.1 Some causes of neonatal and in whom the plasma glucose concentration repeatedly
childhood hypoglycaemia fell to below 2.0 mmol/L despite the absence of clinical
signs. It has been recommended that the plasma glucose
Reduced production of glucose concentration be maintained above 2.0 mmol/L in at-
‘Small for dates’ baby risk infants.
Prematurity If a baby has hypoglycaemia and is asymptomatic,
Birth asphyxia milk feeds are usually given until blood glucose levels
Sepsis are above 2.0 mmol/L. However, in the presence of
Poor nutrition symptoms of hypoglycaemia, intravenous 10 per cent
Hypothermia dextrose (glucose) may need to be given. Nesidioblastosis
Congenital heart disease is due to overgrowth of insulin-producing b-cells in the
Inborn errors of metabolism pancreas and presents with severe hypoglycaemia and
Glycogen storage disease, e.g. type 1 hyperinsulinaemia. Beckwith–Wiedemann syndrome
Organic acidurias is due to a short-arm deletion of chromosome 11
Disorders of fat oxidation, e.g. medium-chain acyl
and is associated with hypoglycaemia, visceromegaly,
coenzyme A dehydrogenase deficiency
Disorders of gluconeogenesis, e.g. pyruvate exomphalos and intellectual disabilities.
carboxylase deficiency Hypoglycaemia determined by near-patient glucose
Galactosaemia and hereditary fructose intolerance tests must be confirmed by proper laboratory testing in
Carnitine deficiency a fluoride sample. Lack of ketonuria in the presence of
Amino acid disorders, e.g. tyrosinaemia hypoglycaemia implies hyperinsulinaemia or a defect
Ketotic hypoglycaemia of infancy of fat oxidation.
Leucine sensitivity The babies of diabetic mothers (including gestational
Hyperinsulinaemia diabetes) tend to be large, jaundiced, hypocalcaemic,
Maternal diabetes hypomagnesaemic, polycythaemic and to have lung
Nesidioblastosis immaturity and congenital defects. These pregnancies are
Beckwith–Wiedemann syndrome associated with greater need for instrumental deliveries
Insulinoma
and caesarean section. Neonatal hypoglycaemia is related
Erythroblastosis fetalis
to the degree of maternal glycaemic control.
Hormone deficiencies
Hypothyroidism Early infancy
Hypopituitarism
Adrenal insufficiency Soon after birth, or the introduction of milk to the
Congenital adrenal hyperplasia diet, hypoglycaemia may be due to one of the following
causes.
Neonates 365
Rickets of prematurity usually becomes clinically metabolite. After treatment with phosphorus, the
evident between the 4th and 12th weeks of life, and plasma calcium concentration may fall rapidly enough
occurs more commonly in very premature, low- to cause clinical symptoms.
birthweight infants than in infants of older gestational Idiopathic infantile hypercalcaemia may occur in
age at birth. Longitudinal growth slows, and the full-term infants receiving inappropriately high-dose
decalcification of the bones predisposes to pathological vitamin D prophylaxis who have abnormalities of the
fractures; in severe cases, respiration is impaired by the CYP24A1 gene, which encodes for a major enzyme
soft ribs. (25-hydroxyvitamin D 24-hydroxylase) that metabolizes
Radiograph changes may be minimal or absent vitamin D. Other conditions include vitamin D excess,
in early cases. In more advanced disease, the classic familial hypocalciuric hypercalcaemia and Williams’
radiological changes appear at the ends of long bones. syndrome. The last mentioned is associated with
Rickets of prematurity may be due to the following: intellectual disabilities, ‘elfin’ facies and cardiac defects.
● Calcium and phosphate depletion: if an infant is born Magnesium
prematurely, unsupplemented breast milk may not
contain enough of these minerals to replace that Hypomagnesaemia often accompanies hypocalcaemia
which should have accumulated in utero during the and may be caused by dietary deficiency or by increased
last 10 weeks of gestation. intestinal or urinary loss. Low plasma magnesium
Phosphate depletion is probably the most concentrations may impair the release and action of PTH
common cause and is indicated by a very low and so delay correction of plasma calcium concentrations.
plasma phosphate concentration, compared with Hypomagnesaemia should be considered if the infant
the appropriate reference range, and low urinary has convulsions despite normocalcaemia.
phosphate excretion. Plasma proteins
● Maternal vitamin D deficiency during pregnancy or in
At birth, the relative concentrations of individual
the infant after birth: in very premature infants, such
plasma proteins differ from those found in adults. The
deficiency may be due to low activity of the renal
concentration of total protein is about 12 g/L lower
1a-hydroxylase needed to convert 25-hydroxyvitamin
than in adults, but that of albumin is only slightly
D to the active 1,25-dihydroxyvitamin D.
lower; as always, the latter may fall during illness. The
● Drugs, such as furosemide, which increase urinary
acute-phase proteins (reflected in the a-globulins and
calcium loss.
b-globulins in the electrophoretic pattern) reach adult
● Renal tubular disorders of phosphate reabsorption,
concentrations by about 6 months, but are affected by
which may cause phosphate depletion: in such cases
illness, again as they are in adults.
the plasma calcium concentration is usually normal,
The immunoglobulin pattern differs significantly
but may even be high because the calcium cannot be
from that of adults. During normal pregnancy, placental
deposited in bone without phosphate.
transfer of maternal immunoglobulin G (IgG) leads to
Treatment a gradual increase in fetal plasma concentrations of this
Vitamin D and calcium and phosphate supplementation immunoglobulin. After birth, maternally derived IgG is
may be monitored by measuring serial plasma alkaline degraded and endogenous immunoglobulin synthesis
phosphatase activities. Despite successful treatment, starts. Adult concentrations of plasma IgM are reached
these may continue to rise for several weeks, when bone by about 9 months, of IgG by between 3 and 5 years,
is being actively laid down, before falling once the bone and of IgA by the age of 15.
is adequately calcified. Measurement of plasma immunoglobulin
concentrations may be indicated if an immune-
Hypercalcaemia in the newborn period deficiency state, whether primary, secondary or transient,
Hypercalcaemia is less common than hypocalcaemia at is suspected. It may also help to detect infection,
this age. It may be associated with phosphate depletion whether it occurred during the intrauterine period or
and hypophosphataemia because calcium cannot be has developed after birth. Results must be compared
deposited in bone without phosphate, and because with age-matched reference ranges, allowance being
hypophosphataemia enhances 1a-hydroxylase activity made for both the time since conception (gestational
and therefore the formation of the active vitamin D age) and age since birth (post-natal age).
368 Clinical biochemistry at the extremes of age
jaundice. (See also Chapter 11 for discussion of thyroid addition, they may well be on numerous medications,
disease.) Neonatal screening is discussed further in and therefore drug interactions are more likely. Ageing
Chapter 27. may be associated with changes in the reference
range of certain analytes; for example plasma alkaline
ELDERLY
phosphatase may increase, probably of bone origin.
Within the next 50 years or so, about one-quarter of Some diseases are more common in the elderly,
the world’s population will be over the age of 65 years. including cardiovascular disease, type 2 diabetes
Just as in the neonate, it is important to understand the mellitus, Paget’s disease, osteoporosis, renal
biochemical changes that occur with age when it comes impairment, thyroid disease and certain tumours.
to interpreting chemical pathology results in the elderly. Multiple myeloma is far more prevalent as age increases
First, it is pertinent to realize that multiple as is other malignant disease.
pathologies are sometimes present in the elderly. In The increased morbidity and mortality of the elderly
are not helped by the fact that nutritional disorders are
also more common. Some elderly people, particularly
CASE 3 if on low incomes, may have poor diets, and deficiency
states have been described, including osteomalacia and
An 83-year-old woman was seen by her general scurvy.
practitioner because of increasing confusion. She was
known to have congestive cardiac failure, hypertension, Cardiovascular disease
diabetes mellitus, osteoporosis and diverticular Ischaemic heart disease and cerebrovascular disease
disease. She was taking 10 different medications. Some constitute the major causes of death in the elderly,
of her biochemical results were as follows: and their risk increases with age. Plasma cholesterol
Plasma concentration also increases with age, although there
Sodium 135 mmol/L (135–145) may be a decline in those over the age of 75 years.
Potassium 5.1 mmol/L (3.5–5.0) Renal function
Urea 9.7 mmol/L (2.5–7.0)
Renal function may deteriorate with age over the age
Creatinine 136 µmol/L (70–110)
of 70 years, Remember that plasma creatinine may
Estimated glomerular filtration rate (eGFR)
be within the adult reference range but estimated
34 mL/min per 1.73 m2
glomerular filtration rate (eGFR) abnormally low.
Albumin 36 g/L (35–45)
This partly relates to the decrease in muscle ‘bulk’ that
Albumin-adjusted calcium 1.90 mmol/L (2.15–2.55)
sometimes occurs with ageing, and a corresponding
Alkaline phosphatase 398 U/L (<250)
decrease in plasma creatinine. This is important to
Phosphate 0.68 mmol/L (0.80–1.35)
remember when it comes to prescribing medications
Glucose 12.6 mmol/L (3.5–5.5)
that are dependent on renal excretion (see Chapter 3).
Glycated haemoglobin (HbA1c) 8.0% (64 mmol/mol)
Thyroid-stimulating hormone 11.9 mU/L (0.2–5.0) Diabetes mellitus
Free thyroxine 8.5 pmol/L (12–25) This is more common in the elderly, in whom it is
DISCUSSION usually type 2. Generally, glucose intolerance increases
The patient was subsequently shown to have had with age (see Chapter 12).
a cerebrovascular accident. Her biochemical tests Thyroid disease
suggest hypothyroidism, hypocalcaemia (found to
Abnormalities of thyroid function also increase with
be due to osteomalacia) and impaired renal function.
age; both hyperthyroidism and hypothyroidism are
The elderly may present with multiple pathologies.
more common in the elderly. The former is more likely
Renal function declines with age. Type 2 diabetes
to be associated with atrial fibrillation and the latter
mellitus is also relatively common in the elderly.
with hypothermia. Because of the increased likelihood
Another clinical problem is ‘poly-pharmacy’, with
of disease in the elderly, sick euthyroidism is also
some elderly patients being treated with multiple
more common (see Chapter 11). The pituitary gland
drugs for multiple conditions and therefore running
diminishes in size in the elderly. Microadenomas are
the risk of dangerous drug interactions.
more likely, as is growth hormone deficiency.
370 Clinical biochemistry at the extremes of age
SUMMARY
● Neonates show a number of biochemical features ● The elderly may present with multiple pathologies.
that are different from those found in adults. Renal function declines with age. Type 2 diabetes
● Unconjugated hyperbilirubinaemia can be mellitus is also relatively common in the elderly.
physiological in neonates, but severely raised plasma Another clinical problem is ‘poly-pharmacy’, with
bilirubin concentration (more than 350 µmol/L many elderly people being treated with multiple
approximately) can result in kernicterus associated drugs for multiple conditions, which runs the risk
with neurological damage. of drug interaction.
● Premature neonates may present with hypocalcaemia, ● Reference ranges for certain analytes may be
jaundice, hypoglycaemia and renal, hepatic and acid– different in the very young and the elderly.
base disorders.
27 Inborn errors of metabolism
Some metabolic consequences of genetic defects 371 When to suspect an inborn error of metabolism 372
Clinical importance of inborn errors of metabolism 371 Principles of treatment of inborn errors of metabolism 373
Neonatal screening 372 Diseases due to inborn errors of metabolism 373
Prenatal screening 372
There is a group of diseases for which recognition in the appropriate place and method of delivery for the
early infancy is of great importance because treatment well-being of the infant or to offer termination, if the
may prevent irreversible clinical consequences or diagnosis is made early enough and if it is acceptable.
death. Some of the more important of these are PKU, Prenatal screening for inherited metabolic
galactosaemia and maple syrup urine disease. disorders most commonly involves demonstrating the
metabolic defect in cultured fetal fibroblasts obtained
NEONATAL SCREENING
by amniocentesis early in the second trimester, or by
Many countries have instituted programmes for chorionic villus sampling during the first trimester.
screening all newborn infants for certain inherited Examples of those groups in whom such screening
metabolic disorders or congenital defects. The criteria may be indicated include women with a previously
should depend on the following characteristics of the affected infant and ethnic groups thought to have a
disorder or of the test: relatively high incidence of the carrier state, such as
● The disease should not be clinically apparent at the of Tay–Sachs disease in Ashkenazi Jews. In these high-
time of screening and should have a relatively high risk populations, screening is often performed before
incidence in the population screened. conception, enabling genetic advice and prenatal
● The disease should be treatable or early treatment diagnosis to be offered to couples who are carriers.
should improve outcome. If, as in cystic fibrosis, the gene defect of the parent
● It must be possible to obtain the result of the of an affected infant is known, there may be a case for
screening test before irreversible damage is likely to selective screening of subsequent pregnancies using
have occurred. molecular biological techniques. Prenatal screening for
● The screening test should be simple and reliable congenital disorders, for example neural tube defects,
and the cost of the programme should, ideally, be or chromosomal abnormalities may also be performed.
at least partly offset by the cost savings resulting WHEN TO SUSPECT AN INBORN
from early treatment. For example, such treatment ERROR OF METABOLISM
may sometimes eliminate the need for prolonged
The possibility of an inherited metabolic defect should
institutional care.
be considered if there are unusual, unexplained clinical
Not all these criteria are necessarily fulfilled in all features (Box 27.1) or abnormal laboratory findings in
screening programmes.
In the UK, at between 5 and 8 days, babies are screened
for certain conditions by taking a small capillary blood Box 27.1 Some clinical findings suggestive
sample from a heel prick (see Chapter 26, Fig. 26.2). of an inborn error of metabolism
Blood spots are placed on a paper card, which can be
posted to the regional laboratory for assay. Early
In the UK, screening is generally carried out for Hypoglycaemia
neonatal hypothyroidism (see Chapters 11 and 26) Metabolic acidosis
and PKU. Other conditions that may be screened for Failure to thrive
Vomiting
in certain regions include cystic fibrosis, sickle cell
Fits or spasticity
disease or thalassaemia, medium-chain acyl coenzyme Hepatosplenomegaly
A dehydrogenase deficiency (MCADD), glucose-6- Prolonged jaundice
phosphatase deficiency, galactosaemia and congenital A peculiar smell, or staining, of the nappies
adrenal hyperplasia. (These conditions are discussed Death of child in family and positive family history
elsewhere in this book.) The use of deoxyribonucleic Cataracts or retinitis pigmentosa
acid (DNA) technology and tandem mass spectroscopy Late
in antenatal screening can be expected to increase in the Intellectual disabilities
future. Refractory rickets
Renal calculi
PRENATAL SCREENING Neuropathy
Short stature
Prenatal screening, of high-risk groups only, may Dysmorphic features
be performed for some disorders in order to plan
Diseases due to inborn errors of metabolism 373
TCA cycle
Figure 27.3 Diagram showing the metabolism of tyrosine and some inborn errors of the aromatic amino acid
pathway. Substances highlighted may be present in abnormal amounts in certain inborn errors of metabolism. (1)
Phenylalanine hydroxylase – phenylketonuria (PKU); (2) homogentisic acid oxidase – alkaptonuria; (3) tyrosinase
– albinism; (4) thyroid enzymes – thyroid dyshormonogenesis. TCA, tricarboxylic acid.
for the synthesis of the cofactor tetrahydrobiopterin are in utero to the high phenylalanine concentrations of
abnormal. undiagnosed or poorly controlled phenylketonuric
Therefore several different inherited deficiencies mothers may have intellectual disabilities, although
may have very similar biochemical and clinical they themselves do not have detectable PKU (maternal
consequences. Phenylalanine cannot be converted to phenylketonuric syndrome).
tyrosine, and accumulates in plasma and is excreted in The aim of treatment is to lower plasma phenylalanine
the urine with its metabolites, such as phenylpyruvic concentrations by giving a low-phenylalanine diet. Such
acid (a phenylketone). treatment should be monitored carefully, especially if
The clinical features include: the patient is planning to conceive or is pregnant. It is
now generally recommended that in proven cases dietary
● intellectual disabilities developing at between 4 and
restriction should be life long, with supervision by a
6 months, with psychomotor irritability,
metabolic physician and expert dietitian. Remember
● a tendency to reduced melanin formation because of
that the artificial sweetener aspartame is metabolized
reduced production of tyrosine – many patients are
to phenylalanine.
pale skinned, fair haired and blue eyed,
● irritability, feeding problems, vomiting and fits Tyrosinaemia
during the first few weeks of life,
Tyrosinaemia presents with renal tubular dysfunction,
● often generalized eczema.
hypoglycaemia and severe liver disease with very
Diagnosis may involve measuring the phenylalanine raised plasma alkaline phosphatase concentration.
concentration in blood taken from a heel prick. The defect is due to abnormal fumarylacetoacetase
The microbiological Guthrie test was used to assay leading to raised tyrosine, succinylacetone and
phenylalanine, but now many laboratories use hydroxyphenylpyruvate. Diagnosis is by showing
chromatography methods or tandem mass spectroscopy. raised urinary succinylacetone concentration and
In the newborn, and especially in preterm infants, the assay of fumarylacetoacetase in cultured leucocytes
enzyme system may not be fully developed and false- or fibroblasts. Treatment can be dietary, by liver
positive results are likely if the test is performed too transplantation or by nitro-trifluoromethylbenzoyl
early. If a positive result is found, the test should be cyclohexanedione, which is thought to reduce the
repeated later, to allow time for development of the accumulation of some of the toxic metabolites.
enzyme. The phenylalanine concentrations may be
greater than 240 µmol/L. Alkaptonuria
Heterozygotes may be clinically normal, but can be Alkaptonuria is an autosomal recessive disorder
detected by biochemical tests. A variant – persistent associated with a deficiency of homogentisic acid
hyperphenylalaninaemia – without intellectual oxidase. Homogentisic acid accumulates in tissues
disabilities has been described. Infants who are exposed and blood, and is passed in the urine. Oxidation
376 Inborn errors of metabolism
and polymerization of homogentisic acid produce involve sulphur-containing amino acids. Patients
the pigment alkapton, in much the same way as may show progressive central nervous system (CNS)
polymerization of dihydroxyphenylalanine results dysfunction, thrombotic disease, eye disease, including
in melanin. The deposition of alkapton in cartilages, cataracts, and cardiovascular problems. The diagnosis
with consequent darkening, is called ochronosis and of homocystinuria is based on the presence of raised
results in visible darkening of the cartilages of the urinary and plasma homocysteine with low plasma
ears and often arthritis in later life. The conversion methionine concentrations. The defective enzyme can
of homogentisic acid to alkapton is accelerated in be assayed in cultured skin fibroblasts.
alkaline conditions, and sometimes the most obvious
abnormality in alkaptonuria is darkening of the urine Maple syrup urine disease
as it becomes more alkaline on standing. Homogentisic In maple syrup urine disease, which is inherited as
acid, a reducing substance, reacts with Clinitest tablets an autosomal recessive condition, there is deficient
(Fig. 27.4). decarboxylation of the oxoacids resulting from
deamination of the three branched-chain amino
Albinism acids, leucine, isoleucine and valine. These amino
A deficiency of tyrosinase in melanocytes causes one acids accumulate in the plasma and are excreted in the
form of albinism; it is inherited as an autosomal recessive urine with their corresponding oxoacids. The sweet
disorder. Pigmentation of the skin, hair and iris is reduced smell of the urine is like that of maple syrup, hence the
and the eyes may appear pink. Reduced pigmentation condition’s name (Fig. 27.5).
of the iris causes photosensitivity, and decreased skin The disease presents during the first week of life
pigmentation is associated with an increased incidence and, if not treated, severe neurological lesions develop
of certain skin cancers. The tyrosinase involved in which cause death within a few weeks or months.
catecholamine synthesis is a different isoenzyme, If a diet low in branched-chain amino acids is given,
controlled by a different gene; consequently, adrenaline normal development is possible. The diagnosis of
(epinephrine) metabolism is normal.
Homocystinuria
Homocystinuria is an autosomal recessive disorder due
to deficiency of cystathionine synthase. These pathways
maple syrup urine disease is made by demonstrating Amino aciduria may also be subdivided according to
raised concentrations of branched-chain amino the pattern of excreted amino acids.
acids in plasma and urine and low plasma alanine
● Specific amino aciduria is due to increased excretion
concentration. It may be confirmed by demonstrating
of either a single amino acid or a group of chemically
the enzyme defect in leucocytes.
related amino acids. It may be overflow or renal in
type.
Histidinaemia
● Non-specific amino aciduria, in which there is
Histidinaemia is associated with deficiency of increased excretion of a number of unrelated amino
histidinase, an enzyme needed for normal histidine acids, is almost always due to an acquired disorder. It
metabolism, and is probably inherited as an autosomal may be overflow in type, as in severe hepatic disease
recessive trait. Some individuals may have intellectual when impaired deamination of amino acids causes
disabilities and speech defects, but others may be raised plasma concentrations; more commonly,
normal. renal amino aciduria results from non-specific
The diagnosis is made by demonstrating raised proximal tubular damage, and other substances that
plasma levels of histidine, and by finding histidine and are usually almost completely reabsorbed by the
the metabolite imidazole pyruvic acid in the urine. proximal tubule are also lost (phosphoglucoamino
aciduria; Fanconi’s syndrome). If it occurs due to
Inherited disorders of amino acid transport an inborn error of metabolism, it is rarely a direct
mechanisms result of the genetic defect, but more commonly
Groups of chemically similar substances are often secondary to tubular damage caused by deposition
transported by shared or inter-related pathways. Such of the substance not metabolized normally, such as
group-specific mechanisms usually affect transport copper in Wilson’s disease (Fig. 27.6).
across all cell membranes, and defects often involve
both the renal tubules and intestinal mucosa. Inborn Cystinuria
errors of the following amino acid group pathways have Cystinuria is the result of an autosomal recessive
been identified: inherited abnormality of tubular reabsorption, with
● the dibasic amino acids (with two amino groups) excessive urinary excretion, of the dibasic amino
cystine, ornithine, arginine and lysine (cystinuria) – acids cystine, ornithine, arginine and lysine. A similar
COAL is a useful mnemonic, transport defect has been demonstrated in the intestinal
● many neutral amino acids (with one amino and one mucosa, but, although dibasic amino acid absorption is
carboxyl group) (Hartnup’s disease), reduced, deficiencies do not occur because they can be
● the imino acids proline and hydroxyproline, which synthesized in the body. Cystine is relatively insoluble
probably share a pathway with glycine (familial and, because of the high urinary concentrations in
iminoglycinuria). homozygotes, may precipitate and form calculi in the
renal tract. In heterozygotes, increased excretion can
Amino aciduria be demonstrated, but concentrations are rarely high
enough to cause precipitation.
Amino acids are usually filtered by the glomeruli, reach
The diagnosis of cystinuria is made by demonstrating
the proximal tubules at concentrations equal to those
excessive urinary excretion of the characteristic amino
in plasma and are almost completely reabsorbed as they
acids. All these amino acids must be identified to
pass through this part of the nephron. Amino aciduria
distinguish this from cystinuria occurring as part of a
may therefore be of two types.
generalized amino aciduria.
● Overflow amino aciduria In which, because of The management of cystinuria aims to prevent calculi
raised plasma concentrations, amino acids reach the formation by reducing urinary concentration. The
proximal tubules at concentrations higher than the patient should drink plenty of fluid. Alkalinizing the
reabsorptive capacity of the cells. urine increases the solubility of cystine. If these measures
● Renal amino aciduria In which plasma prove inadequate, D-penicillamine may be given; this
concentrations are low because of urinary loss due forms a chelate, which is more soluble than cystine alone
to defective tubular reabsorption. (see Chapter 3 for a discussion of renal calculi).
378 Inborn errors of metabolism
Glutamine-
Asparagine
Glycine
Urine 2.0 mL. 150 cm. Column
Serine
Urea
Threonine
Taurine
B
Absorbance 1.0
0.6
0.4
Glutamic acid
Citrulline
0.2
A
Norleucine
standard
1.0
b-Amino-isobutyric
0.6
Tyrosine
Phenylalanine
Homocystine
Absorbance
0.4
Methionine
Cystine
C
Isoleucine
b-Alanine
Leucine
0.2
Valine
0
400 500 600 700
Figure 27.6 Chromatographic pattern of amino aciduria. Reproduced with kind permission from Nyhan WL and
Barshop BA. Atlas of Inherited Metabolic Diseases, 3rd edition. Hodder Arnold, 2012.
Galactose is a reducing substance. The urine may The glycogen storage disorders
give a positive reaction with Clinitest tablets; this Glycogen storage disease type I
feature may be absent if the subject is not receiving milk This is known as von Gierke’s disease and is a deficiency of
and therefore galactose. Tubular damage may cause a glucose-6-phosphatase (see Fig. 27.8 and Chapters 12 and
generalized amino aciduria. 26). Patients may display a lactic acidosis, hypoglycaemia,
The diagnosis is made by identifying galactose by hyperuricaemia and hypertriglyceridaemia.
thin-layer chromatography and by demonstrating
a deficiency of Gal-1-PUT activity in erythrocytes. Glycogen storage disease type II
Urinary reducing substances are usually positive Pompe’s disease or maltase deficiency (a-1,4-
provided the infant is on a lactose-containing milk diet. glucosidase) is a lyosomal defect. It is associated with
Treatment involves eliminating galactose in milk skeletal myopathy, including muscular hypotonia and
and milk products from the diet. Sufficient galactose cardiomyopathy.
for the body’s requirements can be synthesized
endogenously as uridyl disphosphate galactose. This Glycogen storage disease type III
will reverse the acute symptoms but not some of the This is a defect of debranching enzyme and is known
chronic long-term neurological complications. as Forbes–Cori disease. Abnormal glycogen with short
Block in
type III
Fructose 6-phosphate
Pyruvate Lactate
Figure 27.8 Summary of glycogen metabolism in relation to glycogen storage disease types I and III. Glycogen
is converted to limit dextran with four-unit stubs, after which a transferase removes a trisaccharide and attaches
it to a free end, leaving a dextran with single 1,6-linked glucosyl units. If amylo-1,6-glucosidase is lacking, as
in glycogen storage disease type III, the process stops at that point and hypoglycaemia results. In type I, where
the glucose-6-phosphatase is lacking, formation of glucose for the maintenance of euglycaemia is blocked, and
the enhanced alternative pathways tend to produce lactic acidosis and hyperuricaemia. PRPP, 5-phosphoribosyl
pyrophosphate; o, glucose units in the dextrans. Reproduced with kind permission from Candlish JK and Crook
M. Notes on Clinical Biochemistry. Singapore: World Scientific Publishing, 1993.
Diseases due to inborn errors of metabolism 381
Medium-chain acyl coenzyme A dehydrogenase The arterial or venous lactate to pyruvate ratio may
deficiency is autosomal recessive and is one of the most be high (more than 50:1), which suggests a metabolic
common fatty acid oxidation defects (about 1 in 10 000 block in the respiratory chain system. There is often
live births). This potentially fatal condition may present a high plasma lactate at rest. Plasma creatine kinase
with hypoketotic hypoglycaemia, encephalopathy, activity may be raised and rhabdomyolysis can occur
seizures and hepatomegaly following diarrhoea and with myoglobinuria. Specialized muscle histology may
vomiting (reduced food intake). Urinary dicarboxylic be useful and also genetic tests and family studies.
aciduria with glycine conjugates may occur, along with
increased plasma octanoylcarnitine (an acylcarnitine). 7 Peroxisomal disorders
6 Mitochondrial disorders In this group of disorders there is either a deficiency
Mitochondrial DNA (mtDNA) is derived from the of a peroxisomal enzyme or a defect in forming intact
mother. This differs from nuclear DNA in that there are peroxisomes. There are probably about 20 of these
no introns and replication of mtDNA lacks proofreading, disorders affecting about 1 in 30 000 individuals.
and the mutation rate is thus about 10–100 times Peroxisomes are involved in a number of metabolic
greater than that of nuclear DNA. Mitochondria lack processes. The following are some of the defects that
an adequate DNA repair mechanism. A number of have been described: defects of phytanic acid oxidation
clinical features may be present, including neuropathy, (Refsum’s disease), dihydroxyacetone phosphate
intellectual disabilities, lactic acidosis, myopathy, ocular acyltransferase abnormality (Zellweger’s syndrome),
defects, diabetes mellitus, anaemia and hearing loss catalase defects (neonatal adrenoleucodystrophy),
(Fig. 27.9). and abnormal plasmalogen biosynthesis (rhizomelic
There are a number of mitochondrial disorders, chondrodysplasia punctata).
including Leigh’s syndrome, MELAS syndrome These conditions may present with a variety of
(mitochondrial encephalomyopathy, lactic acidosis, features, including dysmorphia, cataracts, liver disease,
stroke), Kearns–Sayre syndrome, NARP syndrome retinitis pigmentosa, adrenal insufficiency, peripheral
(neuropathy, ataxia, retinitis pigmentosa) and LHON neuropathy, deafness and ataxia.
syndrome (Leber’s hereditary optic neuropathy).
8 Drugs and inherited metabolic disorders
The variation in individual response to drugs may be
partly due to genetic variation. There are a number of
OH well-defined inherited disorders that are aggravated by,
PH
or which become apparent only after, the administration
T Cyt b
12s of certain drugs. These disorders may be classified into
V
F P two groups.
16s E
DEAF 1555G PL ND6
ND5 Disorders resulting in deficient metabolism of a drug
LUUR LHON 14448C
LHON 14449A
ND1
MELAS 3243G, 3271C
LHON 3460A L
The muscle relaxant suxamethonium (succinyl choline,
I Q S or scoline) normally has a very brief action because
H
M A QL
LHON 11778A
it is rapidly broken down by plasma cholinesterase.
ND2 N ND4
In suxamethonium sensitivity (see Chapter 18), a
KSS
CY NARP 8993G/C
W
on–
MERRF 8344G
cholinesterase variant of low biological activity impairs
leti
SUCN R ND4L
de
G ND3
54
on
D K COIII Com
COII ATP8ATP6 apnoea’).
Two other inherited disorders are characterized
Figure 27.9 The circular deoxyribonucleic acid (DNA)
by defective metabolism of the drugs isoniazid and
of the human mitochondrial genome. Reproduced phenytoin. In both, toxic effects occur more frequently,
with kind permission from Nyhan WL and Barshop BA. and at lower dosages, than in normal individuals. The
Atlas of Inherited Metabolic Diseases, 3rd edition. genetic differences in the metabolism of azathioprine
Hodder Arnold, 2012. are discussed in Chapter 25.
Diseases due to inborn errors of metabolism 383
Disorders resulting in an abnormal response to a drug safer to prescribe from a ‘White List’ rather than
Deficiency of glucose-6-phosphate dehydrogenase excluding particular drugs.
(G6PD) may cause haemolytic anaemia, and is relatively Some people react to general anaesthetics (most
common in ethnic groups of Mediterranean origin. It commonly halothane with suxamethonium) with
is X-linked. This enzyme catalyses the first step in the a rapidly rising temperature, muscular rigidity and
hexose monophosphate pathway and is needed for acidosis (malignant hyperpyrexia), which is associated
the formation of nicotinamide adenine dinucleotide with high mortality. Many, but not all, susceptible
phosphate, which is important for the maintenance subjects in affected families have a high plasma creatine
of intact red cell membranes. Numerous variants of kinase activity.
G6PD deficiency have been described. Haemolysis may 9 Miscellaneous disorders
be precipitated by certain antimalarial drugs, such as
There are many other IEMs in addition to those
primaquine, and by sulphonamides. In the inherited
mentioned above, including congenital adrenal
hepatic porphyrias (see Chapter 21), acute attacks may
hyperplasia (see Chapter 8), adenosine deaminase
be precipitated by various drugs, such as barbiturates.
deficiency, electron transport chain defects and sulphite
So many drugs have been implicated that it is probably
oxidase deficiency.
SUMMARY
● The incidence of IEMs ranges from about 1 in 100 ● Neonatal screening laboratories use highly skilled
to 1 in 200 000, depending on the disorder and the biochemical and molecular biology techniques.
population involved. ● Some inborn errors can be treated by:
● The presence of an inborn error should be – limiting the dietary intake of precursors
considered in children with family histories of IEM, in the affected metabolic pathway, such
failure to thrive, convulsions and other metabolic as phenylalanine in PKU or lactose in
abnormalities. galactosaemia,
● Neonatal screening programmes based on the – supplying the missing metabolic product, such
analysis of neonate blood spots are used to as cortisol in congenital adrenal hyperplasia,
screen for certain metabolic disorders, including – removing or reducing the accumulated product,
hypothyroidism and PKU. such as ammonia in urea cycle disorders.
Genetics and deoxyribonucleic
28 acid-based technology in
clinical biochemistry
Genetic disorders fall into three main categories: In disorders of single genes (monogenic), the
abnormalities may be of:
● Chromosomal disorders due to the absence, or
abnormal arrangement, of chromosomes affecting ● a structural gene, with production of an abnormal
many genes and therefore many gene products. protein: in this case all the biochemical abnormalities
Examples include Down’s syndrome (trisomy for can be explained by defective synthesis of a single
chromosome 21), Turner’s syndrome (45,XO) and peptide;
Klinefelter’s syndrome (47,XXY). ● a controlling (enhancing) gene, which, by altering the
● Monogenic disorders due to an abnormality of a rate at which one or more structural genes function,
single gene, which is the primary determinant of affects the amounts of one or more structurally
the disorder and which is inherited in a predictable normal peptides.
pattern, such as phenylketonuria.
The affected protein may be an enzyme, but in
● Multifactorial or polygenic disorders due to the
other conditions it may, for example, be a receptor, a
interaction of multiple genes with environmental or
transport or a structural protein, a peptide hormone,
other exogenous factors, such as diabetes mellitus.
an immunoglobulin or a coagulation factor. With the
Human Genome Project, more and more genes and
GENERAL PRINCIPLES
their functions are being discovered.
Genes, located on chromosomes, comprise a sequence
of bases on deoxyribonucleic acid (DNA) and code for
the synthesis of proteins by ribonucleic acid (RNA) PATTERNS OF INHERITANCE
in its ‘messenger’ form (mRNA). All nucleated cells Every inherited characteristic is governed by a pair of
contain an identical complement of genes, but only genes on homologous chromosomes, one gene being
about 1 per cent is expressed. received from each parent. Different genes governing
A genotype is the actual genes present in an the same characteristic are called alleles. An individual
individual; phenotype is the physical expression of with two identical alleles is homozygous for that gene
that genotype, usually via production of a polypeptide or inherited characteristic; an individual with two
or protein. Genes differ in length, sometimes different alleles is heterozygous. Genes may be carried
containing many thousands of bases. However, only on the autosomes (similar in both sexes) or on the sex
10 per cent of the genome incorporates the protein- chromosomes (X and Y); the patterns of inheritance
coding sequences (exons) of genes. Interspersed differ.
within many genes are intron sequences, which have
no coding function. Autosomal inheritance (Fig. 28.1)
The rest of the genome consists of other non-coding If one parent (Parent 1 in the example in Fig. 28.1a) is
regions (such as control sequences and intergenic heterozygous for an abnormal gene (A) and the other
regions). Humans can synthesize about 100 000 varying parent is homozygous for the normal gene (N), the
polypeptides or proteins, which are usually composed possible combinations in the offspring are shown in the
of about 20 different kinds of amino acids. Genes square in this figure.
contain specific sequences of three DNA bases (codons) On a statistical basis, half the offspring will be
instructing the cell’s protein-synthesizing apparatus to heterozygous (AN) for gene A, like Parent 1. None will
add specific amino acids. be homozygous for the abnormal gene (AA).
Patterns of inheritance 385
inheritance, an abnormal X chromosome (Xa) is latent textbook. However, in some disorders, especially those
when combined with a normal X chromosome, but with multiple polymorphisms or mutations or with
active when combined with a Y chromosome. If the incomplete penetrance, a biochemical or phenotypic
mother carries Xa, she will appear to be normal, but diagnosis is still preferable. Epigenetics is the
statistically half her sons will be affected (XaY). Half her inheritance of a characteristic not by a change in DNA
daughters will be carriers (XaX), but all her daughters sequence, such as may occur by histone acetylation
will be clinically unaffected (Fig. 28.1c). or DNA methylation, which modify gene expression.
If the father is affected and the mother carries two See Wenham PR. DNA-based techniques in clinical
normal genes, none of the sons will be affected, but all biochemistry: a beginner’s guide to theory and practice.
the daughters will be carriers (Fig. 28.1d). Ann Clin Biochem 1992; 29: 598–624.
Inherited disease manifesting in male offspring and
carried by females is typical of X-linked inheritance. Basics principles of molecular biology
Females are only clinically affected in the extremely Chromosomes consist of both protein and DNA;
rare circumstance when they are homozygous for chromosomal DNA contains an average of 100–200
the abnormal gene. This will occur only if the female million bases. DNA is a nucleoside polymer arranged
inherited the abnormal genes from an affected father in two strands as a double helix. There are four bases in
and a carrier mother. Haemophilia is the classic example DNA, namely adenine (A), cytosine (C), guanine (G)
of an X-linked recessive disorder. and thymine (T). These nucleotides consist of ester-
linked phosphate residues at the 5¢-hydroxyl residue of
X-linked dominant inheritance a deoxyribose molecule that itself is linked to a purine
In this type of very rare inheritance, both XaX women or pyrimidine base by its 1¢-hydroxyl group. Each of
and XaY men are affected. An example of this very rare the DNA strands has polarity, namely 5¢ and 3¢ ends
type of disorder is familial hypophosphataemia. with the two opposite-running strands in opposite
directions (anti-parallel).
Multiple alleles The two DNA strands are complementary, so that
Occasionally there may be several alleles governing C on one strand bonds by hydrogen bonds with G on
the same characteristic. In such cases, different pair another strand and A pairs with T. These bases are always
combinations may produce different disease patterns, numbered from the 5¢ to the 3¢, with letters indicating
for example some of the haemoglobinopathies, or the the nucleotide bases. Genetic material can copy itself
variant may be detectable only by biochemical testing, (replication) during the S phase of the cell cycle. DNA
such as, for example, some of the plasma protein polymerase needs a pre-existing DNA template and
variants. moves along the two strands and adds the appropriate
In rare cases, spontaneous new mutations may occur nucleotides, following the base pairing rules, to the
and may produce dominant disorders in unaffected growing chain. Two new double strands of DNA are
families. The terms ‘dominant’ and ‘recessive’ are formed, identical to the original double strand and
relative. A dominant gene may fail to manifest itself the strands are anti-parallel. DNA polymerase always
(incomplete penetrance) and may therefore appear moves from 3¢ to 5¢ along the template strand, i.e. the
to skip a generation. A gene may vary in its degree of reverse of the newly forming strand. Variable number
expression, and therefore in the degree of abnormality tandem repeats (VNTR) are short and repetitive
that it produces. A recessive gene, which produces repeats distributed throughout the genome and that are
disease only in homozygotes (AA), may be detectable by grouped near telomeres.
laboratory tests in clinically unaffected heterozygotes. Transcription is the process whereby the polypeptide
or protein-coding instructions from the genes are
GENETIC DIAGNOSIS transcribed indirectly through mRNA; the latter moves
In recent years there has been a huge growth in DNA from the nucleus to the cellular cytoplasm, serving as
technology and research, such that clinical diagnosis the template for protein synthesis. Translation is the
can now be made in certain conditions using small process whereby the ribosomal system then translates
tissue samples. This chapter gives a brief outline of the codons into an amino acid chain that will constitute
such technology; for a more detailed exposition, the the polypeptide or protein molecule for which it codes.
reader is advised to refer to a specific molecular biology A gene is a unique sequence of nucleotides that codes for
Genetic diagnosis 387
a particular protein or peptide and can be represented probes are short, single-stranded DNA probes that differ
by one or more alleles. The genetic code is thus a series in composition by one single nucleotide and are thus
of codons that instruct which amino acids are needed able to detect single DNA point mutations.
to synthesize specific polypeptides or proteins.
Deoxyribonucleic acid polymorphisms
Deoxyribonucleic acid technologies DNA can be cut into smaller fragments by restriction
Deoxyribonucleic acid can easily be prepared from endonucleases derived from bacteria. These are named
very small amounts of biological samples, often by abbreviating the names of the originating bacteria.
blood. This essentially involves three steps: leucocyte These endonucleases often work in a palindromic way,
lysis, chloroform/phenol extraction to remove that is, the sequence of bases on one DNA strand is
contamination by proteins, followed by proteinase and repeated in reverse on the other. A restriction enzyme/
RNAase treatment to remove remaining protein and restriction endonuclease is an enzyme that recognizes a
RNA contamination. specific nucleotide sequence (restriction site) and cuts
Samples of DNA after digestion are separated by (restricts) the nucleic acid at that particular site. An
electrophoresis due to its strong negative charge at example of a restriction endonuclease is EcoRI made
neutral pH and, by running the DNA through agarose by Escherichia coli, which is specific for the following
gels, it can be separated into discrete DNA molecules sequence:
of 100–10 000 base pairs. Smaller DNA pieces can be
● 5¢ … GAATTC … 3¢
separated by polyacrylamide gels, which are a tighter
● 3¢ … CTTAAG … 5¢.
molecular sieve than agarose. DNA probes can be a
piece of DNA of variable length that can be inserted The base pairs on the DNA strands throughout the
into the DNA of a plasmid. These circular pieces of genome differ at particular locations in individuals.
double-stranded DNA can be artificially introduced This is called polymorphism, and, when the differences
into bacteria and incorporated into their DNA by a occur in introns (non-coding sequences of DNA),
process called transformation. By adding divalent no overall change occurs in the final coded protein
cations the bacteria are made permeable to DNA and product. If the polymorphic location occurs at a
a selectable marker such as antibiotic resistance is then particular restriction endonuclease site, either the site
inserted, and only those bacteria containing this will is not recognized by the enzyme or an alternative site
grow in media containing the antibiotic. The probes can may be created elsewhere in the DNA strand. Different
be labelled to locate them in DNA experiments, using sized DNA fragments are produced – restriction
either radiolabelled phosphorus-32 or, for example, fragment length polymorphisms (RFLPs). The RFLPs
non-radiolabelled horseradish peroxidase-enhanced can, thus, be used as markers of certain loci that may
chemiluminescence or digoxigenin. be relevant in disease processes (Fig. 28.2).
RE RE RE RE
RF RF
Figure 28.2 Summary of deoxyribonucleic acid (DNA) fingerprinting techniques. RE, restriction endonuclease; RF,
restriction fragment; VNTR, variable number of tandem repeats. Reproduced with kind permission from Candlish
JK and Crook M. Notes on Clinical Biochemistry. Singapore: World Scientific Publishing, 1993.
optimal stringency, the probe binds only to areas of There is thus a cycle of denaturation, primer
complementary DNA; thus the probe can then be annealing and primer-directed extension.
located for homologous DNA sequences. Taq polymerase is a heat-stable DNA polymerase
derived from the organism Thermus aquaticus.
Northern blotting Polymerase chain reaction (PCR) can amplify
Ribonucleic acid molecules can similarly be separated DNA up to a million-fold. It can be used in the
by electrophoresis and can be hybridized with an RNA diagnosis of known mutations of genomic DNA
or DNA probe. The mRNA from a particular gene using an amplification refractory mutation system
can be identified and characterized. This is based on (ARMS). DNA cannot undergo PCR amplification
the principle that the mRNA will be abnormal either if an oligonucleotide primer contains a 3¢ nucleotide
in amount or size if there is a corresponding gene mismatch. In site-directed mutagenesis, a mutation
mutation. Northern blots can also be used to study the or polymorphism destroys or creates a restriction
tissue-specific expression or development of particular endonuclease cutting site, which can be identified by
genes, as well as identifying and characterizing mRNAs PCR followed by endonuclease digestion (Fig. 28.3).
derived from genes of interest. The ARMS allows diagnosis of single nucleotide
mutations, where specific priming of PCR allows
Microarrays amplification to take place only if the mutation is
These are sometimes called DNA chips and are used to present. Gene sequencing can also be used in some
detect changes in gene expression in different cells. It is cases to diagnose certain mutations.
possible to bind very small amounts of defined DNA
molecules on to a membrane, which is then mixed Variable number tandem repeats
with a sample to see if RNA molecules are present – Variable number tandem repeats have been discussed
somewhat like a reverse Northern blot. above, and embrace microsatellite repeat and
trinucleotide repeat conditions that occur in certain
Polymerase chain reaction diseases, e.g. Huntington’s disease and Friedreich’s
A DNA segment is amplified by two primers using ataxia. The degree of repeats may differ from generation
repeated cycles of denaturation, primer annealing to generation.
and extension by DNA polymerase. The reaction
mixture consists of the original template DNA, the Mitochondrial deoxyribonucleic acid
four nucleotides, and a large excess of oligonucleotide Mitochondrial DNA is discussed in Chapter 27.
primers. The temperature is initially programmed to
95°C to separate the DNA strands. It is then reduced Examples of deoxyribonucleic acid
diagnosis (Fig. 28.4)
to 50°C so that the complementary primers bind and
anneal, and then raised to 72°C for about 1 min, which is Cystic fibrosis
the optimal temperature for the DNA polymerase. The Cystic fibrosis is often due to a 3 base-pair deletion in
polymerase can usually incorporate 50–100 nucleotides codon 508 of the cystic fibrosis chloride channel gene,
per second, following which the temperature is again although other mutations may also occur. The genetic
raised to 95°C to complete the cycle. defect can be detected using PCR and this can be used
Genetic diagnosis 389
First cycle I
Primer extension
1 2
Denaturation annealing II
Second cycle
Primer extension
1 2 3
III
Denaturation, annealing
Third cycle
Primer extension
2.8 kb
Final products
1.6 kb
1.2 kb
SUMMARY
● Genetic disorders fall into three main categories: ● The examples given in this chapter are only a
– chromosomal disorders, selection of the DNA tests that may become
– monogenic disorders, available in the future. However, diseases such as
– multifactorial or polygenic disorders. cystic fibrosis and muscular dystrophy are already
● Deoxyribonucleic acid (DNA) technology will being diagnosed by DNA technology.
undoubtedly increase over the next few years ● The polymerase chain reaction is an important
and its use can be expected to increase in clinical diagnostic molecular biology technique.
biochemistry testing.
29 Patient sample collection and use
of the laboratory
● hospital case number, and/or healthcare number, Effect on results of procedures before venepuncture
● surname and first name(s), correctly and consistently
spelt, ● Some tests may require the patient to fast, for example
● date of birth, rather than age. plasma glucose or triglyceride (see Chapters 12
and 13).
These would usually be considered the minimum ● Oral medication: some assays may be affected by oral
acceptable dataset for patient identification details. medication.
Any of these may be recorded inaccurately on the – Blood should be taken for drug assays at a
form and, unless there is complete agreement with standard time after the dose; misleadingly high
previous details, results may be entered into the wrong plasma concentrations may occur at the time
patient’s record either on a computer or in the patient’s of peak absorption (see Chapter 25).
case notes, causing confusion and possible danger to the
patient. The National Health Service (NHS) number is
being used as a unique individual identifier in the UK. CASE 1
It is important also to include relevant clinical details
so as to facilitate correct interpretation of the results. A 22-year-old man had the following post-operative
biochemical results after an appendectomy:
Location of the patient and identification of
Plasma
the clinician
Sodium 165 mmol/L (135–145)
It should be obvious that, if the ward or department Potassium 1.9 mmol/L (3.5–5.0)
is not stated, it may take time and effort to determine Urea 1.1 mmol/L (2.5–7.0)
where the results should be sent. The requesting doctor Creatinine 38 µmol/L (70–110)
must sign the form legibly, and also state how he or she Glucose 43 mmol/L (3.5–6.0)
can be notified rapidly, for example by ‘bleep number’,
in case abnormal results requiring urgent action are The clinical biochemistry laboratory suggested an
found or advice needs to be sought about treatment. immediate repeat blood sample, which gave the
The doctor must check the completed request form following results:
to be sure that the information given is correct; it is Plasma
also important to include his or her name and contact Sodium 136 mmol/L (135–145)
details. Potassium 3.9 mmol/L (3.5–5.0)
Request forms designed by pathology and other Urea 5.4 mmol/L (2.5–7.0)
departments ask only for information that is essential to Creatinine 89 µmol/L (70–110)
ensure the most efficient possible service to the clinician Glucose 4.5 mmol/L (3.5–6.0)
and therefore to the patient. Now quite widespread DISCUSSION
is the use of electronic test requesting, which should The first sample seems to display profound
improve patient identification and speed up the process hypernatraemia, hyperglycaemia and hypokalaemia.
and may replace ‘paper’ requests. In addition, the plasma urea and creatinine
concentrations are both low. The repeat sample values
COLLECTION OF PATIENT SPECIMENS
are completely different. It later transpired that the
Collection of blood first sample had been taken out of the patient’s drip
If a clinically improbable result has been checked arm, which had a dextrose–saline infusion going into
analytically and the second result is in close agreement it. This had resulted in dilution of the analytes and
with the first, a fresh specimen should be analysed. elevated sodium and glucose concentrations.
Collection of patient specimens 393
– There may be significant hypokalaemia for plasma concentrations. It is sometimes difficult to enter
a few hours after taking potassium-losing ‘bad veins’ without applying stasis. A tourniquet may
diuretics due to rapid clearance of potassium be used and released as soon as the needle is in the vein;
from the extracellular fluid (ECF). The plasma a suitable specimen may be obtained after waiting at
concentration returns to its ‘true’ level as least a further 15 s before withdrawing blood.
equilibration occurs between cells and ECF
Site of venepuncture
(see Chapter 5).
– Interfering substances: previous administration If the patient is receiving an intravenous infusion, the
of a substance may affect a plasma analyte administered fluid in the veins of the same limb, whether
concentration for some time; for example certain proximal or distal to the infusion site, has not mixed
antibiotics may interfere with chemical reactions with the total plasma volume; local concentrations
used in creatinine assays (see Chapter 3). will therefore be unrepresentative of those circulating
● Effect of posture: for example the concentrations of through the rest of the body. Blood taken from the
plasma proteins and of substances bound to them opposite arm will give valid results. Note that glucose
are lower when the patient is supine than when infusion may cause systemic hyperglycaemia and
standing up (see Chapter 19). glycosuria. Only if the hyperglycaemia persists after
● Intravenous infusion: for example a spuriously infusion has stopped should the diagnosis of diabetes
low plasma sodium concentration may result if mellitus be considered (see Chapter 12).
the sample is taken from a dextrose drip arm (see
Containers for blood
Chapter 2).
● Clinical procedures: for example palpation of Most hospital laboratories issue a list of the types of
the prostate by rectal examination may possibly container suitable for different assays (Fig 29.1); this list
release large amounts of prostate-specific antigen may vary from hospital to hospital. For example, most
(PSA) into the circulation; the spuriously elevated laboratories specify that:
concentrations in blood may persist for several days ● Blood for glucose estimation should be put into
(see Chapter 24). a tube containing an inhibitor of erythrocyte
glycolysis, such as fluoride.
● Potassium should ideally be estimated on plasma
Effects on results of the technique of venepuncture
from heparinized blood rather than serum, although
Venous stasis
many laboratories accept serum tubes for potassium
It is usual to apply a tourniquet proximal to the site of on the pragmatic grounds that the differences are
venepuncture to make it easier to enter the vein with
the needle. If occlusion is maintained for more than a
short time, the combined effect of raised intracapillary
pressure and hypoxia of the vessel wall increases the
rate of passage of water and small molecules from the
lumen into the surrounding interstitial fluid. Large
molecules, such as proteins, cannot pass through
the capillary wall at the same rate; their plasma
concentrations therefore rise.
Many plasma constituents are partly bound to
protein. Prolonged venous stasis can raise the plasma
total calcium concentration, sometimes to equivocal
or slightly high levels. Therefore, ideally, blood samples
for plasma calcium estimation should be taken without
stasis, especially if high plasma concentrations have
previously been found (see Chapter 6). Figure 29.1 Blood collection tubes: if in doubt about
Prolonged stasis may also cause local hypoxia; what tubes to use be sure to contact the laboratory for
consequent leakage of intracellular constituents, such advice. Reproduced with kind permission of Greiner
as potassium and phosphate, may cause falsely high Bio-One.
394 Patient sample collection and use of the laboratory
usually non-significant. Potassium is released from the results of calcium estimation. Ethylenediamine
cells, especially platelets, during clotting. Serum tetra-acetic acid is usually in the form of its potassium
potassium concentrations are usually higher than salt; therefore, it renders the sample unsuitable for
those of plasma by a variable amount; this difference potassium analysis.
can be clinically misleading. Marked differences may The use of sodium (instead of lithium) heparin or
be found in patients with leukaemia, in whom the trisodium citrate may give a falsely high plasma sodium
number of white blood cells is usually significantly result. This anticoagulant is often used in specimens
increased. taken for ‘blood gases’; apparent plasma sodium
concentrations of 160–170 mmol/L can result from
Laboratories should accept blood only in the correct
transferring an aliquot of sodium heparinized blood
containers. Even with this precaution, serious errors can
into a lithium heparin vial. The use of lithium heparin
arise if blood is decanted from one container to another,
leads to a falsely high plasma lithium concentration and
although this is less likely to occur if a closed vacuum
it should therefore not be used as an anticoagulant for
system is used for obtaining blood. The anticoagulant
lithium determination.
actions of oxalate and of sequestrene (ethylenediamine
tetra-acetic acid, EDTA) depend on the precipitation Effects of haemolysis and delayed separation of blood
or chelation of calcium, respectively, thus invalidating Haemolysis
The concentration of many substances is very different
in erythrocytes from that in the surrounding plasma.
CASE 2 Haemolysis releases the cell contents into plasma and,
A blood sample had been taken from a 44-year-old consequently, if this occurs in vitro, the concentrations of
woman on the medical ward and the results were as some plasma constituents, such as potassium, phosphate
follows: and aspartate transaminase, may be falsely increased.
The increase is variable and is not related to the intensity
Plasma
of the red colour of plasma due to haemoglobin. It is
Sodium 140 mmol/L (135–145)
uncommon for haemolysis to occur in vivo because
Potassium > 10 mmol/L (3.5–5.0)
these constituents are distributed throughout the total
Urea 4.1 mmol/L (2.5–7.0)
extracellular, not just plasma, volume.
Creatinine 78 µmol/L (70–110)
Haemoglobin may interfere with some chemical
Albumin-adjusted calcium < 0.5 mmol/L (2.15–2.55)
reactions, falsely increasing the apparent plasma
Phosphate 0.92 mmol/L (0.80–1.35)
bilirubin concentration and lowering alkaline
Repeat blood sampling on the same day gave the phosphatase activity. The chance of haemolysis is
following results: minimized if the blood is treated gently and if a closed
Plasma vacuum system is used.
Sodium 139 mmol/L (135–145) Delayed separation of blood
Potassium 3.6 mmol/L (3.5–5.0) It is important to check the sample date. The differential
Urea 4.2 mmol/L (2.5–7.0) concentrations of some analytes across cell membranes
Creatinine 78 µmol/L (70–110) are maintained by energy, derived from glycolysis. In
Albumin-adjusted calcium 2.43 mmol/L (2.15–2.55) vitro, erythrocytes soon use up the available glucose
Phosphate 0.90 mmol/L (0.80–1.35) and therefore the energy source; concentrations of
DISCUSSION these analytes in plasma will then tend to equalize with
The first blood sample had been collected in a those in erythrocytes by passive diffusion across cell
potassium ethylenediamine tetra-acetic acid (EDTA) membranes. If plasma is not separated from blood cells
tube (this is normally used for certain haematology within a few hours, the effect on plasma concentrations
tests such as full blood count) by mistake and then the will be similar to that resulting from haemolysis.
blood had been decanted by the doctor into the correct However, there are a few important differences:
chemistry lithium heparin tube. Note in the first ● Because haemoglobin is not released, the plasma
sample the raised plasma potassium and low calcium colour looks normal; the error is therefore easily
concentrations due to chelation by potassium EDTA. overlooked.
Collection of patient specimens 395
Refrigeration
CASE 3 The refrigeration of whole blood has the effect
A blood sample had been taken in the morning of raising the plasma potassium concentration
from a 43-year-old man and was then sent to the probably by reducing the activity of the adenosine
laboratory from the local health centre. The sample triphosphatase pump. Hence, blood samples for urea
was analysed in the evening of the same day, as there and electrolytes should not be refrigerated. Transport
had been a transport delay, and the following results of samples in cold weather may have a similar effect.
were obtained: Freezing will result in haemolysis. Therefore blood
Plasma
specimens must be centrifuged and the plasma
Sodium 143 mmol/L (135–145) separated from the cells before storing, for example
Potassium 6.0 mmol/L (3.5–5.0) overnight.
Urea 5.1 mmol/L (2.5–7.0) Collection of urine
Creatinine 88 µmol/L (70–110)
Urine estimations performed on timed collections are
The laboratory contacted the patient’s general expressed as units/time (for example mmol/24 h); this
practitioner and an urgent repeat sample showed the figure is calculated by multiplying the concentration
following results. by the volume collected during the timed period. The
accuracy of the final result depends largely on that of
Plasma
the urine collection. Sometimes, if the patient is a child
Sodium 145 mmol/L (135–145)
or incontinent, accurate urine collection is particularly
Potassium 4.2 mmol/L (3.5–5.0)
difficult.
Urea 5.2 mmol/L (2.5–7.0)
For example, a 24-h specimen may be collected
Creatinine 88 µmol/L (70–110)
between 09.00 h on Sunday and 09.00 h on Monday.
DISCUSSION The volume excreted by the kidneys during this period
The first patient sample shows hyperkalaemia, but is the crucial one: urine already in the bladder at 09.00 h
repeat analysis on a ‘fresh’ sample shows a ‘normal’ on Sunday was excreted earlier and should not be
potassium concentration. The spuriously raised included. Therefore the procedure is as follows.
potassium result in the first sample was due to the delay
in sample assay and leakage of intracellular potassium ● 09.00 h on Sunday: the bladder is emptied completely,
ions out of cells, resulting in pseudohyperkalaemia. It whether or not the patient feels the need, and the
is important to transport samples to the laboratory as specimen is discarded.
quickly as possible to avoid storage artefacts. ● Collect all urine passed until 09.00 h on Monday
at which point the bladder is completely emptied,
whether or not the patient feels the need, and the
urine specimen is added to the total collection.
● The plasma potassium concentration rises as the
The shorter the period of collection, the greater the
plasma glucose falls; initially, there may be a slight
error if this procedure is not followed.
fall in the plasma potassium concentration as it
Before collection, a urine container should be
moves into the erythrocytes.
obtained from the laboratory; this may contain
Many plasma constituents, such as bilirubin, a preservative to inhibit bacterial growth (which
deteriorate even if the plasma is correctly separated and might destroy the substance being estimated) or
stored. If the plasma sample is haemolysed or separation acid preservative to prevent precipitation or sample
has been delayed, the plasma potassium concentration degradation, but that does not interfere with the
may still be clinically significant. For example, if the relevant assay. The patient must be told not to discard
sample is visibly haemolysed and the plasma potassium the preservative in the container and that it might be
concentration is only 2.8 mmol/L, this may indicate toxic and harmful if spilt.
profound hypokalaemia; the laboratory staff should
contact the requesting doctor directly to discuss the Collection of faeces
significance of the comment ‘haemolysed specimen’ on Rectal emptying is usually erratic and, unlike that of
the report form. the bladder, can rarely be performed to order. Results
396 Patient sample collection and use of the laboratory
of faecal estimations of, for example, fat may vary by be included, and should be written at the time of
several hundred per cent in consecutive 24-h collections. collection.
If the collection period lasted for weeks, the mean 24-h
Sending the specimen to the laboratory
output would be very close to the true daily loss from
the body into the intestinal tract. Many estimations can be performed rapidly if the result
To render collection more accurate, orally is needed urgently. Non-urgent late blood samples can
administered ‘markers’ are used in certain circumstances. be separated from cells and stored overnight, although
Faecal collections and estimations are time consuming some laboratories work 24/7. In cases of true clinical
and unpleasant for all concerned and are now rarely emergency, the requesting clinician should notify the
required. laboratory, preferably before the specimen is taken,
indicating the reason for the urgency, so that the
Labelling patient specimens laboratory can be ready to deal with the specimen as
All specimens must be accurately labelled and the soon as it arrives. It is the clinician’s responsibility to
information should correspond with that on the indicate the degree of urgency. The misuse of emergency
accompanying request form in every detail, as error services may delay truly urgent results, and some urgent
may cause a medicolegal disaster. The date, and samples may need to be separated immediately and
preferably the time, of specimen collection should kept on ice.
SUMMARY
● It is essential to liaise closely with the laboratory ● Particular attention should be paid to avoiding
when collecting patient samples to help ensure blood samples being collected from the same arm as
correct sampling times and collection conditions. an intravenous infusion, leading to ‘drip arm’ results.
● In vitro haemolysis can lead to a spurious increase ● It is also essential to ensure correct sample labelling
of predominantly intracellular ions in the plasma, and patient identification to avoid potentially life-
such as potassium (pseudohyperkalaemia). threatening errors and medicolegal disasters.
30 Point-of-care testing
Some major advantages of point-of-care testing 397 Some possible clinical settings for point-of-care
testing devices 398
Recently, laboratories have tended to become more Transcutaneous biosensors allow continuous
specialized and centralized and may process millions measurements to be made through the patient’s skin
of samples per year. High throughput, cost-effective without the need for blood collection. Near-infrared
automation (perhaps including the use of robotics), spectroscopy may allow continuous monitoring of more
stringent quality assurance processes, computerization than one analyte as well as in vivo glucose monitoring
with data storage and retrieval systems, and highly with implantable sensors in diabetic patients.
skilled, and trained, personnel are now common. In choosing a POCT analyser system, remember that
In contrast, ‘point-of-care testing’ (POCT) has now duplication may occur within the hospital at separate
developed, which enables clinicians or patients to sites. Different analysers on the same site may result in
perform tests without necessarily using the laboratory the use of different reference ranges and thus difficulties
directly. in comparing patient results. Many of the new POCT
analysers are relatively easy to maintain, but maintenance
SOME MAJOR ADVANTAGES OF POINT- may need to be carried out by non-laboratory staff out
OF-CARE TESTING
of the laboratory setting. It is also likely that results will
Turnaround times need to be interpreted and troubleshooting performed
One of the main advantages of POCT over laboratory by non-laboratory personnel.
testing is the relative immediacy of results. This
Costs
may enable prompt treatment, shortened patient
waiting time and a reduced number of out-patient The reduction in turnaround time may result in a
appointments and clinic visits for the patient. reduction in total costs if patient episodes are shorter
Many POCT devices require minimal specimen and transport costs are reduced, for example courier
preparation or collection (in some cases using a finger costs. However, on a direct charge basis, including
prick of blood). The machine is in the near-patient capital costs, POCT may be more expensive than
setting, thus reducing the delays associated with the central laboratory testing. This can be due in part to
transport of specimens and reports. duplication of tests overall and to economies of scale.
The costs of reagents and machines, quality control
Technological advances and ease of use materials, maintenance, storage of report forms and
The recent increase in POCT has been due partly results and training may all need to be taken into
to technological changes in the design of analysers. consideration when embarking on POCT. Labour
With the advent of microchips, computerization and costs are more difficult to assess in POCT, but may
miniaturization, it has become easier to bring analysis incorporate nursing staff; however, set against this are
nearer to the patient and for it to be performed by possible savings of on-call or out-of-hours costs for
personnel with minimal training or by the patients laboratory staff.
themselves. Some of the modern POCT devices Depending on the structure and organization of
incorporate biosensors, electrodes and dry- and solid- the POCT setting, the overall costs could be less when
phase chemistry reagents. These allow for small sample the merits of rapid therapeutic responses and shorter
and reagent volumes, quick assay reaction times, ease hospital stays are taken into consideration (Box 30.1).
of use and disposal of used reagents, more than one It is essential that there is proper user training and
analyte to be measured simultaneously and probably also maintenance of a complete audit trail for patient
less technical skill. results.
398 Point-of-care testing
SUMMARY
● The use of POCT is increasing in clinical medicine. own blood glucose levels and adjusting their
● POCT allows prompt turnaround of patients’ diabetes therapy accordingly.
results and thus the potential for speedy clinical ● However, POCT also has potential problems, and
management. there should be close liaison with the hospital
● POCT plays a large role in the management of laboratory to ensure optimal usage. Good quality
diabetes mellitus, with some patients testing their control is essential.
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Appendix 1
Units in clinical chemistry
Results in clinical chemistry have been expressed in ● For polyvalent ions, such as calcium and magnesium
a variety of units. For example, the concentrations of (both divalent), the old units are divided by the
electrolytes were formerly usually quoted in mEq/L, valency. For example, a magnesium concentration of
the concentration of protein in g/100 mL and that of 2.0 mEq/L becomes 1.0 mmol/L.
cholesterol in mg/100 mL. The units used might vary
from laboratory to laboratory: calcium concentrations Results previously expressed as mg/100 mL
might be expressed as mg/100 mL or mEq/L; in If results were previously expressed in mg/100 mL
Britain plasma urea concentrations were expressed the method of conversion to mmol/L is to divide by
as mg/100 mL of urea, whereas in the United States it the molecular weight (to convert from mg to mmol)
is usual to report mg/100 mL of urea nitrogen. This and to multiply by 10 (to convert from 100 mL to
situation can be confusing and with patients moving, L). Thus effectively the previous units are divided
not only from one hospital to another, but from one by a tenth of the molecular weight. For example, the
country to another, dangerous misunderstandings have molecular weight of urea is 60, and of glucose 180.
arisen. A urea concentration of 60 mg/100 mL and a glucose
concentration of 180 mg/100 mL are both equivalent
SYSTÈME INTERNATIONAL D’UNITÉS
(SI UNITS) to 10 mmol/L. The factor of 10 is, of course, only
used for concentrations. The total amount of urea
International standardization is obviously desirable, and excreted in 24 h in mg is numerically 60 times that
such standardization has long existed in many branches in mmol.
of science and technology. The main recommendations
for clinical chemistry are as follows: Exceptions
● If the molecular weight (MW) of the substance being ● Units of pressure (for example mmHg) are expressed
measured is known, the unit of quantity should be as pascals [or kilopascals (kPa)]. One kPa equals
the mole or a subunit of a mole: 7.5 mmHg, so that a PO2 of 75 mmHg is 10 kPa.
Pascals are SI units.
weight in g Proteins. Body fluids contain a complex mixture of
Number of moles (mol) = ●
MW proteins of varying molecular weights. It is therefore
In clinical chemistry, in the UK, millimoles recommended that the gram (g) be retained, but
(mmol), micromoles (µmol) and nanomoles (nmol) that the unit of volume be the litre (L). Thus a total
are the most common units. protein of 7.0 g/100 mL becomes 70 g/L.
● 100mL should be expressed as decilitre (dL).
● The unit of volume should be the litre. Units of ● Enzyme units are not yet changed. Note that the
concentration are therefore mmol/L, µmol/L or nmol/L. definition of international units for enzymes does
Examples not state the conditions of the reaction.
Results previously expressed as mEq/L ● Some constituents, such as some hormones, are still
expressed in ‘international’ or other special units.
weight in g
Number of equivalents (Eq) = A conversion table for some of the commoner results is
equivalent weight
listed in Table A.1. Note that:
weight in g ¥ valency
= 1 mol = 1000 mmol (millimoles)
MW ●
Crook
CLINICAL BIOCHEMISTRY CLINICAL BIOCHEMISTRY
AND METABOLIC MEDICINE AND METABOLIC MEDICINE
EIGHTH EDITION
Whether you are following a problem-based, an integrated or a more traditional Martin A Crook
medical course, clinical biochemistry is often viewed as one of the more challenging
Consultant in Chemical
This well-respected text provides comprehensive and measured guidance to this complex
Pathology and Metabolic
area, reflecting the ongoing changes in our understanding of clinical biochemistry
Medicine, Guy’s, St Thomas’
while preserving the acknowledged strengths of previous editions: readability, a firm
and University Hospital
basis in the underlying science and a clear focus on clinical applicability.
Lewisham; and Visiting
Professor, School of Science,
Key features:
University of Greenwich,
Fully integrated approach – clinical practice logically explained in the context of
London, UK
underlying basic science
Clinically oriented – case histories and algorithms direct clinical application of
laboratory findings
Comprehensive – includes common and less common diseases and syndromes
Highly illustrated – with clear and reproducible full colour line diagrams and, new
to this edition, clinical photographs
Companion website – including self assessment questions and image library
EIGHTH EDITION
preparation for MRCP and MRCPath examinations, and trainee clinical scientists.
General practitioners and practising physicians will also find the text helpful in the
diagnosis and management of patients with metabolic disorders.