Medical Biochemistry

Molecular Principles of Structural Organization of Cells

BIOLOGIC SAMPLES
 BLOOD

Blood is a liquid tissue, a heterogeneous system composed of

two phases:
one liquid phase, the plasma, an aqueous solution of mineral
salts and different organic substances (proteins,
carbohydrates, lipids, enzymes, vitamins, hormones)
with pH range 7.35-7.45.
one solid phase, composed of cells, suspended in the liquid
phase:
red blood cells (RBCs, erythrocytes),
white blood cells (WBCs, leukocytes) and
platelets.
 Erythrocytes (RBCs)
Cells Leukocytes (WBCs)
(45%) Platelets (PLT)
BLOOD
Water (91-92%)
Inorganic substances Anions
Plasma Cations
(55%) Dissolved
substances Proteins
Carbohydrates
Organic substances Lipids
Enzymes
Vitamins
Hormones
Final and
intermediary
metabolites
 BLOOD FUNCTIONS

1. Transport function:
Blood is important for the transport of nutrient substances (amino
acids, oses, fatty acids, vitamins) provided by the food digestion
process from the intestinal wall cells to different organs (nutritive
function).
The metabolic residues (urea, uric acid, ammonia) are transported
from the breakdown location to excretory organs (skin, kidneys).
The transport of oxygen from the lungs to tissues and of carbon
dioxide from tissues to lungs is performed by the hemoglobin in the
erythrocytes (respiratory and detoxifying function).
Hormones are transported from the secretory endocrine glands to
specific target organs.
2. Proteins such as gammaglobulins and leukocytes provide the defence
system of the body (immunologic function).
3. Blood clotting system protects against the damage to the vascular
system.
2. Homeostasis control:

Blood participates to the maintenance of ions concentration

and of a constant ratio between different ions (Na+, K+, Cl-,
Ca2+, Mg2+, HCO3-), including the maintenance of acid-base
equilibrium and a constant pH 7.35 (isotonia).
The maintenance of osmotic pressure proportional to the
number of non-dissociated compounds, ions and electrolytes
found in liquid phase (isoosmia).
Heat regulation of the body by its ability to shift water to and
from the surface of the body (isothermia).

The blood is circulating through a closed vascular system, being

in a continuous motion owing to the cardiac pump function; it
acts as an interconnection between the cells of the different
tissues.
 COLLECTING BLOOD SAMPLE
It is recommended to collect the blood “à jeun” (fast conditions)
Except glucose, lipids, amino acids, inorganic phosphorus, the
most chemical constituents reveal no significant change after
standard breakfast.
Lipemia (lactescent) caused by triacylglycerides and
chylomicrons interfere because of turbidity; the blood should be
collected from a patient in the post-absorbtive state (overnight
fast of 12-14 hours) especially for lipids; although 4-6 hours
usually suffice.
Diurnal variations unrelated to eating is associated with a
change in iron and corticosteroids.
Dietary habits may influence uric acid, urea, lipids.
Glucose tolerance test procedure requires an adequate
carbohydrate intake (250 g per day) for 3 days prior to the test.
For the venipuncture (skin puncture for children):

Prepare the puncture site with 70% alcohol or

anhydrous ether when oxygen is being used in the
area.

Use a syringe or vacutainer system.

 When plasma is needed, the blood should be collected in a
vacutainer containing an effective anticoagulant (that prevents
the coagulation process).
– If the anticoagulant is water soluble it may be placed as
dried substance in the collecting tube; the blood is collected
and mixed with it.
– If it is a solution, remember the dilution factor of the solution
formed with the blood and use it in the calculation.

Anticoagulant Preparation Used for analysis Inadequate for analysis

Sodium oxalate oxalate 300 mg/20 ml bidistilled water. Use 0.1 ml/2 ml blood, the fibrinogen enzymes
necessary amount of solution can be evaporated in tubes at metabolites electrolytes, pH
370C.
Sodium citrate 1 part sodium citrate 3.8 %/9 parts blood coagulation tests enzymes
fibrinogen electrolytes, pH

Disodium EDTA 300 mg/20 ml bidistilled water. fibrinogen electrolytes

Use 0.1 ml/2 ml blood. metabolites nitrogen

Sodium (potassium) 60 mg/20 ml bidistilled water. metabolites electrolytes

Use 0.1ml/2 ml blood enzymes
heparinate
Lithium heparinate 60 mg/20 ml bidistilled water. metabolites Alkaline phosphatase
Use 0.1ml/2 ml blood electrolytes

Ammonium heparinate 60 mg/20 ml bidistilled water. electrolytes ammonia

Use 0.1 ml/2 ml blood metabolites, enzymes

ACD 4.7 g citric acid, 16 g trisodium citrate, 25 g glucose diluted with Enzymes in erythrocytes, Electrolytes
bidistilled water up to 1,000 ml. Use 1 part solution/4 parts blood. platelets metabolites
 PROCESSING THE BLOOD
1. Plasma:
centrifuge blood collected on anticoagulant, preferably in the original
container, closed to prevent evaporation, within 1 hour after collection, for
10 minutes at a relative centrifugal force (RCF) 850-1,000
the cells are separated of plasma
store in a refrigerator (4-50C) or freeze (-200C) if the analysis is to be
delayed more than 4 hours.
2. Serum:
allow blood to clot in the original closed container, at room temperature,
for 20-30 minutes;
when clot has formed, gently loosen it at the top with a fine glass rod or
applicator stick;
centrifuge blood 10 minutes, RCF 850-1,000 in the closed container;
label;
store at 4-60C or -200C if the analysis is delayed more than 4 hours.
Plasma is the clear, yellow fluid obtained when blood is collected into a
tube containing an anticoagulant and is centrifuged. The cells are heavier
than the fluid medium and appear as a layer in the bottom half of the
centrifuge tube.
Serum is similar to plasma except that the blood has been allowed to clot
before centrifuging.

The differences between serum and plasma are the following:

– plasma is the blood component existing in the body, whereas serum is
the fluid obtained outside the body, when the blood is allowed to clot;
– plasma obtained outside the body always contains an anticoagulant, a
chemical that prevents the blood from clotting (heparin, oxalate,
citrate, EDTA);
– plasma contains fibrinogen (0.2 to 0.4 g/dl) which is missing from
serum because it has been converted into the insoluble fibrin clot

Plasma - Fibrinogen (- Clotting factors) = Serum

If not appropriately stored, the components may
change their concentration:
glucose decreases value;
inorganic phosphorus increases if stored more than 3 hours;
potassium increases if erythrocytes are not separated of
serum within 1 hour;
chlorine determination is affected if blood is in contact with
air; CO2 is diffusing from the erythrocytes and chlorine takes
its place;
bicarbonate should be determined within 1 hour;
pH is increased from 7.4 to 8 in 4 hours.
The composition may be changed under the bacterial and
enzymatic effect, loss of volatile constituents, interchange of
substances between liquid and intracellular space (K+, Cl-,
H2PO3- from cell to plasma).
Common difficulties:
1. Hemolysis invalidates determination through release of
erythrocyte content (LDH, ACP, K+) or through colour change -
the photometric measurements with 400-500 nm (for example,
hemoglobin interferes the bilirubin dosing).
Causes - collection of blood is not correct:
1. venous stasis is excessive through prolonged application of the
tourniquet;
2. moisture or contamination in the needle, syringe or container.
To avoid hemolysis:
1. use 20 gauge needles,
2. allow the blood to flow into the syringe gently, with minimal suction;
3. do not shake blood to mix with anticoagulant; mix by gentle inversion
6-8 times; agitation of whole blood without anticoagulant induces
hemolysis;
4. mind that the blood is taken from the opposite arm of the intravenous
infusion.
2. Lactescence - milky or lipemic plasma obtained
with blood samples after a meal rich in fats or in
hyperlipemia; it interferes with spectrophotometric
methods, determination of uric acid and enzymes.
3. Icteric serum - the normal straw-yellow colour of
the plasma may vary from intense yellow to brown-
green, due to the presence of increased
concentration of bilirubin; it interferes with all the
colorimetric reactions using the blue light, some of
the methods used to dose the cholesterol and
proteins.
 THE ANALYSIS OF URINE

The kidney is a highly specialized organ that has

evolved to perform two main functions:
eliminate soluble waste products of metabolism,
and
preserve the internal environment of the cells
(maintain water balance, pH, ionic equilibrium, fluid
osmotic pressure).
It also performs other functions such as
synthesis of erythropoietin,
1-hydroxylation of 25-hydroxyvitamin D3.
The active unit of the kidney is the nephron (approximately 1
million per each kidney), composed of
• a glomerulus,
• proximal convoluted tubule,
• long loop of Henle - thin-walled descending limb and
- thick-walled ascending limb,
• distal convoluted tubule,
• collecting tubule that merges with other to form
• collecting ducts.
The urine flows from the collecting ducts into the renal pelvis and
through the ureter (one per kidney) to the bl
adder.
The first step in the formation of urine is the filtration of
plasma through the capillary membrane of glomerulus into the
surrounding Bowman capsule: ions and small molecules as
glucose and water pass, while molecules of molar weight
50,000 Daltons (most proteins and constituents tightly bound
with proteins) are retained in capillaries.
The second step is the passage of the ultrafiltrate (200 L per
day for the average adult; with the same specific gravity as a
protein free filtrate of plasma 1010, pH 7.4) down the proximal
convoluted tubule, where the various selective processes of
absorption begin. About 70% of the water, Na+ and Cl- ions
and all except negligible amounts of glucose, amino acids and
K+ ions are reabsorbed in the proximal tubules. Urea,
phosphates and Ca2+ ions are incompletely reabsorbed. H+
ions are exchanged for the Na+ ions throughout the tubule. K+
ions are exchanged for Na+ ions only in the distal tubule. This
process is regulated by aldosterone.
Laboratory tests play an important role in the diagnosis and
assessment of renal disease because the clinical signs and
symptoms of renal disease may be vague or absent.
The pathologic conditions of the kidney must be considerable
before the tests of renal function become abnormal.
The formation of a normal urine at a normal rate requires the
presence of:
receiving an adequate blood supply;
a sufficiently high blood pressure;
properly functioning kidneys;
no obstruction to urine flow.
EXAMINATION OF THE URINE (URINALYSIS)
A routine urine analysis usually consists of an examination of a morning
specimen (upon arising) for colour, odour, specific gravity, osmolality,
pH, the performance of some qualitative or semiquantitative tests for
proteins, glucose or reducing sugars, ketones, blood, bilirubin,
urobilinogen.
Some hospitals routinely perform a microscopic examination of the
urinary sediment.
The complete analysis is performed on 24 hours urine sample.
To collect it, at a certain hour in the morning, the patient will void,
completely empty his/her bladder, and discard this urine.
Then, the urine is collected into a container until the next day, the same
hour.
To prevent the urine to be altered, preservatives are added (1-2 g thymol,
5-10 ml toluene, xylene, chloroform) in the collection container. The
chloroform is a reducer so it will not be used when glycosuria will be
performed. The preservers will not be used when a bacteriologic exam is
to be performed.
 MACROSCOPIC AND PHYSICAL EXAMINATION

Volume.

The daily output of urine (diuresis) depends largely upon the

fluid intake, exercise, temperature, NaCl intake, hormonal
control.

The average excretion of a normal adult is approximately:

1 ml/min, about 1,400 800 ml/day.
The reference values are
in male 1,500 - 2,000 ml /24 hours,
in female 1,000 - 1,500 ml/24 hours.

Normally, 80% of the urine volume is output during the day.

A decreased urinary output is called oligouria (less than 500
ml/24 hours). It may be caused by:
prerenal affections (low blood pressure, shock, hemorrhage,
fluid deprivation by fever, perspiration, edema);
renal diseases (acute tubular necrosis, poisons, renal
vascular diseases, precipitation of certain compounds in
nephrons);
postrenal (calculi, tumors compressing ureters, prostate
hypertrophy).

Anuria (less than 100 - 150 ml/24 hours) appears in acute renal
failure.
An increased output is referred to as polyuria (more than 2,000
ml/24 hours). It may be caused by:
an increased amount of solutes which have to be excreted,
with obligatory excretion of water (after excessive salt intake,
diabetes mellitus with glycosuria);
an excessive ingestion of fluids or diuretic substances;
a deficiency or depression of antidiuretic hormone (ADH);
renal sclerosis.

When the output during the night is more important than the one
during the day, the situation is called nicturia. It can appear in
heart failure, urinary inflammation.
Polakiuria means that the volume of the daily output is normal
but the number of voids is increased. It is present in cystitis,
pyelitis, prostate hypertrophy.
Colour.
The normal urine has a colour varying from light
yellow to yellow-red, which is determined by the
presence of chromogenous substances or pigments
such as urochroms, urobilin.
It can be influenced by the pH and concentration:
– an alkaline or diluted urine is lighter;
– an acid or concentrated urine is darker.
The diet influences the colour as:
– The vegetarian diet will give an alkaline pH -
lighter colour,
– The diet with animal origin food will give an acidic
pH - darker colour
It is important to call attention to abnormally coloured
urine, even though this does not occur frequently:
during polyuria the colour is very light due to the dilution of
the pigments which are excreted in normal amount;
in acute nephritis the urine is reddish or brown-green due to
the presence of blood; the same colour can be due to the
presence of myoglobin, hemoglobin;
in some hepatic or bile tract diseases, the presence of bile
pigments produces a green-brown or deep yellow colour;
a dark brown urine may be caused by homogentisic acid
excreted in a rare genetic disease, alkaptonuria;
some drugs or dyes may also contribute colour to the urine.
Aspect.

The aspect of urine immediately after emission is clear,

transparent.
Sometimes, the urine has certain turbidity due to the presence
of salts or pus. The differentiation can be performed as it
follows:
if the turbidity disappears after the addition of acetic acid, it is
due to phosphates and urates which are soluble in acidic
medium;
if the turbidity disappears after increasing the temperature, it
shows the presence of urates;
if the turbidity exists after acid addition or heating, it is due to
pus.
The opalescent aspect can be produced by the inappropriate
storage of the urine, permitting the contamination with bacteria.
They produce ammonia and change the pH of urine into
alkaline. In these conditions, ammonia and manganese
phosphates and calcium carbonate precipitate. They become
soluble when adding few drops of acetic acid. Sometimes, few
hours after the collection, the urine can have an opalescent
aspect due to a suspension of cells from the urinary tract,
leukocytes, mucus that can settle in time and sediment
appears.
In pathological conditions, such as glomerulenephritis, cystitis,
pyelitis, the urine contains RBCs, WBCs, mucus, pus, hyaline
and granular casts which determine a more important sediment.
It can be brown-red or white when pus exists.
In chronic renal diseases the urine is “diluted”, looking like
water, due to the insufficient ability of the kidney to concentrate
the urine.
Odour.

Fresh urine has a characteristic odour.

It may be affected by food such as asparagus, garlic, horse-
radish.
Abnormal odour:
In diabetic acidosis there may be a fruit odour (green apple)
caused by ketone bodies.
In maple syrup disease (a rare genetic disease) the urine
has the odour of caramelized sugar or maple syrup.
A strong unusual odour of the new born urine shows an
aminoacidopathia (a metabolic disease).
A putrid odour means that the urine is infected (cystitis) or
has undergone bacterial decomposition because it stood
around too long without refrigeration.
Specific gravity (Density)

SG varies directly with the grams of solutes excreted per liter. It

provides information on the ability of the kidney to concentrate
the glomerulus’s filtrate. It depends on the kind of food and liquid
intake.
The usual range for 24 h specimen: 1.015 - 1.025.
The most concentrated specimen is obtained on arising in the
morning.
The density is increased in oliguria, in heart diseases, fever
Density can be increased when there is high excretion of
glucose as in diabetes mellitus even if there is polyuria; also
when proteins are excreted, in excess.
The density is decreased when polyuria is present. For
example, in renal failure, all the samples of urine of 24 h have
SG 1.010 - 1.011 (izostenuria).
The pH.

The urine has a physiologic pH range of 4.6 - 8.0 (mean 6.0)

It is acidic (5.2-5.3) when the food is of animal origin (due to
the protein metabolism final products) or weak basic (7.0-7.5)
in vegetarian diet.
At midnight the urine is the most acidic. In the morning the
urine is less acidic.
After a meal it becomes more basic because the H+ ions are
used in gastric digestion.
After storage, it can become basic (decomposition of urea in
NH3 by bacteria).
The urine acidity is determined by NaH2PO4 which exists in
excess in comparison with the plasma.
The plasma which is in the afferent arteriole has pH 7.4 (the
glomerulus filtrate has the same pH) and the ratio between
the acidic and the alkaline phosphates is
NaH2PO4/Na2HPO4 = 1/5,
The urine has the pH 6.0, due to the presence of the
following value of the ratio:
NaH2PO4/Na2HPO4 = 9/1.
This way, Na+ (and K+, Mg2+, Ca2+) are maintained in
organism.
Measurement of the pH is usually performed on fresh collected
urine.
1. The pH is measured by means of a paper strip impregnated
with an universal indicator, which is introduced in urine for
some seconds. The colour of the paper will be compared
with the chart of colours which is given, corresponding to
different values of pH.
2. A mixture of indicators can be used (Guillaumin): methyl red
0.06 g, brom-thymol blue 0.20 g, N/20 solution of NaOH 10
ml, distilled water ad 500 ml.
In 5 ml of fresh collected urine in a test tube, add few drops
of indicator mixture. Interpretation of the obtained colours:
– if the colour is red-yellow: the urine is acidic;
– if the colour is green: the urine is neutral;
– if the colour is blue: the urine is alkaline.
Abnormal pH
High values of urine pH (basic) appear in
– alkalosis,
– after the ingestion of alkali over a period of time for the
treatment of ulcer,
– after abundant vomiting of different causes,
– in the presence of bacteria generating ammonia (cystitis,
uretritis, pyelitis, pyelonephritis).
Low values of urine pH (acidic) appear during
– starvation,
– ketosis,
– renal insufficiency.
– acid producing salts are sometimes administrated for the
treatment of urinary tract infections.
 MICROSCOPIC EXAMINATION OF THE URINE SEDIMENT

Microscopic examination of urinary sediment is important

because it yields information that may be helpful in making a
diagnosis.
For best results, obtain a concentrated specimen (upon
arising) that has been clean-voided. The specimen should
be examined within an hour of voiding because cells
deteriorate upon standing; this process may be delayed by
refrigeration or by the addition of formalin (0.2 ml/dl urine).
Procedure: Centrifuge 10 ml of urine for 5 minutes. 9 ml of
the supernatant is discarded by decanting and the remaining
1 ml is used to re-suspend the sediment. One drop is
removed with a pipet, placed on a labelled glass slide and
topped with a cover slip.
Normal Findings:
Red blood cells (RBCS, erythrocytes), occasional or rare have
no pathological significance.
White blood cells (WBCS, leukocytes) have no pathological
significance if occasional or rare.
Epithelial cells:
– squamous epithelial cells (from the lower urinary tract), have
no particular significance;
– transitional epithelial cells (lining the renal pelvis, ureters,
urinary bladder, proximal urethra), few are expected to be
present.
Hyaline casts (containing proteins) may be found particularly
after stress, exercise or fever, in the absence of renal disease.
Bacteria may be present as an external contamination; clean-
voided specimens examined when they are fresh help to
eliminate possible confusion.
Abnormal elements:
Cells:
– Red blood cells more than occasional may originate from
any location in the urinary tract (in women can be of
genital origin);
– White blood cells in large number in freshly voided urine
indicate the presence of an infection in genitourinary tract.
Yeasts are common contaminants but can cause infections
in diabetics with glycosuria, in patients treated vigorously
with antibiotics.
Oval fat bodies, thought to be degenerated tubular epithelial
cells, filled with fat droplets, are usually present in all types
of diseases of renal parenchyma but are characteristic to
nephrotic syndrome.
Casts formed by precipitation of mucoprotein in the lumen of
tubules and collecting ducts pass into urine. They frequently
entrap cells.
RBCs casts, present erythrocytes in the protein matrix, are reddish-brown
or orange and denote glomerular inflammation and bleeding
(glomerulonephritis, systemic lupus erythematosus with kidney
involvement, glomerular diseases.
WBCs casts contain imbedded leukocytes and signify infection
(pyelonephritis).
Hyaline casts contain protein and are found in the urine when there is
proteinuria.
Granular casts contain epithelial cellular debris.
Fatty casts indicate a renal parenchymal disease.
Waxy casts are cellular casts that have degenerated and look like ground
glass and may be present in a number of kidney diseases.
Broad casts formed in the broad collecting tubules and are found only in
renal failure.
Crystals:
urate or uric acid crystals in large amount may indicate
excessive breakdown of the tissue cells (nucleoproteins)
or be present in gout;
aminoacids: leucine and tyrosine in severe liver disease,
cystine in an inherited metabolic affection (cystinuria);
hemosiderin after hemolytic episodes;
sulfonamides, pyridium after medication.