Serum Transthyretin

Transthyretin, known also as prealbumin or thyroxine-binding prealbumin,

serves as a transport protein for thyroxine, and as a carier protein for RBP
(Section 16.2.4). It has a longer half-life (i.e., 2d) (table 16.3) and a slightly larger
body pool (0.010 g/kg body weight), compared to RBP, although the sensitivities
of these two serum proteins to protein deprivation and treatment are similar.
Transthyretin has a high content of the amino acid tryptophan and a high
proportion of essential to nonessential amino acids. Thus it can be used as an
indicator of the availability of essential amino acids in the body (Spiekerman,
1993.).
Serum Transthyretin can be used in hospital settings as a screening tool to
identify patiens at risk for protein-energy malnutrition; more details are given in
Section 27.1. Potter and Luxton (1999) examined the results of using
Transthyretin as a routine diagnostic test for protein-energy malnutrition in
emergency room admissions. They emphasized that Transthyretin was a more
sensitive indicator of protein-energy malnurition than was serum albumin and was
significantly associated with length of hospital stay.
Serum Transthyretin responds rapidly to the short-trem effects of
nutritional therapy. As a result, serum Transthyretin is also used to monitor the
progress of patiens receiving total parenteral nutrition postoperarively and during
the transition from total parenteral nutrition to oral or enteral feeding (Winkler et
al., 1989). Table 16.10 shows results for plasma RBP, Transthyretin, transferrin,
and albumin in 68 patiens receiving total parenteral nutrition (TPN).
Improvements in serum Transthyretin and RBP occurred within the first week of
TPN and persisted throughout its duration, whereas there was no improvement in
transferrin until the end of the therapy. In contrast, there were no significant
differences in mean serum albumin levels between the pre- and post-TPN levels
(Winkler et al., 1989).

Table 16.10. Plasma protein concentrations during total parenteral

nutrition (TPN). Values are percentages of patiens (n = 68) who have
normal concentrations of the proteins. *p < 0.05 vs. pre-TPN by chi-
square. From Winkler et al., Journal of the American DieteticAssociation
89: 684-687, 1989 @ with permission of the Anerican Dietetic
Association.
 Other investigator have also reported a rapid rise in serum Transthyretin
levels in contrast to the much longer time required to show an increase in serum
albumin. Indeed, Bernstein et al. (1995) reported weekly increases in serum
Transthyretin of 40-50 mg/L in response to adequate nutritional support and
suggested that a response of less than 20 mg/L in a week is indicative of either
inadequate nutritional support or a failure to respond to the treatment.
Deficiencies of vitamin A, zinc and iron do not affect the levels of
Transthyretin, unlike those of RBP and tranferrin, as noted previously. In contrast,
the presence of other conditions-such as gastrointestinal diseases, hepatic and
kidney diseases, surgical trauma, stress, inflammation, and infection-leads to
modifications in the metabolism of Transthyretin and reduces its specificity as an
index of protein status. Nevertheless, hepatic disease does not affect Transthyretin
as early or to the same extent as the other serum proteins, particulary RBP.
Further, although serum Transthyretin is moderately elevated in renal diseases due
to decreased catabolism by the kidney, nonetheless, in patients with stable renal
failure, the direction of change in Transthyretin concentration rather than the
equacy of nutritional therapy (Winkler et al., 1989).
The response of serum transthyretin concentrations to dietary protein
intake has also been investigated. Shetty et al. (1979), reported that serum
transthyretin concentrations did not respond to a short-term (10-d) protein
restriction if energy intake was maintained, but levels fell markedly when both
energy and, to a lesser extent. Protein intakes were restricted (Table 16.9).
Ramsey et al. (1992) suggested that because of its very short half-life, serum
transthyretin probably reflects acute protein intake rather than risk of protein
malnutrition, which occurs over about 7 to 10 d. Nevertheless, Ogunshina and
Hussain (1980) noted an inverse relationship of plasma transthyretin
concentrations to the severty of malnutrition as assessed by the anthropometric
classification of Waterlow (1972) (Figure 16.3 Section 13.2.5).

Figure 16.3 Plasma transthyretin (mean ± SE) levels in children classified

as normal or with mild, moderate, and severe protein-energy malnutrition
according to the Waterlow classification of protein-energy malnutrition.
From Ogunshina and Hussain, American Journal of Clinical Nutrition 33 :
794-800, 1980 @ Am J Clin Nutr. American Society for Clinical
Nutrition.
 Table 16.11 : Reference medians and selected percentiles for serum
transthyretin (g/L). stratified by age and gender. Data from Ritchie et al.,
Journal of Clinical Laboratory Analiysis 143 : 273-279, 1999 @ Wileyliss
Inc., with permission.

Certain medications can influence serum transthyretin levels. Levels rise

with anti inflammatory medications including some over-the-counter products
Ingelbleek and Young, 1994). They are also altered by endogenous estrogen (e.g.,
during pregnancy) (Haram et al., 1983) or the use of estrogen containing
preparations (e.g., oral contraceptive agents, estrogen replacement therapy).
Transthyretin values vary according to age and sex (Ritchie et al., 1999b).
Values rise until the second or third decade of life and then decrease thereafter
(Table 16.11). Males have higher values than females during midlife, this
difference being highest at about age 40 y (i.e., 16%).

Interpretive Criteria
Interpretive values for adults based on neph elometry and the extent of the
protein deficit are given in Table 16.8. Nutrition risk is high when serum
transthyretin concentrations fall below 0.11 g/L, whereas poor outcome is
predicted when a level < 0.50 g/L is observed (Logan and Hildebrandt, 2003).
Table 16.11 presents selected percentiles for serum transthyretin by age
and gender, derived from the same Caucasian cohort used for both serum albumin
and serum transferring (Ritchei et al., 1999b).

Measurement of Serum Transthyretin

Serum levels of transthyretin are four to five times higher than those of
RBP and thus are easier to measure. Radial immunodiffusion techniques can also
be used to determine transthyretin using commercial assay kits, as described for
transferring and RBP. Other more sensitive and nephelmetry and turbimetry, both
of which can operate on large automated chemistry analyzers.

Insulin-Like Growth Factor I

Insulin-Like growth factors are growth-hor-mone-dependent serum growth
factors which are produced mainly in the liver. They have a pro-insulin-like
structur and board anabolic properties. The insulin-like growth factors I (IGF-I),
sometimes referred to as somatomedin-C, circulates bound to carrier proteins and
has a half-life of 12-15 h (Thisen et al., 1994).
In studies of children suffering chronic undernutrition, decreased
concentrations of circulating IGF-I in the serum occur, which respond rapidly to
dietary treatment (Smith et al., 1981). When acutely malnourished patiens
received nutritional support for 3-16 d, IGF-I levels increased from initial levels,
although no significant changes in serum albumin, transferring, RBP, or
transthyretin concentrations occurred (Unterman et al., 1985). These results
suggest that serum IGF-I may be more sensitive to acute changes in protein status
than are the other serum proteins. Further, serum IGF-I levels are not subject to
diurnal variation and are not ingluenced by stress, sleep, or exercise, although
levels are decreased in patients with hypothyroidism and with estrogen
administration. in patients with liver disease, kidney failure, and several
autommune diseases, serum IGF-I levels are very variable, unless the carrier
protein is completely removed by the acid chromatography exraction method, as
noted below.
More studies are necessary to establish the sensitivity and specificity of
IGF-I measurements as an index of malnutrition before itcan be used as a routine
biochemical marker of protein status and as marker for the response to nutritional
therapy.

Measurement of insulin-like growth factor I

IGF-I can be assayed using commercially available radioimmunoassay
kits, provided a gamma counter is available. Quality controls are provided with
the assay kits. Other immunoassays inculuding immunoradiometric assays,
enzyme-linked immunsorbent assays (ELISAs), and immunofunctional assays are
also used. A number of different commercial kits are now available (Popii and
Bauman, 2004).
Prior to the assay of IGF-I, any interfering binding proteins must be
removed from the sample by an extraction procedure. Either acid-gel
chromatography or acid-ethanol extraction is the most frequently used method. Of
these, acid-gel chromatography is more difficult technically, but it is preferred
because it removes 98% of the binding protein with a recovery of IGF-I of
approximately 75%.
Either serum or plasma specimens can be used for the assay,but they
separated as soon as possible after collection. Serum must be frozen promptly
after separation to avoid falsey high values (Isley et al., 1990). Samples can be
frozen at - 200C, but repeated freezing and thawing must be avoided.
Plasma Alkaline ribonuclease activity and fibronectin.
The activity of plasma alkaline ribonuclease (EC 3.1.4.2.2) has been
investigated as a measure of protein status. Enzyme activity is reportedly
increased in infants and children with protein malnutrition but returns to normal
within 2-4 wk after rehabilitation (Scott et al., 1984). The measurement may be
especially useful for monitoring the response of malnourished patients to nutrition
interventions. A method for measuring alkaline ribonuclease activity in serum has
been developed by Scoot (1979).
Young et al. (1990) suggest that concentrations of fibronection may also
have potential as an index of protein status, and this warrants further study.
Fibronectin is a glycoprotein which, unlike other serum proteins discussed above,
is not synthesized exclusively by the liver but also by the endothelial cells,
peritoneal macrophages, and fibroblasts. It has a half-life of about 15h.
Fibronectin serves numerous physiological functions, including cell-matrix
interactions, and the binding of macrophages and fibroblasts. In protein
malnutrition, levels of fibronectin are reduced, but they return to normal rapidly
after nutritional rehabilitation (Buonpane et al., 1989). Fibronectin, like the other
serum proteins shown in Table 16.3, alo responds to certain non-nutritional
factors, including infection, trauma, and burns, but again returns to normal on
recovery. Special precautions must be taken when collecting serum samples for
the assay of fibronectin. Analysis can be performed by competitive enzyme
immunoassay (Ylatupa et al., 1993).

Metabolic changes as indices of protein status

Several striking changes in metabolism can develop in response to a
reduced or inadequate supply of dietary protein or of some specific indispensable
amino acids, the changes have been reviewed by Young and Marchini (1990). Of
these, changes in free amino acid profiles in plasma have been described in
children with frank kwashiorkor that are much more pronounced than those for
children with the marasmic form of protein energy malnutrition. Such changes
have been linked to the hyperisulinemia that occurs in kwashiorkor, which, in
turn, results in characteristic alterations in serum amino acid concentrations via its
effect on muscle protein sythesis (Coward and Lunn, 1981). Other metabolic
changes, such as reduced urinary hydroxyproline excretion and increades urinary
nitrogen excretion, occur in both the kwashiorkor and the marasmic forms of
protein energy malnutrition, and these changes are used as less-specific indices of
protein energy malnutrition.

Plasma amino acid ratio

Concentrations of free amino acids in plasma have been extensively
studied in children with maramus and frank kwashiorkor. Earlier work suggested
that the serum amino acid profile of children with frank kwashiorkor was
markedly abnormal (Coward and Lunn, 1981). Concentrations of nonessential
amino acids (NEAA) such as alanine, glycine, serine, and praline were elevated,
whereas those for the essential (indispensable) amino acids (EAA) were lowered,
resulting in a high NEAA to EAA ratio. Such changes in serum amino acid
profiles were much less pronounced in marasmic children.
Based on apparent differences in serum amino acid profiles, Whitehead
and Dean (1964) developed a simplified technique for use in the field to
determine serum amino acid ratios using one dimensional paper chromatography
and a fingerprick blood sample. The method was devised in an effort to
distinguish between subclinical kwashiorkor and marasmus. Unfortunately,
plasma NEAA, EA ratios didn’t show a consistent response in relation to the type
or severity of protein energy malnutrition. Saunders et al. (1967) reported that the
amino acid profile reflected the dietary protein intake immediately prior to the test
and plasma amino acid profiles are no linger used to assess protein status.

Urinary 3-hydroxyproline excretion

Urinary 3- hydroxyproline, principally in the peptide from, is an excretory
product derived from the soluble and insoluble collagens of both soft and calcified
tissues. In malnourished children with impaired growth resulting from
kwashiorkor, urinary 3- hydroxyproline excretion levels are significanly lower
than those in age-matched, well-nourished controls. In adults, levels of 3-
hydroxyproline in the urine can be used as a marker of bone resorption and,
hence. Can help diagnose certain metabolic bone diseases and conective tissue or
endocrine disorders. A variety of extraneous factors influence urinary 3-
hydroxyproline excretion. These are out lined in box 16,4.

Interpretive Criteria
The marked changes in hydroxyproline excretion with age and sex necessitate the
use of age and sex-specific interpretive reference data. investigators have
therefore developed alternative methods for evaluating urinary hydroxyproline
excretion levels which are independent of age. excretion can be expressed as the
hydroxyproline creatinine ratio. this ratio corrects at least partially for differences
adult body size.

Box 16.4 : Factors affecting urinary 3- hydroxyproline excretion.

Figure 16.4 : The hydroxyproline : creatinine ratio of normal individuals from 6
mo to 17 y. From Allison et al., Clinica Chimica Acta 14 : 1729-734, 1966.

As result, ratios in adults are independent of age and are similar in males and
females. For children, however, the ratio changes rapidly with age as shown in
Figure 16.4 : hydroxyproline decreases with age, while creatinine exretion
increases.

As an alternative, hydroxyproline excretion can be expressed as the

hydroxyproline index. Whitehead (1965) developed this index as an age-
independent interpreative criteria for use with children, which additionally
attempts to take body weight into account :

In normal children, between 1 and 6 y, the hydroxyproline index is

relatively constant and is approximately 3.0. In children, however, the
hydroxyproline index is low, irrespective of the type of malnutrition, and
statistically related to the extent of the weight deficit (Whitehead, 1965).
Interpretive guidelines for the hydroxyproline index are shown in table 16.12
(Sauberlich, 1999).

Table 16.12 : Guidelines for the interpretation of urinary hydroxyproline index,

creatinine height index, and urinary urea nitrogen : creatinine ratios. From
Sauberlich (1999)

Measurement of hydroxyproline in urine

The use of 24-h urine specimens is preferred, but it is not practical for
children. Instead, early morning fasting samples can be colected, thereby
minimizing the effects of the deitary ingestion of hydroxyproline. Prockop and
Udenfriend (1960), LeRoy (1967), and Dabev and Struck (1971) have all
described methods for the analysis of hydroxyproline in urine. The method of
Prockop and Udenfriend (1960) requires that the urine samples are frist
hydrolyzed, then decolorized and neutralized prior to oxidation to pyrrole. Pyrole
is estimated colorimetrically after coupling with p-dimethylaminobenzaldehyde.

Nitrogen Balance
Nitrogen balance is a measure of the net status of protein metabolism. It
provides no information on the size of the protein stores or about nutritional
status. Nitrogen balance is most useful when used to monitor changes arising from
nutritional therapy.
The nitrogen balance method is based on the assumption that nearly all of
total body nitrogen is incorporated into protein. As protein contains 16% nitrogen.

In healthy adults with adwquate energy and nutrient intakes, nitrogen

losses are dependent mostly on the amounts and proportions of essential amino
acids in the diet and on the total nitrogen intake. When nitrogen intakes are
adequare to replace the endogenous nitrogen losses and for the growth of hair and
nails, the subject is said to be in nitrogen balance. in such conditions, a correlation
between daily nitrogen intake and daily nitrogen excretion can be expected. The
expression for nitrogen.
 Where I=nitrogen intake (protein/6.25), U=total urinary nitrogen, Ue =
endogenous urinary nitrogen, F = nitrogen voided in feces (as unabsorbed
protein), Fe = endogenous fecal nitrogen losses, and S = dermal nitrogen losses.
Nitrogen intake. is generally estimated from the protein intake. For mixed
diets, approximately 16% of the protein intake can be assumed to be nitrogen. For
parenteral solutions containing free amino acids, specific conversion factors can
be used to calculate the nitrogen content exactly. Alternatively, nitrogen intake
can be determined accurately by analyzing the nitrogen content of the diets or
parenteral/enteral formulas using the micro-Kjeldahl technique.
When the nitrogen intake exceeds nitrogen output, subjects are in positive
balance. This occurs during growth, late pregnancy, athletic training, and recovery
from illiness. in Contrast, when nitrogen output exceeds nitrogen intake, subjects
are in negative nitrogen balance, subjects are in negative nitrogen balance. A
negative nitrogen balance of 1 g/d is equivalent to a reduction in total body protein
of 6.25 g/d. If the negative balance persists, the resultant protein depletion may
have adverse effects on all organ systems.
Several factors may precipitate a negative nitrogen balance. They include
inadequate protein or energy intakes, an imbalance in the NEAA : EAA ratio,
conditions of accelerated protein catabolism (e.g., trauma, sepsis, infection, and
burns), and excessive loss of nitrogen arising from fistulas or excessive diarrhea.
The range of nitrogen balance values observed in hospital patients can vary from
+4 to -20 g of nitrogen per day.
About 90%-95% of daily nitrogen losses are excreted in the urine, the
remainder being lost through nasal secretions, menstrual fluid, semen, and nail
and hair cuttings also occur. As a result, measurements of urinary nitrogen
excretion and nitrogen intake over a defined period of time can be used to estimate
the state of nitrogen balance.
Note that nitrogen balance results tend to be biased toward a positive
balance. This bias arises because intakes tend to be over estimated as a result of
uncosumed food, whereas excretion tends to be underestimated because of
unmeasured nitrogen losses. Some of the sources of error and limitations
associated with nitrogen balance studies are listed in Box 16.5. These must be
kept in mind interpreting nitrogen balance results (Kopple, 1987).

Estimated nitrogen balance and apparent net protein utilization.

Total urinary nitrogen levels are rarely measured in routine hospital
clinical laboratories because the conventional Kjeldahl technique is time
consuming and laborious. Instead, urinary urea nitrogen is more frequently
determined and can be used to replace the estimation of total urinary nitrogen in
some circumstances. This is possible because individuals who are consuming a
normal mixed Western diet, and are in overall nitrogen balance, > 80%-90% of
the total nitrogen in the urine is normally excreted as urea (Bingham et al., 1988).
Moreover, the excretion of the nonurea nitrogen components (e.g., creatinine
nitrogen 6.4%, ammonia nitrogen 7,4%, uric acid nitrogen 2%-3%, and other
minor nitrogenous compounds 1%-2%,) remains fairly stable in such conditions
(Allison and Brid, 1977).
 Box 16.5 : Limitations and sources of erros in nitrogen balance studies.

Figure 16.5 : The relationship of total urinary nitrogen to urinary urea nitrogen for
81 subjects and four groups of hospital patients. From Blackburn et al., Journal of
Parenteral and Enteral Nutrition 1 : 11-22, 1977 @ American Society for
Parenteral and Enteral Nutrition. From Allison and Brid (1977). Elimination of
nitrogen from the body. In : Munro HN, Allison JB (eds) Mammalian Protein
Metabolism, Volume 1. American Medical Association @ Acasemic Press, with
permission.
 Figure 16.5 depicts the relationship between measured total urinary
nitrogen and measured urinary urea nitrogen observed in 81 patiens for 564 study
days and with a variety of clinical and nutritional conditions. The difference
between the total nitrogen and the urea nitrogen in the urine averaged 1.8 g/d
(range 0-5.8 g/d) with a standard deviation of 0.9 g/d (Blackbrun et el., 1977 ;
Mac Kenzie et al., 1985). On the basis of these results, an estimate of 2 g of
nitrogen per day is commonly used for the nonurea nitrogen components of the
urine.
The estimated nitrogen balance can be calvulated from the urinary urea
nitrogen using the following equation :

In this equatin, two correction factors are included : 2 g for dermal and
fecal losses of nitrogen which occur but which are not measured; and 2 g for the
nonurea nitrogen components of the urine (e.g., ammonia, uric acid, and
creatinine), which are also not measured (MacKenzie et al., 1985).
Sereval.