The Tryptophan Load Test

The tryptophtm load test for vitamin Be nutritiontd status (the ability to metabolize a test dose of tryptophem) is one of the oldest metabolic tests for functional vittunin nutritional status. It Wtis developed as a result of observation of the excretion of em abnormed-colored compound, later identified as the tryptophtm metabolite xanthurenic acid. 

Under normal conditions, the rate-limiting enzyme of the pathway is tryptophem dioxygenase (Section 8.3.2), emd there is little accumulation of intermediates. Kynurenine transeiminase, the enzyme which cateilyzes the transamination emd ring closure of kynurenine to kynurenic acid, and of hydroxykynurenine to xanthurenic acid, has a high relative to the normal steady-state concentrations of its substrates in the liver. Kynureninase and kynurenine hydroxylase have lower veilues of K n, so that there is normally little accumulation of kynurenine or hydroxykynurenine. 

Xanthurenic and kynurenic acids, and kynurenine and hydroxykynurenine, are easy to measure in urine, so the tryptophan load test, the ability to metabolize a test dose of 2 to 5 g (150 to 380 /rmol per kg of body weight) of tryptophan, was widely adopted as a convenient and sensitive index of vitamin Be nutritional status. 

In patients suffering from a wide veuiety of unrelated diseases, including Hodgkins lymphoma, rheumatoid arthritis, schizophrenia, porphyria, rened tuberculosis and aplastic anemia, there is abnormed excretion of kynurenine metabolites tdferatest dose of tryptophan (Altman emd Greengend, 1966 Coon and Nagler, 1969). It is unlikely that such disparate conditions would cdl be associated with vitamin Be deficiency. Liver biopsy shows elevated tryptophem 

It is appeuent that abnormally increased excretion of kynurenine metabolites after a test dose of tryptophan cannot necessarily be regarded as evidence of vitamin Be deficiency. This means that the tryptophan load test is unreliable as an index of status in epidemiological studies, although it is (probably) reliable in depletion/repletion studies to determine requirements. 

Induction of extrahepatic mdoleamine dioxygenase (which catalyzes the same reaction as tryptophan dioxygenase, albeit by a different mechanism) by bacterial lipopolysaccharides and mterferon-y may result in the production of relatively large amounts of kynurenine and hydroxykynurenine in tissues that lack the enzymes for onward metabolism. Kidney has kynurenine transaminase activity, and therefore extrahepatic metabolism of tryptophan may result in significant excretion of kynurenic and xanthurenic acids, even when vitamin Bg nutrition is adequate. 

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Metabolic loading tests and the determination of enzyme saturation with cofactor measure the ability of an individual to meet his or her idiosyncratic requirements from a given intake, and, therefore, give a nearly absolute indication of nutritional status, without the need to refer to population reference ranges. A number of factors other than vitamin intake or adequacy can affect responses to metabolic loading tests. This is a particular problem with the tryptophan load test for vitamin Be nutritional status (Section 9.5.4) a number of drugs can have metabolic effects that resemble those seen in vitamin deficiency or depletion, whether or not they cause functional deficiency. 

A preliminary investigation on tryptophan metabolism in aged subjects (over 70 years old) was carried out by Avogaro, Crepaldi, and Parpajola and by Benassi and Allegri, using the tryptophan loading test. 

When the tryptophan loading test was applied in 2 cases of cirrhosis of the liver, the results were the same as in normal persons, with a relatively slight urinary excretion of kynurenine and its acetyl derivative. 

Several tests are available for asses ing vitamin B status measurement of plasma levels of PLP, measurement of the percentage stimulation of red blood cell glula-matc-oxaloacetate aminotransferase, measurement of the daily excretion of urinary pyridoxic acid, and the tryptophan load test. 

At One time it was thought that women taking oral contraceptives were at risk for B deficiency. This notion seem-S to have been in error. The error was due to a misinterpretation of the tryptophan load lest. As mentioned earlier, a deficiency in vitamin B(,can induce the accumulation of specific intermediates of the tryptophan catabolic pathway and enhanced excretion in the urine. Oral contraceptives can also induce ar increase in the formation and excretion of specific intermediates by stimulating the activity of specific enzymes of the tryptophan catabolic pathway, This stimulation was responsible for the false indications of deficiency. Independently of the tryptophan load test, there continues to be some evidence for risk associated with the use of oral contraceptives. Oral contraceptive use may result in lowered levels of plasma vitamin Bf, Tlicsc lowered levels may result in a vitamin deficiency when coupled with pregnancy and lactation. 

H2. Hamfelt, A., Enzymatic determination of pyridoxal phosphate in plasma by decarboxylation of L-tyrosine- C(U) and a comparison with the tryptophan load test. Scand.. Clin. Lab. Invest. 20,1-10 (1967). 

H13. Hughes, P, A. M., and Raine, D. N., In vivo formation of pyridoxal phosphate Schiff s base—an inherent defect in the tryptophan load test. Clin. Chim. Acta 14, 399-402 (1966). 

Xanthurenic and kynurenic acids, and kynurenine and hydroxykynurenine, are easy to measure in urine, so the tryptophan load test (the ability to metabolize a test dose of 2—5 g of tryptophan) has been widely adopted as a convenient and very sensitive index of vitamin nutritional status. However, because glucocorticoid hormones increase tryptophan dioxygenase activity, abnormal results of the tryptophan load test must be regarded with caution, and cannot necessarily be interpreted as indicating vitamin B deficiency. Increased entry of tryptophan into the pathway will overwhelm the capacity of kynureninase, leading to increased formation of xanthurenic and kynurenic acids. Similarly, oestrogen metabolites inhibit kynureninase, leading to results that have been misinterpreted as vitamin B deficiency. 

Figure 11.16 The tryptophan load test for vitamin status.

The metabolism of methionine, shown in Figure 11.22, includes two pyridoxal phosphate-dependent steps cystathionine synthetase and cystathionase. Cystathionase activity falls markedly in vitamin deficiency, and as a result there is an increase in the urinary excretion of homocysteine and cystathionine, both after a loading dose of methionine and under basal conditions. However, as discussed below, homocysteine metabolism is affected more by folate status than by vitamin status, and, like the tryptophan load test, the methionine load test is probably not reliable as an index of... 

All of the studies that suggested that oral contraceptives cause vitamin deficiency used the tryptophan load test (section 11.9.5.1). When other biochemical markers of status were also assessed, they were not affected by oral contraceptive use. Furthermore, most of these studies were performed using the now obsolete high-dose contraceptive pills. 

A water-soluble vitamin of the B group. As pyridoxal phosphate, it is a cofactor for amino acid decarboxylation and transamination reactions. A deficiency produces symptoms of skin roughening. The pyridoxine status of the body can be determined by the tryptophan loading test (qv). 

Inhibition of kynureninase (e.g., by estrogen metabolites) also results in accumulation of kynurenine and hydroxykynurenine, and hence increased formation of kynurenic and xanthurenic acids, again giving results which falsely suggest vitamin Bg deficiency. This has been widely, but incorrectly, interpreted as estrogen-induced vitamin Bg deficiency it is in fact simple competitive inhibition of the enzyme that is the basis of the tryptophan load test by estrogen metabolites. 

While the tryptophan load test is a useful index of status in controlled depletion/repletion studies to determine vitamin Bg requirements, it is not an appropriate index of status in population studies. 

Some 10-25% of the population have a genetic predisposition to hyperhomocysteinemia, which is a risk factor for atherosclerosis and coronary heart disease, as a result of polymorphisms in the gene for methylenetetrahydrofolate reductase. There is no evidence that supplements of vitamin Bg reduce fasting plasma homocysteine in these subjects, and like the tryptophan load test, the methionine load test may be an appropriate index of status in controlled depletion/repletion studies to determine vitamin Bg requirements, but not in population studies. 

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