The Tryptophan Load Test

The tryptophan load test for vitamin B6 nutritional status (the ability to metabolize a test dose of tryptophan) is one of the oldest metabolic tests for functional vitamin nutritional status. It was developed as a result of observation of the excretion of an abnormal-colored compound, later identified as the tryptophan metabolite xanthurenic acid.

Apart from the relatively small amounts that are required for synthesis of the neurotransmitter serotonin (5-hydroxytryptamine), and for net new protein synthesis, essentially the whole of the dietary intake of tryptophan is metabolized by way of the oxidative pathway shown in Figures 8.4 and 9.4, which provides both a mechanism for total catabolism by way of acetyl coenzyme A and a pathway for synthesis of the nicotinamide nucleotide coenzymes (Section 8.3).

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

Kynureninase is especially sensitive to vitamin B6 depletion, because it undergoes self-inactivation as a result of catalyzing the half-reaction of transamination (Section 9.3.1.5). In vitamin B6 deficiency, the activity of kynureninase is lower than that of tryptophan oxygenase, the normal rate-limiting enzyme of the pathway, and there is accumulation of both hydroxykynurenine and kynurenine, permitting greater metabolic flux than usual through kynurenine transaminase, resulting in increased formation of kynurenic and xanthurenic acids.

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 ^mol per kg of body weight) of tryptophan, was widely adopted as a convenient and sensitive index of vitamin B6 nutritional status.

9.5.4.1 Artifacts in the Tryptophan Load Test Associated with Increased Tryptophan Dioxygenase Activity As discussed in Section 8.3.2, tryptophan dioxygenase is subject to both induction by glucocorticoid hormones and also stabilization by tryptophan and heme, and increased activity may well result in a greater rate of entry of tryptophan into the pathway than the capacity of kynureninase or kynurenine hydroxylase, thus leading to increased formation of kynurenic and xanthurenic acids. Because of the problem of enhanced stability of tryptophan oxygenase in the presence of high concentrations of tryptophan, it was suggested that the test dose used should be no more than 150 mol per kg of body weight, or 2 g for adults - a level at which there is only a negligible increase in the rate of tryptophan oxidation.

In patients suffering from a wide variety of unrelated diseases, including Hodgkins' lymphoma, rheumatoid arthritis, schizophrenia, porphyria, renal tuberculosis and aplastic anemia, there is abnormal excretion of kynurenine metabolites after a test dose of tryptophan (Altman and Greengard, 1966; Coon and Nagler, 1969). It is unlikely that such disparate conditions would all be associated with vitamin B6 deficiency. Liver biopsy shows elevated tryptophan dioxygenase activity, presumably because of increased glucocorticoid secretion as a result of the general stress of illness.

Induction of extrahepatic indoleamine dioxygenase (which catalyzes the same reaction as tryptophan dioxygenase, albeit by a different mechanism) by bacterial lipopolysaccharides and interferon-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 B6 nutrition is adequate.

It is apparent that abnormally increased excretion of kynurenine metabolites after a test dose of tryptophan cannot necessarily be regarded as evidence of vitamin B6 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.

9.5.4.2 Estrogens and Apparent Vitamin B6 Nutritional Status Rose (1966a, 1966b) was the first to report apparent vitamin B6 deficiency in women taking combined progestagen-estrogen oral contraceptives. There was increased urinary excretion of xanthurenic acid after a tryptophan load, which was normalized by the administration of relatively high doses of vitamin B6. A great many later reports have confirmed abnormal tryptophan metabolism among women taking the now obsolete high-dose oral contraceptives, and estrogens as menopausal hormone replacement therapy. Although they have been widely interpreted as evidence of estrogen-induced vitamin B6 deficiency or depletion, when other indices of vitamin B6 nutritional status have been measured, they have been unaffected by contraceptive use. This suggests an effect on tryptophan metabolism per se, rather than on vitamin B6 nutritional status.

Modern low-dose estrogen oral contraceptives do not affect tryptophan metabolism, although they may cause increased plasma and erythrocyte concentrations ofpyridoxal (Masse et al., 1996).

As discussed in Section 8.3.3, estrogen metabolites inhibit kynureninase and reduce the activity of kynurenine hydroxylase to such an extent that, even without induction of tryptophan dioxygenase (Section 9.5.4.1), the activity of these enzymes is lower than is needed for the rate of flux through the pathway, thus leading to increased formation of xanthurenic and kynurenic acids.

The gender difference in pellagra (Section 8.5) suggests that endogenous estrogens may have an effect on tryptophan metabolism similar to that of exogenous estrogens used as contraceptives. It implies that not only is the

Tryptophan Degradation Test

Figure 9.5. Methionine load test for vitamin B6 status. Methionine synthetase, EC 2.1.1.13 (vitamin B12-dependent); 2.1.1.5 (betaine as methyl donor); cystathionine synthetase, EC 4.2.1.22; and cystathionase, EC 4.4.1.1. Relative molecular masses (Mr): methionine, 149.2; homocysteine, 135.2; cystathionine, 222.3; and cysteine, 121.2.

Figure 9.5. Methionine load test for vitamin B6 status. Methionine synthetase, EC 2.1.1.13 (vitamin B12-dependent); 2.1.1.5 (betaine as methyl donor); cystathionine synthetase, EC 4.2.1.22; and cystathionase, EC 4.4.1.1. Relative molecular masses (Mr): methionine, 149.2; homocysteine, 135.2; cystathionine, 222.3; and cysteine, 121.2.

tryptophan load test unreliable as an index of vitamin B6 nutritional status in women taking estrogens, but also that it may be inappropriate for women in general (Bender, 1987).

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