Tryptophan Xanthurenic acid

R4. Raskin, I. M., Tryptophan-xanthurenic acid test in acute and chronic hepatitis. Vopr. Pitaniya 21, 33-38 (1962) Chem. Ahstr. 57, 14347 (1962). 

Cl. Gassmann, B., Knapp, A., and Gartner, L. L., Vitamin Be deficiency and urinary excretion of xanthurenic acid and other tryptophan metabolites in disease. Klin. Wochschr. 37, 189-195 (1959). 

The amount of vitamin B6 required by humans is not well established,73 and only recently has evidence been obtained that the needs are variable. Hansen and Bessey74 have found that in some babies 3 or 4 times as much vitamin B6 is needed to prevent the excretion of xanthurenic acid after a test dose of tryptophane than in others. It is these particular babies who develop clinical vitamin B6 deficiency when the intake is low. These findings seem to indicate strongly that some babies have vitamin B6 requirements 3 or 4 times as high as others. 

Purines such as xanthine (91), hypoxanthine (92), guanine (93), and uric acid (95) are found in excreta of many insects (Table VI) 48). Uric acid (95) is known to be the main end product of nitrogen metabolism in almost all insects. Various purines are found in the wasp Vespa) and the sawfly Gilpinia) in common with other insects (Table VI). In addition, various pteridines occur in Vespa and in the honeybee (Table VI). The latter also contains xanthurenic acid (52) or kynurenic acid (53), xanthurenic acid 4,8-digiucoside (56), and a yellow pigment, xanthommatin (58), as tryptophan metabolites (Table V). 

A predominant toxin (51) from water beetles of the genus llybius (Table V) shows a UV absorption corresponding to hydroxyquinoline or hydroxyiso-quinoline. The H-NMR spectrum exhibits, beside signals of methyl ester and phenol, signals of five aromatic protons as both ABC and AB systems, the latter indicating two protons at C-3 and C-4 in quinoline. Since electron pyrolysis of 51 gives radioactive 8-hydroxyquinoline, its structure is identified as methyl 8-hydroxyquinoline-2-carboxylate (51) and confirmed by synthesis from xanthurenic acid (52) (Scheme 48) (101). The precursor of this alkaloid was shown to be tryptophan (444). 

Tryptophan catabolism is also associated with several dead-end pathways, for example the formation of kynurenic and xanthurenic acids. Normal urine contains small amounts of hydroxykynurenine, kynurenine, kynurenic acid, and xanthurenic add. When large amounts of tryptophan are fed to animals, the excretion of these compounds increases. Xanthurenic acid is excreted in massive quantities in vitamin B6 deficiency. 

Figure 8.4. Pathways of tryptophan metaholism. Tryptophan dioxygenase, EC 1.13.11.11 formylkynurenine formamidase, EC 3.5.1.9 kynurenine hydroxylase, EC 1.14.13.9 kynureninase, EC 3.7.1.3 3-hydroxyanthranilate oxidase, EC 1.10.3.5 picolinate carboxylase, EC 4.1.1.45 kynurenine oxoglutarate aminotransferase, EC 2.6.1.7 kynurenine glyoxylate aminotransferase, 2.6.1.63 tryptophan hydroxylase, EC 1.14.16.4 and 5-hydroxytryptophan decarboxylase, EC 4.1.1.26. Relative molecular masses (Mr) tryptophan, 204.2 serotonin, 176.2 kynurenine, 208.2 3-hydroxykynurenine, 223.2 kynurenic acid, 189.2 xanthurenic acid, 205.2 and quinolinic acid 167.1. CoA, coenzyme A.

Urine xanthurenic acid after 2 g tryptophan load <65 xmol/24 h increase... 

The tryptophan load test for vitamin Bg 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. 

Under normal conditions, the rate-limiting enzyme of the pathway is tryptophan dioxygenase (Section 8.3.2), and there is hide 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 relative to the normal steady-state concentrations of its substrates in the liver. Kynureninase and kynurenine hydroxylase have lower values of K, so that there is normally litde accumuladon of kynurenine or hydroxykynurenine. 

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. 

The second quinoline derivative produced in animal metabolism is xanthurenic acid, which was isolated by Musajo (M12). Xanthurenic acid (4,8-dihydroxyquinoline-2-carboxylic acid) also originates from tryptophan through kynurenine (M13). 

In 1942 it was shown that the urine of pyridoxine-deficient rats contained large amounts of xanthurenic acid (L2). This was the starting point for studies on the interrelationship among vitamin Be, tryptophan, and protein metabolism. 

The diazo reaction, not given to kynurenine, is clearly produced by xanthurenic acid when present in urine. In view of this, Chiancone in 1935 administered 3 g L-tryptophan to a young man with bilateral tuberculosis and 1.5 g L-kynurenine to another young patient. By the methods ai ailable at that time, no xanthurenic acid was found in the urine of either individual (C4). 

In the same year paper chromatography was first attempted by Benassi (B4) for the simultaneous analysis of 8 tryptophan metabolites (kyn-urenine, 3-hydroxykynurenine, kynurenic acid, xanthurenic acid, anthra-nilic acid, 3-hydroxyanthranilic acid, 2-aminoacetophenone, and 2-amino-3-hydroxyacetophenone), separated by means of a mixtiue of methanol, n-butanol, benzene, and water and revealed through the fluorescence in ultraviolet light of 3655 A. Each compound elicits a different fluorescent color (cf. Table 1). 

Fic. 3. Chromatographic fractionation of a mixture of tryptophan metabolites on an ion-exchange column of Amberlite IR-120 ( 28 X 0.9 cm). The temperature was held at 37 °C and the flow rate was adjusted at 12 ml per hour with formic acid-pyridine buffers. The metabolite concentration is given as (ig/ml after fluorometric readings. Effluent was collected in 2-ml fractions. The following abbreviations are used KA, kynurenic acid XA, xanthurenic acid AHA, o-aminohippuric acid K, kynurenine 30HAA, 3-hydroxyanthranilic acid 30HK, 3-hydroxykynurenine. 

Another line of research was initiated by Chiancone (C4a) in 1950, using a tryptophan loading test. The xanthurenic index of Chiancone expresses, as a percentage of the administered tryptophan, the amount of xanthurenic acid excreted within 24 hours after tryptophan ingestion. 

We carried out several experiments by loading with 100 or 50 mg/kg body weight of L-tryptophan and determining kynurenine, N-a-acetyl-kynurenine, 3-hydroxykynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, and free anthranilic acid. These tests were performed in a variety of conditions and the results are discussed in the following sections. 

Boyland and Williams (B18) loaded 10 normal controls with 10 g DL-tryptophan. In spite of the fact that it is essential to use L-tryptophan in studies of this type and although data on excretion of kynurenic and xanthurenic acids are lacking, at least 5% was recovered as increased kynurenine, 3-hydroxykynurenine, anthranilic acid, and 3-hydroxyan-thranilic acid, free and conjugated. It should be pointed out that in these studies only a low percentage of the ingested dose of tryptophan was found in the form of urinary metabolites. The remainder, approximately 93-94%, of the load could not be recovered. 

Similar results were observed, using a different amount of DL-trypto-phan, in the xanthurenuria of women at the end of normal pregnancy (E3). However, in one investigation, of eight women well advanced in pregnancy, only one excreted excessive xanthurenic acid after a test dose of DL-tryptophan and this pregnancy was complicated by disease (H5). The excessive excretion in this case was reduced with pyridoxine. 

Total and % Excretion of Kynubenic and Xanthurenic Acids by 18 Pregnant Women AT Different Stages of Pregnancy after Tryptophan Loading ... 

Various reports in the literature indicate the influence of endocrine organs on tryptophan metabolism. Chiancone and co-workers (C5, V2) reported that ovariectomy or hypophysectomy of rats caused increased excretion of xanthurenic acid and that adrenalectomy caused a decrease. An adrenal mechanism is suggested for the regulation of 3-hydroxy-anthranilic acid conversion to nicotinic and picolinic acids (M7). 

Brown et al. (B25) confirmed the results of Coppini and Camurri (C9) on excretion of kynurenic and xanthurenic acids and, at the same time, examined the excretion of other tryptophan metabolites. Their data indicate that the high levels of all urinary metabolites excreted by pregnant subjects were lowered by pyridoxine administration. It must be remembered that the requirement for pyridoxine in pregnancy varies in the different animal species (C6). It was also found that the levels of pyridoxine in the fetal blood are elevated whereas those of maternal blood decrease (GIO). 

Weller and Fichtenbaum (WIO) followed the urinary excretion of xanthurenic acid in 48 arteriosclerotic patients after an oral dose of 10 g DL-tryptophan. On the basis of urinary xanthurenic acid above the range of normal controls, 29% of these patients showed a deficiency of vitamin Bg. Using the same criterion, pyridoxine deficiency was found in 60% of diabetic patients with arteriosclerotic complications. 

Tryptophan metabolism was investigated in neurological diseases (V5, Cl) in which the xanthurenic acid excretion was measured after ingestion of L-tryptophan (100 mg/kg). The greater amounts of xanthurenic acid excreted by certain patients suggested an alteration of tryptophan metabolism, probably related to pyridoxine deficiency. 

In patients suffering from schizophrenia urinary excretion of both N-methyl-nicotinamide and xanthiurenic acid was determined (L6) after 10 g DL-tryptophan loadings. Excretion of the former was significantly decreased, in comparison with that of normal controls, whereas an increase was found in that of xanthurenic acid. 

Harris et al. (H4) reported a case of an adult patient with hematological abnormalities unresponsive to the usual hematopoietic agents and characterized by a hypochromic anemia, leucocytosis, high serum iron, and high percent iron-binding protein saturation. Measurements of the urinary excretion of kynurenine, kynurenic acid, acetylkynurenine, xanthurenic acid, o-aminohippuric acid before and after an oral dose of 4 g L-tryptophan indicated abnormalities of tryptophan metabolism. This alteration was partially normalized on a 1-mg pyridoxine dose and completely normalized at the 10-mg level. Also the clinical and laboratory abnormalities disappeared and hematological remission followed the pyridoxine administration. 

By determining only xanthurenic acid, Gehrmann (G2) fonnd that patients with neoplastic anemias excreted large amounts of this metabolite after a 10-g load of nL-tryptophan. Such a xanthurenuria was slightly positive in some cases of leukemia and multiple myeloma and fairly normal in patients with Hodgkin s disease. 

Xanthurenic acid excretion was followed in a group of 20 patients with diflFerent forms of anemia after a 10-g load of DL-tryptophan (R2). Abnormal increasing of urinary xanthurenic acid was considered by Rade-maker and Verloop an early diagnostic signal of a pyridoxine deficiency. A case of hypochromic anemia, unreactive to any therapy but pyridoxine, showed a normal excretion of xanthurenic acid. On the contrary, an increase on excretion of xanthurenic acid was observed in some iron-deficiency anemia patients whose anemia was not Bg-dependent (R2). 

---