3-Hydroxyanthranilic acid from 3-hydroxykynurenine

Alkaloids derived from nicotinic acid contain a pyridine nucleus. Nicotinic acid itself is synthesized from L-tryptophan via A-formylkynurenine, L-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid. 

It was furthermore reported (K20) that nicotinic acid-deficient animals would grow only if given tryptophan, thus suggesting the conversion of tryptophan to nicotinic acid. Not only is tryptophan converted to nicotinic acid but also kynurenine and 3-hydroxyanthranilic acid. The peculiar degradation of the latter to pyridine derivatives gave rise to many interesting investigations. 3-Hydroxyanthranilic acid is derived from 3-hydroxykynurenine, another important tryptophan metabolite, the his-... 

The pieces of paper with 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and xanthurenic acid are each placed in an Erlenmeyer flask fitted with a ground-glass stopper and containing 3.8 ml distilled water. After 15-16 hours, 1 ml diazotized sulfanilic acid (0.5% in 2% hydrochloric acid mixed immediately before using with an equal volume of 0.5% solution of sodium nitrite in water) and 0.2 ml pyridine are added. The temperature must be held constant at 15° to obtain reproducible results. The color resulting from xanthurenic acid is read immediately at 510 mp, and of the other two derivatives after 60-80 minutes at 450 mp. 

A column of Amberlite IR-120, 0.9 X 28 cm, held constant at 37°C and formic acid-pyridine buffers are employed. With a buffer formic acid-pyridine 0.2 N, pH 2.50-2.60, kynurenic, xanthurenic, and o-amino-hippuric acids are eluted successively from the column. By increasing molarity and pH, respectively, to 0.3 N and 4.20 there emerge kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine, which are collected automatically in fractions of 2 ml. Figure 3 gives an example of chromatographic separation. 

From our investigation it is evident that abnormal excretion of tryptophan metabolites is not a typical feature of bladder tumor subjects, since human beings with neoplastic and nonneoplastic extrabladder urinary diseases have also been found to excrete spontaneously elevated amounts of tryptophan derivatives. It seems that the metabolic abnormality is not restricted to bladder tumors, but is rather more specific for patients with tumors of the upper urinary tracts and of the renal parenchyma. Actually 59% of these patients (Fig. 4) excreted abnormal amounts of kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid. 

This conversion has been found to be catalyzed by an enzyme isolated from the liver and kidney of several mammalian species (6Jir67), from tryptophan-adapted Pseudomonas (68, 69), and from Neurospora (60). The enzyme appears to be identical with the system which causes the hydrolysis of 3-kynurenine to anthranilic acid. The products of the hydrolysis of hydroxykynurenine are hydroxyanthranilic acid and alanine. Pyri-doxal phosphate is required for the reaction. 

Kynurenine is oxidized (again by atmospheric oxygen) to yield S-hydroxy-kynurenine and subsequently is cleaved to 3-hydroxyanthranilic acid and alanine. The fate of alanine has already been presented. Sidepaths from kynurenine and 3-hydroxykynurenine lead to kynurenic axid and xanthurenic acid] this reaction is explained as a transamination followed by spontaneous ring closure. 

Most of the kynurenine formed is, however, hydroxylated to xanthurenic acid and 8-hydroxyquinaldic acid. The enzyme kynureninase yields from 3-hydroxykynurenine 3-hydroxyanthranilic acid, which is then degraded via the glutarate pathway. 

Tryptophan is an essential amino acid involved in synthesis of several important compounds. Nicotinic acid (amide), a vitamin required in the synthesis of NAD+ and NADP+, can be synthesized from tryptophan (Figure 17-24). About 60 mg of tryptophan can give rise to 1 mg of nicotinamide. The synthesis begins with conversion of tryptophan to N-formylkynurenine by tryptophan pyrrolase, an inducible iron-porphyrin enzyme of liver. N-Formylkynurenine is converted to kynurenine by removal of formate, which enters the one-carbon pool. Kynurenine is hydroxylated to 3-hydroxykynurenine, which is converted to 3-hydroxyanthranilate, catalyzed by kynureninase, a pyridoxal phosphate-dependent enzyme. 3-Hydroxyanthranilate is then converted by a series of reactions to nicotinamide ribotide, the immedi-... 

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