Hydroxyanthranilic acid, tryptophan

Administration of quinolinic acid promotes the growth of niacin-deficient rats, but in this respect it is far less effective than either niacin or tryptophan. 3-Hydroxyanthranilic acid can be converted to quinolinic acid by rat liver slices and homogenates, and by enzyme extracts. However, it has not been found possible to convert quinolinic acid to nicotinic acid by incubation with liver slices. Neither has it been possible to convert tryptophan or kynurenine to quinolinic acid by these in vitro procedures. ... 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

The biosynthesis and metabolism of nicotinic acid in disease has received little attention metabolic studies deal mainly with normal animals and man (01, R5). After a tryptophan load dose, the main catabolites in the urine are nicotinuric acid, N1-methylnicotinamide, nicotinamide, quinolinic acid, kynurenine, 6-pyridone, anthranilic acid, and 3-hydroxyanthranilic acid. These excretory products were estimated... 

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. 

Precursors in the biosynthesis of niacin In animals and bacteria, tryptophan and in plants, glycerol and succinic acid. Intermediates in the synthesis include kynurenine, hydroxyanthranilic acid, and quinolinic acid. In animals, the niacin storage sites are liver, heart, and muscle. Niacin supplements are prepared commercially by (1) Hydrolysis of 3-cyanopyndine or (2) oxidation of nicotine, quinoltne, or collidine. 

Kynureninase is involved in the oxidative metabolism of tryptophan. It catalyzes the conversion of L-kynurenine to anthranilic acid. The enzyme also converts L-3-hydroxykyneurenine to 3-hydroxyanthranilic acid. The latter compound has a high fluorescence, which is the basis for detection in this assay. 

Animals and yeasts can synthesize nicotinamide from tryptophan via hydroxyanthranilic acid (52) and quinolinic acid (53, Fig. 6A) (31), but the biosynthetic capacity of humans is limited. On a diet that is low in tryptophan, the combined contributions of endogenous synthesis and nutritional supply of precursors, such as nicotinic acid, nicotinamide, and nicotinamide riboside, may be insufficient, which results in cutaneous manifestation of niacin deficiency under the clinical picture of pellagra. Exogenous supply of nicotinamide riboside was shown to promote NAD+-dependent Sir2-function and to extend life-span in yeast without calorie restriction (32). 

B) in plants and bacteria. 50, dihydroxyacetone phosphate 51 tryptophan 52, hydroxyanthranilic acid 53, quinolinic acid 54, aspartate 55, nicotinic acid mononucleotide 56, NAD, 57, NADP. 

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-... 

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. 

As previously mentioned, 3-hydroxyanthranilic acid was found chro-matographically by Musajo et al. (M18) in the urine of tuberculous patients. This was the starting point for an extensive investigation of tryptophan metabolites excreted spontaneously, i.e., by normal subjects or patients with different diseases all fed a normal diet without added tryptophan. 

Bladder and Kidney Cancer and Other Urological Diseases. The excretory pattern in cases of bladder tumor has been studied for many years in our laboratory (B5, B8) after Boyland and Williams (B18) had suspected that o-aminophenolic metabolites of tryptophan (i.e., 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and 2-amino-3-hydroxy-acetophenone) might be endogenous agents of bladder cancer simi-... 

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. 

It was recently observed (K2) that a kidney with a small tumor excretes more of the so-called carcinogenic o-aminophenols (3-hydroxykynurenine and 3-hydroxyanthranilic acid) than the opposite healthy kidney after a 100 mg/kg loading dose of L-tryptophan. The small size of the hypernephroma encountered at nephrectomy and also the higher excretion of carcinogenic metabolites of tryptophan previously thought to be restricted to tumors of transitional-cell epithelium (K2) are emphasized. 

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. 

It is generally accepted that under normal conditions human subjects convert the largest part of an ingested dose of L-tryptophan to non-aromatic products. The last identified benzene derivative on this pathway is 3-hydroxyanthranilic acid when its ring is opened to form unstable intermediates, apparently only a small percentage of them are converted to niacin (H8). 

These patients appear to excrete a slightly higher amount (8.9-12.3%) of tryptophan derivatives than normals (average 6.7%), The excretion ratios are different for each metabolite the highest ratio is found for 3-hydroxykynurenine and the lowest for 3-hydroxyanthranilic acid. 

Paper chromatographic analysis indicated that the abnormal excretion of xanthurenic acid always corresponded with higher amounts of other tryptophan metabolites such as kynurenine, acetylkynurenine, 3-hydroxy-kynurenine, and kynurenic and 3-hydroxyanthranilic acids after the usual load of tryptophan. 

Chizhova and Ivanova (C7) studied 20 children, aged 1-12 years, under therapy for leukemia and 10 healthy children as control. A total of 15-20 g of tryptophan was administered during 5-10 days (1.5-3 g/day) to 7 children whereas 13 were given a single dose of 2-3 g. Daily determinations of urinary metabolites by paper chromatography demonstrated a disturbance of tryptophan metabolism in 19 of the 20 leukemic children before and after tryptophan loading. Kynurenine, 3-hydroxykynurenine, and anthranilic and 3-hydroxyanthranilic acids appeared in urine, whereas 5-hydroxyindoleacetic acid was absent in the majority of the young patients. The disturbances of tryptophan metabolism were similar in all of them. Administration of vitamin Be restored tryptophan metabolism to normal in the majority of the patients. 

Alloxan-diabetic animals, in which the conversion of tryptophan to methylnicotinamide was greatly impaired, excreted increased amounts of methylnicotinamide when very large doses of tryptophan were given, indicating that the defect may be due to changes in the kynurenine pathway of tryptophan (M5). Moreover, diabetic rats excreted much more xanthurenic acid and less anthranilic and 3-hydroxyanthranilic acids than did nondiabetics, following large doses (200-400 mg) of tryptophan. 

Also Tojo and Uenoyama (T2) by means of paper chromatography detected neither 5-hydroxyanthranilic acid nor its conjugate forms in urine of senile cataract patients. They found, however, an ethanol-extractable substance giving a positive ninhydrin reaction and Rf value coinciding with that of tryptophan. The oral administration of a dose of anthra-nilic acid did not alter their findings. 

In independent studies, Aprison et al. (A7) found among tryptophan metabolites that 3-hydroxyanthranilic acid was capable of inhibiting the oxidation of N,N-dimethyl-p-phenylendiamine by purified ceruloplasmin and serum oxidase. 

T3. Tompsett, S. L., The determination in urine of some metabolites of tryptophan-kynurenine, anthraniUc acid and 3-hydroxyanthranilic acid—and reference to the presence of o-aminophenol in urine. CZin. Chim. Acta 4, 411-419 (1959). 

Interest then moved to animals. Both isotopic and nutritional experiments showed that the pathway established in microorganisms applied equally to mammals. Thus hydroxyanthranilic acid was converted to nicotinic acid (9, 604), which it could replace as a growth factor (944), whereas there was no similar conversion of anthranilic acid (343). An outstanding series of isotopic experiments, especially by Heidelberger and co-workers, showed that the 8-carbon atom of the tryptophan side chain became the 8-carbon atom of the kynurenine side chain and that the side chain was lost in conversion of kynurenine to nicotinic acid (369, 371, 427). Moreover the carbon in the 3-position of the indole nucleus became the carboxyl carbon of nicotinic acid (370 this experiment proved conclusively the reality of the tryptophan-nicotinic acid conversion) and the indole nitrogen appeared with only slight dilution in kynurenine, kynurenic acid, and xanthurenic acid (759). All these relations are those to be expected for the pathway tryptophan —+ kynurenine —> hydroxykynurenine (or its phosphate) —> hydroxyanthranilic acid (or its phosphate) — nicotinic acid, illustrated in diagrams 17 and 18. 

Both anthranilic acid and hydroxyanthranilic acid can be formed from tryptophan in insect mutants, and both are conjugated with glycine to give substituted hippuric acids (796), whereas in plants or bacteria anthranilic acid tends to be conjugated as the /3-glucoside (848). 

Isotopic experiments (763) with tryptophan labeled with N and deuterium in the indole ring have shown that quinolinic acid nitrogen is probably entirely derived from the indole nitrogen of tryptophan, and that scission of the benzene ring probably occurs between carbons 3 and 4. Presumably, therefore, the hydroxyanthranilic acid is converted to intermediate A without participation of a catechol-type intermediate, and it is possible that the phosphate-bond energy of hydroxyanthranilic acid phosphate (if this is in fact an intermediate) may contribute to the transformation. It is known... 

If a large dose of hydroxyanthranilic acid is given to an animal a small proportion is excreted unchanged (104). Hydroxyanthranilic acid is also excreted by man after a large dose of tryptophan (696), and has been found in human urine in tuberculosis (624). The latter is probably due to high endogenous protein breakdown (c/. 178). 

In addition many stages in knowm pathways of tryptophan metabolism require further investigation, in particular, the intermediate lying between tryptophan and formylkynurenine, the hydroxylation reaction in conversion of kynureine to hydroxykynurenine, the intermediates in the conversion of hydroxyanthranilic acid to nicotinic acid, and the site of synthesis and hormonal function of 5-hydrox3dryptamine. 

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