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The subcellular distribution of the first three enzymes in the ommochrome eye pigment pathway were studied by means of differential centrifugation. Tryptophan pyrrolase and kynurenine formamidase are found in the soluble fraction of the homogenate. A relatively small amount of tryptophan pyrrolase, 10 to 15% of the total, is sedimentable. The head region of pupae contains approximately 15% of the total activity of the animal. The subcellular distribution of the activity of the head is not appreciably different from the distribution of activity in the rest of the animal. Therefore no evidence suggests that the enzyme exists in a particulate form specifically in the head. Kynurenine hydroxylase is judged to be a mitochondrial enzyme on the basis of its co-sedimentation with particles containing cytochrome oxidase and by the demonstration of mitochondria in the sedimented fractions containing kynurenine hydroxylase activity. Kynurenine hydroxylase was judged not to be present in pigment granules since developmental and genetic changes in pigment granule morphology do not affect the sedimentation of kynurenine hydroxylase containing particles. These results on the three enzymes lead us to conclude that they are not organized into a single multi-enzyme complex as has been previously suggested. A consideration of the six ommochrome deficient mutants indicates that four of these can be explained by existing data. A hypothesis which does not involve enzyme complexes is presented to account for the remaining two.

The interrelationship between female hormones associated with reproduction and kynurenine metabolism was studied in the whole liver homogenates from male mice. These in vitro studies were planned to investigate the possible mechanisms by which β-oestradiol, ethinyloestradiol, mestranol and progesterone may affect the vitamin-B 6 -dependent enzymes, kynurenine aminotransferase and kynurenine hydrolase. It was found that the chemical structure of the oestrogenic hormone may be an important factor in determining the occurrence or not of inhibition of these enzymes since mestranol and progesterone have no inhibitory effect on both enzyme systems. Whereas β-oestradiol and ethinyloestradiol inhibit preferably the kynurenine aminotransferase enzyme. The inhibitory effect of β-oestradiol is of the competitive type while that of ethinyloestradiol is a non-competitive one.

The metabolism of orally administered 8-methyl ether of xanthurenic acid-carboxy-14C and methoxy-14C was studied in rabbits. There was no evidence for decarboxylation of the 8-methyl ether of xanthurenic acid-carboxy-14C. However, rabbits O-demethylated nearly 20% of administered 8-methyl ether of xanthurenic acid-methoxy-14C, giving rise to 14CO2. This O-demethylation was accompanied by the presence of xanthurenic acid and its further metabolites, 8-hydroxyquinaldic acid and an 8-hydroxyquinaldic acid conjugate, in urine samples from rabbits receiving the 8-methyl ether of xanthurenic acid-carboxy-14C. The 8-methyl ether of xanthurenic acid-carboxy-14C and methoxy-14C were dehydroxylated in rabbits, as are xanthurenic acid and kynurenic acid, giving rise to a small amount of urinary 8-methoxyquinaldic acid. Thus, dehydroxylation represents a significant metabolic pathway for all 4-hydroxyquinoline-2-carboxylic acid derivatives studied to date. Pretreatment of rabbits with oral Neomycin sulfate in amounts sufficient to inhibit the multiplication of gastrointestinal microorganisms severely inhibited the O-demethylation and dehydroxylation of the 8-methyl ether of xanthurenic acid in rabbits. These results suggest that all further metabolism of the 8-methyl ether of xanthurenic acid in rabbits is mediated by or requires the presence of the gastrointestinal flora.

Paper chromatographic examination on urinary tryptophan metabolites following an oral dose of L-tryptophan, 100mg/kg, was carried out on infants and children with convulsive disorders including infantile spasm.The incidence in cases with abnormal tryptophan metabolism was found to be higher in cases with convulsion than in those without convulsion.A detailed study on an infant with typical infantile spasm revealed that there was a marked increase in urinary excretion of kynurenine, kynurenic acid and xanthurenic acid following the oral dose of tryptophan, and that the glutamic oxaloacetic transaminase activity of erythrocytes, vitamin B6 levels of serum, and the kynureninase activity of the liver were within normal limits. From the present finding it was assumed that the abnormality in tryptophan metabolism observed in some cases with convulsive disorders might not be a cause of convulsion, but a sequential phenomenon associated with convulsion.

1. 6-Hydroxykynurenic acid (4,6-dihydroxyquinoline-2-carboxylic acid, 6-HKA) was isolated in crystalline form from both green and cured tobacco leaves. 2. A method for the determination of 6-HKA by paper chromatography and fluorimetry is described. 3. The content of 6-HKA in the flowers, stem and roots of the tobacco plant was much lower than that in the leaf. 4. The 6-HKA content increased throughout leaf development and senescence. 5. 6-HKA was detected in the leaves of plants representing 11 out of 27 families sampled. 6. 6-HKA was found to be devoid of antibacterial and antifungal activity, and was inactive in the Avena-coleoptile and cress-seed-germination tests. 7. The presence of 6-HKA is taken as evidence in plants of the tryptophan-catabolic pathway already known in mammals and micro-organisms.

I NTEREST IN ABNORMALITIES relating to the metabolism of certain amino acids in rheumatoid arthritis was first aroused in 1958, when Nishimura et al.50 reported on the presence of an abnormal metabolite of tyrosine in the urine of patients with collagen diseases. A repetition of this work by McMillatV3 in patients with rheumatoid arthritis did not confirm this findng but noted instead an increased excretion of 3-hydroxyanthranilic acid, a normal metabolite of tryptophan. These observations were later confirmed by Bett4 with the added observation that there was also an increased excretion of kynurenine, another metabolite of tryptophan. 6 Tryptophan loading was followed by an abnormally large increase in the excretion of these and other metabolites, which could be partially blocked by the simultaneous administration of pyridoxine, suggesting that there may be a functional deficiency of pyridoxine. Several other studies in recent years3~‘g~3”~42~52~54~74 have demonstrated similar abnormalities in one or more of the metabolites of tryptophan excreted in the urine. In most of these studies there has been a wide scatter of values for the various metabolites in healthy and diseased subjects, with considerable overlap between the two groups. Abnormalities in the metabolism of tryptophan are not specific for the collagen diseases. However, among this group of disorders, there has been some indication that patients with rheumatoid arthritis and scleroderma showed these abnormalities to the greatest degree. 31,32*40,44 At the same time, the wide overlap of values has made it difficult to distinguish between these two diseases on the basis of the data collected. The clinical significance of the changes observed is still unknown. Furthermore, the degree of abnormality has not correlated well with the clinical and laboratory parameters of disease activity. Despite this lack of correlation, studies in several patients with rheumatoid arthritis on low tryptophan diets, have revealed a tendency toward improvement in the clinical state with a drop in the sedimentation rate and plasma fibrinogen (Fig. 1). Whether this is a function simply of tryptophan deprivation or the negative nitrogen balance associated with this deprivation is not yet clear, although it does appear to influence the course of the disease in a favorable way.“”

Spontaneous excretion of tryptophan metabolites was followed in 68 patients affected by haemoblastosis. Abnormal levels of kynurenine were found to be excreted by 8 out of 13 subjects with malignant lymphoma, by 8 out of 22 subjects with leukaemia, and by 3 out of 5 cases with multiple myeloma. In all 28 Hodgkin's disease patients examined, kynurenine was excreted consistently above normal levels. Excretion values varied among the patients and were roughly correlated to the severity of the disease. Abnormal amounts of 3-hydroxy-kynurenine and 3-hydroxyanthranilic acid were excreted by a few of the same patients. Administration of vitamin B6, as pyridoxine hydrochloride or pyridoxal-5-phosphate, In io patients with Hodgkin's disease normalized kynurenine and 3-hydroxykynure-nine excretion whereas it caused a contemporaneous increase of urinary 3-hydroxy-anthranilic acid. The effects of several therapeutic agents on the excretion were also observed. Tryptophan loading tests (50 mg/kg) were performed in n Hodgkin's disease patients: excretion of kynurenine, N-α-acetyl-kynurenine, 3-hydroxykynurenine, kynurenic, xanthurenic, and 3-hydroxyanthranilic acids was determined in comparison with that of 5 normal controls. The total amount of these substances was 3.3 times larger in these patients than in the controls. A week after the first load a second tryptophan test was carried out with contemporaneous administration of 3 large doses of vitamin B6. Pyridoxine was unable to normalise the patients' excretion. The vitamin acted by increasing the amount of 3-hydroxyanthranilic acid and by decreasing the amounts of the other metabolites.

The enzymic synthesis of kynurenic acids-2- and 3-C14 from the correspondingly labeled tryptophans is described. The method utilized successive treatment with tryptophan pyrrolase, formamidase, and l-amino acid oxidase. 7,8-Dihydroxykynurenic acid has been synthesized by application of the Conrad-Limpach method to 2,3-dimethoxyaniline and diethyloxalacetate. These compounds are of importance in the elucidation of the pathway of tryptophan metabolism by certain bacteria of the Pseudomonas species group.

The effect of dietary copper on selected metabolic systems requiring pyridoxal phosphate was investigated by comparing certain urinary excretory products of pair-fed copper-deficient and copper-adequate rats. Kynurenic acid and xanthurenic acid, two tryptophan metabolites, were in lower concentrations in the urines of the copper-deficient animals in the pretryptophan-load period. Following a tryptophan load, these metabolites increased markedly in the urines of both groups, but the in crements were much larger for the copper-deficient animals. The increases in kynurenic acid were greater than those of xanthurenic acid in both groups, but the increase was disproportionately higher in the copper-deficient rats. The excretion of taurine and urea by the copper-deficient rats was significantly less. At the dilution used, several amino acids were found in significant amounts in the urines of the copper-deficient animals, whereas only taurine was detected in the urines of those fed copper. Several possible explanations of these findings are discussed. J. Nutr. 103: 1764-1771, 1973. INDEXING KEYWORDS urinary amino acids •ure copper deficiency •urinary tryptophan metabolites

The metabolism of the essential amino acid tryptophan has been the subject of numerous investigations in a variety of research disciplines over the last 20 years. Prior to this, techniques had not been fully developed to allow adequate quantitative measurements. Elucidation of the tryptophan-niacin pathway, the enzymes involved, dietary requirements, and various metabolic studies have been well reviewed previously (1-5), as well as in other papers of this con

Tryptophan-loading tests were performed on two normal subjects before and after diiodohydroxyquin treatment and on three acrodermatitis enteropathica patients being treated with diiodohydroxyquin. Patients who received a loading dose of tryptophan had higher urinary levels of kynurenine, kynurenic acid, and acetylkynurenine in comparison with normal subjects; 3-hydroxykynurenine and N-methylnicotinamide values were lower in patients than in control subjects. A possible explanation for these results would be a decreased activity of kynurenine hydroxylase in the patients.

The effect of the new antischistosomal drug methanesulphonate derivative of hycanthone(1- {[2-(diethylamino)ethyl]amino}-4-(hydroxymethyl)-thioxanthen-9-one)-(etrenol) on the metabolism of kynurenine was compared with that of lead acetate. Etrenol produced inhibition of both kynureninase and kynurenine transaminase enzymes whereas lead acetate inhibited the former enzyme only. It has been suggested that the inhibitory effects of etrenol were brought about by inactivation of the sulfhydryl groups of these enzymes since these inhibitions were not reversed by the supplementation of exogenous pyridoxal phosphate and that lead acetate protected both enzymes from the inhibitory effects of etrenol.

There have been only two brief reports which presented direct evidence of the existence of more than one monoamine oxidase in mitochondria although this has been suggested by many workers. Gorkin (1963) partially separated p-nitrophenylethylamine oxidase activity from m-nitro-p-hydroxybenzylamine oxidase activity by ion exchange chromatography of mitochondrial extracts in the presence of nonionic detergent (OP-10). Youdim and Sandler (1967) report the appearance of two or three bands of enzyme activity upon polyacrylamide gel electrophoresis of partially purified monoamine oxidase from rat liver or human placental mitochondria. This paper describes the separation of solubilized liver mitochondrial monoamine oxidases from three species by gel filtration in the presence of detergent. Based on substrate specificity there appear to be at least three different monoamine oxidases present in rat and rabbit liver and at least two in beef liver.

A fluorometric method for determining monoamine oxidase in human serum with kynuramine as substrate is described. The optimal experimental condi tions for the determination are given.

Metabolism of labeled enantiomers of tryptophan, kynurenine and hydroxykynurenine into C 14 O 2 and nine tryptophan metabolites was studied in five patients with scleroderma. The D-isomers of tryptophan, kynurenine and hydroxykyurenine were poorly converted to CO 2 but did give rise to small amounts of activity in several of the urinary metabolites. These observations suggest that man may have D-amino acid oxidase and hydroxylase enzyme systems capable of converting small amounts of the D-isomers into metabolites found in the urine. However, on the basis of previously published animal data, which showed a delayed absorption of the D-isomers from the intestine, it is not possible to exclude metabolites arising from action of the intestinal flora. With both the D- and L-isomers, the C 14 activity isolated in known urinary metabolites accounted for only a small part of the total urinary C 14 activity. In several patients given 2 gms of L-tryptophan with C 14 labeled tryptophan, expiratory C 14 O 2 was depressed and urinary labeled quinolinic acid activity was elevated when compared to patients in previous studies. These factors suggest that some scleroderma patients may have an alteration of a metabolic step in the pathway after hydroxyanthranilic acid. Conclusive evidence of a relationship between amount of C 14 O 2 produced after a 2 gm load of L-tryptophan and metabolic abnormalities was not observed. The experiments showed that the racemic mixture of a labeled tryptophan compound could be used for a C 14 O 2 metabolism study, but that the use of pure, natural L-isomers is necessary for meaningful results in the study of tryptophan metabolism in man.

Extract: Tryptophan oxidation was evaluated in 6 institutionalized phenylketonuric subjects (age range 5–13 years) by measuring the urinary excretion of oxidation metabolites following administration of L-tryptophan loads by mouth. Observations were made when subjects were on a general diet, a low-phenylalanine diet and a low-phenylalanine diet containing added L-phenylalanine. At some time during each dietary regime, the subjects also received tryptophan by mouth, 100 mg/kg body weight. Measurement was made in urine of: 1. 3-hydroxykynurenine; 2. kynurenine; 3. acetyl kynurenine; 4.3-hydroxyanthranilic acid; 5. xanthurenic acid; 6. kynurenic acid; 7. anthranilic acid; 8.3-indole-acetic acid; 9. o-hydroxyphenylacetic acid; and 10. phenylpyruvic acid. Excretion of tryptophan-kynurenine metabolites (products 1–9) amounted to 3.78 μmoles/kg/24 h and was similar to that of control subjects while on all diets. With tryptophan loading on normal diets, excretion of metabolic products was lower than for control subjects. With tryptophan loading on low-phenylalanine diets, the excretion of tryptophan metabolites was 2–3 times higher than the mean value of 22.7 μmoles/kg/24 h observed in normal subjects. The response to loading when the diet contained added phenylalanine was variable, since two patients excreted normal amounts of metabolites and three excreted amounts several times greater than the controls. Excretion of indole and 3-indoleacetic acid was abnormally elevated with and without tryptophan loading on normal diets and when phenylalanine was added to the low-phenylalanine diets. Values in μmoles/kg/24 h were: 3-indoleacetic acid 1.7 (normal); 2.5–9.4 (PKU); indican 6.24 (normal); 14.9–24.6 (PKU). Speculation: Limited metabolism of tryptophan may be of importance to central nervous system development and function in certain phenylketonuric children. Malabsorption of tryptophan coupled with an inhibition of the tryptophan-kynurenine synthesis pathway for nicotinic acid could deprive the central nervous system of important cofactors essential to central nervous system metabolism. The subtle effects of secondary disturbances on enzymatic pathways for tryptophan, tyrosine and other amino acids may be more important than the direct influence of the metabolites of phenylalanine on the central nervous system in phenylketonuric subjects.

Two children (siblings) with mental retardation revealed an excessive output of xanthurenic acid, kynurenic acid, 3-hydroxykynurenine and kvnurenine in urine following tryptophan loading. The disturbance was normalized tem-porarily by treatment with large doses of vitamin B6. The levels of pyridoxal phosphate in sera from both the patients were found to be within normal limits. It was found that the activity of kynureninase in the liver of the patient was markedly reduced without addition of pyridoxal phosphate but the activity was restored to a considerable extent by the addition of an excess of pyridoxal phosphate. These findings suggest that the basic defect in the patients is an inability of apoenzyme (kynureninase) to combine normally with the coenzyme (pyridoxal phosphate). The present studies, in combination with the work of Frimpter et al. on cystathionuria, may give us biochemical understanding for so-called vitamin B6 dependency.

The range of aromatic compounds that support the growth ofAlcaligenes eutrophushas been determined, and the pathways used for the dissimilation of these substrates have been explored, largely by enzymatic analyses. The β-ketoadipate pathway operates in the dissimilation of benzoate andp-hydroxybenzoate; the genetisate pathway, in the dissimilation ofm-hydroxybenzoate; and themetacleavage pathway, in the dissimilation of phenol andp-cresol.l-Tryptophan is oxidized via anthranilate; but the metabolic fate of anthranilate was not established. The metabolism of the three stereoisomers of muconic acid was also examined.

In the course of our study on kynurenic acid metabolism we observed that very concentrated sonic extracts of a pseudomonad, employing the so-called “quinoline pathway”, were able to degrade D-tryptophan. In this communication we wish to describe a new enzyme system which catalyzes the conversion of D-tryptophan to D-kynurenine, together with a new enzyme, D-kynurenine oxidase, which catalyzes the oxidative deamination of D-kynurenine to kynurenic acid. Recently Behrman and Cullen (1961) found tryptophan racemase in another strain of pseudomonad, which metabolizes D-tryptophan via the so-called “aromatic pathway”, but in our strain of pseudomonad D-tryptophan is metabolized without racemization to the L-isomer.

Three patients who had hypoglycemia associated with neoplasia were studied. In one, total tryptophan and protein-free metabolites of tryptophan in blood and tryptophan metabolites in urine were increased during hypoglycemia. In another, total tryptophan and protein-free tryptophan metabolites in serum were increased markedly and tryptophan metabolites in urine were increased mostly during hypoglycemia; during normoglycemia, however, concentration of both total tryptophan and protein-free tryptophan metabolites in serum and of protein-free tryptophan metabolites in urine was mostly normal. In the third, during normoglycemia, concentrations of total tryptophan and protein-free tryptophan metabolites in serum and of protein-free tryptophan metabolites in urine were normal or nearly so. Thus, patients with hypoglycemia associated with neoplasia seemingly have increased concentrations of tryptophan and tryptophan metabolites in serum and urine during hypoglycemic periods. Intraperitoneal administration of indole-3-propionic acid, indole-3-butyric acid, and indole-3-aretic acid caused profound hypoglycemia in normal and alloxandiabetic mice while L-tryptophan and kynurenic acid had no effect.