Microanalysis of tryptophan metabolites

In tryptophan metabolites, 3-hydroxykynurenine and 3-hydroxyanthranilic acid have been reported to show a carcinogenic action to mice bladder and the relation of the metabolites to human bladder cancer has been discussed. We developed methods for the fluorometric assay of these compounds and showed that the excretion of 3-hydroxyanthranilic acid increased in the patients with bladder cancer. We also devised methods for the fluorometric assay of glucuronide and sulfate of 3-hydroxyanthranilic acid and showed that the minor excretion of these conjugated forms was shown in humans. The distribution of these compounds was also studied and the obtained data suggests that 3-hydroxykynurenine has affinity for the pancreas. We then developed methods for the determination of other metabolites of tryptophan. A fluorescence reaction with UV irradiation was found and applied to the determination. This method is the most sensitive to kynurenic acid but can be applied to kynurenine, nicotinamide and quinolinic acid. Furthermore, this methods also applied to the determination of some medicines, e.g. indomethacin, isoniazid, nalidixic acid, nicorandil and disodium cromoglicate in the serum or urine. We further devised other methods for the determination of xanthurenic acid and 5-hydroxyindoles.     

Kynurenine pathway enzymes in a rat model of chronic epilepsy: immunohistochemical study of activated glial cells

The kynurenine pathway metabolites quinolinic acid and kynurenic acid have been hypothetically linked to the occurrence of seizure phenomena. The present immunohistochemical study reports the activation of astrocytes containing three enzymes responsible for the metabolism of quinolinic acid and kynurenic acid in a rat model of chronic epilepsy. Rats received 90 min of patterned electrical stimulation through a bipolar electrode stereotaxically positioned in one hippocampus. This treatment induces non-convulsive limbic status epilepticus that leads to chronic, spontaneous, recurrent seizures. One month after the status epilepticus, the rats showed neuronal loss and gliosis in the piriform cortex, thalamus, and hippocampus, particularly on the side contralateral to the stimulation. Astrocytes containing the kynurenic acid biosynthetic enzyme (kynurenine aminotransferase) and the enzymes for the biosynthesis and degradation of quinolinic acid (3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase, respectively) became highly hypertrophied in brain areas where neurodegeneration occurred. Detailed qualitative and quantitative analyses were performed in the hippocampus. In CA1 and CA3 regions, the immunostained surface area of reactive astrocytes increased up to five-fold as compared to controls. Enlarged cells containing the three enzymes were mainly observed in the stratum radiatum, whereas the stratum pyramidale, in which neuronal somata degenerated, showed relatively fewer reactive glial cells. Hypertrophied kynurenine aminotransferase- and 3-hydroxyanthranilic acid oxygenase-immunoreactive cells were comparable in their morphology and distribution pattern. In contrast, reactive quinolinic acid phosphoribosyl transferase-positive glial cells displayed diversified sizes and shapes. Some very large quinolinic acid phosphoribosyl transferase-immunoreactive cells were noticed in the molecular layer of the dentate gyrus. In the hippocampus, the number of immunoreactive glial cells increased in parallel to the hypertrophic responses. In addition, pronounced increases in immunoreactivities, associated with hypertrophied astrocytes, occurred around lesioned sites in the thalamus and piriform cortex. These findings indicate that kynurenine metabolites derived from glial cells may play a role in chronic epileptogenesis.     

The effect of cycloserine on pyridoxine-dependent metabolism in tuberculosis

Measurements were made of urinary tryptophan metabolites of 13 tuberculosis patients in order to reveal characteristics of pyridoxine-dependent metabolism before and during cycloserine treatment. The abnormally high level of xanthurenic acid excretion in untreated patients suggests a decreased availability of pyridoxal phosphate related to the disease process. Although plasma cycloserine levels were kept high once therapy began, xanthurenic acid excretion before and after tryptophan load became progressively more normal as symptoms diminished. This observation suggests that the convulsions which may sometimes accompany cycloserine administration are not due to a direct pyridoxine antagonism by the drug. Throughout the study, no significant changes in 5-hydroxyindoleacetic acid excretion were observed. Presumably, the metabolic pathway of serotonin is unaffected by tryptophan loading, cycloserine administration, or the apparent pyridoxine depletion associated with tuberculosis.     

Abnormal tryptophan metabolism in a family with a history of bladder cancer

Tryptophan metabolism was studied in a family in which a sister and brother had bladder cancer. Urinary tryptophan metabolites after administration of 2 g L-tryptophan were measured in 4 sisters and 1 brother, all free of disease when studied. One sister with and 2 without histories of bladder cancer had significantly elevated levels of kynurenine, acetylkynurenine, and 3-hydroxykynurenine. Administration of 100 mg pyridoxine hydrochloride returned the tryptophan metabolism to normal in these 3 individuals. One brother with a 1 sister without bladder cancer had normal metabolism. A repeat study 2 years later confirmed the abnormal metabolism in the 3 sisters. Two sisters with abnormal tryptophan metabolism were given 200 mg L-kynurenine sulfate orally to bypass the effects of tryptophan oxygenase activity. Both excreted elevated levels of kynurenine and 1 excreted elevated levels of 3-hydroxykynurenine.     

Pressor responses to distension of the ureter in anaesthetised rats: characterisation of a model of acute visceral pain

HIV-associated dementia is a frequent consequence of HIV infection and relates to neuronal damage, possibly as a result of increased neurotoxic kynurenine metabolites. Indoleamine-2,3-dioxygenase (IDO) activity, which regulates kynurenine metabolism, may thus be increased in HIV infection. We measured IDO activity in post-mortem brain tissue from AIDS patients, including a subgroup that exhibited dementia, and age-matched control subjects. IDO activity was increased, but not significantly, in the AIDS group as well as the non-dementia group, compared to controls. Enzyme activity was significantly increased in the dementia group when compared to control values. IDO activity is increased in HIV-associated dementia and is thus likely to increase kynurenine pathway metabolites, such as 3-hydroxykynurenine and quinolinic acid, and elevated levels of these neurotoxins may contribute to the neuronal deficits underlying HIV-associated dementia.     

Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito

Malaria is transmitted from vertebrate host to mosquito vector by mature sexual blood-living stages called gametocytes. Within seconds of ingestion into the mosquito bloodmeal, gametocytes undergo gametogenesis. Induction requires the simultaneous exposure to at least two stimuli in vitro: a drop in bloodmeal temperature to 5 degrees C below that of the vertebrate host, and a rise in pH from 7.4 to 8.0-8.2. In vivo the mosquito bloodmeal has a pH of between 7.5 and 7.6. It is thought that in vivo the second inducer is an unknown mosquito-derived gametocyte-activating factor. Here we show that this factor is xanthurenic acid. We also show that low concentrations of xanthurenic acid can act together with pH to induce gametogenesis in vitro. Structurally related compounds are at least ninefold less effective at inducing gametogenesis in vitro. In Drosophila mutants with lesions in the kynurenine pathway of tryptophan metabolism (of which xanthurenic acid is a side product), no alternative active compound was detected in crude insect homogenates. These data could form the basis of the rational development of new methods of interrupting the transmission of malaria using drugs or new refractory mosquito genotypes to block parasite gametogenesis.     

Differential effects of kynurenine and tryptophan treatment on quinolinate immunoreactivity in rat lymphoid and non-lymphoid organs

Quinolinate is a tryptophan metabolite and an intermediary in nicotinamide adenine dinucleotide (NAD+) synthesis in hepatocytes. Kynurenine is an upstream metabolite in the same biochemical pathway. Under normal physiological conditions, kynurenine is thought to be produced primarily in the liver as an NAD+ precursor. However, during immune stimulation or inflammation, numerous extrahepatic tissues convert systemic tryptophan to kynurenine, and its concentration subsequently rises dramatically in blood. The fate and role of extrahepatic kynurenine are uncertain. In order to begin addressing this question, the present study was performed to determine which cell types can produce quinolinate from either systemic tryptophan or kynurenine. By using highly specific antibodies to protein-coupled quinolinate, we found that intraperitoneal injections of tryptophan led to increased quinolinate immunoreactivity primarily in hepatocytes, with moderate increases in tissue macrophages and splenic follicles. In contrast, intraperitoneal injections of kynurenine did not result in any significant increase in hepatocyte quinolinate immunoreactivity, but rather led to dramatic increases in immunoreactivity in tissue macrophages, splenic white pulp, and thymic medulla. These findings suggest that hepatocytes do not make significant use of extracellular kynurenine for quinolinate or NAD+ synthesis, and that, instead, extrahepatic kynurenine is preferentially metabolized by immune cells throughout the body. The possible significance of the preferential metabolism of kynurenine by immune cells during an immune response is discussed.     

Isolation and characterization of a mutant of Pseudomonas aureofaciens ATCC 15926 with an increased capacity for synthesis of pyrrolnitrin

A mutant having a 30-fold increased ability to synthesize pyrrolnitrin was isolated from Pseudomonas aureofaciens ATCC 15926 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant strain also differed from the parent strain in pigmentation and in its inability to catabolize anthranilic acid and the benzene moiety of tryptophan and kynurenine.     

Secretory patterns of tryptophan metabolites in midgut carcinoid tumor cells

Hormonal overproduction is a significant problem in patients with disseminated midgut carcinoid tumors. Serotonin (5-HT) is one major product secreted from such tumors and the urinary excretion of its metabolite (5-hydroxyindoleacetic acid, 5-HIAA) serves as an important tumor marker. The present study aimed at elucidating mechanisms of tryptophan metabolite secretion to facilitate the treatment of the carcinoid syndrome. When midgut carcinoid tumors were studied in primary cell cultures, several similarities with adrenergic neurons could be demonstrated. A marked dose-dependent depletion of intracellular 5-HT could be induced by reserpine, and monoamine oxidase-activity was revealed both in functional studies and by immunocytochemistry. Differences between tumors in the ratios of tryptophan metabolites released indicated that enzymes for synthesis and degradation of 5-HT were individually expressed. Treatment with the somatostatin analogue octreotide or with dexamethasone decreased the extracellular levels of tryptophan metabolites, but the mechanisms were partly different. In some tumors octreotide also decreased the synthesis of 5-HT, while dexamethasone markedly increased the intracellular 5-HIAA levels. It is of clinical interest to further elucidate these mechanisms, since the two drugs may have complementary actions in carotid crisis reactions.     

Expression, structure and chromosomal localization of the human cGMP-binding cGMP-specific phosphodiesterase PDE5A gene

1. Nicotinylalanine, an inhibitor of kynurenine metabolism, has been shown to elevate brain levels of endogenous kynurenic acid, an excitatory amino acid receptor antagonist. This study examined the potential of nicotinylalanine to influence excitotoxic damage to striatal NADPH diaphorase (NADPH-d) and gamma-aminobutyric acid (GABA)ergic neurones that are selectively lost in Huntington's disease. 2. A unilateral injection of the N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid, into the rat striatum produced an 88% depletion of NADPH-d neurones. Intrastriatal infusion of quinolinic acid also produced a dose-dependent reduction in striatal GABA content. 3. Nicotinylalanine (2.3, 3.2, 4.6, 6.4 nmol 5 microl(-1), i.c.v.) administered with L-kynurenine (450 mg kg(-1)), a precursor of kynurenic acid, and probenecid (200 mg kg(-1)), an inhibitor of organic acid transport, 3 h before the injection of quinolinic acid (15 nmol) produced a dose-related attenuation of the quinolinic acid-induced loss of NADPH-d neurones. Nicotinylalanine (5.6 nmol 5 microl(-1)) in combination with L-kynurenine and probenecid also attenuated quinolinic acid-induced reductions in striatal GABA content. 4. Nicotinylalanine (4.6 nmol, i.c.v.), L-kynurenine alone or L-kynurenine administered with probenecid did not attenuate quinolinic acid-induced depletion of striatal NADPH-d neurones. However, combined administration of kynurenine and probenecid did prevent quinolinic acid-induced reductions in ipsilateral striatal GABA content. 5. Injection of nicotinylalanine, at doses (4.6 nmol and 5.6 nmol i.c.v.) which attenuated quinolinic acid-induced striatal neurotoxicity, when combined with L-kynurenine and probenecid produced increases in both whole brain and striatal kynurenic acid levels. Administration of L-kynurenine and probenecid without nicotinylalanine also elevated kynurenic acid, but to a lesser extent. 6. The results of this study demonstrate that nicotinylalanine has the potential to attenuate quinolinic acid-induced striatal neurotoxicity. It is suggested that nicotinylalanine exerts its effect by increasing levels of endogenous kynurenic acid in the brain. The results of this study suggest that agents which influence levels of endogenous excitatory amino acid antagonists such as kynurenic acid may be useful in preventing excitotoxic damage to neurones in the CNS.     

High performance liquid chromatographic profiling of tryptophan and related indoles in body fluids and tissues of carcinoid patients

A high performance liquid chromatographic method with quaternary gradient elution and fluorometric detection was developed for profiling of tryptophan (TRP), 5-hydroxytryptophan, serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in urine, platelet-rich plasma and (tumour) tissue of patients with carcinoid tumours. Prior to injection, urine samples were diluted and filtered. Platelet-rich plasma and tissue homogenates were prepurified by C18 solid phase extraction. Detection limits were approx. 2 pmol. Results of urinary 5-HT and 5-HIAA compared favourably with those of single component analyses. No consistent diurnal variations were found for TRP, 5-HT and 5-HIAA in 12-h urine samples from 15 healthy adults. Abstinence of 5-HT-rich foods reduced urinary levels of 5-HT and 5-HIAA. C18 extraction of indoles from protein-containing matrices was studied in platelet-rich plasma. Although time-consuming and complicated for daily routine use, the present approach offers particular advantages over single component analyses in the study of TRP metabolism in patients with carcinoid tumours.     

Familial cerebellar degeneration with slow eye-movements, mental deterioration and incidental nevus of ota (oculo-dermal melanocytosis)

In a bipartite rearing experiment (day 1-24 and 24-45) 72 early-weaned piglets were used to study the effect of varying dietary vitamin B6 contents on renal excretion of xanthurenic and kynurenic acid after a tryptophan load, on urea concentration and activities of two transaminases in serum at the end of each period. The animals, divided into 6 groups, were fed ad libitum a prestarter and a starter in period I and II, respectively, each containing 0.5, 1.2, 2.0, 2.8, 3.5 or 6.6 mg vitamin B6 per kg dry matter. The urinary xanthurenic acid excretion was elevated especially at the vitamin B6 supply of 0.5 ppm and rose severalfold with increasing depletion time (period II). In both periods, the smallest amount was excreted by piglets supplemented with 2.8 ppm. In comparison to groups B (1.2 ppm) and C (2.0 ppm), their average excretion rate was reduced by 29% and 15%, respectively, in period I and by 50% and 22%, respectively, in period II. Analogously to xanthurenic acid, the smallest amount of kynurenic acid was excreted by group D (2.8 ppm). Starting from the lowest vitamin B6 supply, the activity of SGPT showed an almost linear increase in both experimental periods. In contrast, SGOT already reached an upper activity level with the dietary vitamin B6 content of 3.5 and 2.8 ppm at the end of period I and II, respectively. The concentration of serum urea was influenced only by the lowest vitamin B6 supply of 0.5 ppm.     

Synthesis and biochemical evaluation of N-(4-phenylthiazol-2-yl)benzenesulfonamides as high-affinity inhibitors of kynurenine 3-hydroxylase

In this paper we describe the synthesis, structure-activity relationship (SAR), and biochemical characterization of N-(4-phenylthiazol-2-yl)benzenesulfonamides as inhibitors of kynurenine 3-hydroxylase. The compounds 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulfonamide 16 (IC50 = 37 nM, Ro-61-8048) and 4-amino-N-[4-[2-fluoro-5-(trifluoromethyl)phenyl]-thiazol-2-yl] benzenesulfonamide 20 (IC50 = 19 nM) were found to be high-affinity inhibitors of this enzyme in vitro. In addition, both compounds blocked rat and gerbil kynurenine 3-hydroxylase after oral administration, with ED50's in the 3-5 mumol/kg range in gerbil brain. In a microdialysis experiment in rats, 16 dose dependently increased kynurenic acid concentration in the extracellular hippocampal fluid. A dose of 100 mumol/kg po led to a 7.5-fold increase in kynurenic acid outflow. These new compounds should allow detailed investigation of the pathophysiological role of the kynurenine pathway after neuronal injury.     

Role of tryptophan in the elevated serotonin-turnover in hepatic encephalopathy

The increase of the brain levels of 5-hydroxyindoleacetic acid (5-HIAA) in hepatic encephalopathy (HE) suggests an increased turnover of serotonin (5-HT). To study the role of tryptophan on the increased brain 5-HT metabolism in HE, we attempted to monitor brain levels of tryptophan in rats with thioacetamide-induced acute liver failure by intravenous infusion of branched-chain amino acids (BCAA). The effect of this treatment on 5-HT synthesis and metabolism was investigated in five brain areas. BCAA-infusions (1 and 2 gm/kg/24 h) increased the ratio BCAA/aromatic amino acids in plasma two- and fourfold, respectively, and lowered both plasma and brain levels of tryptophan. At the higher BCAA-dose all parameters suggesting an altered brain 5-HT metabolism (increased brain levels of 5-HT and 5-HIAA, increased 5-HIAA/5-HT ratio) were almost completely normalized. These results provide further evidence for the role of tryptophan in the elevation of brain 5-HT metabolism and for a potential role of BCAA in the treatment of HE.     

Mutagenic activity of tryptophan metabolites produced by rat intestinal microflora

The catabolism of tryptophan by rat intestinal microflora was studied for the production of mutagenic metabolites that might be involved in the etiology of colon cancer. Various tryptophan metabolites were assayed for mutagenic and comutagenic activity in the Ames bacterial test system. These included metabolites that were identified by thin-layer chromatography in cultures of rat fecal bacteria, other compounds structurally related to tryptophan, whole unfractionated mixed fecal bacteria culture filtrates, and concentrated solvent extracts. A total of 27 materials were tested with 5 Salmonella strains in the mutagenesis assay. Most substances were inactive, and only one compound, o-aminoacetophenone, which was unlikely to be produced in the intestine, showed weak comutagenic activity. Our results did not support the hypothesis that tryptophan metabolites produced by intestinal microflora are major etiologic factors in cancer of the colon.     

Renal excretion of xanthurenic acid as an index of vitamin B6 allowance in infants

Renal excretion of xanthurenic acid without any tryptophan load, passage of 4-pyridoxic acid with diurnal urine and its excretion with urine collected during 1 hour in the morning on an empty stomach were investigated in 86 practically healthy infants and in 77 others with acute respiratory viral infections aged from two weeks to one year. Investigations of the tryptophan tolerance in infants yielded negative results, viz. on administering to them of D,L-tryptophan in a load dose the infants started vomiting. Practically healthy infants did not excrete xanthurenic acid, while the renal excretion of 4-pyridoxic acid remained within normal limits. In patients at the height of the disease the passage of 4-pyridoxic acid steeply increased. In 9 of them xanthurenic acid appeared in the diurnal urine. In the quiscent stage of the affection in two infants xanthurenuria continued against the general background of diminished excretion of 4-pyridoxic acid. There is no reason to relate the disclosed xanthurenuria in sick infants with the state of hypovitaminosis in them.     

Melatonin's role as an anticonvulsant and neuronal protector: experimental and clinical evidence

The pineal gland classically has been considered as a vestigial and mystic organ. In the last decades, and with the incorporation of new methodologic procedures, it could be proved that it also has physiologic actions that vary depending on the level of the phylogenetic scale. Its best-known secretion, melatonin, has been related to many different actions, such as sleep promotion, control of biologic rhythms, hormonal inhibition, and an inhibiting action on central nervous system regulation mechanisms. In animal experimentation, there are papers even accepting an anticonvulsant effect. In humans, evidence is reduced to few experiences. In addition to this clinical experience, there is other evidence that clearly relates melatonin to convulsive phenomena. This relationship must be mediated by the following mechanisms attributed to melatonin: altered brain GABAergic neurotransmission, its known interaction with benzodiazepinic brain receptors, through tryptophan metabolite activity (kynurenine, kynurenic acid), or even by its efficacy as a free-radical scavenger.     

Classification of dorsal horn neurons based on somatic receptive fields in cats with intact and transected spinal cords: neural plasticity

Effects of various amino acids and tryptophan metabolites on the synthesis of nerve growth factor (NGF) in cultured mouse astroglial cells were evaluated. L-Tryptophan stimulated NGF production in a dose-dependent manner. Serotonin and quinolinic acid slightly increased NGF synthesis. L-Kynurenine had a marked stimulatory effect on NGF production at a dose of 10 microM. In contrast, kynurenic acid had no effect on the reaction. L-Glutamine, L-glutamic acid, L-phenylalanine and L-methionine produced no significant change in NGF synthesis. These results were discussed in relation to the treatment of Alzheimer's diseases.     

Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment: spectroscopic studies

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been biosynthetically incorporated into Staphylococcal nuclease, its V66W mutant, and the Delta 137-149 fragment of the latter mutant. The guanidine-HCl induced unfolding and thermal unfolding of these proteins were studied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three proteins have tryptophan residues at positions 140 (in wild type) and 66 (in the Delta 137-149 fragment of V66W) and at both positions (in V66W). The unfolding data show that 5-hydroxytryptophan does not perturb the stability of wild-type nuclease, but it destabilizes the fragment and causes the V66W mutant to unfold in a more cooperative manner. 7-Azatryptophan is found to destabilize all three proteins. 4-Fluorotryptophan is slightly stabilizing of the three proteins, but the other two fluorotryptophans do not alter the stability of the proteins.