A Homozygous Deletion of Exon 5 of KYNU Resulting from a Maternal Chromosome 2 Isodisomy (UPD2) Causes Catel-Manzke-Syndrome/VCRL Syndrome.

Vertebral, Cardiac, Renal and Limb Defect Syndrome (VCRL), is a very rare congenital malformation syndrome. Pathogenic variants in HAAO (3-Hydroxyanthranilate 3,4-dioxygenase), NADSYN1 (NAD+ Synthetase-1) and KYNU (Kynureninase) have been identified in a handful of affected individuals. All three genes encode for enzymes essential for the NAD+ de novo synthesis pathway. Using Trio-Exome analysis and CGH array analysis in combination with long range PCR, we have identified a novel homozygous copy number variant (CNV) encompassing exon 5 of KYNU in an individual presenting with overlapping features of VCRL and Catel-Manzke Syndrome. Interestingly, only the mother, not the father carried the small deletion in a heterozygous state. High-resolution SNP array analysis subsequently delineated a maternal isodisomy of chromosome 2 (UPD2). Increased xanthurenic acid excretion in the urine confirmed the genetic diagnosis. Our findings confirm the clinical, genetic and metabolic phenotype of VCRL1, adding a novel functionally tested disease allele. We also describe the first patient with NAD+ deficiency disorder resulting from a UPD. Furthermore, we provide a comprehensive review of the current literature covering the genetic basis and pathomechanisms for VCRL and Catel-Manzke Syndrome, including possible phenotype/genotype correlations as well as genetic causes of hypoplastic left heart syndrome.

Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism.

BACKGROUND: Autism spectrum disorder (ASD) is still diagnosed through behavioral observation, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics on human biofluids provides a sensitive tool to identify metabolite profiles potentially usable as biomarkers for ASD. Initial metabolomic studies, analyzing urines and plasma of ASD and control individuals, suggested that autistic patients may share some metabolic abnormalities, despite several inconsistencies stemming from differences in technology, ethnicity, age range, and definition of "control" status. METHODS: ASD-specific urinary metabolomic patterns were explored at an early age in 30 ASD children and 30 matched controls (age range 2-7, M:F = 22:8) using hydrophilic interaction chromatography (HILIC)-UHPLC and mass spectrometry, a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway. RESULTS: Urinary metabolites displaying the largest differences between young ASD and control children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B6, riboflavin, phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding increased levels of indolyl 3-acetic acid and indolyl lactate. CONCLUSIONS: The metabolic pathways most distinctive of young Italian autistic children largely overlap with those found in rodent models of ASD following maternal immune activation or genetic manipulations. These results are consistent with the proposal of a purine-driven cell danger response, accompanied by overproduction of epileptogenic and excitotoxic quinolinic acid, large reductions in melatonin synthesis, and gut dysbiosis. These metabolic abnormalities could underlie several comorbidities frequently associated to ASD, such as seizures, sleep disorders, and gastrointestinal symptoms, and could contribute to autism severity. Their diagnostic sensitivity, disease-specificity, and interethnic variability will merit further investigation.

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced by Semen Strychni.

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure, 12 typical biomarkers are selected and a simple LC-MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3-indoxyl sulfate, indole-3-acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C18 column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3-indoxyl sulfate, and indole-3-acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.

Metabonomics evaluation of urine from rats administered with phorate under long-term and low-level exposure by ultra-performance liquid chromatography-mass spectrometry.

The purpose of this study was to investigate the toxic effect of long-term and low-level exposure to phorate using a metabonomics approach based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Male Wistar rats were given phorate daily in drinking water at low doses of 0.05, 0.15 or 0.45 mg kg⁻¹ body weight (BW) for 24 weeks consecutively. Rats in the control group were given an equivalent volume of drinking water. Compared with the control group, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), urea nitrogen (BUN) and creatinine (CR) were increased in the middle- and high-dose groups whereas albumin (ALB) and cholinesterase (CHE) were decreased. Urine metabonomics profiles were analyzed by UPLC-MS. Compared with the control group, 12 metabolites were significantly changed in phorate-treated groups. In the negative mode, metabolite intensities of uric acid, suberic acid and citric acid were significantly decreased in the middle- and high-dose groups, whereas indoxyl sulfic acid (indican) and cholic acid were increased. In the positive mode, uric acid, creatinine, kynurenic acid and xanthurenic acid were significantly decreased in the middle- and high-dose groups, but 7-methylguanine (N7G) was increased. In both negative and positive modes, diethylthiophosphate (DETP) was significantly increased, which was considered as a biomarker of exposure to phorate. In conclusion, long-term and low-level exposure to phorate can cause disturbances in energy-related metabolism, liver and kidney function, the antioxidant system, and DNA damage. Moreover, more information can be provided on the evaluation of toxicity of phorate using metabonomics combined with clinical chemistry.

Novel spectrophotometric method for the quantitation of urinary xanthurenic acid and its application in identifying individuals with hyperhomocysteinemia associated with Vitamin B6 deficiency.

A novel spectrophotometric method for the quantification of urinary xanthurenic acid (XA) is described. The direct acid ferric reduction (DAFR) procedure was used to quantify XA after it was purified by a solid-phase extraction column. The linearity of proposed method extends from 2.5 to 100.0 mg/L. The method is precise, yielding day-to-day CVs for two pooled controls of 3.5% and 4.6%, respectively. Correlation studies with an established HPLC method and a fluorometric procedure showed correlation coefficients of 0.98 and 0.98, respectively. Interference from various urinary metabolites was insignificant. In a small-scale screening of elderly conducted at Penghu county in Taiwan (n = 80), we were able to identify a group of twenty individuals having hyperhomocysteinemia (>15 µ mole/L). Three of them were found to be positive for XA as analyzed by the proposed method, which correlated excellently with the results of the activation coefficient method for RBC's AST/B6 functional test. These data confirm the usefulness of the proposed method for identifying urinary XA as an indicator of vitamin B6 deficiency-associated hyperhomocysteinemic condition.

The niacin required for optimum growth can be synthesized from L-tryptophan in growing mice lacking tryptophan-2,3-dioxygenase.

In mammals, nicotinamide (Nam) is biosynthesized from l-tryptophan (l-Trp). The enzymes involved in the initial step of the l-Trp→Nam pathway are l-Trp-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO). We aimed to determine whether tdo-knockout (tdo(-/-)) mice fed a diet without preformed niacin can synthesize enough Nam to sustain optimum growth. Wild-type (WT) and tdo(-/-) mice were fed a chemically defined 20% casein diet with or without preformed niacin (30 mg nicotinic acid/kg) for 28 d. Body weight, food intake, and liver NAD concentrations did not differ among the groups. In the groups of mice fed the niacin-free diet, urinary concentrations of the upstream metabolites kynurenine (320% increase, P < 0.0001), kynurenic acid (270% increase, P < 0.0001), xanthurenic acid (770% increase, P < 0.0001), and 3-hydroxyanthranilic acid (3-HA; 450% increase, P < 0.0001) were higher in the tdo(-/-) mice than in the WT mice, while urinary concentrations of the downstream metabolite quinolinic acid (QA; 50% less, P = 0.0010) and the sum of Nam and its catabolites (10% less, P < 0.0001) were lower in the tdo(-/-) mice than in the WT mice. These findings show that the kynurenine formed in extrahepatic tissues by IDO and subsequent enzymes can be metabolized up to 3-HA, but not into QA. However, the tdo(-/-) mice sustained optimum growth even when fed the niacin-free diet for 1 mo, suggesting they can synthesize the minimum necessary amount of Nam from l-Trp, because the liver can import blood kynurenine formed in extrahepatic tissues and metabolize it into Nam via NAD and the resulting Nam is then distributed back into extrahepatic tissues.

Untargeted metabolomics identifies enterobiome metabolites and putative uremic toxins as substrates of organic anion transporter 1 (Oat1).

Untargeted metabolomics on the plasma and urine from wild-type and organic anion transporter-1 (Oat1/Slc22a6) knockout mice identified a number of physiologically important metabolites, including several not previously linked to Oat1-mediated transport. Several, such as indoxyl sulfate, derive from Phase II metabolism of enteric gut precursors and accumulate in chronic kidney disease (CKD). Other compounds included vitamins (pantothenic acid, 4-pyridoxic acid), urate, and metabolites in the tryptophan and nucleoside pathways. Three metabolites, indoxyl sulfate, kynurenine, and xanthurenic acid, were elevated in the plasma and interacted strongly and directly with Oat1 in vitro with IC50 of 18, 12, and 50 µM, respectively. A pharmacophore model based on several identified Oat1 substrates was used to screen the NCI database and candidate compounds interacting with Oat1 were validated in an in vitro assay. Together, the data suggest a complex, previously unidentified remote communication between the gut microbiome, Phase II metabolism in the liver, and elimination via Oats of the kidney, as well as indicating the importance of Oat1 in the handling of endogenous toxins associated with renal failure and uremia. The possibility that some of the compounds identified may be part of a larger remote sensing and signaling pathway is also discussed.

Metabolomic study of a rat fever model induced with 2,4-dinitrophenol and the therapeutic effects of a crude drug derived from Coptis chinensis.

This study describes the metabonomics of fevers in animal models and the therapeutic effects of Rhizoma coptidis extract (RCE) on them. The rat urinary samples were analyzed by UPLC/ ESI-Q-TOF/MS, combined with principal component analysis (PCA). Nine ions were chosen to characterize the similarities and differences in the responses to fever. The ion at m/z 206.0278 was unambiguously identified to be xanthurenic acid. This study demonstrated that the metabonomic approach can readily distinguish between febrile and healthy individuals. This data support the contention that the metabonomic approach represents a promising new technology for the development of rapid-throughput in vivo fever screening. Furthermore, this approach can detect the interfering effects of RCE. This investigation has led the authors to believe that metabonomics is a valid approach for explaining the therapeutic effects of traditional Chinese medicine on fevers.

Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry.

The separation and detection of 11 urinary aromatic acids was developed using HPLC-MS/MS. The method features a simple sample preparation involving a single-step dilution with internal standard and a rapid 8 min chromatographic separation. The accuracy was evaluated by the recovery of known spikes between 87 and 110%. Inter- and intra-assay precision (CV) was below 11% in all cases and the analytes were observed to be stable for up to 8 weeks when stored at -20 degrees C. The method was validated based upon linearity, accuracy, precision and stability and was used to establish reference intervals for children and adults.

Identification of xanthurenic acid 8-O-beta-D-glucoside and xanthurenic acid 8-O-sulfate as human natriuretic hormones.

Hormonal regulation of salt excretion and water balance by the kidneys is well documented. Before 1961, it was widely believed that the glomerular filtration rate and the steroid hormone aldosterone controlled sodium balance in the body. In 1961, deWardener et al. [de Wardener HE, Mills IH, Clapham WF, Hayter CJ (1961) Clin Sci 21:249-258] showed that when these two variables were controlled, the kidney was still able to increase sodium excretion in response to a salt load. Several lines of evidence argued for a small-molecule signal as a definitive modulator of sodium excretion by the kidney. However, the chemical nature of the suspected natriuretic agent remained unknown. Here we report the identification and natriuretic activity of two closely related small molecules isolated from human urine, xanthurenic acid 8-O-beta-d-glucoside and xanthurenic acid 8-O-sulfate. The two compounds were partially purified by activity-guided fractionation and subsequently identified by using NMR spectroscopic analyses of enriched active fractions. Both compounds caused substantial and sustained (1- to 2-h) natriuresis in rats and no or minimal concomitant potassium excretion. We believe these compounds constitute a class of kidney hormones that also could influence sodium transport in nonkidney tissues given that these tryptophan metabolites presumably represent evolutionarily old structures.

Xanthurenic aciduria due to a mutation in KYNU encoding kynureninase.

Massive urinary excretion of xanthurenic acid, 3-hydroxykynurenine and kynurenine, known as xanthurenic aciduria or hydroxykynureninuria, in a young Somali boy suggested kynureninase deficiency. Mutation analysis of KYNU encoding kynureninase of the index case revealed homozygosity for a c.593 A > G substitution leading to a threonine-to-alanine (T198A) shift. A younger brother was found to have a similar excretion pattern and the same genotype. At present, neither of the two boys has symptoms of niacin deficiency. This is the first report linking xanthurenic aciduria to a mutation in the gene encoding kynureninase.

Effect of nicotinamide administration on the tryptophan-nicotinamide pathway in humans.

The vitamin nicotinamide is synthesized in the liver from tryptophan, and distributed to non-hepatic tissues. Although it is generally accepted that 60 mg tryptophan is equivalent to 1 mg nicotinamide in humans, the conversion ratio of tryptophan to nicotinamide is changeable. To determine if de novo nicotinamide synthesis from tryptophan is influenced by nicotinamide intake itself, six young women consumed controlled diets containing 30.4 or 24.8 mg niacin-equivalent nicotinamide supplements with 0, 89, 310, or 562 micromol/day (0, 10.9, 37.8, or 68.6 mg/day, respectively), and urinary excretion of intermediates and metabolites of the tryptophan-nicotinamide pathway were measured. Urinary excretion of nicotinamide metabolites increased linearly in a dose-dependent manner. None of the intermediates, including anthranilic acid, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, and quinolinic acid, changed at all, even when up to 562 micromol/day nicotinamide was given. That is, exogenous nicotinamide did not affect de novo nicotinamide synthesis. Therefore, when niacin equivalent is calculated, the intake of nicotinamide itself need not be considered as a factor that changes the tryptophan-nicotinamide conversion ratio.

The effects of phthalate esters on the tryptophan-niacin metabolism.

Several phthalate esters (PhE), used as a plasticizer for numerous plastic devices, induce liver tumors and testicular atrophy, although the precise nature and mechanism for the action of PhE on these organs have remained unclear. We have previously reported that the administration of a large amount of di(n-butyl)phthalate (DBP) increased the conversion ratio of tryptophan to niacin in rats. To clarify the mechanism for the toxicity of PhE, we investigated the effects of di(2-ethylhexyl) phthalate (DEHP), one of the most frequently used additives, on the conversion ratio and how altering the conversion ratio of tryptophan to niacin depended on the concentration of DEHP. Rats were fed with a diet containing 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, or 3.0% DEHP for 21 days. To assess the conversion ratio of tryptophan to niacin, urine samples were collected at the last day of the experiment and measured for metabolites on the tryptophan-niacin pathway. The conversion ratio increased with increasing dietary concentration of DEHP above 0.05%; the conversion ratio was about 2% in the control group, whereas it was 28% in the 3.0% DEHP group. It is suggested that the inhibition of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) by DEHP or its metabolites caused this increase in the conversion ratio. We conclude that PhE such as DEHP and DBP disturbed the Tryptophan-niacin metabolism.

Growth-promoting activity of pyrazinoic acid, a putative active compound of antituberculosis drug pyrazinamide, in niacin-deficient rats through the inhibition of ACMSD activity.

We have recently reported that the antituberculosis drug, pyrazinamide (PZA), caused a significant increase in the conversion ratio of tryptophan to niacin in rats. In the present work, we investigated whether or not pyrazinoic acid (POA), a putative metabolite of PZA, increased the conversion ratio of tryptophan to niacin. Weaning rats were fed with a niacin-free and tryptophan-limited diet (negative control diet), or with the negative control diet supplemented with 0.003% nicotinic acid (positive control diet) or 1% POA (test diet) for 27 days. The growth rate was almost same between the groups fed on the positive control diet and the test diet. Dietary POA significantly increased the conversion ratio of tryptophan to niacin. Although POA did not directly inhibit the activity of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), the rate-limiting enzyme in the tryptophan-niacin pathway, liver ACMSD activity was only not detected in the test diet group. These results suggest that a derivative of POA metabolized by rats inhibited the ACMSD activity.

Influence of adenine-induced renal failure on tryptophan-niacin metabolism in rats.

To discover the role of the kidney in tryptophan degradation, especially tryptophan to niacin, rat kidneys were injured by feeding a diet containing a large amount of adenine. The kidney contains very high activity of aminocarboxymuconate-semialdehyde decarboxylase (ACMSD), which leads tryptophan into the glutaric acid pathway and then the TCA cycle, but not to the niacin pathway. On the other hand, kidneys contain significant activity of quinolinate phosphoribosyltransferase (QPRT), which leads tryptophan into the niacin pathway. The ACMSD activity in kidneys were significantly lower in the adenine group than in the control group, while the QPRT activity was almost the same, however, the formations of niacin and its compounds such as N1-methylnicotinamide and its pyridones did not increase, and therefore, the conversion ratio of tryptophan to niacin was lower in the adenine group than in the control group. The contents of NAD and NADP in liver, kidney, and blood were also lower in the adenine group. The decreased levels of niacin and the related compounds were consistent with the changes in the enzyme activities involved in the tryptophan-niacin metabolism in liver. It was concluded from these results that the conversion of tryptophan to niacin is due to only the liver enzymes and that the role of the kidney would be extremely low.

Konjac mannan improves the vitamin B-6 status of rats fed a vitamin B-6-deficient diet.

To investigate how dietary fiber in the diet affects vitamin B-6 nutriture of rats which have been deprived of vitamin B-6, rats were made vitamin B-6-deficient by feeding a vitamin B-6-deficient 70% casein diet. They were fed 2% cellulose powder-based vitamin B-6-deficient diets supplemented with 3% of additional dietary fiber sources (agar, konjac mannan, pectin and cellulose powder) for subsequent 18 days. Vitamin B-6 status was evaluated according to several biological criteria (weight gain, urinary excretion of xanthurenic acid after tryptophan loading, plasma pyridoxal 5'-phosphate, apparent pyridoxal 5'-phosphate-saturation of liver kynureninase, urinary excretion of 4-pyridoxic acid and fecal output of vitamin B-6). Vitamin B-6 status evaluated by these criteria was considerably improved in the konjac mannan-fed group, when compared with the respective data of the vitamin B-6 supplemented group. The relative mean effect of the konjac mannan diet was about 40% of the vitamin B-6 supplemented diet. In conclusion, konjac mannan was effective for improving the vitamin B-6 nutritional state in vitamin B-6-deprived rats.

Increased conversion ratio of tryptophan to niacin in severe food restriction.

The effect of food restriction on the conversion ratio of tryptophan to niacin was investigated, because it is known that the conversion ratio is influenced by nutritional factors. A 20% casein diet was fed to rats ad libitum (control), 1/2 the food of the control. 1/4 the food of the control, or starved for 9 days, and urine samples were collected to measure the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, and nicotinamide. The conversion ratio in the 1/2, 1/4, or starving group increased at day 1 of the experiment, but returned to the original value from day 2. Only in the starving group did the conversion ratio extremely increase from day 6 to day 9, being about 5-times higher than that of the original value on day 9. The possible mechanism by which the conversion ratio increased during food restriction is discussed.

Zinc, copper and iron levels in tissues of the vitamin B6 deficient rat.

Tissue zinc (Zn), copper (Cu) and iron (Fe) were determined in three groups of young male Wistar rats that received a daily pyridoxine hydrochloride (PN.HCl) intake of 45, 23 and 0 micrograms respectively in their diets over 8 weeks. No significant differences were found in the Zn and Cu levels in the liver, kidney, skeletal and cardiac tissue of all 3 groups. The Fe levels were significantly higher in the heart and liver and significantly lower in the skeletal muscle of the group receiving no PN.HCl in the diet (P < 0.05). This study indicates that the increased fecal excretion of Zn and Cu observed during a previous balance study on the above vitamin B6 deficient group of animals may be due to a decreased absorption of these elements from the diet rather than their excretion from tissue stores. The changes in Fe levels in the heart, liver and skeletal muscle points towards some alteration in tissue stores of this element during a vitamin B6 deficiency.

Feeding experiments of pyridoxine derivatives as vitamin B6.

In order to compare the nutritional effect of vitamin B6 derivatives, long-term feeding experiments with rats were carried out using pyridoxine-alpha-D-glucoside (PN-alpha-Glc), pyridoxine-beta-D-glucoside (PN-beta-Glc) or epsilon- (N-phosphopyridoxyl)lysine (PNP-Lys) with test diets consisting of basically the AIN-76 composition, except for the addition of 0.1 mg pyridoxine equivalent (PN eq.)/100 g diet. During 21 days of pair-feeding against the vitamin B6-deficient diet group, body weight gain, urinary excretion of xanthurenic acid and pyridoxic acid were measured. After the feeding experiment, rats were killed and examined in terms of liver kynureninase activity (EC 3.7.1.3) with and without adding exogenous pyridoxal 5'-phosphate (PLP), erythrocyte aspartate aminotransferase activity (EC 2.6.1.1), as well as PLP concentration in blood. Rats fed with PN-alpha-Glc grew well, relative to the PN group. On the contrary, PN-beta-Glc poorly served as vitamin B6 source, because average bioavailability was only about 22% in comparison to that of PN (100%). From this long-term feeding experiments, we have shown that PN-alpha-Glc (average bioavailability about 84%) is a good source of vitamin B6 similar to PN.

Relationships between methoxyindole and kynurenine pathway metabolites in plasma and urine in children suffering from febrile and epileptic seizures.

OBJECTIVE: The methoxyindole pathway metabolite, melatonin (aMT), and the kynurenine pathway metabolites, kynurenic acid (KYNA), xanturenic acid (XA) and 3-hydroxyantranilic acid (3HANA) are anticonvulsants, whereas the kynurenine pathway metabolites, L-kynurenine (KYN) and 3-hydroxykynurenine (3HK), are proconvulsants. It is thought that alterations in the concentrations of these compounds may be responsible for the excitotoxic aspect of human seizures. The aim of this study was to determine whether alterations in tryptophan metabolism might be related to the occurrence and type (febrile or non-febrile) of seizures in children. DESIGN: One hundred and eighteen children from the University of Granada Hospital were studied. They were divided into two main groups (febrile or epileptic convulsive) depending upon their clinical diagnosis. An age-, weight- and gender-matched control group was also studied. Each group was then divided into two subgroups of patients sampled between 0900 h and 2100 h (diurnal groups) and patients sampled between 2100 h and 0900 h (nocturnal groups). MEASUREMENTS: Plasma melatonin was measured in samples obtained from both the diurnal and nocturnal groups. Urinary excretion of melatonin and kynurenine metabolities were measured in an aliquot of 12-h urine samples collected from both the diurnal and nocturnal groups. RESULTS: Besides the typical circadian rhythm of melatonin we also found diurnal/nocturnal differences in the concentrations of all the kynurenines, which reached significantly higher levels during the day. In normal humans the production of methoxyindoles is lower during the day and rises at night, whereas the production of kynurenines is higher during the day and decreases at night. In patients suffering from febrile and epileptic convulsions, however, there was a significant increase in the nocturnal production of KYN, 3HK, KYNA and XA. Thus we found the circadian rhythm of kynurenines to be altered in convulsive patients. Furthermore, while the various kynurenine metabolites increased by the same amount during the night in febrile convulsive children, in epileptic children the increase in KYN and 3HK was significantly lower than the increase in KYNA and XA. During the day the proconvulsant KYN decreased significantly and the anticonvulsant XA increased in both convulsive groups. Moreover, plasma aMT increased during the day in febrile convulsive group and also during the night in both febrile and epileptic groups although showing no significant change in their urinary excretion levels. CONCLUSIONS: Our results point to the existence of an imbalance in the tryptophan metabolite pathways during convulsions, blunting the normal diurnal-nocturnal rhythm of kynurenines. They also support the idea of a difference in the production of tryptophan metabolites between febrile and epileptic patients, suggesting that the tryptophan pathways follow different routes depending upon the type and duration of the convulsion.