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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.

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 B 6 , 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.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Measurement of urinary xanthurenic acid (XA) has been used clinically to study a variety of disorders caused by vitamin B6 deficiency. To obviate some cumbersome steps of current methods for measuring XA in human urine, we have developed a simple fluorometric method. We apply the urine sample to a solid-phase extraction column containing trimethylaminopropyl group bound to silica, which enables us to purify and concentrate the XA from the urine without contamination from various tryptophan metabolites. The XA in the acidic eluate can then be quantified fluorometrically. The linearity of the proposed method extends from 0.2 to 10.0 mg/L. The method is precise, yielding day-to-day CVs for two pooled control specimens (1.08 and 1.90 mg/L) of 1.2% and 2.6%, respectively. Correlation studies with an established HPLC method and with a spectrophotometric procedure showed correlation coefficients of 0.99 and 0.98, respectively. Interference from vitamin C, uric acid, salicylate, acetaminophen, vanillylmandelic acid, and homovanillic acid was insignificant. The proposed method for urinary XA is rapid, simple, and suitable for routine use in the clinical laboratory.

To investigate how vitamin B6 (B6) deficiency affects the whole metabolism of tryptophan-niacin, rats were fed for 19 days with each of the following four kinds of diets; a complete 20% casein diet (control diet), the control diet without B6, the control diet without nicotinic acid, and the control diet without nicotinic acid and B6, and the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide each and the enzyme activities involved in tryptophan-niacin pathway were measured. The urinary excretion of kynurenic acid decreased while that of xanthurenic acid increased drastically in the two B6-deficient groups, when compared with the B6-containing groups. These results indicate that the rats fed with the B6-free diets were in the vitamin-deficient state. The conversion ratio was calculated from the ratio of the urinary excretion of sum of nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, to the Trp intake. The ratio was statistically lower in the B6-free diet than in the B6-containing diet under the niacin-free conditions.

The vitamin B-6 requirement of young women consuming a high-protein diet (1.55 g/kg body wt) and the effect of protein quality on this requirement was studied. In addition, the response of clinical, functional, and biochemical measures of vitamin B-6 nutriture to short-term depletion and step-wise repletion of vitamin B-6 were evaluated. Eight healthy young women resided in a metabolic unit and were fed a formula depletion diet (< 0.05 mg vitamin B-6/d) for 11-28 d followed by either an animal-protein (AP) or plant-protein (PP) diet with successively increasing vitamin B-6 intakes (0.5, 1.0, 1.5, and 2.0 mg/d) for periods of 14-21 d. Animal proteins were primarily from dairy and poultry sources and plant proteins were primarily from legumes. Vitamin B-6 status measures were assessed at weekly intervals. Results showed that a PP diet does not elevate the vitamin B-6 requirement over that required for an AP diet given the high amount of dietary protein used in this study. It was also found that 0.015 mg vitamin B-6/g protein intake normalized most biochemical indexes of vitamin B-6 status (including those indicative of functional status), and that 0.020 mg/g protein normalized all biochemical measures except total urinary vitamin B-6. Adding a margin of safety to either the 0.015 or 0.020 mg/g protein intake would raise the vitamin B-6 requirement for young women above the currently recommended dietary allowance of 0.016 mg/g protein.

This double-blind randomised phase III trial was designed to assess the effect of pyridoxine administration on the recurrence of Ta and T1 transitional cell tumours of the bladder. The trial accrued 291 patients and showed no significant difference between the pyridoxine and placebo treatment groups with respect to the time to first recurrence or the recurrence rate. Adjustment for the main prognostic factors, namely the recurrence rate prior to entry, the number of tumours at entry, the G grade and the levels of the tryptophan metabolites kynurenine plus acetyl kynurenine at entry do not change the overall conclusions.

We developed a simple and sensitive assay for the urinary 8-methyl ether of xanthurenic acid (XA-OMe) by high-performance liquid chromatography with fluorescence detection (excitation at 340 nm; emission at 450 nm). Urine samples were diluted with 0.03 M potassium phosphate buffer (pH 6.0) and applied to an octadecylsilane-bonded column (Nucleosil 5C18, 150 x 4 mm I.D.). The mobile phase used was a mixture of this same buffer and acetonitrile (1000:140, v/v). Both direct injection of urine and solvent extraction prior to HPLC were tested and showed a good correlation and sensitivity, although the peak of XA-OMe was occasionally less distinguishable from close peaks in urine from normal controls by the direct injection method. The quantification limit was 5 x 10(-14) mol which was sensitive enough to detect XA-OMe in urine from normal subjects. The method was applied to samples from patients with a deficiency in tryptophan catabolism, xanthurenic acid/3-hydroxykynurenine-uria and showed a striking elevation in urinary XA-OMe excretion.

A simple enzymatic method for the spectrophotometric determination of xanthurenic acid (XA) in urine is described. The method is based on the formation of a pigment derived from an oxidative coupling reaction with XA and N,N-diethyl-p-phenylenediamine (DE-PPD) in the presence of hydrogen peroxide and horseradish peroxidase. The color of the reaction mixture was developed with a peak absorbance at 750 nm. The recoveries against XA added to urine ranged from 91.0 to 96.5%. With this method, XA can be determined within 5 min, without a time-consuming sample preparation. The detection range of this method was between 5 and 150 microM, with a coefficient of variation of 2.7% or less. The correlation between this and the fluorometric method was good.

This study defined structural requirements for organophosphorous and methylcarbamate insecticides for altering the L-kynurenine pathway of L-tryptophan metabolism in mice. Kynurenine formamidase inhibition by organophosphorous acid triesters and methylcarbamates is the proposed primary event resulting in increase in xanthurenic acid urinary excretion and plasma L-kynurenine. Alteration of the L-kynurenine pathway occurred with compounds that inhibited liver kynurenine formamidase by more than 80%. Pyrimidinyl phosphorothioates followed by crotonamide phosphates were the most potent compounds that changed L-tryptophan metabolism, i.e., pirimiphos-ethyl (20 mg/kg) inhibited liver kynurenine formamidase by 99%, and increased xanthurenic acid urinary excretion and plasma L-kynurenine by 576 +/- 195 and 330 +/- 44%, respectively. Replacement of sulphur by oxygen in the phosphorothioate diazinon reduced in vivo liver kynurenine formamidase inhibition. Consequently, xanthurenic acid urinary excretion and plasma L-kynurenine were not elevated. Atropine, cycloheximide, 2-PAM and phenylmethylsulfonyl fluoride did not alleviate diazinon-altered L-tryptophan metabolism. Because of the potential of the majority of organophosphorous acid triesters and methylcarbamates to inhibit kynurenine formamidase, this novel noncholinergic mechanism warrants consideration in assessment of organophosphorous and methylcarbamate toxicity in occupational and accidental exposures.

Diazinon [O,O-diethyl O-(2-isopropyl-6-methyl-4- pyrimidinyl)phosphorothioate] altered the formation of several L-tryptophan metabolites associated with the L-kynurenine pathway in mice. Liver kynurenine formamidase was inhibited almost completely by diazinon (10 mg/kg). The enzyme inhibition resulted in reduced L-kynurenine biosynthesis in livers with a concomitant accumulation of N-formyl-L-kynurenine. In contrast to the liver, plasma L-kynurenine increased up to 5-fold in diazinon-treated mice. Consequently, the urinary excretion of xanthurenic acid and kynurenic acid was raised 5- to 15-fold. The revelation of this novel mechanism of diazinon action is an important piece of information needed for a better understanding of the noncholinergic toxicity of organophosphorous acid triesters and methylcarbamates.

The catabolism of homocysteine through cystathionine synthesis requires pyridoxal-5'-phosphate, thus the effect of vitamin B-6 deficiency on plasma homocysteine concentrations was evaluated. Total fasting plasma homocysteine concentrations were measured in 11 elderly subjects aged 64.4 +/- 1.7 y (mean +/- SE) who consumed a vitamin B-6-deficient diet for less than or equal to 20 d. Only 1 of the 11 subjects was found to have elevated homocysteine concentrations even though all subjects exhibited high urinary xanthurenic acid concentrations after a tryptophan load, a measure indicative of vitamin B-6 deficiency. In a supporting study, fasting plasma homocysteine concentrations were measured in 3- and 23-mo-old rats fed vitamin B-6-deficient diets and were compared with those of vitamin B-6-replete, pair-fed controls. There was no difference in homocysteine concentrations between deficient and pair-fed animals after 6 wk of the dietary regimen for either age group; after 9 wk a modest elevation was observed in the 3-mo-old deficient rats whereas no difference was observed for the 23-mo-old rats. It is concluded that fasting plasma homocysteine concentrations are not initially elevated in vitamin B-6 deficiency and therefore fasting plasma homocysteine concentrations are not a good indicator of vitamin B-6 status.

The tryptophan-load test for vitamin B-6 nutritional status was administered to adult female Long-Evans rats fed graded levels of pyridoxine hydrochloride (PN.HCl) in two experiments, and its sensitivity to marginal vitamin B-6 intake was evaluated. In Experiment 1, rats were 4-h meal-fed an AIN-76A (20% casein) diet devoid of PN.HCl for 3 wk, then repleted (n = 12) for 6 wk with 4-h pair-fed meals of either 0.25, 0.5, 1.0 or 7.0 (control) mg PN.HCl/kg diet. In Experiment 2, rats (n = 16) were pair-fed for 10 wk either 0.0, 0.5, 1.0 or 7.0 (control) mg PN.HCl/kg diet, with 24-h access to food. Vitamin B-6 nutritional status was assessed at the end of each experiment. Except in rats fed 0 mg PN.HCl/kg diet, mean body weights were not significantly different among diet groups of either experiment. Plasma pyridoxal phosphate (PLP), pyridoxal and total vitamin B-6 concentrations, determined by HPLC, were very sensitive to gradations in dietary PN.HCl concentrations (P less than 0.05). Red blood cell endogenous and PLP-stimulated alanine and aspartate aminotransferase activity did not statistically differentiate all levels of dietary vitamin B-6, although the calculated activity coefficient for each enzyme (stimulated/endogenous activity) did. Urinary xanthurenic acid excretion following a tryptophan load [24.5 mumol (5 mg) L-tryptophan/100 g body weight, injected intraperitoneally] was significantly (P less than 0.05) elevated compared with controls only in the group fed 0 mg PN/HCl/kg diet. At the tryptophan dose used here, the tryptophan-load test was not useful in detecting marginal vitamin B-6 intake in rats.

The vitamin B-6 requirements of 12 men and women over 60 y old were studied. The protocol consisted of a 5-d baseline period and four experimental periods during which the subjects successively received 0.003, 0.015, 0.0225 and 0.03375 mg of vitamin B-6/(kg body wt.d). Dietary protein was 1.2 or 0.8 g/(kg body wt.d). At 5- or 6-d intervals, xanthurenic acid (XA) after a 5-g L-tryptophan load and 4-pyridoxic acid (4-PA) in 24-h urine, erythrocyte aspartate aminotransferase activity coefficient (EAST-AC) and plasma pyridoxal-5'-phosphate (PLP) were measured. These measurements were abnormal during vitamin B-6 depletion but returned to normal during repletion. Men who ingested approximately 120 g protein/d required 1.96 +/- 0.11 mg of vitamin B-6 to normalize XA; women who ingested 78 g protein/d required 1.90 +/- 0.18 mg of vitamin B-6 to normalize XA. To attain normal levels of EAST-AC and 4-PA in men, 2.88 +/- 0.17 mg of vitamin B-6 were needed; to normalize PLP, 1.96 +/- 0.11 mg of vitamin B-6 were required. Women required 1.90 +/- 0.18 mg or more of vitamin B-6 to normalize these measurements. Vitamin B-6 requirements were not decreased in two of three subjects who ingested 54 g of protein daily. Thus, vitamin B-6 requirements of elderly men and women are about 1.96 and 1.90 mg/d, respectively.

In 43 patients with presenile cataracts an oral tryptophan loading test with 5 g L-tryptophan was performed and the 24-hour urinary excretion of kynurenine and xanthurenic acid was determined. 5 cases showed pathological deviations and an excretion pattern of tryptophan metabolites via kynurenine, similar as in vitamin B6-dependent xanthurenic aciduria.

Our previous research showed that tryptophan is an important precursor in the biogenesis of melanins. Therefore, with the purpose of observing whether a relationship exists between tryptophan metabolism and diseases characterized by an altered process of skin pigmentation in man, we studied the metabolism of this aminoacid along the kynurenine pathway in 29 vitiliginous patients (11 males and 18 females) and in 21 control subjects (11 males and 10 females) by determining 10 urinary metabolites after an oral loading of 50 mg/kg body weight L-tryptophan. The mean total excretion of the metabolites in patients resulted similar to that of the controls. However, considering the individual metabolites one can observe a decreased excretion of 3-hydroxykynurenine, o-aminohippuric acid and 3-hydroxyanthranilic acid and an increased excretion of xanthurenic acid and of its 8-methyl ether in the group of vitiliginous patients in respect to the controls. These results seem to indicate a decreased formation of nicotinic acid from tryptophan. Moreover, in relation to the fact that 3-hydroxykynurenine could be the metabolite through which tryptophan is involved in melanin biosynthesis, this study supports the hypothesis of a connection of tryptophan metabolism with the lack of pigmentation in vitiligo.

Two groups of pregnant women belonging to a low socioeconomic group, one with oral lesions and the other without lesions, were studied in the 3rd trimester of pregnancy for vitamin B6 nutritional status. Both the groups showed evidence of abnormal tryptophan metabolism, and in pregnant women with clinical signs the postload xanthurenic acid excretion in urine was higher than in the other group, suggesting a severe deficiency of vitamin B6. Treatment with vitamin B6 cured the oral lesions and also corrected the abnormalities in tryptophan metabolism. Excretion of 4-pyridoxic acid was low in both the groups and more so in women with oral lesions. Administration of tryptophan load significantly elevated the excretion of 4-pyridoxic acid in urine. These results indicated that pregnant women belonging to a low socioeconomic group were, in general, deficient in vitamin B6. In pregnant women with oral lesions the deficiency of vitamin B6 was more severe than in those pregnant women without oral lesions. Whether the severity of vitamin B6 deficiency would precipitate clinical signs like oral lesions is not yet known.

Oral tryptophan loading tests were performed in a patient with photosensitive pellagra-like skin rash and cerebellar ataxia but without hyperaminoaciduria. Plasma tryptophan concentrations after loading were similar in the patient and control subjects. Average urinary excretion of tryptophan in the patient from 0 to 6 and 6 to 12 hr was 2.69 and 2.58 mumol/kg, respectively; that in the control subjects was 0.82 and 0.34 mumol/kg, respectively. However, the average renal clearance of tryptophan during the first 6 hr of the loading tests in the patient was 0.757 ml plasma/1.73 m2 and that in the control subjects was 0.706 ml plasma/1.73 m2. Renal excretion of kynurenine in the patient was markedly decreased. The average from 0 to 6 and 6 to 12 hr in the patient was 1.90 and 1.13 mumol/kg, respectively; that in the control subjects was 12.90 and 18.15 mumol/kg, respectively. Under ultraviolet light, paper chromatograms of urine from the patient showed a deficiency of xanthurenic acid, kynurenic acid, kynurenine, and formylkynurenine. The deficiency of formylkynurenine in the patient's urine was confirmed by staining the paper chromatograms with Ehrlich's reagent. The patient was "sensitive" to oral nicotinic acid treatment; however, oral nicotinamide was well tolerated with improvement in the photosensitive skin rash.

The parallelism between xanthurenuria and liver affections caused by protein and choline deficiency was studied. At the stages marking the development of lipohepatosis and fibrosis there occurred an intensive passage of xanthurenic acid. The intensity of xanthurenuria at the stage marking the appearance of hyperplastic nodes continued to gain strength. With progressive advance of pathological changes, which by the 9--12th months of the experiment reached the stage of a fully developed nodular cirrhosis xanthurenuria gradually stopped.

The excretion of xanthurenic acid in urine after oral intake of 5 grams of L-tryptophan was studied and correlated with test and test-person parameters. The xanthurenic acid was determined by a colorimetric method, which gives reliable and reproducible results. Two groups, of 11 healthy volunteers each, participated. The first group started the test at 08.00 hours and the second group at 22.00 hours. Both groups repeated the same test, within one weed of the first test, at 22.00 hours. The xanthurenic acid excretion in the 24-hour urine correlated significantly (p = 0.04) with urine volume, but did not correlated with age, sex, height, body-weight, urine pH and urinary output of creatinine or 17-hydroxysteroids. The intraindividual variability of the test is not significant. The test should be started in the evening and the urine should be collected over 24 hours. The new reference values for the xar thurenic acid excretion under these conditions are 68.8 +/- 19.0 mumols/24 h.

Female Wistar rats were exposed to 100 ppm or 600 ppm carbon disulfide for 6 hours a day, 5 days a week for 12 weeks. During the exposure period, their urinary excretion of 4-pyridoxic acid was determined. The urinary excretions of xanthurenic acid and kynurenic acid after tryptophan loading were also determined. At the end of the exposure period, the rats were sacrificed and the levels of the five major forms of vitamin B6, pyridoxine, pyridoxal, pyridoxamine, pyridoxal phosphate and pyridoxamine phosphate, in the liver, kidneys and brain were determined by HPLC fluorometry. During the exposure the urinary excretions of xanthurenic acid and kynurenic acid after i.p. administration of tryptophan increased significantly in both experimental groups. However, urinary excretion of 4-pyridoxic acid decreased only slightly in the group exposed to carbon disulfide at 600 ppm and did not decrease in the group exposed to 100 ppm carbon disulfide. Furthermore, no significant changes were observed in the contents of vitamins B6 in any tissues examined. These results indicate that carbon disulfide does not cause vitamin B6 deficiency and thus that the disorders of tryptophan metabolism induced by carbon disulfide intoxication are not due to vitamin B6 deficiency.

Seventeen out of 21 children with severe asthma had an abnormal increase in urinary Kynurenic (KA) and Xanthurenic (XA) acid following oral tryptophane administration. In a second study 11 asthmatic children had significantly greater increases in XA and KA than nine normal healthy children. A micro-assay of KA and XA by column chromatography was performed. The data suggest the presence of a partial block in the metabolism of tryptophane in some children with bronchial asthma.

5-hydroxyindole acetic acid, xanthurenic acid and kynurenin, --that is to say the major metabolites of serotonin, were measured in the urines of 31 patients with dumping syndrome. Serotonin concentrations were measured as well in peripheral venous blood before and after a glucose load in 47 patient with dumping syndrome. All values measured were in the normal range. Comparison of serotonin levels in peripheral and portal venous blood yielded normal results as well. It is therefore concluded, that serotonin does not play an essential role in the etiology of the dumping syndrome.

The excretion of the tryptophan metabolites L-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and xanthurenic acid, by normal Japanese and British pre-menopausal, menopausal and post-menopausal women was measured after a loading dose of L-tryptophan. No significant difference was found between the mean excretion levels of the two races. A correlation with the level of plasma oestradiol and the excretion of L-kynurenine and also with the total of the four metabolites was found in pre-menopausal British women but not in Japanese women.

Monoamineoxidase (MAO) activity in some regions of brain and urinary xanthurenic acid were investigated in female rats administered with oral contraceptive (OC) steroids-ethynyl estradiol (Ees), lynestrenol (Ly) and both in combination (Cm) for 75 days. The MAO activity was reduced significantly in most regions specially in cortex-the extent of reduction being 65 per cent with Ees, 51 per cent with Ly and 69 per cent with Cm treatments. In the hypothalamus, the activity was decreased by 50 per cent with Ees, 38 per cent with Ly and by 40 per cent with Cm treatments. In the corpus striatum the activity was reduced by 14 per cent with Ly treatment, 25 per cent with Cm treatment and in the midbrain by 59 per cent with Ees treatment only. The concentration of xanthurenic acid in urine was higher by 55 per cent with Ees, 109 per cent with Ly and by 120 per cent with Cm treatments. These changes in MAO activity and level of xanthurenic acid excretion indicate the possible alteration in the metabolism of neurotransmitter, associated with prolonged use of OC.

In phenylalanine administered rat, tryptophan chiefly metabolized to xanthurenic acid and in nicotinic acid administration to 5-hydroxy indole acetic acid mainly. MAO reaction inhibited by quinoline compound and the inhibition of kynurenine aminotransferase activity through the injection of epinephrine or serotonin were observed. And also the induction of tryptophanpyrrolase in starvation was discussed.