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Although migraine belongs to the main causes of disability worldwide, the mechanisms of its pathogenesis are poorly known. As migraine diagnosis is based on the subjective assessment of symptoms, there is a need to establish objective auxiliary markers to support clinical diagnosis. Tryptophan (TRP) metabolism has been associated with the pathogenesis of neurological and psychiatric disorders. In the present work, we investigated an association between migraine and the urine concentration of TRP and its metabolites 5-hydroxyindoleacetic acid (5-HIAA), kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QA) in 21 low-frequency episodic migraine patients and 32 controls. We chose the interictal phase as the episodic migraine patients were recruited from the outpatient clinic and had monthly migraine days as low as 1-2 in many cases. Migraine patients displayed lower urinary levels of 5-HIAA ( p < 0.01) and KYNA ( p < 0.05), but KYN and QA were enhanced, as compared with the controls ( p < 0.05 and 0.001, respectively). Consequently, the patients were characterized by different values of the 5-HIAA/TRP, KYN/TRP, KYNA/KYN, and KYNA/QA ratios ( p < 0.001 for all). Furthermore, urinary concentration of 5-HIAA was negatively correlated with Migraine Disability Assessment score and monthly migraine and monthly headache days. There was a negative correlation between Patient Health Questionnaire 9 scores assessing depression. In conclusion, the urinary 5-HIAA level may be further explored to assess its suitability as an easy-to-determine marker of migraine.

The biosynthetic routes leading to de novo nicotinamide adenine dinucleotide (NAD+) production are involved in acute kidney injury (AKI), with a critical role for quinolinate phosphoribosyl transferase (QPRT), a bottleneck enzyme of de novo NAD+ biosynthesis. The molecular mechanisms determining reduced QPRT in AKI, and the role of impaired NAD+ biosynthesis in the progression to chronic kidney disease (CKD), are unknown. We demonstrate that a high urinary quinolinate-to-tryptophan ratio, an indirect indicator of impaired QPRT activity and reduced de novo NAD+ biosynthesis in the kidney, is a clinically applicable early marker of AKI after cardiac surgery and is predictive of progression to CKD in kidney transplant recipients. We also provide evidence that the endoplasmic reticulum (ER) stress response may impair de novo NAD+ biosynthesis by repressing QPRT transcription. In conclusion, NAD+ biosynthesis impairment is an early event in AKI embedded with the ER stress response, and persistent reduction of QPRT expression is associated with AKI to CKD progression. This finding may lead to identification of noninvasive metabolic biomarkers of kidney injury with prognostic and therapeutic implications.

Background: Quinolinic acid (QA), a neuroactive metabolite of the Kynurenine Pathway (KP), is an excitotoxin that is implicated in the pathogenesis of many neurological disorders. KP is the main tryptophan degradation pathway. Phthalates can structurally mimic tryptophan metabolites and diets containing phthalates in rats enhanced the production and excretion of QA. However, there are no human studies that have examined the association between phthalates and QA., Objectives: Taking advantage of different mesalamine formulations with/without dibutyl phthalate (DBP), we assessed whether DBP from mesalamine (>1000x background) altered the urinary concentrations of QA., Methods: Men with inflammatory bowel disease participated in a prospective crossover pilot study. 15 Men were on non-DBP mesalamine (background) at baseline crossed-over for 4 months to high-DBP mesalamine (high) (B 1 H-Arm) and vice versa for 15 men who were on high-DBP mesalamine at baseline (H 1 B-Arm). Men provided 60 urine samples (2/man). We estimated crossover and cross-sectional changes in the creatinine normalized-QA using multivariable linear mixed effect models with random intercepts., Results: At baseline, men who were on high-DBP mesalamine (H 1 B-Arm) had 72%, (95% confidence interval (CI): 18, 151) higher normalized-QA than men who were on background exposure and when high-DBP mesalamine was removed for four months, normalized-QA decreased with 32%, (95% CI: -45.0, -15.1). Consistently, when men in B 1 H-Arm were newly-exposed to high-DBP mesalamine, normalized-QA increased with 11%, (95% CI: -11, 38)., Conclusions: High-DBP exposure from mesalamine increased the urinary concentrations of QA, which was largely reversed after removal of the high-DBP exposure for four months. This novel hypothesis should warrant new promising research considering the KP and QA concentrations as a plausible mediator for the neurotoxicity possibly linked with phthalate exposures., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nicotinamide adenine dinucleotide (NAD + ) extends longevity in experimental organisms, raising interest in its impact on human health. De novo NAD + biosynthesis from tryptophan is evolutionarily conserved yet considered supplanted among higher species by biosynthesis from nicotinamide (NAM). Here we show that a bottleneck enzyme in de novo biosynthesis, quinolinate phosphoribosyltransferase (QPRT), defends renal NAD + and mediates resistance to acute kidney injury (AKI). Following murine AKI, renal NAD + fell, quinolinate rose, and QPRT declined. QPRT +/- mice exhibited higher quinolinate, lower NAD + , and higher AKI susceptibility. Metabolomics suggested an elevated urinary quinolinate/tryptophan ratio (uQ/T) as an indicator of reduced QPRT. Elevated uQ/T predicted AKI and other adverse outcomes in critically ill patients. A phase 1 placebo-controlled study of oral NAM demonstrated a dose-related increase in circulating NAD + metabolites. NAM was well tolerated and was associated with less AKI. Therefore, impaired NAD + biosynthesis may be a feature of high-risk hospitalizations for which NAD + augmentation could be beneficial.

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.

Major depressive disorder (MDD) is a prevalent and debilitating mental disorder. Yet, there are no objective biomarkers available to support diagnostic laboratory testing for this disease. Here, gas chromatography-mass spectrometry was applied to urine metabolic profiling of 126 MDD and 134 control subjects. Orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to identify the differential metabolites in MDD subjects relative to healthy controls. The OPLS-DA analysis of data from training samples (82 first-episode, drug-naïve MDD subjects and 82 well-matched healthy controls) showed that the depressed group was significantly distinguishable from the control group. Totally, 23 differential urinary metabolites responsible for the discrimination between the two groups were identified. Postanalysis, 6 of the 23 metabolites (sorbitol, uric acid, azelaic acid, quinolinic acid, hippuric acid, and tyrosine) were defined as candidate diagnostic biomarkers for MDD. Receiver operating characteristic analysis of combined levels of these six biomarkers yielded an area under the receiver operating characteristic curve (AUC) of 0.905 in distinguishing training samples; this simplified metabolite signature classified blinded test samples (44 MDD subjects and 52 healthy controls) with an AUC of 0.837. Furthermore, a composite panel by the addition of previously identified urine biomarker (N-methylnicotinamide) to this biomarker panel achieved a more satisfactory accuracy, yielding an AUC of 0.909 in the training samples and 0.917 in the test samples. Taken together, these results suggest this composite urinary metabolite signature should facilitate development of a urine-based diagnostic test for MDD.

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.

Valproic acid (VPA) is a short-chained, branched fatty acid that is widely used in humans as an anticonvulsant and mood stabilizer, and has been reported to increase the liver NAD concentration. We investigated the effects of VPA on the conversion of tryptophan to nicotinamide. Rats were fed diets containing various amounts of VPA (0, 0.5, and 1.0% in the diets) for 14 d, 24-h urine samples were collected, and tryptophan and its catabolites were measured. We found that the conversion of tryptophan to nicotinamide was increased by feeding a diet containing VPA (p<0.01; 0% vs. 1.0% VPA). Of the intermediates formed during the conversion of tryptophan to nicotinamide, the tryptophan to 3-hydroxyanthranilic acid step was not affected by the administration of VPA, while such metabolites beyond quinolinic acid as nicotinamide and its catabolites were significantly increased (p<0.01; 0% vs. 1.0% VPA). This increase was dependent on the intake of VPA.

Pyridine nucleotide coenzymes are involved in >500 enzyme reactions and are biosynthesized from the amino acid L-tryptophan (L-Trp) as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp. We generated mice that are missing the quinolinic acid (QA) phosphoribosyltransferase (QPRT) gene. QPRT activity was not detected in qprt(-/-)mice. The qprt(+/+), qprt(+/-), or qprt(-/-) mice (8 wk old) were fed a complete diet containing 30 mg nicotinic acid (NiA) and 2.3 g L-Trp/kg diet or an NiA-free diet containing 2.3 g L-Trp/kg diet for 23 d. When qprt(-/-)mice were fed a complete diet, food intake and body weight gain did not differ from those of the qprt(+/+) and qprt(+/-) mice. On the contrary, in the qprt(-/-) mice fed the NiA-free diet, food intake and body weight were reduced to 60% (P < 0.01) and 70% (P < 0.05) of the corresponding values for the qprt(-/-) mice fed the complete diet at d 23, respectively. The nutritional levels of niacin, such as blood and liver NAD concentrations, were also lower in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice. Urinary excretion of QA was greater in the qprt(-/-) mice than in the qprt(+/+) and qprt(+/-) mice (P < 0.01). These data suggest that we generated truly niacin-deficient mice.

Induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 μmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 μmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 μmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.

To determine the tolerable upper intake level of nicotinic acid in humans, we investigated the effects of excess nicotinic acid administration on body weight gain, food intake, and urinary excretion of water-soluble vitamins and the metabolism of tryptophan in weaning rats. The weaning rats were freely fed a niacin-free 20% casein diet (control diet) or the same diet with 0.1%, 0.3% or 0.5% nicotinic acid for 23 days. The excess nicotinic acid intake did not affect body weight gain, food intake, serotonin contents in the brain, stomach and small intestine, or the urinary excretions of water-soluble vitamins. Although excess nicotinic acid did not affect the upper part of the tryptophan-nicotinamide pathway, 0.5% nicotinic acid diet increased the urinary excretion of quinolinic acid. The diet containing more than 0.3% nicotinic acid also increased the urinary excretion of nicotinic acid, which is usually below the limit of detection. As determined from the results of body weight gain and food intake as indices for apparent adverse effects, the no-observed-adverse-effect-level (NOAEL) for nicotinic acid was 0.5% in diet, corresponding to 450 mg/kg body weight/day. As judged from in increase of urinary quinolinic acid and nicotinic acid as indices of metabolic change, NOAEL was 0.1% in diet, corresponding to 90 mg/kg body weight/day, and the lowest-observed-adverse-effect-level (LOAEL) was 0.3% in diet, corresponding to 270 mg/kg body weight/day.

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.

Tryptophan is metabolized to alpha-amino-beta-carboxymuconate-epsilon-semialdehyde (ACMS) via 3-hydroxyanthranilate (3-HA). ACMS decarboxylase (ACMSD) directs ACMS to acetyl CoA; otherwise ACMS is non-enzymatically converted to quinolinate (QA), leading to the formation of NAD and its degradation products. Thus, ACMSD is a critical enzyme for tryptophan metabolism. Phthalate esters have been suspected of being environmental endocrine disrupters. Because of the structural similarity of phthalate esters with tryptophan metabolites, we examined the effects of phthalate esters on tryptophan metabolism. Phthalate esters containing diets were orally given to rats and the urinary excreted tryptophan metabolites were quantified. Of the phthalate esters with different side chains tested, di(2-ethylhexyl)phthalate (DEHP) and its metabolite, mono(2-ethylhexyl)phthalate (MEHP), most strongly enhanced the production of QA and degradation products of nicotinamide, while 3-HA was unchanged. This pattern of metabolic change led us to assume that these esters lowered ACMSD protein or its activity. Although DEHP could not be tested because of its low solubility, MEHP reversibly inhibited ACMSD from rat liver and mouse kidney, and also the recombinant human enzyme. Correlation between inhibition of ACMSD by phthalate esters with different side chains and urinary excretion of QA supports the notion that phthalate esters perturb tryptophan metabolism by inhibiting ACMSD. Quinolinate is a potential endogenous toxin and has been implicated in the pathogenesis of various disorders. Although toxicity of phthalate esters through accumulation of QA remains to be investigated, they may be detrimental by acting as metabolic disrupters when intake of a tryptophan-rich diet and exposure to phthalate esters occur coincidentally.

We have reported the effect of di(2-ethylhexyl)phthalate (DEHP) on the tryptophan (Trp)-niacin pathway in rats. To clarify the universal effect of DEHP on rodents, we studied whether DEHP also has an effect on Trp metabolism in mice. Mice were fed a niacin-free, 20% casein diet supplemented with DEHP for 21 days. Feeding with DEHP decreased the body weight gain and increased the liver weight in correlation with the dose level of DEHP. The administration of DEHP significantly increased the formation of quinolinic acid and the lower metabolites of the Trp-niacin pathway. The flux of niacin in the lower part of the Trp-niacin pathway in mice was enhanced by feeding with DEHP.

We investigated a useful chemical index for an excessive nicotinamide intake and how this excessive nicotinamide intake affects the tryptophan-nicotinamide metabolism in rats. Weaning rats were fed on a tryptophan-limited and nicotinic acid-free diet containing no, 0.003%, 0.1%, 0.2%, or 0.3% nicotinamide for 21 days. Urine samples were collected on the last day and analyzed the intermediates and metabolites on the tryptophan-nicotinamide pathway. Nicotinamide N-oxide, nicotinic acid and nicotinuric acid, metabolites of nicotinamide, were detected when nicotinamide at more than 0.1% had been taken. An intake of nicotinamide of more than 0.1% increased the urinary excretion of quinolinic acid, an intermediate on the pathway. Nicotinamide N-oxide and nicotinuric acid increased with increasing dietary concentration of nicotinamide. These results show that the measurements of nicotinamide N-oxide and nicotinuric acid in urine would be useful indices for an excessive nicotinamide intake.

We have reported that the administration of di(2-ethylhexyl)phthalate (DEHP) increased the formations of quinolinic acid (QA) and its lower metabolites on the tryptophan-niacin pathway. To discover the mechanism involved in disruption of the tryptophan-niacin pathway by DEHP, we assessed the daily urinary excretion of QA and its lower metabolites, and enzyme activities on the tryptophan-niacin pathway. Rats were fed with a niacin-free, 20% casein diet or the same diet supplemented with 0.1% DEHP or 0.043% phthalic acid and 0.067% 2-ethylhexanol added for 21 days. Feeding of DEHP increased the urinary excretions of QA and its lower metabolites in a time-dependent manner, and the increase of these excretions reached a peak at 11 days, but feeding of phthalic acid and 2-ethylhexanol had no effect. Feeding of DEHP, however, did not affect any enzyme activity including alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), affecting the formation of QA, on the tryptophan-niacin pathway.

Objective: We evaluated the utility of neopterin and quinolinic acid (QUIN) as surrogate measures of disease activity in juvenile idiopathic inflammatory myopathies (IIMs)., Methods: Plasma and first morning void urine samples were measured for neopterin and QUIN using commercial ELISA, HPLC, or gas chromatography-mass spectrometry in 45 juvenile IIM patients and 79 healthy controls. Myositis disease activity assessments were obtained., Results: Plasma and urine neopterin and QUIN concentrations were increased in juvenile IIM patients compared with healthy controls (P <0.017). Urine neopterin and QUIN highly correlated with each other (r(s) = 0.73; P <0.0001). Urine neopterin and QUIN correlated moderately with myositis disease activity assessments, including physician and parent global activity assessments, muscle strength testing, functional assessments (Childhood Myositis Assessment Scale, Childhood Health Assessment Questionnaire), skin global activity, and edema on magnetic resonance imaging (r(s) = 0.42-0.62; P <0.05), but generally not with muscle-associated enzymes in serum. Urine neopterin or QUIN, in combination with either serum lactate dehydrogenase (LD) or aspartate aminotransferase (AST), significantly predicted global disease activity (R(2) =0.40-0.56; P <0.002), and both were more sensitive to change than these serum enzymes (standardized response means, -0.41 to -0.48)., Conclusions: Urinary neopterin and QUIN are candidate measures of disease activity in juvenile IIM patients and add significantly to the prediction of global disease activity in combination with serum LD or AST values. Measurement of these markers in first morning void urine specimens appears to be as good as, or possibly better than, measurements of their concentrations in plasma.

The effects of dietary orotic acid on the metabolism of tryptophan to niacin in weaning rats was investigated. The rats were fed with a niacin-free, 20% casein diet containing 0% (control diet) or 1% orotic acid diet (test diet) for 29 d. Retardation of growth, development of fatty liver, and enlargement of liver were observed in the test group in comparison with the control group. The concentrations of NAD and NADP in liver significantly decreased, while these in blood did not decrease compared to the control group. The formation of the upper metabolites of tryptophan to niacin such as anthranilic acid, kynurenic acid, and 3-hydroxyanthranilic acid were not affected, but the quinolinic acid and beyond, such as nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, were significantly reduced by the administration of orotic acid. Therefore, the conversion ratio of tryptophan to niacin significantly decreased in the test group in comparison with the control group.

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.

A chemometric approach was applied for determining quinolinic acid in human plasma by differential pulse polarography after solid phase extraction. A fractional factorial design was used to examine the significant experimental variables for the peak height maximization. A Doehlert design, which allowed a sequential response surface methodology to be performed, was applied to the variables scan rate and drop size. The results indicated that the scan rate had the greatest effect on the response peak height. The linear range was extended from 8.52 x 10(-8) to 1.34 x 10(-5) M and the limit of detection was 2.9 x 10(-8) M. The validation process consisted of a pre-validation study followed by the main validation in the plasma matrix. The robustness and the intermediate precision were evaluated by means of experimental design. A 3(4)//9 screening symmetric matrix and a central composite design were used to optimize the solid phase extraction procedure of the analyte from human plasma using anion exchange cartridges. The goal was to select the best retention, wash and elution solvents and their volumes in order to maximize the extraction efficiency using as the response the polarographic peak height. An extraction efficiency of 90% was found. The method was also applied to the determination of quinolinic acid in urine and the mean concentration in human plasma and urine, was found to be 3.7 x 10(-7) and 4.9 x 10(-5) M respectively.

Capillary zone electrophoresis with UV absorbance detection was used to separate tryptophan and ten of its metabolites. Run buffers of pH 4.0-10.0 were evaluated for their effect on resolution; a pH 9.6 buffer was found to give optimum separation of all components. Ethylenediaminetetraacetic acid (EDTA), which prevents complexation of some analytes with polyvalent cations, was included in the run buffer to insure good peak shape and reproducible mobilities. The resulting method was used to detect the presence of quinolinic acid in a urine sample.

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.