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.

Supplementing healthy women with up to 5.0 g/d of L-tryptophan has no adverse effects.

Because of the frequent use of L-tryptophan (L-Trp) in dietary supplements, determination of the no-observed-adverse-effect-level is desirable for public health purposes. We therefore assessed the no-observed-adverse-effect-level for L-Trp and attempted to identify a surrogate biomarker for excess L-Trp in healthy humans. A randomized, double-blind, placebo-controlled, crossover intervention study was performed in 17 apparently healthy Japanese women aged 18-26 y with a BMI of ≈ 20 kg/m(2). The participants were randomly assigned to receive placebo (0 g/d) or 1.0, 2.0, 3.0, 4.0, or 5.0 g/d of L-Trp for 21 d each with a 5-wk washout period between trials. Food intake, body weight, general biomarkers in blood and urine, and amino acid composition in blood and urine were not affected by any dose of L-Trp. Administration of up to 5.0 g/d L-Trp had no effect on a profile of mood states category measurement. The urinary excretion of nicotinamide and its catabolites increased in proportion to the ingested amounts of L-Trp, indicating that participants could normally metabolize this amino acid. The urinary excretion of L-tryptophan metabolites, including kynurenine (Kyn), anthranilic acid, kynurenic acid, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and quinolinic acid (QA), all of which are intermediates of the L-TRP→Kyn→QA pathway, was in proportion to L-Trp loading. The response of 3-HK was the most characteristic of these L-Trp metabolites. This finding suggests that the urinary excretion of 3-HK is a good surrogate biomarker for excess L-Trp ingestion.

An investigation of the antidepressant action of xiaoyaosan in rats using ultra performance liquid chromatography-mass spectrometry combined with metabonomics.

A rapid, highly sensitive, and selective method was applied in a non-invasive way to investigate the antidepressant action of Xiaoyaosan (XYS) using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and chemometrics. Many significantly altered metabolites were used to explain the mechanism. Venlafaxine HCl and fluoxetine HCl were used as chemical positive control drugs with a relatively clear mechanism of action to evaluate the efficiency and to predict the mechanism of action of XYS. Urine obtained from rats subjected to chronic unpredictable mild stress (CUMS) was analyzed by UPLC-MS. Distinct changes in the pattern of metabolites in the rat urine after CUMS production and drug intervention were observed using partial least squares-discriminant analysis. The results of behavioral tests and multivariate analysis showed that CUMS was successfully reproduced, and a moderate-dose XYS produced significant therapeutic effects in the rodent model, equivalent to those of the positive control drugs, venlafaxine HCl and fluoxetine HCl. Metabolites with significant changes induced by CUMS were identified, and 17 biomarker candidates for stress and drug intervention were identified. The therapeutic effect of XYS on depression may involve regulation of the dysfunctions of energy metabolism, amino acid metabolism, and gut microflora changes. Metabonomic methods are valuable tools for measuring efficacy and mechanisms of action in the study of traditional Chinese medicines.

¹H NMR-based metabolic profiling of naproxen-induced toxicity in rats.

The dose-dependent perturbations in urinary metabolite concentrations caused by naproxen toxicity were investigated using ¹H NMR spectroscopy coupled with multivariate statistical analysis. Histopathologic evaluation of naproxen-induced acute gastrointestinal damage in rats demonstrated a significant dose-dependent effect. Furthermore, principal component analysis (PCA) of ¹H NMR from rat urine revealed a dose-dependent metabolic shift between the vehicle-treated control rats and rats treated with low-dose (10 mg/kg body weight), moderate-dose (50 mg/kg), and high-dose (100 mg/kg) naproxen, coinciding with their gastric damage scores after naproxen administration. The resultant metabolic profiles demonstrate that the naproxen-induced gastric damage exhibited energy metabolism perturbations that elevated their urinary levels of citrate, cis-aconitate, creatine, and creatine phosphate. In addition, naproxen administration decreased choline level and increased betaine level, indicating that it depleted the main protective constituent of the gastric mucosa. Moreover, naproxen stimulated the decomposition of tryptophan into kynurenate, which inhibits fibroblast growth factor-1 and delays ulcer healing. These findings demonstrate that ¹H NMR-based urinary metabolic profiling can facilitate noninvasive and rapid diagnosis of drug side effects and is suitable for elucidating possible biological pathways perturbed by drug toxicity.

Urinary excretory ratio of anthranilic acid/kynurenic acid as an index of the tolerable amount of tryptophan.

Some people may take excessive tryptophan as a supplement in the expectation that the tryptophan metabolite, melatonine, will help to induce sufficient sleep. We investigated the basis for a useful index to assess the risk of a tryptophan excess. Young rats were fed on a 20% casein diet with 0, 0.5, 1.0, 2.0 or 5.0% added tryptophan for 30 d the apparent toxicity and growth retardation was observed in the 5.0% tryptophan-added group. Metabolites of the Tryptophan-nicotinamide pathway and such intermediates as kynurenic acid (KA), anthranilic acid (AnA), xanthurenic acid, 3-hydroxyanthranilic acid and quinolinic acid in 24-h urine increased in a dose-dependent manner. Of those metabolites and intermediates, the urinary excretion of KA progressively increased, and that of AnA dramatically increased in the 2.0 and 5.0% tryptophan-added groups. The urinary excretory ratio of AnA/KA was a high value for both the groups. These results suggest that the urinary ratio of AnA/KA could be a useful index to monitoran excessive tryptophan intake.

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.

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.

Differences in vitamin B6 status indicator responses between young and middle-aged women fed constant diets with two levels of vitamin B6.

The responses of adult women to two levels of vitamin B6, was conducted with five young and eight middle-aged subjects. A constant diet (2.3-2.4 mg vitamin B6 per day) was fed for four weeks followed by three weeks of the same diet supplemented with 8.0 mg pyridoxine. Plasma pyridoxal 5'-phosphate (PLP), plasma and urinary vitamin B6, and urinary 4-pyridoxic acid (4-PA) were determined. The older women had significantly lower plasma PLP, plasma and urinary vitamin B6 and slightly higher urinary 4-PA values on normal vitamin B6 intakes. With supplementation, only the difference in urinary total vitamin B6 remained significant. Tryptophan load tests revealed no significant between-group differences in xanthurenic acid or kynurenic acid excretion. These results demonstrate an age-related difference in vitamin B6 status indicators in women under controlled dietary intake of vitamin B6.

Vitamin B6 status and static muscle function. 2 case reports.

2 young adult females, identified as vitamin B6 deficient based on xanthurenic acid excretion levels following a loading dose of tryptophan, were tested twice during each of 3 menstrual cycles for static muscle strength and endurance of the handgrip muscles. During each of the last 2 cycles either a 25-mg dose of pyridoxine hydrochloride or a placebo were administered daily in the double-blind fashion. Measurement of 24-hour xanthurenic acid and 4-pyridoxic acid excretion levels indicated correction of the biochemical indicators of vitamin B6 deficiency. Results of the tests of static muscle strength and endurance indicated no substantial improvement following vitamin B6 supplementation.

Therapy of side effects of oral contraceptive agents with vitamin B6.

Studies carried out in different countries during the last 15 years have provided evidence that supplementation with (or excess of) estro-progestational hormones may be accompanied by an increased urinary excretion of tryptophan metabolites, as happens in pyridoxine deficiency. Further methods of assessment of vitamin B6 in humans have confirmed an impaired status in women using hormonal contraception. Disturbances in the metabolism of tryptophan have been shown to be responsible for such symptoms as depression, anxiety, decrease of libido and impairment of glucose tolerance occurring in some of the OCA users. Administration of 40 mg of vitamin B6 daily not only restores normal biochemical values but also relieves the clinical symptoms in those vitamin B6 deficient women taking OCA's. Further studies are justified to clarify whether vitamin B6 supplementation may contribute to improving depression also in other situations with hyperoestrogenism (pregnancy, puerperium, estro-progestational treatments, etc.), as well as correcting metabolic impairments, such as a minor alteration of glucose tolerance.

PIP: Studies carried out in different countries over the last 15 years have provided evidence that supplementation with or excess of estrogen-progestogen hormones may be accompanied by an increased urinary excretion of tryptophan metabolites, as occurs in pyroxidine deficiency. Further methods of assessment of vitamin B6 in humans have confirmed that there is impaired status in women using oral contraceptives (OCs). Disturbances in the tryptophan metabolism have been shown to be responsible for such symptoms as depression, anxiety, decrease in libido, and impairment of glucose tolerance occurring in some OC users. Administration of 40 mg vitamin B6 daily not only restores the normal biochemical values but also relieves the clinical symptoms in those vitamin B6 deficient women taking OCs. Further studies are justified to clarify whether vitamin B6 supplementation may contribute to improving depression in other situations where there is hyperestrogenism (pregnancy, puerperium, estrogen-progestogen treatments), as well as correcting metabolic impairments, such as a minor alteration in glucose tolerance. (author's modified)

The vitamin B6 requirement in oral contraceptive users. II. Assessment by tryptophan metabolites, vitamin B6, and pyridoxic acid levels in urine.

PIP: The requirement for vitamin B6 in oral contraceptive users was studied in 8 college-age women who used combined (7) or sequential (1) oral contraceptives. The subjects and 8 controls consumed a basal diet supplemented to result in daily intake of 2.06 mg pyridoxine hydrochloride for 10 days (predepletion) and then containing only .36 mg of vitamin B6 for 32 days. After the depletion period, the diet was supplemented with pyridoxine hydrochloride to increase the intake of B6 to .96, 1.56, and 5.06 mg for 8, 9, and 7 days respectively. Complete 14-hour urine collections were analyzed for xanthurenic acid, kynurenic acids, kynurenine, and 3-hydroxykynurenine after administration of a l load-dose of 2 gm L-trytophan on days 2, 11, 18, 25, 32, 39, 43, 50 , 59, and 66 for the subjects and days 2 and 10 for the controls. Pretryptophan urine was analyzed for vitamin B6. Posttryptophan urine was analyzed for 4-pyridoxic acid. It was found that during the depletion phase the excretion of tryptophan metabolites increased significantly. Excretion dropped significantly upon supplementation with 1.56 or 5.06 mg of vitamin B6, returning values to normal. Levels of vitamin B6 and 4-pyridoxic acid in the urine decreased during depletion to be restored to normal upon supplementation with 1.56 mg/day. Since an intake of 5.0 mg vitamin B6 caused a loss of the vitamin in the urine and all levels were returned to normal with an intake of 1.56 mg, it is suggested that 1.5 mg of vitamin B6 is sufficient to meet the needs of most oral contraceptive users and that there is no significant difference in the vitamin B6 requirement of oral contraceptive users and nonusers.^ieng

Effect of varying vitamin B6 intake of early-weaned piglets on urinary xanthurenic and kynurenic acid excretion, serum transaminase activity and urea concentration.

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

Plasma pyridoxal phosphate levels and tryptophan metabolism in children with burns and scalds.

Depression of the pyridoxal phosphate concentration in the plasma was observed in eight children after burning or scalding. The concentrations remained low for several weeks with the usual dietary supplements but could be restored rapidly with large doses of pyridoxine; this does not seem to alter the excretion of xanthurenic acid but causes an increased loss of kynurenic acid and kynurenine.

Pyridoxine treatment of childhood bronchial asthma.

Urinary xanthurenic and kynurenic acid levels were measured in five patients while they were receiving 50 mg and 100 mg of pyridoxine. The levels of tryptophane metabolite decreased progressively as the dose was increased but remained above basal levels. There was marked clinical improvement in these patients while receiving the higher dose only. The double-blind study with 76 asthmatic children followed for five months indicated significant improvement in asthma following pyridoxine therapy (200 mg daily) and reduction in dosage of bronchodilators and cortisone. The data suggest that these children with severe bronchial asthma had a metabolic block in tryptophane metabolism, which was benefitted by long-term treatment with large doses of pyridoxine.