4-pyridoxic Acid Research Articles

Vitamin B-6

Vitamin B-6 is a water-soluble vitamin first discovered as a factor that cured dermatitis in rats. There are 6 forms of vitamin B-6 found in mammals. The naturally occurring forms of the vitamin include pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their respective monophosphorylated derivatives (PNP, PLP, and PMP). PN, PL, and PM are converted to pyridoxal 5′-phosphate (PLP) through the consecutive actions of 1) a kinase that phosphorylates the 5′hydroxymethyl group and 2) pyridoxamine phosphate oxidase (PNPO), which acts on PNP and PMP. PLP is the biologically active form of the vitamin that functions as an enzyme cofactor and/or regulator for >140 enzyme-catalyzed reactions. PLP-dependent proteins account for ∼4% of total cellular enzymes. PLP is covalently bound to the active sites of aminotransferases, decarboxylases, racemases, and dehydratases, among other enzymes, through a Schiff’s base between the aldehyde of PLP and the e-amino of an active site lysine. PLP-dependent enzymes play essential roles in amino acid metabolism, glycolysis, gluconeogenesis, glycogenolysis, transsulfuration, polyamine biosynthesis, and synthesis of sphingoid bases (required for myelin formation) and the heme precursor δ-aminolevulinic acid. In energy metabolism, PLP-dependent transaminases allow interconversion of amino acids and intermediates in energy-generating pathways. In the brain, PLP is required for the synthesis of the neurotransmitters serotonin, norepinephrine, epinephrine, and γ-aminobutyrate (GABA), and as such is involved in both neuronal excitation and inhibition. Vitamin B-6 is catabolized through the oxidation of pyridoxal to 4-pyridoxic acid, which is excreted in urine.

Markers of vitamin B6 status and metabolism as predictors of incident cancer: the Hordaland Health Study.

Dietary intake and/or circulating concentrations of vitamin B6 have been associated with risk of cancer, but results are inconsistent and mechanisms uncertain. Pyridoxal 5'-phosphate (PLP) is the most commonly used marker of B6 status. We recently proposed the ratio 3-hydroxykynurenine/xanthurenic acid (HK/XA) as an indicator of functional vitamin B6 status, and the 4-pyridoxic acid (PA) /(pyridoxal (PL) +PLP) ratio (PAr) as a marker of vitamin B6 catabolism during inflammation. We compared plasma PLP, HK/XA and PAr as predictors of cancer incidence in a prospective community-based cohort in Norway. This study included 6,539 adults without known cancer at baseline (1998-99) from the Hordaland Health Study (HUSK). HR and 95% CI were calculated for the risk of overall and site-specific cancers using multivariate Cox proportional hazards regression with adjustment for potential confounders. After a median follow-up time of 11.9 years, 963 cancer cases (501 men and 462 women) were identified. Multivariate-adjusted Cox-regression showed no significant relation of plasma PLP or HK/XA with risk of incident cancer. In contrast, PAr was significantly associated with risk of cancer with HR (95% CI) = 1.31 (1.12-1.52) per two standard deviation (SD) increment (p < 0.01). Further analysis showed that PAr was a particular strong predictor of lung cancer with HR (95% CI) = 2.46 (1.49-4.05) per two SD increment (p < 0.01). The present results indicate that associations of vitamin B6 with cancer may be related to increased catabolism of vitamin B6, in particular for lung cancer where inflammation may be largely involved in carcinogenesis.

Crystal structure of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase, an NAD+-dependent dismutase from Mesorhizobium loti

5-Formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase (FHMPCDH) from Mesorhizobium loti is the fifth enzyme in degradation pathway I for pyridoxine. The enzyme catalyzes a dismutation reaction: the oxidation of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) to 3-hydroxy-2-methylpyridine 4,5-dicarboxylic acid with NAD+ and reduction of FHMPC to 4-pyridoxic acid with NADH. FHMPCDH belongs to the l-3-hydroxyacyl-CoA dehydrogenase (HAD) family. The crystal structure was determined by molecular replacement and refined to a resolution of 1.55Å (R-factor of 16.4%, Rfree=19.4%). There were two monomers in the asymmetric unit. The overall structure of the monomer consisted of N- and C-terminal domains connected by a short linker loop. The monomer was similar to members of the HAD family (RMSD=1.9Å). The active site was located between the domains and highly conserved to that of human heart l-3-hydroxyacyl-CoA dehydrogenase (HhHAD). His-Glu catalytic dyad, a serine and two asparagine residues of HhHAD were conserved. Ser116, His137 and Glu149 in FHMPCDH are connected by a hydrogen bonding network forming a catalytic triad. The functions of the active site residues in the reaction mechanism are discussed.

Vitamin B status in patients with type 2 diabetes mellitus with and without incipient nephropathy

AimTo investigate the vitamin B status, with particular focus on vitamin B6, in adults with and without incipient nephropathy secondary to type 2 diabetes mellitus. MethodsPlasma and/or urine concentrations of vitamins B6, B1, B12, related vitamers and biomarkers (including total homocysteine, methylmalonic acid) were measured in 120 adults with type 2 diabetes (including 46 patients with microalbuminuria) and 52 non-diabetic control subjects. ResultsPlasma concentrations of pyridoxal 5′-phosphate (PLP) were significantly lower in patients with type 2 diabetes than in control subjects (median: 22.7nmol/L, diabetes with microalbuminuria; 26.8nmol/L, diabetes without microalbuminuria; 39.5nmol/L, non-diabetic control; p<0.0001). The prevalence of low PLP (<30nmol/L) was 63%, 58%, and 25% in the diabetes groups with and without microalbuminuria and the control group, respectively. Plasma levels of pyridoxine and pyridoxal were also lower (p<0.0001), but levels of pyridoxamine, pyridoxamine 5′-phosphate, and pyridoxic acid were higher in both groups with diabetes compared to the control group (p<0.001). Thiamine deficiency was highly prevalent in all groups, whereas low vitamin B12 and elevated methylmalonic acid were rare. Increased levels of C-reactive protein and soluble vascular cell adhesion molecule-1 were observed in the groups with diabetes (p<0.05, versus healthy control). ConclusionsDeficiency of vitamin B6 (PLP, pyridoxine, pyridoxal) and vitamin B1 (thiamine) was prevalent in type 2 diabetes. Incipient nephropathy was associated with more pronounced alterations in vitamin B6 metabolism and stronger indications of endothelial dysfunction and inflammation.

Vitamin B-6 vitamers in human plasma and cerebrospinal fluid

Vitamin B-6 comprises a group of 6 interrelated vitamers and is essential for numerous physiologic processes, including brain functioning. Genetic disorders disrupting vitamin B-6 metabolism have severe clinical consequences. To adequately diagnose known and novel disorders in vitamin B-6 metabolism, a reference set is required containing information on all vitamin B-6 vitamers in plasma and cerebrospinal fluid (CSF). Concentrations of vitamin B-6 vitamers in the plasma and CSF of 533 adult subjects were measured by ultra high-performance liquid chromatography-tandem mass spectrometry. The relative vitamin B-6 vitamer composition of plasma [pyridoxal phosphate (PLP) > pyridoxic acid (PA) > pyridoxal] differed from that of CSF (pyridoxal > PLP > PA > pyridoxamine). Sex influenced vitamin B-6 vitamer concentrations in plasma and CSF and should therefore be taken into account when interpreting vitamin B-6 vitamer concentrations. The strict ratios and strong correlations between vitamin B-6 vitamers point to a tight regulation of vitamin B-6 vitamer concentrations in blood and CSF. Given the unique design of this study, with simultaneously withdrawn blood and CSF from a large number of subjects, reliable CSF:plasma ratios and correlations of vitamin B-6 vitamers could be established. We provide an extensive reference set of vitamin B-6 vitamer concentrations in plasma and CSF. In addition to providing insight on the regulation of individual vitamers and their intercompartmental distribution, we anticipate that these data will prove to be a valuable reference set for the diagnosis and treatment of conditions associated with altered vitamin B-6 metabolism.

A metabonomic study of adjuvant-induced arthritis in rats using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory and autoimmune disease accompanied by the destruction and deformities of joints. Adjuvant-induced arthritis (AIA) is one of the excellent animal models of RA used to understand disease pathogenesis and screen potential drugs. In this paper, a urinary metabonomics method based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) has been established to investigate the disease progression of AIA and find potential biomarkers of secondary inflammation in AIA rats. 24 potential biomarkers were identified, including xanthurenic acid, kynurenic acid, 4-pyridoxic acid, and phenylalanine, which revealed that tryptophan metabolism, phenylalanine metabolism, gut microbiota metabolism and energy metabolism were disturbed in AIA rats. These potential biomarkers and their corresponding pathways are helpful to further understand the mechanisms of AIA and pathogenesis of RA. This study demonstrates that metabonomics based on UPLC-Q-TOF-MS is a powerful methodology to analyze the underlying disease pathogenesis.

Evidence for increased catabolism of vitamin B-6 during systemic inflammation

Plasma concentrations of PL 5'-phosphate (PLP), which is the active coenzyme form of vitamin B-6, are reduced during inflammation. The underlying mechanisms may include altered tissue distribution or increased catabolism via pyridoxal (PL) to pyridoxic acid (PA). Recently, we showed that catabolic enzyme activity could be assessed by substrate product ratios measured in plasma. We evaluated the ratios PA:PL, PA:PLP, and PA:(PL + PLP) as possible markers of vitamin B-6 catabolism. Cross-sectional and longitudinal data were derived from the Western Norway B-Vitamin Intervention Trial. We analyzed associations of ratios with inflammatory markers and other clinical variables by using multiple linear regression and partial correlation. In addition, intraclass correlation coefficients (ICCs) were used to assess the ability of plasma indexes to differentiate between subjects. PA:(PL + PLP) had the highest ICC of all vitamin B-6 metabolites and ratios tested. In regression models, the inflammatory markers C-reactive protein, white blood cell count, neopterin, and kynurenine:tryptophan collectively accounted for 28% of the total and > 90% of the explained variation in PA:(PL + PLP). For individual B-6 metabolites, corresponding numbers were 19-25% and 20-44%, respectively, with vitamin supplement intake, smoking, and kidney function (estimated glomerular filtration rate) as additional predictors. In an analysis of receiver operating characteristics, PA:(PL + PLP) discriminated high inflammatory concentrations with an area under the curve (95% CI) of 0.85 (0.81, 0.89). Broad-specificity enzymes upregulated to reduce oxidative and aldehyde stress could explain increased catabolism of vitamin B-6 during inflammation. The ratio PA:(PL + PLP) may provide novel insights into pathologic processes and potentially predict risk of future disease.

Most Blood Biomarkers Related to Vitamin Status, One-Carbon Metabolism, and the Kynurenine Pathway Show Adequate Preanalytical Stability and Within-Person Reproducibility to Allow Assessment of Exposure or Nutritional Status in Healthy Women and Cardiovascular Patients1–3

Knowledge of stability during sample transportation and changes in biomarker concentrations within person over time are paramount for proper design and interpretation of epidemiologic studies based on a single measurement of biomarker status. Therefore, we investigated stability and intraindividual vs. interindividual variation in blood concentrations of biomarkers related to vitamin status, one-carbon metabolism, and the kynurenine pathway. Whole blood (EDTA and heparin, n = 12) was stored with an icepack for 24 or 48 h, and plasma concentrations of 38 biomarkers were determined. Stability was calculated as change per hour, intraclass correlation coefficient (ICC), and simple Spearman correlation. Within-person reproducibility of biomarkers was expressed as ICC in samples collected 1–2 y apart from 40 postmenopausal women and in samples collected up to 3 y apart from 551 patients with stable angina pectoris. Biomarker stability was similar in EDTA and heparin blood. Most biomarkers were essentially stable, except for choline and total homocysteine (tHcy), which increased markedly. Within-person reproducibility in postmenopausal women was excellent (ICC > 0.75) for cotinine, all-trans retinol, cobalamin, riboflavin, α-tocopherol, Gly, pyridoxal, methylmalonic acid, creatinine, pyridoxal 5′-phosphate, and Ser; was good to fair (ICC of 0.74–0.40) for pyridoxic acid, kynurenine, tHcy, cholecalciferol, flavin mononucleotide, kynurenic acid, xanthurenic acid, 3-hydroxykynurenine, sarcosine, anthranilic acid, cystathionine, homoarginine, 3-hydroxyanthranilic acid, betaine, Arg, folate, total cysteine, dimethylglycine, asymmetric dimethylarginine, neopterin, symmetric dimethylarginine, and Trp; and poor (ICC of 0.39–0.15) for methionine sulfoxide, Met, choline, and trimethyllysine. Similar reproducibilities were observed in patients with coronary heart disease. Thus, most biomarkers investigated were essentially stable in cooled whole blood for up to 48 h and had a sufficient within-person reproducibility to allow one-exposure assessment of biomarker status in epidemiologic studies. The Western Norway B Vitamin Intervention Trial was registered at clinicaltrials.gov> as NTC00354081.

Structure of 4-pyridoxolactonase from Mesorhizobium loti.

4-Pyridoxolactonase from Mesorhizobium loti catalyzes the zinc-dependent lactone-ring hydrolysis of 4-pyridoxolactone (4PAL) to 4-pyridoxic acid (4PA) in vitamin B6 degradation pathway I. The crystal structures of 4-pyridoxolactonase and its complex with 5-pyridoxolactone (5PAL; the competitive inhibitor) were determined. The overall structure was an αβ/βα sandwich fold, and two zinc ions were coordinated. This strongly suggested that the enzyme belongs to subclass B3 of the class B β-lactamases. In the complex structure, the carbonyl group of 5PAL pointed away from the active site, revealing why it acts as a competitive inhibitor. Based on docking simulation with 4PAL, 4PA and a reaction intermediate, 4-pyridoxolactonase probably catalyzes the reaction through a subclass B2-like mechanism, not the subclass B3 mechanism.

A simple high-performance liquid chromatography (HPLC) method for the measurement of pyridoxal-5-phosphate and 4-pyridoxic acid in human plasma

BackgroundLow concentration of plasma pyridoxal-5-phosphate (PLP) is associated with hyperhomocysteinemia and inflammation. Most methods for the measurement of plasma PLP require large specimen volume and involve the use of toxic reagents. MethodsWe have developed a HPLC method for the measurement of PLP and 4-pyridoxic acid (4-PA) in plasma, which requires small specimen volume. The samples are prepared without adding any toxic reagents. Furthermore, we have examined whether intake of vitamin B6 affects the concentration of plasma PLP and 4-PA. ResultsThe coefficient of variation of the method was 6% and the recovery of the added vitamin in plasma was about 100%. The concentrations of plasma PLP and 4-PA in 168 healthy subjects were 40.6 (8.4–165.0) nmol/L, median and (range) and 17.5 (3.7–114.79) nmol/L, median and (range) respectively. In the multiple regression analyses, the concentration of plasma PLP was associated with the concentration of plasma 4-PA (p<0.0001), BMI, (p=0.02) and sex, (p=0.0008). The concentration of plasma 4-PA was associated with plasma PLP (p<0.0001), serum folate (p=0.004), smoking (p=0.03) and vitamin B6 intake (p=0.01). ConclusionThe present method is suitable for large clinical studies for the measurement of plasma PLP and 4-PA. Our findings demonstrate that plasma 4-PA, BMI and sex are the major determinants of plasma PLP in healthy individuals.

One‐carbon metabolite levels in mid‐pregnancy and risks of conotruncal heart defects

Evidence exists for an association between use of vitamin supplements with folic acid in early pregnancy and reduced risk for offspring with conotruncal heart defects. A few observations have been made about nutrients related to one-carbon metabolism other than folate. Our prospective study attempted to extend information on nutrition and conotruncal heart defects by measuring analytes in mid-pregnancy sera. This study included data from a repository of women's mid-pregnancy serum specimens based on screened pregnancies in California from 2002-2007. Each woman's specimen was linked with delivery information to determine whether her fetus had a conotruncal heart defect or another structural malformation, or was nonmalformed. We identified 140 conotruncal cases and randomly selected 280 specimens as nonmalformed controls. Specimens were tested for a variety of analytes, including homocysteine, methylmalonic acid, folate, vitamin B12 , pyridoxal phosphate, pyridoxal, pyridoxic acid, riboflavin, total choline, betaine, methionine, cysteine, cystathionine, arginine, asymmetric and symmetric dimethylarginine. We did not observe statistical evidence for substantial differences between cases and controls for any of the measured analytes. Analyses specifically targeting B-vitamins also did not reveal differences between cases and controls.

Cirrhosis associated with pyridoxal 5'-phosphate treatment of pyridoxamine 5'-phosphate oxidase deficiency.

We report the case of an 8-year-old boy with pyridoxamine 5'-phosphate oxidase (PNPO) deficiency. He developed seizures at 24h of age that were refractory to standard anticonvulsant therapy and a trial of pyridoxine but responded to pyridoxal phosphate (PLP) at 28days of life. Genetic testing identified compound heterozygous mutations in the PNPO gene. Management of encephalopathic episodes required escalation of PLP dose to 100 mg/kg/day by 2 years of age. Routine blood tests at this time showed significantly deranged liver function tests (LFTs). A wedge liver biopsy showed early cirrhosis with marked elevation of pyridoxal and pyridoxic acid levels in the liver sample. Despite extensive investigation, no cause other than PLP therapy could be identified for the cirrhosis. The PLP dose was weaned to 50mg/kg/day before episodes of encephalopathy recurred. Concurrent with the reduction of his PLP dose, LFTs showed improvement. However, at 8 years of age, there is persistent evidence of hepatic fibrosis and early portal hypertension. We hypothesise that hepatic toxicity due to PLP or its degradation products is the cause of cirrhosis in this boy. Until further evidence becomes available, we would suggest that people with PNPO deficiency are treated with the minimum dose of PLP required to prevent episodes of encephalopathy.

Attention-deficit hyperactivity disorder (ADHD) as a pyridoxine-dependent condition: Urinary diagnostic biomarkers

The data obtained in children with different forms of epilepsy allowed us to consider epilepsy as an inborn error of pyridoxine (vitamin B6) metabolism (Dolina et al., 2012). Mutual interconnections between ADHD and epilepsy indicate that such an approach is reasonable for ADHD.To check such an assumption we analyzed in ADHD patients the same parameters of pyridoxal phosphate (PLP)-dependent tryptophan (TRP) degradation, which were analyzed in epileptic children. The level of TRP and concentrations of compounds formed or metabolized by TRP degradation, the ratios between some of them, and the level of 4-pyridoxic acid were HPLC detected in ADHD children and healthy controls. The data obtained, including low values of 4PA/TRP, IND/TRP and IND/KYN ratios, have evidenced dramatically impaired activity of pyridoxine-dependent enzymes in ADHD patients.Ritalin treatment did not change the general pattern of TRP degradation, but still created a kind of balance between some of detected metabolites. However, the 4PA/TRP, IND/TRP and IND/KYN ratios remained as low as in untreated patients, keeping the importance of diagnostic markers.Almost identical parameters of TRP degradation in untreated ADHD and epileptic patients allow to assume that inborn disorders of vitamin B6 metabolism are the common biochemical background of both diseases. The disturbed activity of PLP dependent enzymes apparently forms those profound disturbances of neurotransmitter systems, which are inherent in ADHD: low concentrations of monoamines and disordered amino acid metabolism. If vitamin B6 disorders are the core biochemical disturbances inherent in ADHD, then the long-term pyridoxine treatment is pathogenetically based replacement therapy of the disease. According to our data, multi-year pyridoxine treatment normalizes completely the pattern of ADHD behavior, without causing any serious side effects.

Values for evaluating the nutritional status of water-soluble vitamins in humans

Previously, we clarified that the amount of urinary excretion of water-soluble vitamins closely reflects the surplus amount of water-soluble vitamins in the body stores of rats and humans. We tried to set a tentative amount of urinary excretion of eight water-soluble vitamins of nine water-soluble vitamins (except vitamin B 12 ) for maintaining health based on experiments in healthy young females administered a semi-chemically defined diet according to Japanese Dietary Reference Intakes and related data. We proposed a tentative value for the amount of urinary excretion of water-soluble vitamins for maintaining health. The values were: 200–2000 nmol/d for vitamin B 1 ; 200–2000 nmol/d for vitamin B 2 ; 2–15 µ mol/d for 4-pyridoxic acid (a catabolite of vitamin B 6 ); 50–300 µ mol/d for the sum of the nicotinamide catabolites N 1 -methylnicotinamide, N 1 -methyl-2-pyridone-5-carboxamide, and N 1 -methyl-4-pyridone-3-carboxamide; 10–30 µ mol/d for pantothenic acid; 15–100 nmol/d for folate; 50–200 nmol/d for biotin; and 100–2000 µ mol/d for vitamin C. By using these values, we attempted to evaluate the nutritional status of water-soluble vitamins for 709 young Japanese females. The percentage within the tentative value of urinary excretion of water-soluble vitamin for maintaining health was 73.6% for vitamin B 1 , 63.5% for vitamin B 2 , 90.0% for vitamin B 6 , 85.6% for niacin, 58.1% for folate, 85.6% for pantothenic acid, 70.2% for biotin, and 65.4% for vitamin C. The percentage beyond the lower limit of detection was 22.4% for vitamin B 1 , 31.3% for vitamin B 2 , 6.2% for vitamin B 6 , 14.0% for niacin, 40.9% for folate, 12.4% for pantothenic acid, 26.2% for biotin, and 33.0% for vitamin C. The percentage over the upper limit of detection was 4.1% for vitamin B 1 , 5.2% for vitamin B 2 , 3.8% for vitamin B 6 , 0.4% for niacin, 1.0% for folate, 2.0% for pantothenic acid, 3.6% for biotin, and 1.6% for vitamin C. Nutritional assessment using urinary excretion amounts of water-soluble vitamins is persuasive, and leads to the transformation of habitual dietary intakes.

Dietary Supplement Use and Smoking Are Important Correlates of Biomarkers of Water-Soluble Vitamin Status after Adjusting for Sociodemographic and Lifestyle Variables in a Representative Sample of U.S. Adults

Biochemical indicators of water-soluble vitamin (WSV) status were measured in a nationally representative sample of the U.S. population in NHANES 2003–2006. To examine whether demographic differentials in nutritional status were related to and confounded by certain variables, we assessed the association of sociodemographic (age, sex, race-ethnicity, education, income) and lifestyle (dietary supplement use, smoking, alcohol consumption, BMI, physical activity) variables with biomarkers of WSV status in adults (aged ≥20 y): serum and RBC folate, serum pyridoxal-5′-phosphate (PLP), serum 4-pyridoxic acid, serum total cobalamin (vitamin B-12), plasma total homocysteine (tHcy), plasma methylmalonic acid (MMA), and serum ascorbic acid. Age (except for PLP) and smoking (except for MMA) were generally the strongest significant correlates of these biomarkers (|r| ≤ 0.43) and together with supplement use explained more of the variability compared with the other covariates in bivariate analysis. In multiple regression models, sociodemographic and lifestyle variables together explained from 7 (vitamin B-12) to 29% (tHcy) of the biomarker variability. We observed significant associations for most biomarkers (≥6 of 8) with age, sex, race-ethnicity, supplement use, smoking, and BMI and for some biomarkers with PIR (5 of 8), education (1 of 8), alcohol consumption (4 of 8), and physical activity (5 of 8). We noted large estimated percentage changes in biomarker concentrations between race-ethnic groups (from –24 to 20%), between supplement users and nonusers (from –12 to 104%), and between smokers and nonsmokers (from –28 to 8%). In summary, age, sex, and race-ethnic differentials in biomarker concentrations remained significant after adjusting for sociodemographic and lifestyle variables. Supplement use and smoking were important correlates of biomarkers of WSV status.

Application of Ultraperformance Liquid Chromatography/Mass Spectrometry–Based Metabonomic Techniques to Analyze the Joint Toxic Action of Long-term Low-Level Exposure to a Mixture of Organophosphate Pesticides on Rat Urine Profile

In previously published articles, we evaluated the toxicity of four organophosphate (OP) pesticides (dichlorvos, dimethoate, acephate, and phorate) to rats using metabonomic technology at their corresponding no observed adverse effect level (NOAEL). Results show that a single pesticide elicits no toxic response. This study aimed to determine whether chronic exposure to a mixture of the above four pesticides (at their corresponding NOAEL) can lead to joint toxic action in rats using the same technology. Pesticides were administered daily to rats through drinking water for 24 weeks. The above mixture of the four pesticides showed joint toxic action at the NOAEL of each pesticide. The metabonomic profiles of rats urine were analyzed by ultraperformance liquid chromatography/mass spectrometry. The 16 metabolites statistically significantly changed in all treated groups compared with the control group. Dimethylphosphate and dimethyldithiophosphate exclusively detected in all treated groups can be used as early, sensitive biomarkers for exposure to a mixture of the OP pesticides. Moreover, exposure to the OP pesticides resulted in increased 7-methylguanine, ribothymidine, cholic acid, 4-pyridoxic acid, kynurenine, and indoxyl sulfate levels, as well as decreased hippuric acid, creatinine, uric acid, gentisic acid, C18-dihydrosphingosine, phytosphingosine, suberic acid, and citric acid. The results indicated that a mixture of OP pesticides induced DNA damage and oxidative stress, disturbed the metabolism of lipids, and interfered with the tricarboxylic acid cycle. Ensuring food safety requires not only the toxicology test data of each pesticide for the calculation of the acceptable daily intake but also the joint toxic action.

The Intestine Plays a Substantial Role in Human Vitamin B6 Metabolism: A Caco-2 Cell Model

BackgroundVitamin B6 is present in various forms (vitamers) in the diet that need to be metabolized to pyridoxal phosphate (PLP), the active cofactor form of vitamin B6. In literature, the liver has been reported to be the major site for this conversion, whereas the exact role of the intestine remains to be elucidated.ObjectiveTo gain insight into the role of the intestine in human vitamin B6 metabolism.Materials and MethodsExpression of the enzymes pyridoxal kinase (PK), pyridox(am)ine phosphate oxidase (PNPO) and PLP-phosphatase was determined in Caco-2 cells and in lysates of human intestine. Vitamin B6 uptake, conversion and excretion were studied in polarized Caco-2 cell monolayers. B6 vitamer concentrations (pyridoxine (PN), pyridoxal (PL), PLP, pyridoxamine (PM), pyridoxamine phosphate (PMP)) and pyridoxic acid (PA) were quantified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using stable isotope-labeled internal standards.ResultsThe enzymatic system involved in vitamin B6 metabolism (PK, PNPO and PLP-phosphatase) is fully expressed in Caco-2 cells as well as in human intestine. We show uptake of PN, PM and PL by Caco-2 cells, conversion of PN and PM into PL and excretion of all three unphosphorylated B6 vitamers.ConclusionWe demonstrate, in a Caco-2 cell model, that the intestine plays a substantial role in human vitamin B6 metabolism.

Contents of All Forms of Vitamin B6, Pyridoxine-^|^beta;-Glucoside and 4-Pyridoxic Acid in Mature Milk of Japanese Women According to 4-Pyridoxolactone-Conversion High Performance Liquid Chromatography

The contents of six vitamin B6 forms, pyridoxine-β-glucoside, and 4-pyridoxic acid in mature milk of 20 Japanese lactating women consuming ordinary Japanese foods were determined by a 4-pyridoxolactone-conversion HPLC method. These compounds were determined with the average recovery rate of 83.9% or more. The average total content of vitamin B6 forms was 1.01 ± 0.32 (µmol/L). Pyridoxal and pyridoxal 5'-phosphate were found in all of the samples, and their average contents were 0.71 ± 0.28 (µmol/L) and 0.16 ± 0.07 (µmol/L), respectively. Pyridoxamine, pyridoxine, pyridoxamine 5'-phosphate, pyridoxine 5'-phosphate, and pyridoxine-β-glucoside were found in 15, 14, 13, 9, and 7 samples, respectively. The presence of pyridoxine 5'-phosphate was for the first time found in human milk. A method for the determination of 4-pyridoxic acid, which is the excretion form of vitamin B6, was modified to quantitate it by isocratic HPLC. 4-Pyridoxic acid was found in all samples, and its average content was 0.094 ± 0.040 (µmol/L), which was only 12% of its content in cow (Holstein) milk. The total content of vitamin B6 forms, and predominant presence of pyridoxal among other vitamin B6 forms in the Japanese women's milk samples shared similar characteristics with American women's milk samples.

Urinary excretion of B-group vitamins reflects the nutritional status of B-group vitamins in rats.

We have reported previously that the urinary excretion of B-group vitamins reflects recent dietary intakes of these vitamins. We also proposed reference values for the urinary levels of B-group vitamins for human subjects, and used these for evaluating human nutritional status. However, the question arises as to whether the urinary excretion of B-group vitamins in animals or human subjects decreases immediately before they become B-group vitamin insufficient or when fed a diet low in vitamins. In the present study, rats were fed a vitamin-free diet for 5d, and changes in the levels of B-group vitamins in urine and blood were monitored. Urinary excretion of vitamin B1, vitamin B2, 4-pyridoxic acid (a catabolite of vitamin B6), pantothenic acid, folate and biotin steeply decreased, and all of the values reached zero within 1-2d. With respect to blood, the concentrations of only three of the eight B-group vitamins (vitamin B1, pyridoxal phosphate and biotin) decreased to 15% (P<0·0001), 7% (P<0·0001) and 2% (P<0·0001) on day 5, respectively, compared with the values at the beginning of the experiment. The decrease was more rapid and the changes were greater in the urine samples than in the blood samples. The present data complement our previous proposal that the urinary excretion of B-group vitamins reflects the nutritional status of these vitamins.

Fluorescence characteristics of human urine from normal individuals and ovarian cancer patients

Early diagnostics of ovarian cancer is difficult, because there are no symptoms until the disease has progressed to an advanced stage. As urine contains many intrinsic fluorophores, modern fluorescence techniques are perspective candidates for new routine urine tests. The presented work deals with differences in the fluorescence of metabolites in urine of ovarian cancer patients comparing to healthy volunteers using the fluorescence excitation-emission matrices. The most serious differences were found in undiluted urine at the fluorescence emission wavelengths from 400 nm to 460 nm when excited at 310 - 390 nm. Statistical analyses of our data have shown a 5-fold reduction in the intensity of the peak at 330/420 nm (excitation/emission wavelength) for undiluted urine samples excreted by cancer patients as compared to those of normal donors. Moreover, the ratio of intensities of the peaks at 370/440 nm and at 330/420 nm is 18-times elevated in urine excreted by patients with ovarian cancer as compared to healthy urine samples. The observed changes could be interpreted as reduction of the presence of pyridoxic acid, whereas blue-fluorescing pteridines becomes dominant in excitation-emission matrices of cancer urine samples in comparison to healthy donors. We suggest pteridines, which are related to cellular metabolism, as suitable candidates for neoplasia-associated fluorescent markers in human urine. Our work showed that monitoring of human urine fluorescent metabolites offers an alternative for ovarian cancer screening.