Plasma B6 vitamer and plasma and urinary 4-pyridoxic acid concentrations of middle-aged obese black women.

Plasma B6 vitamer and plasma and urinary 4-pyridoxic acid (4-PA) concentrations of fifteen middle-aged obese black women were determined by high-performance liquid chromatography (HPLC). Estimated protein and vitamin B6 intakes of the subjects, aged 27-52 years, were 64.5 +/- 15.6 g and 1.21 +/- 0.68 mg (mean +/- S.D.), respectively. Mean HPLC-derived plasma B6 vitamer and 4-PA concentrations for these subjects were 68.9, 3.1, 1.2, 4.1, 3.4, 7.2 and 2.0 nmol/l for pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate, pyridoxamine 5'-phosphate, pyridoxal, pyridoxine, pyridoxamine and 4-PA, respectively. The mean urinary 4-PA/creatinine ratio of the women was 0.88 mumol/mmol. All subjects had plasma PLP levels indicative of adequate vitamin B6 status. Vitamin B6 status parameters of the middle-aged obese black women were similar to those previously reported for white nonobese women having adequate vitamin B6 status.     

Quantities of B6 vitamers in human milk by high-performance liquid chromatography. Influence of maternal vitamin B6 status

A rapid, sensitive procedure is described for the analysis of the B6 vitamers pyridoxal, pyridoxamine, and pyridoxine in human milk from women taking and not taking supplements containing the vitamin using high-performance liquid chromatography with fluorometric detection. Vitamer values represent the sum of their phosphorylated and unphosphorylated forms. Minimum detectable quantities were 1-3 ng. Excellent recoveries of these vitamers in milk were obtained. Similar B6 vitamer concentrations of milk were obtained using the developed high-performance liquid chromatographic and the accepted microbiological techniques. Pyridoxal, actually consisting of pyridoxal plus pyridoxal phosphate, was the predominant B6 vitamer in human milk. The concentration of B6 vitamers in milk was reflective of the maternal vitamin B6 status.     

Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.     

Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in Human Hair Using LC-MS/MS

Water-soluble B vitamins participate in numerous crucial metabolic reactions and are critical for maintaining our health. Vitamin B deficiencies cause many different types of diseases, such as dementia, anaemia, cardiovascular disease, neural tube defects, Crohn’s disease, celiac disease, and HIV. Vitamin B3 deficiency is linked to pellagra and cancer, while niacin (or nicotinic acid) lowers low-density lipoprotein (LDL) and triglycerides in the blood and increases high-density lipoprotein (HDL). A highly sensitive and robust liquid chromatography–tandem mass spectroscopy (LC/MS-MS) method was developed to detect and quantify a vitamin B3 vitamer (nicotinamide) and vitamin B6 vitamers (pyridoxial 5′-phosphate (PLP), pyridoxal hydrochloride (PL), pyridoxamine dihydrochloride (PM), pridoxamine-5′-phosphate (PMP), and pyridoxine hydrochloride (PN)) in human hair samples of the UAE population. Forty students’ volunteers took part in the study and donated their hair samples. The analytes were extracted and then separated using a reversed-phase Poroshell EC-C18 column, eluted using two mobile phases, and quantified using LC/MS-MS system. The method was validated in human hair using parameters such as linearity, intra- and inter-day accuracy, and precision and recovery. The method was then used to detect vitamin B3 and B6 vitamers in the human hair samples. Of all the vitamin B3 and B6 vitamers tested, only nicotinamide was detected and quantified in human hair. Of the 40 samples analysed, 12 were in the range 100–200 pg/mg, 15 in the range 200–500 pg/mg, 9 in the range of 500–4000 pg/mg. The LC/MS-MS method is effective, sensitive, and robust for the detection of vitamin B3 and its vitamer nicotinamide in human hair samples. This developed hair test can be used in clinical examination to complement blood and urine tests for the long-term deficiency, detection, and quantification of nicotinamide.     

Plasma B6 vitamer and 4-pyridoxic acid concentrations of men fed controlled diets

A rapid and sensitive procedure is described for the analysis of all the B6 vitamers and 4-pyridoxic acid in human plasma utilizing reversed-phase high-performance liquid chromatography with ultraviolet and fluorometric detection. Pyridoxal phosphate values obtained by radiometric and chromatographic, ultraviolet and fluorometric, assays were highly correlated as were pyridoxine phosphate values determined using both detectors. Plasma B6 vitamer and 4-pyridoxic acid concentrations of 22 men fed diets containing 0.75-0.98 mg of vitamin B6 daily for eight weeks were in the range of reported values; pyridoxal phosphate was their predominant plasma B6 vitamer. This methodology should be useful in the assessment of vitamin B6 requirements.     

Plasma B-6 vitamer and plasma and urinary 4-pyridoxic acid concentrations in young women as determined using high performance liquid chromatography.

Plasma B-6 vitamer and plasma and urinary 4-pyridoxic acid concentrations of 21 young white women, 21-27 years, having radiomonitored pyridoxal 5'-phosphate and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate vitamin B-6 status were determined in an effort to establish normal ranges for plasma B-6 vitamers. B-6 vitamers and 4-pyridoxic acid were quantitated using reversed phase high performance liquid chromatography with fluorometric and ultraviolet detection. Pyridoxal phosphate values obtained by radioenzymatic and chromatographic, fluorometric and ultraviolet, assays were highly correlated as were pyridoxine phosphate values determined using both detectors. The B-6 vitamer and 4-pyridoxic acid values of these subjects should be of use in the establishment of normal ranges of these congeners in women.     

采用高效液相色谱技术分析生物体内维生素B6

维生素B6(VB6)是一类2-甲基-3-羟基吡啶类化合物的总称，基本类型有吡哆醇(PN)、吡哆醛(PL)和吡哆胺(PM)，磷酸酯型有磷酸吡哆醇(PNP)、磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)，其中磷酸吡哆醛和磷酸吡哆胺为活性形式，是多种酶的辅酶，哺乳动物尿中VB6的代谢产物主要是不具有生理活性的吡哆酸(PIC)，在植物体内还发现有数种吡哆醇的糖衍生物和氨基酸衍生物。     

Stability of pyridoxal-5-phosphate semicarbazone: applications in plasma vitamin B6 analysis and population surveys of vitamin B6 nutritional status

The determination of pyridoxal-5-phosphate (PLP) and pyridoxal (PL) in plasma requires the availability of dark room facilities, due to the photosensitivity of these vitamin B6 vitamers. The fact that the semicarbazone forms of PL and PLP are more strongly fluorescent than the underivatized B6 vitamers has been exploited in plasma analyses, but it was not previously realised that these semicarbazone forms are also very stable even under conditions that lead to rapid decomposition of free PL and PLP. The stabilisation of PLP and PL obtained in this manner is sufficient and fully adequate to meet the practical requirements of clinical field studies. We report a high-performance liquid chromatographic method for plasma PLP and PL determinations based on precolumn semicarbazone formation and fluorescence detection. The method is sensitive enough for quantitative plasma PLP determinations even in B6-deficient patients.     

Quantitative profiling of biomarkers related to B‐vitamin status,tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry

Vitamins B₂ and B₆ serve as cofactors in enzymatic reactions involved in tryptophan and homocysteine metabolism. Plasma concentrations of these vitamins and amino acids are related to smoking and inflammation, and correlate with other markers of immune activation. Large‐scale studies of these relations have been hampered by lack of suitable analytical methods. The assay described includes riboflavin, five vitamin B₆ forms (pyridoxal 5′‐phosphate, pyridoxal, 4‐pyridoxic acid, pyridoxine and pyridoxamine), tryptophan and six tryptophan metabolites (kynurenine, kynurenic acid, anthranilic acid, 3‐hydroxykynurenine, xanthurenic acid and 3‐hydroxyanthranilic acid), cystathionine, neopterin and cotinine. Trichloroacetic acid containing 13 isotope‐labelled internal standards was added to 60 µL of plasma, the mixture was centrifuged, and the resulting supernatant used for analysis. The analytes were separated within 5 min on a stable‐bond C8 column by a gradient‐type mobile phase containing acetonitrile, heptafluorobutyric acid and high concentration (650 mmol/L) of acetic acid, and detected using electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The mobile phase ensured sufficient separation and high ionization efficiency of all analytes. Recoveries were 75–123% and within‐day and between‐day coefficients of variance (CVs) were 2.5–9.5% and 5.4–16.9%, respectively. Limits of detection ranged from 0.05 to 7 nmol/L. The method enables quantification of endogenous plasma concentrations of 16 analytes related to B‐vitamin status and inflammation, and may prove useful in large‐scale epidemiological studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of water-soluble vitamins by liquid chromatography with a parallel dual-electrode electrochemical detector

A method for the determination of water-soluble vitamins (ascorbic acid, pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxamine dihydrochloride, p-aminobenzoic acid, folic acid) by liquid chromatography, with a parallel dual-electrode electrochemical detector, is described. One electrode was controlled at +0.80 V (vs. SCE), the other at +1.20 V (vs. SCE). The possibility of interference by eight other water-soluble vitamins (riboflavin, nicotinamide, cyanocobalamin, menadione, dextro calcium pantothenate, thiamine, nicotinic acid, dextro biotin) was studied. These vitamins did not interfere when a parallel dual-electrode detector system was used. The estimation of five of the vitamins was studied in detail. The linear ranges found were 10 ng-1.2 mug for pyridoxine hydrochloride, 2 ng-2mug for pyridoxal hydrochloride, 10 ng-3 mug for pyridoxamine dihydrochloride, 5-200 ng for folic acid and 0.6-200 ng for p-aminobenzoic acid, the limits of detection being 3, 0.6, 1, 2 and 0.06 ng respectively. Application of the technique to the estimation of vitamin B(6) in tablets is illustrated. The results indicate that the vitamin B(6) in these tablets existed in the pyridoxine hydrochloride form and the B(6) content agreed well with liquid chromatograph by spectrophotometric analysis.     

A qualitative and quantitative color reaction for pyridoxal with sulfuric acid

The carbonyl-phenol-acid reaction system yields color reactions with phenols, carbonyl compounds (aldehydes and ketones), and inorganic acids. 'I'o test for one of the components of this reaction system, the remaining two components constitute the specific reagents.The four related compounds, pyridoxine, pyridoxamine, pyridoxal, and pyridoxic acid each possess a phenolic hydroxyl. Pyridoxal possesses an aldehyde group in addition to the phenolic hydroxyl. Pyridoxal yields an intense yellow color on treatment with concentrated sulfuric acid. Pyridoxine, pyridoxamine and pyridoxic acid prove not to be chromogenic on treatment with concentrated sulfuric acid. Pyridoxal can therefore be differentiated by means of the sulfuric acid reaction from the other three compounds related to vitamin B₆.The colored product obtained by the interaction of pyridoxa1 and concentrated sulfuric acid yields a characteristic absorption spectrum and follows the Beer-Lambert law in the concentrations of pyridoxal tested (l0—100 μg).     

Liquid chromatography of vitamin B6 with electrochemical detection using a carbon fibre electrode

A flow-through electrochemical detector with a carbon fibre electrode was fabricated. A chromatographic method for the determination of vitamin B₆ using this detector is described, and the results are compared with those obtained by detection with a thin-layer glassy carbon electrode or spectrophotometry. The detection limit of vitamin B₆ is 2.5 ng for pyridoxine, 1 ng for pyridoxal and 1 ng for pyridoxamine. The technique was applied to the determination of vitamin B₆ in tablets.     

A 13C n.m.r. study of the B6 vitamins and of their aldimine derivatives

The vitamins, pyridoxine, pyridoxal, pyridoxamine, pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate, have been studied in aqueous solution over a pH range of 2–12 by ¹³C nuclear magnetic resonance spectroscopy. Resonance assignments are made primarily by the spin–spin coupling constants of carbons with protons and with phosphorus. The proton–carbon coupling constants show a marked conformational dependence in the hemiacetal form of pyridoxal. Furthermore, the H-6? C-5 coupling constant in the vitamins is much smaller than the corresponding constant in pyridine. This may be due either to an effect of the C-5 substituent in vitamins or to a different electronic configuration of the zwitterionic hydroxypyridine ring. The addition of manganese to a solution of pyridoxal phosphate causes line broadenings consistent with the interaction of the metal ion with this vitamin at the formyl and phenolic oxygens. The chemical shifts of the aromatic carbons of pyridoxine have been calculated, as a function of pH, by summing shielding parameters which were estimated empirically from pyridine derivatives. The calculated shifts agree well with the experimental data for C-3, C-5 and C-6, less well for C-2, and poorly for C-4. The deviation from additivity for C-4 indicates a preferred orientation for the 4-hydroxymethyl substituent caused by internal hydrogen bonding between the substituents at C-3 and C-4. Evidence is presented for the existence of the free aldehyde form of pyridoxal at alkaline pH. Aldimine complexes of pyridoxal and pyridoxal phosphate with amines and amino acids have also been studied. Characteristic chemical shift changes caused by both pyridinium and aldimine nitrogen deprotonations are seen. Additionally, the chemical shifts of carbons of the pyridine ring are dependent upon the structure of the imine, especially when the aldimine nitrogen is protonated. We conclude that this dependency is due to steric effects in an aldimine complex which is constrained by internal hydrogen bonding. We also discuss the merits of carbons 3 and 4 as possible sites of cofactor labeling for enzymatic studies.     

Chemistry of pyridoxine in drug design

Russian Chemical Bulletin - Pyridoxine and its derivatives, pyridoxamine and pyridoxal, are the three main forms of vitamin B6, which play exceptionally important biological roles in living...     

Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants

Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1-4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 x 10(7) M-1 s-1 for 1; 7.5 x 10(7) M-1 s-1 for 2, 6.2 x 10(7) M-1 s-1 for 3 and 7.5 x 10(7) M-1 s-1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased approximately 10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased approximately 10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1-4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.     

Etude des Dérivés de la Vitamine B6. Conformation des Complexes de Lanthanides avec le Pyridoxal-5′-phosphate et la Pyridoxamine-5′-phosphate en Phase Aqueuse à pD = 1,3

A suitable procedure is described for establishing the conformational analysis of flexible molecules coordinated to lanthanide cations from ¹H and ¹³C NMR paramagnetic shift data. The examples of pyridoxal 5′-phosphate (PLP) and pyridoxamine 5′-phosphate (PMP) in aqueous solution at pD = 1.3 values are discussed. In the two cases, a hindered rotation around P? O-5′, with Gaussian distribution is proposed. The values of angles and distances between rare earth cations and ligand nuclei are determined.     

The B6‐vitamer Pyridoxal is a Sensitizer of UVA‐induced Genotoxic Stress in Human Primary Keratinocytes and Reconstructed Epidermis

UVA‐driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3‐hydroxypyridine‐derived chromophores including B₆‐vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA‐induced photooxidative stress in human skin cells. Here, we report that the B₆‐vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA‐induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ‐H2AX (Ser139)], comet analysis indicated the formation of Fpg‐sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA‐driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B₆‐vitamers.     

Spectrophotometric,potentiometric, and conductometric studies of binary complex formation between copper(II) and three forms of vitamin B6 in aqueous solutions

This article reports the detailed study concerning the mode of binding of three forms of vitamin B₆, pyridoxamine (pm), pyridoxine (pn), and pyridoxal (pl), with Cu(II) in aqueous solutions using three independent methods: potentiometry, conductometry, and UV–vis spectroscopy. The stability constants of complexes formed between copper(II) and vitamin B₆ were investigated by potentiometric titration in 0.1 M KNO₃ ionic medium at 25 °C. While drawing the relations between molar conductance and the ratio of metal to ligand concentrations, different types of lines were obtained indicating the formation of 1 : 1 and 1 : 2 stoichiometric compounds. The stability constants have been determined using EQUID and CVEQUID computer programs and the obtained results were in agreement. The relatively high values of stability constants of Cu(II)-vitamin B₆ complexes obtained from three independent methods in comparison to those with other competing cations suggest that the complexes studied are relatively stable in aqueous solutions.     

Metallation of Ligands with Biological Activity: Synthesis and X‐Ray Characterization of [UO2(PN)2(H2O)]Cl2{PN = vitamin B6 pyridoxine[2‐methyl‐3‐hydroxy‐4, 5‐bis(hydroxymethyl) pyridine]}

Chloridrate of pyridoxine (vitamin B₆) reacts with UO₂(NO₃)₂·6H₂O in acetonitril containing triethylamine to give the complex salt [UO₂(PN)₂(H₂O)]Cl₂. The structure of the novel compound was analyzed by single crystal X‐ray diffraction affording the centrosymmetric triclinic space group . In [UO₂(PN)₂(H₂O)]²⁺ two zwitterionic pyridoxine molecules complex the uranium atom in a planar manner with a water molecule achieving the coordination of a semi planar pentagon. The two uranyl oxo ligands set the axis of a distorted pentagonal bipyramide. The ability of vitamin B₆ pyridoxine to react with UO₂²⁺ allowing the chelation of one uranium atom represents a very specific model of assimilation of uranium by living beings. It could also explain the serious damages caused by heavy or radioactive metals like uranium since their complexation “in vivo” by enzymatic systems like pyridoxal phosphate‐containing enzymes would lead to a modification of the prosthetic groups of the metalloenzymes with loss of their catalytic activities.