Zur Analytik der Pyridoxine

Zusammenfassung Es werden 4 spezifische Nachweis- und Bestimmungsreaktionen für Pyridoxal und Pyridoxamin mitgeteilt. In Kombination mit dem Nachweis mit Gibbss'chem Reagens können nunmehr Pyridoxol, Pyridoxal und Pyridoxamin in Gemischen nebeneinander bestimmt werden. Die Überführbarkeit der 3 Komponenten ineinander wird aufgezeigt.

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On the analysis of pyridoxins

4 selective tests for pyridoxal and pyridoxamine are described. In combination with the test by means of 2,6-dichloroquinone-4-chloroimine it is possible to determine pyridoxol, pyridoxamine and pyridoxal in presence of each other in mixtures. It is shown that the three components are able to be transformed into one another.

     

Kinetic and mechanism of reactions of L-α-Glutamic acid and L-Glutamine with pyridoxal

The kinetics and mechanisms of condensation of pyridoxal with L-α-glutamic acid and L-glutamine were studied by UV spectroscopy and polarimetry. L-α-Glutamic acid reacts with pyridoxal to form a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and α-ketoglutaric acid. The reaction of Lglutamine with pyridoxal involves the Γ-NH₂ group and affords a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and L-α-glutamic acid.     

Simultaneous determination of pyridoxal and pyridoxamine by different spectrofluorimetric techniques

Binary mixtures of pyridoxal and pyridoxamine can be resolved by using zero-crossing first derivative spectrofluorimetry, first devivative constant wavelength synchronous luminescence spectrometry and first derivative constant energy synchronous luminescence spectrometry. These methods do not require any previous separation steps. The lowest quantization limit is obtained with first derivative constant energy synchronous fluorescence (13.0 and 9.0 mug/1. for pyridoxal and pyridoxamine, respectively). The measurements were performed in aqueous medium at pH 7.0 provided by adding 0.05M phosphate buffer solution. In order to demonstrate the validity of these methods a complete and exhaustive statistical analysis of the experimental data was performed. Pyridoxal and pyridoxamine were determined by these methods in synthetic and real mixtures with good results.     

Simple isocratic high-performance liquid chromatographic method for measuring pyridoxine kinase activity in crude biological extracts

To measure the enzymatic activity of pyridoxine kinase (EC 2.7.1.35) when pyridoxine or pyridoxamine are the substrates an additional enzymatic step is usually required. The products of the kinase activity, pyridoxine 5'-phosphate or pyridoxamine 5'-phosphate, are oxidized, enzymatically, to pyridoxal 5'-phosphate (co-enzyme) which is then measured either spectrophotometrically or using apo-enzymes. In this report the enzymatic activity of pyridoxine kinase, in crude biological extracts, is assayed by a simple high-performance liquid chromatographic method which determines the amount of pyridoxine 5'-phosphate formed when pyridoxine is the substrate. The same method could be used when pyridoxamine is the substrate.     

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。     

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...     

Pyridoxamine,a scavenger agent of carbohydrates

Pyridoxamine has been found to inhibit protein glycation and to avoid the formation of advanced glycation end‐products (AGEs). One of the mechanisms by which pyridoxamine can inhibit glycation involves the scavenger of carbonyl groups with glycation capacity. In this work, we conducted a kinetic study of the reactions of pyridoxamine with various carbohydrates under physiological pH and temperature. The reactions involving hexoses were found to give a tricyclic compound ( 5 ) in addition to pyridoxal and pyridoxine. Such a tricyclic compound inhibits the Amadori rearrangement and the formation of other carbonyl compounds with glycating properties. The reactions involving pentoses gave compound 7 and pyridoxal—by transamination of the Schiff base. The transamination reaction enhances the inhibitory action of pyridoxamine. The formation rate constants for the Schiff base, k₃, were found to be similar to those for the reactions of D ‐glucose with amino acids, which suggests competition between pyridoxamine and terminal amino residues in proteins for glycating sites in sugars. These constants are dependent on the electrophilic character of the carbonyl carbon in the carbohydrate. © 2007 Wiley Periodicals, Inc. 39: 154–167, 2007     

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.     

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.     

Kinetics and mechanism of the condensation of pyridoxal hydrochloride with L-tryptophan and D-tryptophan,and the chemical transformation of their products

The kinetics and mechanism of interaction between pyridoxal and L-tryptophan, D-tryptophan, and their derivatives are studied. It is found that condensation reactions proceed via three kinetically distinguishable stages: (1) the rapid intraplanar addition of the NH₂ groups of the amino acids to pyridoxal with the formation of amino alcohols; (2) the rotational isomerism of amino alcohol fragments with their subsequent dehydration and the formation of a Schiff base with a specific configuration; (3) the abstraction of α-hydrogen in the product of condensation of pyridoxal with L-tryptophan, or the abstraction of СО₂ in the product of condensation of pyridoxal with D-tryptophan with the formation of quinoid structures, hydrolysis of which results in the preparation of pyridoxamine and keto acid or pyridoxal and tryptamine, respectively. Schiff bases resistant to further chemical transformations are formed in the reaction with tryptophan methyl ester.     

Vitamin b6 analogs

The synthesis of 2- and 5-modified analogs of pyridoxine from which analogs of pyridoxal and pyridoxal 5-phosphate were obtained is described. The transition to analogs of pyridoxamine and pyridoxamine 5-phosphate is realized by hydrogenation of the oximes of these aldehydes.See [1] for communication XV.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 655–660, May, 1974.     

Chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids

The mechanism of chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids is studied by kinetic measurements. The Schiff bases are shown to be fairly stable in neutral media. In acid media, the Schiff bases are hydrolyzed into the initial components. In alkaline media, cleavage of α-hydrogen from the amino acid fragment and structural rearrangement into the quinoid form followed by hydrolysis of the latter with elimination of pyridoxamine and keto acid take place. The rate constants of the chemical transformations of the Schiff bases are found to depend on the pH of the medium. It is shown for the first time that the phosphate group in the pyridoxal 5′-phosphate fragment catalyzes the α-hydrogen cleavage and strongly accelerates alkaline decomposition of the Schiff bases.     

Separation and quantification of the B6 vitamers in plasma and 4-pyridoxic acid in urine of adolescent girls by reversed-phase high-performance liquid chromatography

The vitamin B6 status of seemingly healthy adolescent girls was determined using several accepted and proposed parameters in an effort to establish guidelines for status evaluation. High-performance liquid chromatography-derived plasma B6 vitamers (pyridoxal phosphate, PLP; pyridoxine phosphate. PNP; pyridoxamine phosphate, PMP; pyridoxal, PL; pyridoxine, PN; and pyridoxamine, PM) and 4-pyridoxic acid (4-PA) concentrations and urinary 4-PA levels of 28 white adolescent females, 12-15 years, having radiomonitored plasma PLP concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate status were determined. Mean vitamin B6 and protein intakes were 1.48 mg and 78.3 g. Ranges for plasma B6 vitamer and 4-PA concentrations (nmol/l) were: PLP, 40.9-122.2; PNP, non-detectable (ND)-16.1; PMP, ND-8.1; PL, ND-15; PN, ND-21.9; PM, ND-17.8; and 4-PA, ND-55.7. PLP was the only vitamer found in plasma of all subjects. Urinary 4-PA concentrations ranged from 0.11 to 2.50 mumol/mmol of creatinine. B6 vitamer values of these girls should be of use in the establishment of normal ranges for vitamin B6 status parameters.     

Simultaneous fluorometric resolution of pyridoxal, pyridoxamine and 4-pyridoxic acid using chemometric methods.

An alternatively minimizing covariant matrix error (AMCME) algorithm, newly proposed by the present authors, was applied to the simultaneous fluorometric determination of pyridoxal, pyridoxamine and 4-pyridoxic acid without loss of sensitivity. The experimental results illustrate that the profiles of spectra and concentration can be accurately resolved using the AMCME algorithm with a high sensitivity and stable repeatability. That is to say, the closely overlapping problem of the spectra could be resolved owing to the characteristic features of the AMCME algorithm.     

Chemical transformations of the condensation products of pyridoxal with L-α-alanine and D-α-alanine

The kinetics and mechanism of the reactions of pyridoxal with L- and D-α-alanine were studied. Under comparable conditions, the condensation of L- and D-α-alanines with pyridoxal includes three kinetically different steps. The first fast step is addition of the amino acid to pyridoxal with formation of the corresponding amino alcohol, the second (slower) step is dehydration of the amino alcohol to give Schiff base, and the third (very slow) step is elimination of α-hydrogen atom from the L-α-amino acid fragment or decarboxylation of the D-α-amino acid fragment, followed by isomerization of the Schiff base to quinoid structure whose subsequent hydrolysis yields pyridoxamine and pyruvic acid or acetaldehyde, respectively. A scheme was proposed for chemical transformations of the pyridoxal condensation products with L- and D-α-alanines.     

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).     

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.     

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.     

Kinetics and Mechanism of the Condensation of Pyridoxal with Amino Acids

The kinetics and mechanism of the condensation of amino acids with pyridoxal were studied in relation to the amino acid structure, solvent, pH, and temperature. A spectrophotometric study revealed several kinetically discernible reaction steps. The condensation rate as a function of pH passes through a maximum, which is caused by formation of two intermediates of different structures. The final products of the condensation and subsequent hydrolysis are pyridoxamine and α-keto acids. The reaction mechanism was suggested.