Pyridoxine assay

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

Among various methods reported, we wish to describe an assay technique with Saccharomyces carlsbergensis 4228. This yeast had been used by Atkin et al. (All) for the determination of vitamin B6. We have omitted inositol from the medium and have added an excess of pyridoxine (S19). The composition of the medium is given in Table 8. The method has an accuracy of 0.1 mpg/ml. 

Pyridoxine, HC1 has been assayed by non-aqueous titrations, using 0.1 N perchloric acid in the presence of mercuric acetate and crystal violet indicator (4,6, 10, 87). 

Several colorimetric assays have been developed for pyridoxine. The U.S.P. method (4) for the determination of pyridoxine HC1, is based on coupling of pyridoxine with 2,6-dichloroquinone chloroimide to give a blue color, according to the method described by Kuhn and Low (121). The absorbance of the blue color is measured at 650 nm and compared with standard solutions. The... 

Wada and Snell (150) have developed a method for the assay of pyridoxine and pyridoxamine phosphate, based on enzymatic oxidation to pyridoxal which is allowed to react with phenylhydrazine. 

Three enzymes play an active role in the metabolism of vitamin B6 in human erythrocytes. Pyridoxal kinase uses ATP to phosphorylate pyridoxine, pyri-doxamine, and pyridoxal. Pyridoxamine oxidase oxidizes pyridoxamine-5 -phosphate and pyridoxine-5 -phosphate to pyridoxal-5 -phosphate. The phosphatase activity produces pyridoxal from pyridoxal-5 -phosphate. The assay of the three enzymes required separation of the semicarbazone derivatives of pyridoxal-5 -phosphate and pyridoxal. The mobile phase used by Ubbink and Schnell (1988) contained 2.5% acetonitrile. Detection was by fluorescence. 

All three forms of vitamin B6 [pyridoxal, pyridoxine, and pyridoxamine] are phosphorylated by a single kinase that uses ATP as the phosphate donor. This assay describes the use of pyridoxamine as the substrate. 

The assay contained in a volume of 1 mL 20 mM potassium phosphate buffer (pH 5.75), 0.08 mM ZnCl2,0.06 mM KG, 0.02 mM isopyridoxal (internal standard), 1.2 mM ATP, 0.1 mM pyridoxine, and liver extract as the source of enzyme. To assay the yeast enzyme, ZnQ2 was replaced by 0.1 mM MgG2 and KC1 was omitted. The reaction was started by adding enzyme, and incubations were continued in the dark at 37°C for 90 minutes. The reaction was stopped by heating the test tubes in a boiling water bath for 3 minutes. After centrifugation, an aliquot of the supernate was injected into the HPLC system. The reaction was linear for at least 90 minutes when the rate of pyridoxine phosphate formation was not more than 13 nmol/h. 

One patient has been found with this deficiency (All). Hie patient, an infant, was mentally retarded, had a megaloblastic anemia and abnormally high levels of serum and erythrocyte folate. In spite of the high serum folate concentration there was a marked rise in the reticulocyte count when the patient was treated with folate. It was thought that the patient had impaired utilization of -methyltetrahydrofolate. Assay of liver W -methyltetrahy-drofolate transferase showed it to be reduced. It was suggested that folate accumulated at the N -methyltetrahydrofolate block and could therefore not be further utilized. Treatment with pteroylglutamic acid provided a means of producing active folate up to the point of the block. Unfortunately this patient was also treated with pyridoxine, and it is not clear which vitamin was responsible for the reticulocyte response. Further studies are required to determine the precise nature of this metabolic disorder. 

Biological assay of pyridoxal, pyridoxamine and pyridoxine. J. biol. Chem. 165, 55 (1946). 

Several B vitamins, including folic acid, niacin, pyridoxine, and pantothenic acid, are routinely determined using microbiological assays, details of which can be found in the AOAC Official Methods of Analysis. Standard methods for thiamine determination using fluorimetric detection are also detailed in the AOAC methods in addition, LC techniques are now being used routinely for thiamine and other B vitamins, e.g., riboflavin. 

Ethoxy-4-methyloxaole has been used to form pyridoxine (MI-286, R = H). With maleic anhydride it gives an adduct that, on treatment with ethanolic hydrogen chloride, forms the products W-287 and XII-288 (R = R - Et) and a monoester (XII-288 R = Et, R = H or R = H, R = Et). The diethyl ester MI-288 is also formed from ethyl maleate or ethyl fumarate and 5-ethoxy-4-methyloxazole. Fumaronitrile and ethoxy-4inethyloxazol ve 4,5-dicyano-2-methyl-3-pyridinol, ° also a known precursor to pyridoxine. 5-Ethoxy 4-irethyloxaolesand 2-butene-1,4-diol give pyridoxine (XII-286, R = H), which is difficult to purify when prepared in this way (assay, 23%). 

Coverage includes B vitamins and folate in the context of a historical background, disease, cardiovascular effects and the importance of vitamins in biochemistry as illustrated by a single vitamin. Thereafter there are chapters on the chemistry and biochemistry of thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folate and cobalamin. Methodical aspects include characterization and assays of B vitamins and folate in foods of all kinds, dietary supplements, biological fluids and tissues. The techniques cover solid-phase extraction, spectrofluorimetry, mass spectrometry, HPLC, enzymatic assay, biosensor and chemiluminescence. In terms of fimction and effects or... 

Yagi, T., Murayama, R., Do, H.T.V., Ide, Y., Mugo, A.N., and Yoshikane, Y., 2010b. Development of a simultaneous enzymatic assay method for all six individual vitamin Bg forms and pyridoxine-p-glucoside. Journal of Nutritional Science and Vitaminology. 56 157-163. 

The structure of the isolated compound was determined by degradation and synthesis. to be 2-methyl-3-hydroxy-4,5-bis(hydroxymethyl)p3n idine (I, Fig. i) this compound was named p3nidoxine by Gyorgy . This latter term largely displaced the name vitamin Be from the literature during the period between 1939 and 1942. By use of certain lactic acid bacteria for assay of vitamin Be, SnelD showed in 1942 that compounds other than pyridoxine contributed to the vitamin Be activity of natural materials, that... 

In 2000, Chen et al. [8] developed a method based on CE-ECD for the determination of melatonin and pyridoxine in pharmaceutical preparations for health-caring purposes. CE was performed in a 25 mM phosphate buffer (pH 8.3). The two compounds were separated and detected within 12 min. The working electrode used was a carbon electrode operated in a wall-jet configuration. Excellent linearity was observed between peak current and concentration of analytes with the detection limits ranging from 1.3 to 2.7 pM. The proposed method was successfully applied to analyze the actual samples with satisfactory assay results. 

The first successful separation of calcium pantothenate in multivitamin preparations that allowed the quantitative determination in <5 min was reported by Jonvel et al. (65). They used a 20-cm Nucleosil 7 Cig reversed-phase column, an acetic acid-water (5 95, v/v) mobile phase at a flow rate of 2 mL/min, and a refractometer as a detector. Samples were simply dissolved in the mobile phase and analyzed without any internal standard. Mean recoveries of the assays of commercially available calcium pantothenate tablets containing also thiamine, riboflavin, niacinamide, and pyridoxine ranged Ifom 96.8% to 104.4%. The detection limit was 50 ng injected. 

MEASUREMENT/ASSAY. No international standard or unit system of vitamin B-6 is in current usage. Analytical results are expressed in weight units of pyridoxine hydrochloride. One milligram of pyridoxine hydrochloride is equivalent to 0.82 mg pyridoxine or 0.81 mg pyridoxal, or 0.82 mg pyridoxamine. 

Although the fluorimetric method given above is applicable to this preparation, Foster and Murfin successfully applied alginic acid to the quantitative separation of aneurine, nicotinamide and pyridoxine when present in admixture, with subsequent assay of these separated components spectrophotometrically. Preparation of the alginic acid is described under Nux Vomica (p. 461). 

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