Pyridoxal Kinase, Pyridoxamine Oxidase, and

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. 

Enzymes were assayed in hemolysates of erythrocytes isolated from venous blood collected with EDTA as anticoagulant. 

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. 

Pyridoxine, pyridoxine-5 -phosphate, isopyridoxal (internal standard), ATP, and ADP were separated on a Whatman Partisil-lOSCX column (4.6 mm x 250 mm). The method also resolved pyridoxamine and pyridox-amine-5 -phosphate. The mobile phase was 0.1 M ammonium dihydrogen 

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. 

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