A simple fluorimetric assay for pyridoxamine phosphate oxidase in erythrocyte haemolysates: effects of riboflavin supplementation and of glucose 6-phosphate dehydrogenase deficiency.
The formation of pyridoxal and its phosphate from pyridoxamine phosphate by red cell haemolysates was measured in a centrifugal analyser by the formation of the fluorescent adduct with semicarbazide. Pyridoxal phosphate was found to react more rapidly than pyridoxal, thus permitting a distinction between the two products, and hence the measurement of phosphatase activity. Activity of the enzyme, pyridoxamine phosphate:oxygen oxidoreductase (deaminating) EC 1.4.3.5 (PPO) was measured in haemolysates from 72 Gambian women with evidence of riboflavin deficiency, and was repeated after 6 weeks of placebo or riboflavin supplementation. Those who received the riboflavin supplement responded with a marked increase in PPO activity, which was matched by a decrease in the activation coefficient (AC) of erythrocyte NAD(P)H2:glutathione oxidoreductase, EC 1.6.4.2 (glutathione reductase, EGR). No difference between the supplemented and unsupplemented groups was observed in the capacity of haemolysates to hydrolyse pyridoxal 5-phosphate, nor in the extent of activation of erythrocyte L-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1. by pyridoxal phosphate. Although the three subjects with low levels of D-glucose 6-phosphate: NADP 1-oxidoreductase EC 1.1.1.49 (G6P-D) had, as expected, correspondingly low AC's of EGR, their unsupplemented activities of PPO were in the same low range as those of the G6P-D-normal subjects, and they responded as G6P-D-normal subjects did to riboflavin supplementation. PPO thus does not appear to resemble EGR in retaining its flavin coenzyme during riboflavin depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
D000293:Adolescent; D001219:Aspartate Aminotransferases / Q000097:blood; D002648:Child; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D005470:Fluorometry; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000201:enzymology*; D005980:Glutathione Reductase / Q000097:blood; D006461:Hemolysis; D006801:Humans; D008297:Male; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000097:blood*; D011247:Pregnancy; D011734:Pyridoxaminephosphate Oxidase / Q000097:blood*; D012256:Riboflavin / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D012257:Riboflavin Deficiency / Q000201:enzymology; D012805:Sickle Cell Trait / Q000097:blood / Q000201:enzymology
D011734:Pyridoxaminephosphate Oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D005980:Glutathione Reductase; D001219:Aspartate Aminotransferases; D012256:Riboflavin