Enzymatic fluorometric assay for plasma pyridoxal 5'-phosphate.
An enzymatic fluorometric assay to quantitate plasma pyridoxal 5'-phosphate (PLP) is described. PLP is preincubated for 30 min with purified tyrosine decarboxylase apoenzyme (TDA) in acetate buffer and is then incubated with L-tyrosine for 60 min. The decarboxylated metabolite, tyramine, is extracted into ethyl acetate, air dried, dissolved in borate buffer and reacted with fluorescamine to form a fluorophor (395/475 nm). Recovery of exogenous PLP was 83%. After log-normal transformation of 129 plasma PLP levels obtained from 3 groups of subjects (in-patients, out-patients, staff), a normal distribution curve was obtained yielding a mean of 83 nmol/L. With 95% confidence level, the reference limits were 32-170 nmol/L. There were no age or sex differences in mean plasma PLP levels between any of the 3 groups of subjects studied.
Poon
H C
HC
Children's Psychiatric Research Institute, London, Ontario, Canada.
Schmidt
B M
BM
D000293:Adolescent; D000328:Adult; D000704:Analysis of Variance; D001051:Apoenzymes / Q000378:metabolism; D002138:Calibration / Q000592:standards; D002648:Child; D002675:Child, Preschool; D005260:Female; D005450:Fluorescamine; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D008297:Male; D011732:Pyridoxal Phosphate / Q000097:blood*; D013379:Substrate Specificity; D014445:Tyrosine Decarboxylase / Q000378:metabolism
D001051:Apoenzymes; D005450:Fluorescamine; D011732:Pyridoxal Phosphate; D014445:Tyrosine Decarboxylase