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.