Radioenzymatic assay for direct measurement of plasma pyridoxal 5'-phosphate.
A radioenzymatic assay for measurement of pyridoxal 5'-phosphate (PLP) is described, based on the incubation of L-[3H]tyrosine (10(6) cpm, spec. acty. 1.88 Ci/mol) in the presence of the apoenzyme tyrosine decarboxylase (EC 4.1.1.25) from Streptococcus faecalis and PLP in phosphate buffer (0.1 mol/L, pH 5.5) at 37 degrees C for 60 min. The decarboxylated metabolite formed, [3H]tyramine, was selectively extracted into ethyl acetate, and the tritium radioactivity in the sample was determined by liquid scintillation counting. As little as 0.5 nmol of PLP can be detected per liter. The assay is specific, no cross reactivity having been noted for several compounds closely related to PLP. With this we could directly measure the concentrations of PLP in plasma without prior deproteinization and ether washing of the samples. Using the assay to determine plasma concentrations of PLP in healthy adult populations, we found results that were comparable with previously reported data.
D001051:Apoenzymes / Q000302:isolation & purification; D006801:Humans; D011732:Pyridoxal Phosphate / Q000097:blood*; D012588:Scintillation Counting; D014439:Tyramine / Q000302:isolation & purification; D014445:Tyrosine Decarboxylase / Q000302:isolation & purification
D001051:Apoenzymes; D011732:Pyridoxal Phosphate; D014445:Tyrosine Decarboxylase; D014439:Tyramine