A Direct Assay for Pyridoxal 5′-Phosphate Using Pig Heart Apo-Aspartate Transaminase

Abstract

The development of a simple enzymatic method for the determination of blood and plasma pyridoxal 5′-phosphate (PLP) using pig heart apo-aspartate transaminase (apo-AST) is described. The technique requires three steps: (1) sample extraction using perchloric acid, (2) a binding step in which PLP in the sample extract is attached to the apo-AST and (3) the enzymatic assay of the reconstituted holo-AST. PLP extracts were analysed with and without a known concentration of added PLP to correct for variation in recovery between different specimens. Procedures are outlined for manual and automatic analysis of the PLP extracts. Using the KONE Clinical Analyser after the extraction step, it is possible to measure enzyme activity in 100 specimens (i.e. 400 tubes) in a 5 h period. Results are shown from 185 healthy women aged 20–45 years, in which plasma PLP concentrations ranged from 5 to 165 nmol/L, and 142 men and 56 women in Government service aged 17–64 years, whose plasma PLP ranged from 8 to 169 nmol/L. Values less than 20 nmol/L are believed to indicate vitamin B6 deficiency and the method is able to measure 5 nmol/L.