Homogeneous, Nonradioactive, Enzymatic Assay for Plasma Pyridoxal 5-Phosphate

Abstract

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B(6). Clinical studies suggest that low PLP concentrations are an independent risk factor for cardiovascular and other diseases. However, PLP concentrations are not routinely diagnosed because of the lack of a homogeneous, nonradioactive assay. We describe a homogeneous, nonradioactive, enzymatic PLP assay that uses the apo form of the PLP-dependent recombinant enzyme, homocysteine-alpha,gamma-lyase (rHCYase). PLP was removed from holoenzyme rHCYase by incubation with hydroxylamine to obtain apo-rHCYase. The restoration of enzymatic activity by reconstitution of the holoenzyme was linearly related to the amount of PLP bound to the enzyme. The amplification principle of the assay allowed nanomolar concentrations of PLP to be measured by the conversion (by reconstituted holo-rHCYase) of millimolar concentrations of homocysteine to H(2)S. N,N-Dibutylphenylenediamine (DBPDA) was used for determination of H(2)S, the combination of which forms a chromophore with high absorbance. The assay was initiated by incubation of 5 microL of plasma with apo-rHCYase in a binding buffer for 60 min at 37 degrees C. Homocysteine (2.5 mmol/L) was added to the assay buffer and incubated at 37 degrees C for 20 min. The DBPDA reaction was allowed to progress for 10 min and then read at 675 nm. The PLP enzymatic assay has a lower limit of detection of 5 nmol/L and is linear to 200 nmol/L. The recovery of PLP was 98%. The mean within- and between-run CVs were 9.6% and 12%, respectively. Correlation of 45 samples in the PLP enzymatic assay and the B(6) (3)H radioenzymatic assay (American Laboratory Products Co., Ltd.) yielded: y = 0.9367x + 10.569 (R(2) = 0.9201). This new PLP assay is the first homogeneous, nonradioactive, vitamin B(6) diagnostic method. The assay is applicable to chemistry automated analyzers and may have wide clinical use.