RP-HPLC determination of paraoxonase 3 activity in human blood serum

Abstract

The aim of the present work was to establish conditions for paraoxonase 3 (PON3) activity determination in human blood serum with simvastatin (SV) as a substrate. The activity of PON3 is considered as a good early predictor of susceptibility to premature atherosclerosis as well as of statin therapy effectiveness. The method used quantifies the SV and β,δ-dihydroxyacid simvastatin (SVA) liberated from SV after incubation with blood serum, followed by deproteinization of the reaction mixture. Separation of SV and SVA was performed on an LC(18) column by isocratic elution with acetonitrile-K-phosphate buffer of pH 4.5 (v/v, 70:30) as a mobile phase at flow rate of 1.5 ml min −1. Detection based on ultraviolet absorption at a wavelength of 239 nm was reliable for the simultaneous assay of SV and SVA. The applied method was sufficiently sensitive, precise and accurate for determination of low simvastatin lactone hydrolase (statinase) activity in blood serum of children (1.97–6.86 pmol min −1ml −1). The method is characterized by good linearity over the measurement range of 0.5–6 μg ml −1 (1.194–14.3 nmol ml −1). Limits of detection (LOD) and quantitation (LOQ) for SV were 3.1 and 10.4 ng ml −1, respectively. In case of SVA, LOD and LOQ were 4.7 and 14.44 ng ml − for a 20 μl sample, respectively. Precision and accuracy of PON3 statinase activity determination in human blood serum with SV as substrate were satisfactory and acceptable for bioanalytical methods