The effect of homocysteine thiolactone on paraoxonase and aryl esterase activity of human serum purified paraoxonase 1 in vitro experiments.

Abstract

The important role of lipoproteins, particularly low-density lipoprotein (LDL) and high-density lipoprotein (HDL), has been highly regarded among the known causes of cardiovascular disease (CVD). A wide range of risk factors may cause structural and functional changes in lipoprotein particles, resulting in deposition and formation of atherosclerotic plaques. Homocysteine is one of the most important risk factors in heart disease, and its atherosclerotic properties appear to be related to its intermediate metabolite called homocysteine thiolactone (HCTL). The major aim of the present investigation was to study the effect of HCTL in different concentrations (10, 50, and 100 μM) on paraoxonase and aryl esterase activities of purified human serum paraoxonase 1 (PON1) antioxidant enzyme related to HDL, as an extracellular hydrolyzing enzyme of HCTL. In order to purify PON1 enzyme from human serum, three-step chromatographic methods including DEAE Sephadex A50, Sephadex G100, and DEAE Sephadex A50 were used. Protein concentration and paraoxonase and aryl esterase activities of each fraction were measured separately and the highest activities fractions were collected and subsequently pooled together for the next steps. Ultimately, both activities of PON1 in the presence of different concentrations of HCTL were measured in triplicate by spectrophotometry technique. HCTL at concentrations of 50 and 100 μM decreased both paraoxonase and aryl esterase activities (P < 0.05) in comparison with the control group, which is directly related to the increase in HCTL concentration. However, at a concentration of 10 μM HCTL, no significant difference was observed in both paraoxonase and aryl esterase activities compared to the control group. HCTL is a highly toxic and reactive compound that is produced in all cells. Extracellular enzyme PON1 causes its hydrolysis with high efficiency. The results obtained from the present study showed that paraoxonase and aryl esterase activities decreased in vitro in the presence of HCTL and therefore, HCTL may cause changing in the protein structure of this enzyme. Previous in vivo studies have also shown decrease of PON1 activity in patients with hyperhomocysteinemia.