Usefulness of plasma and apolipoprotein B-depleted serum samples in paraoxonase 1 assessment

Abstract

Paraoxonase 1 (PON1) is closely associated with antioxidant, anti-inflammatory, and antiatherosclerotic functions of high-density lipoprotein (HDL). Although many clinical studies have evaluated relationships between PON1 activity and various diseases based on its multiple functions, their results were contradictory because of the difference of sample preparation methods. Therefore, we investigated an optimal preanalytical method for PON1 analysis by measuring three different PON1 activities in various types of specimens. Samples were prepared from healthy human serum, plasma with or without calcium addition, HDL isolated by ultracentrifugation, and apolipoprotein B-depleted serum (BDS). Using these samples, PON1 protein concentration and activities using three substrate types (p-nitrophenyl acetate, paraoxon, and γ-thiobutyrolactone) were evaluated. PON1 distributions in HDL subfractions from serum and BDS were also investigated. Although PON1 activities in plasma were lower than those in serum, removing EDTA and adding calcium rescued PON1 activities in plasma similar to levels comparable to those in serum. In contrast, HDL isolated by ultracentrifugation had significantly lower PON1 activities and protein concentrations, indicating that many PON1 proteins were not bound to the HDL particle in the HDL fractions collected from serum and plasma by ultracentrifugation. PON1 protein concentration and distributions in BDS showed similar to those in serum sample than those in HDL sample. Furthermore, three types of PON1 activities were differentially affected by sample preparation procedures. The reduction of PON1 activity in BDS differed among individuals and by the activity type. Focusing on each of three different PON1 activities might further enhance the clinical significance of PON1 testing.