Reverse-phase high-performance liquid chromatographic assay of phospholipases: application of spectrophotometric detection to rat phospholipase A2 isozymes

Abstract

This paper appraises an HPLC method for assaying phospholipase A2 (PLA2). The procedure is based on heptane-isopropanol-H2SO4 extraction of fatty acids released by the enzyme in the presence of margaric acid as an internal standard, and precolumn derivatization with 9-anthryldiazomethane. The derivatives of naturally occurring long-chain fatty acids were accurately determined by reverse-phase HPLC with ultraviolet detection at 254 nm; the fatty acids were identical with margaric acid in terms of their extraction efficiency in the presence or absence of a bile salt, reactivity with the labeling reagent, and molar extinction coefficients of their derivatives. HPLC conditions were optimized so as to separate the derivatives of palmitic and oleic acids completely within 7 min. The use of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/cholate system as substrate proved useful for the sensitive detection of PLA2 activities in rat tissue homogenates. Distribution of immunoreactive pancreatic and group II phospholipases A2 was estimated from the degree of inhibition of enzyme activities by specific antibodies raised against either forms of phospholipase A2 isozymes. The results were consistent with those of immunoblot analyses.