Abstract
Phospholipase A2 (PLA2) activity is usually assayed with expensive radioactive or chromogenic substrates unsuitable for performing large numbers of assays. We have designed a simple microplate assay for human serum PLA2 using the chromogenic substrate 4-nitro-3-octanoyloxybenzoic acid. Using this substrate, serum PLA2 activity was similar to that measured with the previously characterized chromogenic phospholipid substrate 1,2-bis-heptanoylthioglycerophosphocholine. However, the assay described here appears to be more sensitive. The mean PLA2 activity in serum from healthy volunteers (n = 30) measured by this assay was 10.4 ± 1.6 μmol · h−1 · ml−1. The assay is reproducible and is suitable for the analysis of large numbers of samples in a clinical setting. We have also demonstrated that 94% of the PLA2 activity in normal human serum is associated with high-density lipoproteins and that serum PLA2 activity is positively correlated with the lipoprotein parameters total triglyceride (P < 0.0001), total cholesterol (P < 0.0001), and atherogenic index (P = 0.008). The serum PLA2 activity was calcium dependent and was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin (EC50 = 0.4 mM).The PLA2 activity characterized here is unlikely to be due to plasma platelet-activating factor acetylhydrolase or low molecular weight His-Asp sPLA2, and may represent a new sPLA2 type.
Highlights
Phospholipase A2 (PLA2) activity is usually assayed with expensive radioactive or chromogenic substrates unsuitable for performing large numbers of assays
To determine its maximum solubility, known amounts of 4-nitro-3octanoyloxy-benzoic acid (4N3OBA) were resuspended in PLA2 reaction buffer
In subsequent assays for PLA2 activity, 2 mM 4N3OBA was chosen as the standard substrate concentration
Summary
Phospholipase A2 (PLA2) activity is usually assayed with expensive radioactive or chromogenic substrates unsuitable for performing large numbers of assays. The studies performed to date on plasma PLA2 vary in their conclusions partly because plasma was taken from a variety of organisms (including humans) and in different physiological states. These include samples from patients with severe inflammatory diseases [5], sepsis, rheumatoid arthritis [6], and acute pancreatitis [7] or from rats treated intravenously with heparin [8]. In these reports, the presence of several different PLA2 enzymes in plasma has been suggested.
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