A simple assay for a human serum phospholipase A2 that is associated with high-density lipoproteins.
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-octanoyloxy-benzoic acid. Using this substrate, serum PLA2 activity was similar to that measured with the previously characterized chromogenic phospholipid substrate 1,2-bis-heptanoylthio-glycerophosphocholine. 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 micromol x h(-1) x 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 (EC(50) = 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.
Petrovic
N
N
Asthma and Allergy Research Institute, Ground Floor, E Block, Sir Charles Gairdner Hospital, Nedlands, WA 6009, Australia.
Grove
C
C
Langton
P E
PE
Misso
N L
NL
Thompson
P J
PJ
D002850:Chromatography, Gel; D003365:Costs and Cost Analysis; D004791:Enzyme Inhibitors; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood / Q000378:metabolism*; D008403:Mass Screening / Q000191:economics; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000097:blood* / Q000378:metabolism*; D054467:Phospholipases A2; D013053:Spectrophotometry / Q000379:methods; D013223:Statistics as Topic
D004791:Enzyme Inhibitors; D008075:Lipoproteins, HDL; D010741:Phospholipases A; D054467:Phospholipases A2