A simple assay for a human serum phospholipase A2 that is associated with high-density lipoproteins

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A simple assay for a human serum phospholipase A₂ that is associated with high-density lipoproteins

Nenad Petrovic^a,b, Carolyn Grove^a,b, Paul E. Langton^a,b, Neil L. A. Misso^a,b, and Philip J. Thompson^a,b
^a Asthma and Allergy Research Institute, Ground Floor, E Block, Sir Charles Gairdner Hospital, Nedlands WA 6009, Australia
^b Department of Medicine, The University of Western Australia, Perth WA 6009, Australia

Correspondence to: Philip J. Thompson, To whom correspondence should be addressed., pjthomps{at}cyllene.uwa.edu.au (E-mail)

Phospholipase A₂ (PLA₂) activity is usually assayed with expensiveradioactive or chromogenic substrates unsuitable for performinglarge numbers of assays. We have designed a simple microplateassay for human serum PLA₂ using the chromogenic substrate 4-nitro-3-octanoyloxy-benzoicacid. Using this substrate, serum PLA₂ activity was similarto that measured with the previously characterized chromogenicphospholipid 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 µmol· h^-¹ · ml^-¹. The assay is reproducible and issuitable for the analysis of large numbers of samples in a clinicalsetting. We have also demonstrated that 94% of the PLA₂ activityin normal human serum is associated with high-density lipoproteinsand that serum PLA₂ activity is positively correlated with thelipoprotein parameters total triglyceride (P < 0.0001), totalcholesterol (P < 0.0001), and atherogenic index (P = 0.008).The serum PLA₂ activity was calcium dependent and was inhibitedby the serine protease inhibitor 3,4-dichloroisocoumarin (EC₅₀= 0.4 mM).

The PLA₂ activity characterized here is unlikely to be due toplasma platelet-activating factor acetylhydrolase or low molecularweight His-Asp sPLA₂, and may represent a new sPLA₂ type. —Petrovic, N., C. Grove, P. E. Langton, N. L. A. Misso, and P.J. Thompson. A simple assay for a human serum phospholipaseA₂ that is associated with high-density lipoproteins. J. LipidRes. 2001. 42: 1706–1713.

Supplementary key words: enzyme, substrate, cholesterol, atherogenic

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