Novel enzymatic method for assaying Lp-PLA2 in serum

Background: Measurement of lipoprotein-associated Phospholipase A₂ (Lp-PLA₂) can be used as an adjunct to traditional cardiovascular risk factors for identifying individuals at higher risk of cardiovascular events. This can be performed by quantification of the protein concentration using an ELISA platform or by measuring Lp-PLA₂ activity using platelet-activating factor (PAF) analog as substrate. Here, an enzymatic Lp-PLA₂ activity assay method using 1-O-Hexadecyl-2-acetyl-rac-glycero-3-phosphocholine (rac C₁₆ PAF) was developed.

Methods: The newly revealed substrate specificity of lysoplasmalogen-specific Phospholipase D (lysophospholipase D (LysoPLD)) was exploited. Lp-PLA₂ hydrolyzes 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C₁₆ PAF) to 1-O-Hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF). LysoPLD acted on LysoPAF, and the hydrolytically released choline was detected by choline oxidase.

Results: Regression analysis of Lp-PLA₂ activity measured by the enzymatic Lp-PLA₂ activity assay vs. two chemical Lp-PLA₂ activity assays, i.e. LpPLA₂ FS and PLAC® test, and ELISA, gave the following correlation coefficients: 0.990, 0.893 and 0.785, respectively (n = 30).

Conclusion: Advantages of this enzymatic Lp-PLA₂ activity assay compared with chemical Lp-PLA₂ methods include the following; (i) only requires two reagents enabling a simple two-point linear calibration method with one calibrator (ii) no need for inhibitors of esterase-like activity in serum.

1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; Enzymatic assay method; Lp-PLA(2); Lysophospholipase D; PAF; PAF-AH (platelet-activating factor-acetylhydrolase).