Saki Yamaura1, Shin-Ichi Sakasegawa2, Emisa Koguma1, Shigeru Ueda1, Yuzo Kayamori3, Daisuke Sugimori4, Ken Karasawa5. 1. Asahi Kasei Pharma Corporation, Shizuoka 410-2321, Japan. 2. Asahi Kasei Pharma Corporation, Shizuoka 410-2321, Japan. Electronic address: sakasegawa.sb@om.asahi-kasei.co.jp. 3. Department of Health Sciences, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. 4. Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, Fukushima 960-1296, Japan. 5. Laboratory of Molecular Pharmaceutics, Faculty of Pharmaceutical Sciences, Teikyo University, Tokyo 173-0003, Japan.
Abstract
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.
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. LpPLA2FS 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.