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Journal of Lipid Research, Vol. 48, 453-457, February 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

A novel method for measuring human hepatic lipase activity in postheparin plasma

S. Imamura^*, J. Kobayashi^1,, S. Sakasegawa^*, A. Nohara, K. Nakajima, M. Kawashiri^**, A. Inazu, M. Yamagishi^**, J. Koizumi and H. Mabuchi

^* Diagnostics Research & Development Department, Diagnostics Division, Asahi Kasei Pharma Corporation, Izunokuni City, Japan
Department of Lipidology, Kanazawa University Graduate School of Medical Science, Kanazawa University Hospital, Kanazawa City, Japan
Nakajima & Associates, Co., Ltd., Kanazawa University Hospital, Kanazawa City, Japan
^** Division of Cardiology, Kanazawa University Graduate School of Medical Science, Kanazawa University Hospital, Kanazawa City, Japan
Kanazawa University, Faculty of Medicine, School of Health Science, Laboratory Sciences, Kanazawa University Hospital, Kanazawa City, Japan
Department of General Medicine, Kanazawa University Hospital, Kanazawa City, Japan

Published, JLR Papers in Press, November 7, 2006.

¹ To whom correspondence should be addressed. e-mail: junji{at}med.kanazawa-u.ac.jp

The objective of this study was to establish a hepatic lipase (HL) assay method that can be applied to automatic clinical analyzers. Seventy-four hyperlipidemic subjects (men/women 45/29) were recruited. Lipase activity was assayed measuring the increase in absorbance at 546 nm due to quinonediimine dye production. Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM glycerol-1,2-dioleate, 0.4% (unless otherwise noted) polyoxyethylene-nonylphenylether, 3 mM ATP, 3 mM MgCl₂, 1.5 mM CaCl₂, monoacylglycerol-specific lipase, glycerol kinase, glycerol-3-phosphate oxidase, 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase, ascorbic acid oxidase. Reaction mixture R-2 contained 50 mM Tris-HCl (pH9.5), 0.15% 4-aminoantypirine. Automated assay for activity was performed with a Model 7080 Hitachi analyzer. In the lipase assay, 160 µl of R-1 was incubated at 37°C with 3 µl of samples for 5 min, and 80 µl of R-2 was added. Within-run coefficient of variations was 0.9–1.0%. Calibration curve of lipase activity was linear (r = 0.999) between 0 and 320 U/l. Analytical recoveries of purified HL added to plasma were 96.6–99.8%. HL activity in postheparin plasma measured in this method had a closer correlation with HL mass by a sandwich ELISA (r = 0.888, P < 0.0001) than those in the conventional method using [¹⁴C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer.

Supplementary key words dioleoylglycerol • quinonediimine dye • automatic clinical analyzer

Abbreviations: BMI, body mass index; EL, endothelial lipase; IDL, intermediate-density lipoprotein; LPL, lipoprotein lipase; PHP, postheparin plasma; TG, triglyceride

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				S. Imamura, J. Kobayashi, K. Nakajima, S. Sakasegawa, A. Nohara, T. Noguchi, M. A. Kawashiri, A. Inazu, S. S. Deeb, H. Mabuchi, et al. A novel method for measuring human lipoprotein lipase and hepatic lipase activities in postheparin plasma J. Lipid Res., July 1, 2008; 49(7): 1431 - 1437. [Abstract] [Full Text] [PDF]