A novel method for measuring human hepatic lipase activities in post-heparin plasma

Shigeyuki Imamura, Junji Kobayashi, Shinichi Sakasegawa, Atsushi Nohara, Katsuyuki Nakajima, Masa-aki Kawashiri, Akihiro Inazu, Masakazu Yamagishi, Junji Koizumi, and Hiroshi Mabuchi

Department of lipidology, Kanazawa University Graduate School of Medical Science, Kanazawa city, Ishikawa 920-8640

Corresponding Author: junji{at}med.kanazawa-u.ac.jp

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

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