A novel method for measuring human hepatic lipase activity in postheparin plasma.
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 MgCl(2), 1.5 mM CaCl(2), 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 microl of R-1 was incubated at 37 degrees C with 3 microl of samples for 5 min, and 80 microl 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 [(14)C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer.
Imamura
S
S
Diagnostics Research and Development Department, Diagnostics Division, Asahi Kasei Pharma Corporation, Izunokuni City, Japan.
Kobayashi
J
J
Sakasegawa
S
S
Nohara
A
A
Nakajima
K
K
Kawashiri
M
M
Inazu
A
A
Yamagishi
M
M
Koizumi
J
J
Mabuchi
H
H
D000328:Adult; D044466:Asian People; D005260:Female; D006493:Heparin / Q000097:blood*; D006801:Humans; D006949:Hyperlipidemias / Q000097:blood; D008049:Lipase / Q000097:blood*; D008297:Male; D008875:Middle Aged; D011092:Polyethylene Glycols / Q000494:pharmacology
C504173:LIPC protein, human; D011092:Polyethylene Glycols; D006493:Heparin; D008049:Lipase