Simple, reproducible procedure for selective measurement of lipoprotein lipase and hepatic lipase.
Existing methods for determining the release of lipoprotein lipase (EC 3.1.1.34) and hepatic lipase (EC 3.1.1.3) into plasma after heparin injection give highly variable results, primarily traceable to errors in the isolation of labeled oleate from the substrate, triolein. Methods involving anion-exchange resin to bind oleate show high variability and have a low yield. Introducing a strong base in the last step of the assay may spuriously increase the counts from oleate, whereas a detergent such as Triton X-100, used to minimize this problem, has a strong quenching effect. We report a simple and rapid method in which we eliminate rather than correct for the sources of variation. The substrate, tri[1-14C]oleoyl-glycerol, is sonicated under strictly standardized conditions with gum arabic, 50 g/L. Incubation is stopped by addition of a benzene/chloroform/methanol mixture and NaOH, 0.2 mol/L. Labeled oleic acid is extracted with hexane after acidification of the alkaline aqueous (upper) phase, so that no alkali is introduced into the scintillation liquid. For lipoprotein lipase measurement, hepatic lipase is inactivated by a specific antiserum, whereas hepatic lipase is measured after lipoprotein lipase is inactivated by NaCl, 1.0 mol/L. The method is efficient and specific, and quenching and chemiluminescence artifacts are avoided.
D000818:Animals; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008074:Lipoproteins / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008722:Methods; D009829:Oleic Acids / Q000302:isolation & purification; D051381:Rats; D014304:Triolein
D008074:Lipoproteins; D009829:Oleic Acids; D014304:Triolein; D006493:Heparin; D008071:Lipoprotein Lipase