A stable , radioactive substrate emulsion for assay of I i poprotein I ipase

A method is described for the assay of lipoprotein lipase, using a stable, radioactive substrate emulsion. Fatty acid-labeled trioleoylglycerol was emulsified by homogenization in glycerol with lecithin as detergent. This anhydrous emulsion was stable for at least six weeks. Substrate solutions for enzyme assay were prepared b y diluting the emulsion with buffer containing serum and albumin. The fatty acid produced on hydrolysis was isolated in a one-step liquid-liquid partition system. Incubations with extracts of acetone powders from adipose tissue displayed ch;n-;icteristics of lipoprotein lipase activity, i.e., serum dependence and inhibition by NaCl and protamine. The method is rapid (< 1 hour), sensitive and reproducible, and suitable f o i . routine use. Supplementary key words adipose lipoprotein lipase . tri[3H]oleoylglycerol . triglyceride * postheparin serum A variety of substrates have been employed for the determination of lipoprotein lipase (LPL) activity, reflecting the inherent difficulties in lipase assays. Chylomicrons, a natural substrate, are relatively unstable and difficult to standardize ( 1). Commercially available emulsions (2-4) are stable but suffer from lack of specificity due to the presence of partial glycerides and water-soluble esters (3, 5), which may constitute substrates for esterolytic activities other than LPL (5). This problem is avoided in assays using triacylglycerol emulsions prepared by sonication in the presence of a detergent (5-7). Incorporation of radioactive tracers in such substrate preparations greatly increases the sensitivity and speed of operation (811); however, the instability of these emulsions precludes repeated use and necessitates daily sonication under strictly controlled conditions to minimize interassay variation (1 1 13). The present report describes the preparation of a stable, radioactive trioleoylglycerol (TO) emulsion, which allows repeated assay of LPL with high sensitivity, specificity, and reproducibility. A one-step liquid-liquid partition system for isolation of reaction products ( 14) contributes to the simplicity and rapidity of the method. MATERIALS Tri-[9, 10-3H]oleoylglycerol ( L3H]TO) (414 mCil mmole), obtained from Amersham-Searle, Arlington Heights, Ill., was purified by column chromatography using 2 g of silicic acid covered by a layer of Florisil (200 mg) to improve separation of fatty acid (15). Ten mCi of [3H]T0 were applied to the columns in petroleum ether (bp 30-60°C) with 50 mg of unlabeled TO as carrier. After passage of 50 ml of petroleum ether, T O was eluted with 5% diethyl ether in petroleum ether (80-90% recovery). Radiopurity of the product, as determined by thin-layer chromatography, was >99.9%. Unlabeled TO (NuChek Prep, Chicago, Ill.) was purified in the same way. The purification of TO substrates resulted in substantially lower blank values (about 300 cpm compared to about 1200 cpm when nonpurified TO was used as substrate). Also, the removal of monoacylglycerol (frequently constituting 0.51.2% of total acylglycerols in the commercial TO preparations) minimized the risk of interference in the assay of other lipolytic activities, e.g., monoacylglycerol-hydrolyzing enzymes (5, 11). [9, 10-3H]Oleic acid, obtained from AmershamSearle, had a stated purity of >99%. Unlabeled oleic acid was purchased from NuChek Prep. Lecithin and lysolecithin, both from egg yolk, were from Sigma Chemical Co., St. Louis, MO. Crystalline bovine serum albumin, protamine sulfate (salmine) and heparin (160 U/mg) were obtained from Calbiochem, La Jolla, Calif. Glycerol (spectrophotometric grade, <0.05% HzO) was obtained from Mallinckrodt, St. Louis, MO. All other substances and solvents used were reagent grade. Serum was obtained from fasting rats and heated to 62°C for 10 min to eliminate endogenous lipolytic ilbbrevkations: LPL, lipoprotein lipase; TO, trioleoylglycerol; ['H ]TO, tri-[3H]oleoylglycerol. 536 Journal of Lipid Research Volume 17, 1976 by gest, on S etem er 6, 2017 w w w .j.org D ow nladed fom activity (16). Acetone powders of rat epididymal adipose tissue were prepared as previously described (1 7). The powders were extracted (2-5 mg of powder per ml of buffer) with 0.05 M NH,OH-NH,CI buffer (pH 8.1) or 0.05 M Tris-HC1 (pH 8.0) containing 1M ethylene glycol (1 7). The supernates obtained after centrifugation for 40,000 g-min were employed as enzyme source in most of the experiments. Postheparin serum, used to check stability of the concentrated substrate emulsion, was obtained from blood drawn 10 min after the injection of 10 U heparin per kg body wt into fasting rats. Lipolytic activity of postheparin serum was stable for at least 2 months at -20°C. As reference method for the measurement of LPL activity, our earlier described assay system (12) was employed. This substrate was prepared by sonication of T O in Tris-HC1 buffer, using lysolecithin as emulsifier. The final concentrations in the assay were TO, 1.47 pmoledml; lysolecithin, 0.085 pmoledml; albumin, 0.1% (w/v); and serum, 3% (v/v); and 0.1 M Tris-HC1 (pH 8.0). Incubations, isolation of released fatty acid, and radioactivity measurements were performed as described below under Procedure.