A method of determining lipoprotein-lipase activity in human adipose tissue

Abstract

A method of determining lipoprotein-lipase activity (LPLA) in human adipose tissue specimens (weighting 5-50 mg) is described. The specimens were incubated at 37 degrees C in a reaction medium based on a glycine buffer (pH 8.3, ionic strength 0.08), in which the enzyme was stablized and the velocity of release of fatty acids was constant during the incubation. The enzyme activity was increased, as is characteristic of lipoprotein-lipase (LPL), three to four-fold by the addition of serum. The inhibitions by NaCl, protamine sulphate and apolipoprotein C-III were as for LPL, when analysed in both a serum-activated and non-activated reaction medium. The apparent LPLA was about six times greater when analysed in a reaction medium based on a glycine buffer in the presence of heparin (1 g/l) than when analysed in a reaction medium based on a Tris buffer. An analysis of the influence of a high (1 g/l) and a low (0.05 g/l) concentration of heparin on the properties of the enzyme activity was carried out, using LPL of bovine skim milk as a reference enzyme. A phospholipid/soybean-oil emulsion was used as substrate, with [3H]triolein as a trace substance. The emulsion was stable for 5 months. The adipose tissue specimens were stored in liquid nitrogen. The analytical error was 15%, which was reduced to 11% (=within-day variation) when intra-individual comparisons were made.