Selective measurement of lipoprotein lipase and hepatic triglyceride lipase in heparinized plasma from horses

Summary Affinity chromatography on heparin sepharose was used to identify 2 lipolytic enzymes in heparinized plasma from horses. One enzyme was typical of hepatic triglyceride lipase ( htgl ), because it was resistant to inactivation by high concentrations of NaCl, and it did not require the addition of serum for activity. The other enzyme was identified as lipoprotein lipase ( lpl ), because of its inactivation at NaCl concentrations in excess of 0.2 M , and its dependency on addition of serum as a source of apolipoprotein C-II activator. The enzymes were purified by 347- ( htgl ) and 442- ( lpl ) fold, with yields of 54 and 58%, respectively. The partially purified enzymes were used to design incubation conditions that gave optimal activities for each enzyme in vitro. A selective assay was then developed for direct measurement of lpl and htgl activities in heparinized plasma from horses. Analysis of htgl took advantage of the almost complete inactivition of lpl when serum cofactor was excluded from the assay at the NaCl concentration that gave optimal htgl activity. Prior incubation of heparinized plasma with sodium dodecyl sulfate to inhibit htgl was necessary for measurement of lpl , because htgl retained 67% of its activity at the NaCl concentration required for optimal lpl activity. Activity of each enzyme was measured in heparinized plasma from 12 Shetland ponies. The mean activity ± sd for lpl was 3.22 ± 1.04 μmol of fatty acids/ml of heparinized plasma/h (μmol of fa /ml/h). The mean activity for htgl was 4.9 ± 1.56 μmol of fa /ml/h. The performance of the assay was assessed by replicate analysis of pools of each enzyme with high and low activities. The intra-assay coefficient of variation ranged between 3.4 and 8.7% (n = 10), and the interassay coefficient of variation ranged between 5.2 and 10.7% (n = 7) for the same pools analyzed over 7 weeks.