Identification of lipoprotein lipase immunoreactive protein in pre- and postheparin plasma from normal subjects and patients with type

Postheparin plasma is a convenient source for the measurement of lipoprotein lipase (LPL) in humans. Previous studies have focused on the measurement of LPL catalytic activity, and have been unable to conveniently measure the LPL protein or identify possibly different plasma forms of the enzyme. Preand postheparin plasma was treated with a highly specific antibody raised against bovine milk LPL and the immunoprecipitate was analyzed by Western blotting. In normal subjects there were several species of LPL in plasma. A 56 kD protein increased after heparin injection, and likely represented active LPL. The anti-LPL antibody reacted specifically with this 56 kD protein, and also reacted specifically with proteins at 52 kD, 69 kD, as well as a 20 kD breakdown product. In addition, using peptide mapping, the 56 kD protein was structurally similar to the 52 and 69 kD LPL proteins. The antibodies were affinity purified, biotinylated, and used to quantitate LPL immunoreactive mass using an enzyme-linked immunosorbant assay (ELISA). LPL immunoreactive mass was present in all subjects in preheparin plasma. In postheparin plasma, five patients with type I hyperlipoproteinemia displayed decreased LPL immunoreactive mass when compared to normal subjects, although there was a wide range of specific activity of the small amount of enzyme present. When the LPL from the plasma of the patients was immunoprecipitated and Western blotted, there was considerable heterogeneity in the appearance of the LPL forms, and an overall decrease in LPL protein. Thus, several different immunoreactive LPL proteins were present in pre-and postheparin plasma. In preheparin plasma, as well as in patients with type I hyperlipoproteinemia, there was decreased immunoreactive LPL protein, and the LPL protein that was present was of low specific activity. -Kern, P. A,, R. A. Mar tin, J. Carty, I. J. Goldberg, and J. M. Ong. Identification of lipoprotein lipase immunoreactive protein in preand postheparin plasma from normal subjects and patients with type I hyperlipoproteinemia. J. Lipid Res. 1990. 31: 17-26. Supplementary key w o r d s peptide mapping bovine milk lipoprotein lipase ELISA triglyceride-rich lipoproteins (1). After LPL is produced by parenchymal cells, the enzyme is transported to the capillary endothelium, where it is bound to glycosaminoglycans. It has long been known that an injection of heparin releases LPL activity from its glycosaminoglycan binding sites, and postheparin plasma LPL activity has been a useful measurement in clinical studies. The measurement of LPL activity, however, only detects catalytically active LPL protein, which may not be representative of actual LPL protein mass if alterations in LPL specific activity occur. Enzyme-linked immunosorbant assays (ELISA) for human postheparin plasma LPL have been reported previously (2-4) using monoclonal antibodies to LPL. However, a monoclonal antibody may not recognize an LPL molecule that is modified due to degradation, mutation, or denaturation. Hence, an assay that detects both active and inactive LPL protein has been needed for the study of LPL regulation, and to characterize the different forms of circulating LPL. The absence of measurable LPL activity is essential for the diagnosis of familial LPL deficiency (type I hyperlipoproteinemia) (5, 6). This disorder is found in approximately 1 in 1 million in the population (5-8), is autosomal recessive in inheritance, and typically presents in childhood with chylomicronemia, pancreatitis, and eruptive xanthomata. Although the type I hyperlipoproteinemia phenotype occasionally occurs due to apoC-I1 deficiency (9) or a circulating inhibitor of LPL (10, ll), the majority of patients with the type I phenotype have no measurable LPL activity in adipose tissue and/or postheparin plasma (7, 12, 13). Whether this decreased enzyme activity results from a decreased mass of normal Lipoprotein lipase (LPL) is an enzyme found in a number of tissues and is responsible for the hydrolysis of Abbreviations: LPL, lipoprotein lipase; ELISA, enzyme-linked immunosorbant assay; BSA, bovine serum albumin; PBSAT, phosphatebuffered saline, 0.1 % BSA, 0.1 % Triton X-100; FFA, free fatty acids. Journal of Lipid Research Volume 31, 1990 17 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom enzyme or the production of a defective LPL protein could not be answered by measuring only the catalytically Enzyme-linked immunosorbant assay (ELISA) for LPL active LPL protein. We report herein the characterization of plasma LPL with a polyclonal antibody that specifically recognizes several molecular forms of LPL in plasma. In addition to studying LPL immunoreactive mass in preand postheparin plasma, the plasmas from five patients with type I hyperlipoproteinemia were analyzed.