Abstract
BackgroundDetermination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. Consequently by using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles.MethodsPost-heparin plasma was added to the VLDL substrate prepared by ultracentrifugation of heat inactivated normolipidemic human serums, diluted in buffer, pH 8.15. Following incubation at 37°c, the NEFA (non esterified fatty acids) produced were assayed hourly for 4 hours. LPL activity was expressed as µmol/l/min after subtraction of hepatic lipase (HL) activity, obtained following LPL inhibition with NaCl 1.5 mmol/l. Molecular analysis of LPL, GPIHBP1, APOA5, APOC2, APOE genes was available for 62 patients.ResultsOur method was reproducible (coefficient of variation (CV): intra-assay 5.6%, inter-assay 7.1%), and tightly correlated with the conventional radiolabelled triolein emulsion method (n = 26, r = 0.88). Normal values were established at 34.8±12.8 µmol/l/min (mean±SD) from 20 control subjects. LPL activities obtained from 71 patients with documented history of major hypertriglyceridemia showed a trimodal distribution. Among the 11 patients with a very low LPL activity (<10 µmol/l/min), 5 were homozygous or compound heterozygous for LPL or GPIHBP1 deleterious mutations, 3 were compound heterozygous for APOA5 deleterious mutations and the p.S19W APOA5 susceptibility variant, and 2 were free of any mutations in the usual candidate genes. No homozygous gene alteration in LPL, GPIHBP1 and APOC2 genes was found in any of the patients with LPL activity >10 µmol/l/min.ConclusionThis new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.
Highlights
Lipoprotein lipase (LPL) plays a crucial role in the metabolism of very low density lipoproteins (VLDL) and chylomicrons [1,2]
Optimization of substrate First, we verified that similar lipoprotein profiles were obtained from different VLDL substrate pools (n = 40, the coefficient of variation (CV) was,5% for cholesterol and TG concentrations and,10% for apoB, C-II and C-III)
In order to determine the optimal TG concentration chosen for the mixture, we tested 2 patient samples (PHLA activity 17.4 and 49.2 mmol/l/min) with 7 different TG concentrations in the mixture; the activity was decreased (244, 267%) with low TG levels (, 0.90 mmol/l) and slightly increased (+17, +24%) with high TG concentration (. 2.7 mmol/l) in the mixture
Summary
Lipoprotein lipase (LPL) (glycerol ester hydrolase, EC 3.1.1.34) plays a crucial role in the metabolism of very low density lipoproteins (VLDL) and chylomicrons [1,2]. Basu D et al [15] used a commercially fluorescent substrate (EnzChek), esterified by BODIPY-C12 at the sn-1 position of glycerol, which was stable and solubilised with a specific detergent (Zwittergent). Highly appealing, this method has not been tested in human post-heparin plasmas. Determination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. By using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles
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