Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects

• Published in: PloS one 2014-05, Vol.9 (5), p.e96482-e96482
• Main Authors: Di Filippo, Mathilde, Marçais, Christophe, Charrière, Sybil, Marmontel, Oriane, Broyer, Martine, Delay, Mireille, Merlin, Micheline, Nollace, Axel, Valéro, René, Lagarde, Michel, Pruneta-Deloche, Valérie, Moulin, Philippe, Sassolas, Agnès
• Format: Article
• Language: English
• Subjects: Adult; Aged; Aged, 80 and over; Analysis; Anticoagulants; Anticoagulants - pharmacology; Apolipoprotein E; Apolipoproteins; Apolipoproteins E - blood; Apolipoproteins E - genetics; Apolipoproteins E - metabolism; Assaying; Biology and Life Sciences; Blood Coagulation - drug effects; Coefficient of variation; Defects; Diabetes; Diagnosis; Dilution; DNA Mutational Analysis; Enzyme Assays - methods; Enzymes; Esterification; Fatty acids; Fatty Acids, Nonesterified - blood; Fatty Acids, Nonesterified - metabolism; Female; Fluorescence; Genes; Glycerol; Heparin; Heparin - pharmacology; Humans; Hypertriglyceridemia; Hypertriglyceridemia - blood; Hypertriglyceridemia - diagnosis; Hypertriglyceridemia - genetics; Kinetics; Life Sciences; Lipase; Lipids; Lipolysis; Lipolysis - genetics; Lipoprotein lipase; Lipoprotein Lipase - blood; Lipoprotein Lipase - genetics; Lipoprotein Lipase - metabolism; Lipoproteins; Lipoproteins (very low density); Lipoproteins, VLDL - metabolism; Liver; Male; Measurement; Medicine and Health Sciences; Metabolism; Methods; Middle Aged; Mutation; Nutrition; Patients; pH effects; Physical Sciences; Receptors, Lipoprotein - blood; Receptors, Lipoprotein - genetics; Receptors, Lipoprotein - metabolism; Reproducibility of Results; Sensitivity and Specificity; Serum; Sodium chloride; Sonication; Substrate Specificity; Substrates; Subtraction; Triglycerides; Triglycerides - blood; Triolein; Ultracentrifugation; Young Adult
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0096482

Determination 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. Post-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. Our 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. This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.