Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects
Résumé
BACKGROUND: 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. METHODS: 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 degrees c, the NEFA (non esterified fatty acids) produced were assayed hourly for 4 hours. LPL activity was expressed as micromol/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. RESULTS: 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 micromol/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 (\textless10 micromol/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 \textgreater10 micromol/l/min. CONCLUSION: This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.
Mots clés
Adult
Female
Humans
Male
Aged
Middle Aged
Reproducibility of Results
Sensitivity and Specificity
80 and over
Young Adult
Kinetics
Triglycerides/*blood
Anticoagulants/pharmacology
Apolipoproteins E/blood/genetics/metabolism
Blood Coagulation/drug effects
DNA Mutational Analysis
Enzyme Assays/*methods
Fatty Acids
Nonesterified/blood/metabolism
Heparin/pharmacology
Hypertriglyceridemia/blood/diagnosis/genetics
Lipolysis/genetics
Lipoprotein Lipase/blood/genetics/*metabolism
Lipoproteins
VLDL/*metabolism
Receptors
Lipoprotein/blood/genetics/metabolism
Substrate Specificity
Domaines
Sciences du Vivant [q-bio]Origine | Fichiers éditeurs autorisés sur une archive ouverte |
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