title: Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects.
abstract: 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°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.
  
  
  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 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.
  
  
  CONCLUSIONS
  This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Di Filippo
authors.forename: Christophe
authors.initials: C
authors.lastname: Marçais
authors.forename: Sybil
authors.initials: S
authors.lastname: Charrière
authors.forename: Oriane
authors.initials: O
authors.lastname: Marmontel
authors.forename: Martine
authors.initials: M
authors.lastname: Broyer
authors.forename: Mireille
authors.initials: M
authors.lastname: Delay
authors.forename: Micheline
authors.initials: M
authors.lastname: Merlin
authors.forename: Axel
authors.initials: A
authors.lastname: Nollace
authors.forename: René
authors.initials: R
authors.lastname: Valéro
authors.forename: Michel
authors.initials: M
authors.lastname: Lagarde
authors.forename: Valérie
authors.initials: V
authors.lastname: Pruneta-Deloche
authors.forename: Philippe
authors.initials: P
authors.lastname: Moulin
authors.forename: Agnès
authors.initials: A
authors.lastname: Sassolas
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000072040:Apolipoprotein A-V; D053304:Apolipoprotein C-II / Q000378:metabolism; D001054:Apolipoproteins A / Q000378:metabolism; D001057:Apolipoproteins E / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D005260:Female; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D015228:Hypertriglyceridemia / Q000097:blood / Q000175:diagnosis / Q000378:metabolism; D008066:Lipolysis; D008071:Lipoprotein Lipase / Q000737:chemistry*; D008079:Lipoproteins, VLDL / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D010949:Plasma / Q000737:chemistry* / Q000378:metabolism; D018110:Receptors, Lipoprotein / Q000378:metabolism; D014280:Triglycerides / Q000097:blood / Q000737:chemistry*; D055815:Young Adult
chemical_list: C437704:APOA5 protein, human; D000072040:Apolipoprotein A-V; D053304:Apolipoprotein C-II; D001054:Apolipoproteins A; D001057:Apolipoproteins E; C523701:GPIHBP1 protein, human; D008079:Lipoproteins, VLDL; D018110:Receptors, Lipoprotein; D014280:Triglycerides; D006493:Heparin; D008071:Lipoprotein Lipase