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



This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.
UF Dyslipidémies Cardiobiologie, Département de Biochimie et Biologie Moléculaire, Centre de Biologie et de Pathologie Est, Laboratoire de Biologie Médicale Multi Sites, Hospices Civils de Lyon, Bron, France; INSERM U1060, INSA de Lyon, INRA U1235, Univ Lyon-1, Université de Lyon, Villeurbanne, Oullins, France.
Mathilde
M
Di Filippo
INSERM U1060, INSA de Lyon, INRA U1235, Univ Lyon-1, Université de Lyon, Villeurbanne, Oullins, France; Laboratoire de Biochimie spécialisée, Centre de Biologie Sud, Centre Hospitalier Lyon-Sud, Laboratoire de Biologie Médicale Multi Sites, Hospices Civils de Lyon, Pierre-Bénite, France.
Christophe
C
Marçais
INSERM U1060, INSA de Lyon, INRA U1235, Univ Lyon-1, Université de Lyon, Villeurbanne, Oullins, France; Fédération d'endocrinologie, maladies métaboliques, diabète et nutrition, Hôpital Louis Pradel, Hospices Civils de Lyon, Bron, France.
Sybil
S
Charrière
UF Dyslipidémies Cardiobiologie, Département de Biochimie et Biologie Moléculaire, Centre de Biologie et de Pathologie Est, Laboratoire de Biologie Médicale Multi Sites, Hospices Civils de Lyon, Bron, France.
Oriane
O
Marmontel
UF Dyslipidémies Cardiobiologie, Département de Biochimie et Biologie Moléculaire, Centre de Biologie et de Pathologie Est, Laboratoire de Biologie Médicale Multi Sites, Hospices Civils de Lyon, Bron, France.
Martine
M
Broyer
Laboratoire de Biochimie spécialisée, Centre de Biologie Sud, Centre Hospitalier Lyon-Sud, Laboratoire de Biologie Médicale Multi Sites, Hospices Civils de Lyon, Pierre-Bénite, France.
Mireille
M
Delay
Laboratoire de Biochimie spécialisée, Centre de Biologie Sud, Centre Hospitalier Lyon-Sud, Laboratoire de Biologie Médicale Multi Sites, Hospices Civils de Lyon, Pierre-Bénite, France.
Micheline
M
Merlin
UF Dyslipidémies Cardiobiologie, Département de Biochimie et Biologie Moléculaire, Centre de Biologie et de Pathologie Est, Laboratoire de Biologie Médicale Multi Sites, Hospices Civils de Lyon, Bron, France.
Axel
A
Nollace
Département de Nutrition, Maladies Métaboliques, Endocrinologie, APHM, Hôpital de la Timone, Aix-Marseille Université, UMR_S 1062, UMR_A1260, Marseille, France.
René
R
Valéro
INSERM U1060, INSA de Lyon, INRA U1235, Univ Lyon-1, Université de Lyon, Villeurbanne, Oullins, France.
Michel
M
Lagarde
INSERM U1060, INSA de Lyon, INRA U1235, Univ Lyon-1, Université de Lyon, Villeurbanne, Oullins, France.
Valérie
V
Pruneta-Deloche
INSERM U1060, INSA de Lyon, INRA U1235, Univ Lyon-1, Université de Lyon, Villeurbanne, Oullins, France; Fédération d'endocrinologie, maladies métaboliques, diabète et nutrition, Hôpital Louis Pradel, Hospices Civils de Lyon, Bron, France.
Philippe
P
Moulin
UF Dyslipidémies Cardiobiologie, Département de Biochimie et Biologie Moléculaire, Centre de Biologie et de Pathologie Est, Laboratoire de Biologie Médicale Multi Sites, Hospices Civils de Lyon, Bron, France; INSERM U1060, INSA de Lyon, INRA U1235, Univ Lyon-1, Université de Lyon, Villeurbanne, Oullins, France.
Agnès
A
Sassolas
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000925:Anticoagulants / Q000494:pharmacology; D001057:Apolipoproteins E / Q000097:blood / Q000235:genetics / Q000378:metabolism; D001777:Blood Coagulation / Q000187:drug effects; D004252:DNA Mutational Analysis; D057075:Enzyme Assays / Q000379:methods*; D005230:Fatty Acids, Nonesterified / Q000097:blood / Q000378:metabolism; D005260:Female; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D015228:Hypertriglyceridemia / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D007700:Kinetics; D008066:Lipolysis / Q000235:genetics; D008071:Lipoprotein Lipase / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D018110:Receptors, Lipoprotein / Q000097:blood / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014280:Triglycerides / Q000097:blood*; D055815:Young Adult
D000925:Anticoagulants; D001057:Apolipoproteins E; D005230:Fatty Acids, Nonesterified; C523701:GPIHBP1 protein, human; D008079:Lipoproteins, VLDL; D018110:Receptors, Lipoprotein; D014280:Triglycerides; D006493:Heparin; D008071:Lipoprotein Lipase