Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects.
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 o...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4008628?pdf=render |
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| author | Mathilde Di Filippo Christophe Marçais Sybil Charrière Oriane Marmontel Martine Broyer Mireille Delay Micheline Merlin Axel Nollace René Valéro Michel Lagarde Valérie Pruneta-Deloche Philippe Moulin Agnès Sassolas |
| author_facet | Mathilde Di Filippo Christophe Marçais Sybil Charrière Oriane Marmontel Martine Broyer Mireille Delay Micheline Merlin Axel Nollace René Valéro Michel Lagarde Valérie Pruneta-Deloche Philippe Moulin Agnès Sassolas |
| author_sort | Mathilde Di Filippo |
| collection | DOAJ |
| description | 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. CONCLUSION: This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects. |
| first_indexed | 2024-12-12T03:36:44Z |
| format | Article |
| id | doaj.art-52db34c3708f4327920442435ee41040 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-12T03:36:44Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-52db34c3708f4327920442435ee410402022-12-22T00:39:46ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0195e9648210.1371/journal.pone.0096482Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects.Mathilde Di FilippoChristophe MarçaisSybil CharrièreOriane MarmontelMartine BroyerMireille DelayMicheline MerlinAxel NollaceRené ValéroMichel LagardeValérie Pruneta-DelochePhilippe MoulinAgnès SassolasBACKGROUND: 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. CONCLUSION: This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.http://europepmc.org/articles/PMC4008628?pdf=render |
| spellingShingle | Mathilde Di Filippo Christophe Marçais Sybil Charrière Oriane Marmontel Martine Broyer Mireille Delay Micheline Merlin Axel Nollace René Valéro Michel Lagarde Valérie Pruneta-Deloche Philippe Moulin Agnès Sassolas Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects. PLoS ONE |
| title | Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects. |
| title_full | Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects. |
| title_fullStr | Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects. |
| title_full_unstemmed | Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects. |
| title_short | Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method for routine assessment of plasma triglyceride lipolysis defects. |
| title_sort | post heparin lpl activity measurement using vldl as a substrate a new robust method for routine assessment of plasma triglyceride lipolysis defects |
| url | http://europepmc.org/articles/PMC4008628?pdf=render |
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