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Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays
Lipoprotein lipase (LPL) plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides (TGs) in packaged lipoproteins. Since hypertriglyceridemia (HTG) is a major risk factor for cardiovascular disease (CVD), the leading cause of death worldwide, methods that accurately quantify the h...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9329559/ https://www.ncbi.nlm.nih.gov/pubmed/35911520 http://dx.doi.org/10.3389/fcvm.2022.926631 |
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author | Oldham, Dean Wang, Hong Mullen, Juliet Lietzke, Emma Sprenger, Kayla Reigan, Philip Eckel, Robert H. Bruce, Kimberley D. |
author_facet | Oldham, Dean Wang, Hong Mullen, Juliet Lietzke, Emma Sprenger, Kayla Reigan, Philip Eckel, Robert H. Bruce, Kimberley D. |
author_sort | Oldham, Dean |
collection | PubMed |
description | Lipoprotein lipase (LPL) plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides (TGs) in packaged lipoproteins. Since hypertriglyceridemia (HTG) is a major risk factor for cardiovascular disease (CVD), the leading cause of death worldwide, methods that accurately quantify the hydrolytic activity of LPL in clinical and pre-clinical samples are much needed. To date, the methods used to determine LPL activity vary considerably in their approach, in the LPL substrates used, and in the source of LPL activators and inhibitors used to quantify LPL-specific activity, rather than other lipases, e.g., hepatic lipase (HL) or endothelial lipase (EL) activity. Here, we describe methods recently optimized in our laboratory, using a synthetic ApoC-II peptide to activate LPL, and an n-terminal Angiopoietin-Like 4 fragment (nAngptl4) to inhibit LPL, presenting a cost-effective and reproducible method to measure LPL activity in human post-heparin plasma (PHP) and in LPL-enriched heparin released (HR) fractions from LPL secreting cells. We also describe a modified version of the triolein-based assay using human serum as a source of endogenous activators and inhibitors and to determine the relative abundance of circulating factors that regulate LPL activity. Finally, we describe how an ApoC-II peptide and nAngptl4 can be applied to high-throughput measurements of LPL activity using the EnzChek™ fluorescent TG analog substrate with PHP, bovine LPL, and HR LPL enriched fractions. In summary, this manuscript assesses the current methods of measuring LPL activity and makes new recommendations for measuring LPL-mediated hydrolysis in pre-clinical and clinical samples. |
format | Online Article Text |
id | pubmed-9329559 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-93295592022-07-29 Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays Oldham, Dean Wang, Hong Mullen, Juliet Lietzke, Emma Sprenger, Kayla Reigan, Philip Eckel, Robert H. Bruce, Kimberley D. Front Cardiovasc Med Cardiovascular Medicine Lipoprotein lipase (LPL) plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides (TGs) in packaged lipoproteins. Since hypertriglyceridemia (HTG) is a major risk factor for cardiovascular disease (CVD), the leading cause of death worldwide, methods that accurately quantify the hydrolytic activity of LPL in clinical and pre-clinical samples are much needed. To date, the methods used to determine LPL activity vary considerably in their approach, in the LPL substrates used, and in the source of LPL activators and inhibitors used to quantify LPL-specific activity, rather than other lipases, e.g., hepatic lipase (HL) or endothelial lipase (EL) activity. Here, we describe methods recently optimized in our laboratory, using a synthetic ApoC-II peptide to activate LPL, and an n-terminal Angiopoietin-Like 4 fragment (nAngptl4) to inhibit LPL, presenting a cost-effective and reproducible method to measure LPL activity in human post-heparin plasma (PHP) and in LPL-enriched heparin released (HR) fractions from LPL secreting cells. We also describe a modified version of the triolein-based assay using human serum as a source of endogenous activators and inhibitors and to determine the relative abundance of circulating factors that regulate LPL activity. Finally, we describe how an ApoC-II peptide and nAngptl4 can be applied to high-throughput measurements of LPL activity using the EnzChek™ fluorescent TG analog substrate with PHP, bovine LPL, and HR LPL enriched fractions. In summary, this manuscript assesses the current methods of measuring LPL activity and makes new recommendations for measuring LPL-mediated hydrolysis in pre-clinical and clinical samples. Frontiers Media S.A. 2022-07-14 /pmc/articles/PMC9329559/ /pubmed/35911520 http://dx.doi.org/10.3389/fcvm.2022.926631 Text en Copyright © 2022 Oldham, Wang, Mullen, Lietzke, Sprenger, Reigan, Eckel and Bruce. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cardiovascular Medicine Oldham, Dean Wang, Hong Mullen, Juliet Lietzke, Emma Sprenger, Kayla Reigan, Philip Eckel, Robert H. Bruce, Kimberley D. Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays |
title | Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays |
title_full | Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays |
title_fullStr | Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays |
title_full_unstemmed | Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays |
title_short | Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays |
title_sort | using synthetic apoc-ii peptides and nangptl4 fragments to measure lipoprotein lipase activity in radiometric and fluorescent assays |
topic | Cardiovascular Medicine |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9329559/ https://www.ncbi.nlm.nih.gov/pubmed/35911520 http://dx.doi.org/10.3389/fcvm.2022.926631 |
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