Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry
The measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipit...
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| Format: | Article |
| Language: | English |
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Elsevier
2000-07-01
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| Series: | Journal of Lipid Research |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520320241 |
| _version_ | 1818385413952241664 |
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| author | Laurent Beghin Nathalie Duhal Philippe Poulain Philippe Hauw Brigitte Lacroix Jean-Michel Lecerf Jean-Paul Bonte Jean-Charles Fruchart Gérald Luc |
| author_facet | Laurent Beghin Nathalie Duhal Philippe Poulain Philippe Hauw Brigitte Lacroix Jean-Michel Lecerf Jean-Paul Bonte Jean-Charles Fruchart Gérald Luc |
| author_sort | Laurent Beghin |
| collection | DOAJ |
| description | The measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipitation of apoB followed by quantification of the precipitate. In this study, we present the measurement of apoB concentration in lipoproteins purified by ultracentrifugation by combining isopropanol precipitation and gas chromatography/mass spectrometry. Very low density lipoprotein (VLDL; d < 1.006 g/mL); VLDL plus intermediate density lipoprotein (VLDL + IDL; d < 1.019 g/mL); and VLDL, IDL, and low density lipoprotein (VLDL + IDL + LDL; d < 1.063 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was selectively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine as internal standard. Knowledge of the number of leucine molecules in one apoB-100 molecule makes it possible to calculate the plasma concentration of apoB in the various lipoprotein fractions. ApoB in IDL (d 1.006–1.019 g/mL) and LDL (d 1.019–1.063 g/mL) were then determined by subtracting VLDL-apoB from apoB in lipoproteins d < 1.019 and apoB in lipoproteins d < 1.019 g/mL from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol precipitate was verified as pure apoB (>97%) in lipoprotein fractions isolated from normo- and hyperlipidemic plasma and the method appeared reproducible.The combination of isopropanol precipitation and the GC/MS method appears therefore to be a precise and reliable method for kinetic and epidemiological studies. |
| first_indexed | 2024-12-14T03:37:46Z |
| format | Article |
| id | doaj.art-8db1b4bf80ac40d49f72cfb86320fb2c |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-14T03:37:46Z |
| publishDate | 2000-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-8db1b4bf80ac40d49f72cfb86320fb2c2022-12-21T23:18:34ZengElsevierJournal of Lipid Research0022-22752000-07-0141711721176Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometryLaurent Beghin0Nathalie Duhal1Philippe Poulain2Philippe Hauw3Brigitte Lacroix4Jean-Michel Lecerf5Jean-Paul Bonte6Jean-Charles Fruchart7Gérald Luc8Department of Atherosclerosis, Institut Pasteur de Lille, 59019 Lille Cedex, FranceCentre Universitaire de Mesures et d'Analyses, Faculté de Pharmacie, Lille, France; Université de Lille II, Lille, FranceDepartment of Atherosclerosis, Institut Pasteur de Lille, 59019 Lille Cedex, FranceDepartment of Atherosclerosis, Institut Pasteur de Lille, 59019 Lille Cedex, FranceCentre Universitaire de Mesures et d'Analyses, Faculté de Pharmacie, Lille, France; Université de Lille II, Lille, FranceService de Médecine Interne A, University Hospital of Lille, 59037 Lille, FranceCentre Universitaire de Mesures et d'Analyses, Faculté de Pharmacie, Lille, France; Université de Lille II, Lille, FranceDepartment of Atherosclerosis, Institut Pasteur de Lille, 59019 Lille Cedex, France; Department of Atherosclerosis, Institut Pasteur de Lille, 59019 Lille Cedex, FranceTo whom correspondence should be addressed.; Department of Atherosclerosis, Institut Pasteur de Lille, 59019 Lille Cedex, France; Service de Médecine Interne A, University Hospital of Lille, 59037 Lille, FranceThe measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipitation of apoB followed by quantification of the precipitate. In this study, we present the measurement of apoB concentration in lipoproteins purified by ultracentrifugation by combining isopropanol precipitation and gas chromatography/mass spectrometry. Very low density lipoprotein (VLDL; d < 1.006 g/mL); VLDL plus intermediate density lipoprotein (VLDL + IDL; d < 1.019 g/mL); and VLDL, IDL, and low density lipoprotein (VLDL + IDL + LDL; d < 1.063 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was selectively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine as internal standard. Knowledge of the number of leucine molecules in one apoB-100 molecule makes it possible to calculate the plasma concentration of apoB in the various lipoprotein fractions. ApoB in IDL (d 1.006–1.019 g/mL) and LDL (d 1.019–1.063 g/mL) were then determined by subtracting VLDL-apoB from apoB in lipoproteins d < 1.019 and apoB in lipoproteins d < 1.019 g/mL from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol precipitate was verified as pure apoB (>97%) in lipoprotein fractions isolated from normo- and hyperlipidemic plasma and the method appeared reproducible.The combination of isopropanol precipitation and the GC/MS method appears therefore to be a precise and reliable method for kinetic and epidemiological studies.http://www.sciencedirect.com/science/article/pii/S0022227520320241apolipoprotein Blipoproteinsmass spectrometry |
| spellingShingle | Laurent Beghin Nathalie Duhal Philippe Poulain Philippe Hauw Brigitte Lacroix Jean-Michel Lecerf Jean-Paul Bonte Jean-Charles Fruchart Gérald Luc Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry Journal of Lipid Research apolipoprotein B lipoproteins mass spectrometry |
| title | Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry |
| title_full | Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry |
| title_fullStr | Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry |
| title_full_unstemmed | Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry |
| title_short | Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry |
| title_sort | measurement of apolipoprotein b concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry |
| topic | apolipoprotein B lipoproteins mass spectrometry |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520320241 |
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