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• W1968112386 abstract "Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. The aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. Human serum lipoproteins VLDL, LDL, and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 μl serum. Subsequently, phosphatidylcholine (PC), lysophosphatidylcholine, sphingomyelin (SM), ceramide (CER), phosphatidylethanolamine (PE), PE-based plasmalogen (PE-pl), cholesterol, and cholesteryl ester (CE) content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analysis of FPLC fractions with PAGE demonstrated that albumin partially coelutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant coelution with the albumin containing fractions. Approximately 60% of lipoprotein CER were found in LDL fractions and 60% of PC, PE, and plasmalogens in HDL fractions. VLDL, LDL, and HDL displayed characteristic lipid class and species pattern. The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort. Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. The aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. Human serum lipoproteins VLDL, LDL, and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 μl serum. Subsequently, phosphatidylcholine (PC), lysophosphatidylcholine, sphingomyelin (SM), ceramide (CER), phosphatidylethanolamine (PE), PE-based plasmalogen (PE-pl), cholesterol, and cholesteryl ester (CE) content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analysis of FPLC fractions with PAGE demonstrated that albumin partially coelutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant coelution with the albumin containing fractions. Approximately 60% of lipoprotein CER were found in LDL fractions and 60% of PC, PE, and plasmalogens in HDL fractions. VLDL, LDL, and HDL displayed characteristic lipid class and species pattern. The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort. Major lipoprotein components beside free cholesterol (FC), cholesteryl esters (CE), and triglycerides are phospholipids, particularly phosphatidylcholine (PC) and sphingomyelin (SM). These lipid classes are not only important structural components, but also modulate the function of lipoproteins including their metabolism or activity of related enzymes. Moreover, PC and SM serve as precursors for a variety of regulatory molecules including lysophosphatidylcholine (LPC) (1Meyer zu H.D. Jakobs K.H. Lysophospholipid receptors: signalling, pharmacology and regulation by lysophospholipid metabolism.Biochim. Biophys. Acta. 2007; 1768: 923-940Crossref PubMed Scopus (309) Google Scholar, 2Mueller R.B. Sheriff A. Gaipl U.S. Wesselborg S. Lauber K. Attraction of phagocytes by apoptotic cells is mediated by lysophosphatidylcholine.Autoimmunity. 2007; 40: 342-344Crossref PubMed Scopus (41) Google Scholar) and ceramide (CER) (3Tani M. Ito M. Igarashi Y. Ceramide/sphingosine/sphingosine 1-phosphate metabolism on the cell surface and in the extracellular space.Cell. Signal. 2007; 19: 229-237Crossref PubMed Scopus (122) Google Scholar). The LCAT reaction in reconstituted HDL is inhibited by SM addition (4Bolin D.J. Jonas A. Sphingomyelin inhibits the lecithin-cholesterol acyltransferase reaction with reconstituted high density lipoproteins by decreasing enzyme binding.J. Biol. Chem. 1996; 271: 19152-19158Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 5Subbaiah P.V. Horvath P. Achar S.B. Regulation of the activity and fatty acid specificity of lecithin-cholesterol acyltransferase by sphingomyelin and its metabolites, ceramide and ceramide phosphate.Biochemistry. 2006; 45: 5029-5038Crossref PubMed Scopus (19) Google Scholar), and CER has the ability to alter the substrate specificity of LCAT and favors the synthesis of unsaturated CE at the expense of saturated esters (5Subbaiah P.V. Horvath P. Achar S.B. Regulation of the activity and fatty acid specificity of lecithin-cholesterol acyltransferase by sphingomyelin and its metabolites, ceramide and ceramide phosphate.Biochemistry. 2006; 45: 5029-5038Crossref PubMed Scopus (19) Google Scholar). An increased SM to PC ratio enhances the susceptibility of LDL to secretory sphingomyelinase, which leads to CER generation and the formation of aggregated LDL exhibiting a high atherogenic potential (6Schissel S.L. Jiang X. Tweedie-Hardman J. Jeong T. Camejo E.H. Najib J. Rapp J.H. Williams K.J. Tabas I. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development.J. Biol. Chem. 1998; 273: 2738-2746Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). In contrast, the antiatherogenic potential of HDL (i.e., the cholesterol uptake capacity) may be enhanced by an enrichment of PC and SM (7Yancey P.G. de L.-M. Swarnakar S. Monzo P. Klein S.M. Connelly M.A. Johnson W.J. Williams D.L. Rothblat G.H. High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI.J. Biol. Chem. 2000; 275: 36596-36604Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Additionally, there is a relationship between HDL mediated cellular cholesterol efflux, phospholipid acyl chain length, and degree of unsaturation (8Davidson W.S. Gillotte K.L. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. The effect of high density lipoprotein phospholipid acyl chain composition on the efflux of cellular free cholesterol.J. Biol. Chem. 1995; 270: 5882-5890Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Recently, LPC plasma levels were discussed as a biomarker in ovarian (9Okita M. Gaudette D.C. Mills G.B. Holub B.J. Elevated levels and altered fatty acid composition of plasma lysophosphatidylcholine(lysoPC) in ovarian cancer patients.Int. J. Cancer. 1997; 71: 31-34Crossref PubMed Scopus (134) Google Scholar) and colorectal (10Zhao Z. Xiao Y. Elson P. Tan H. Plummer S.J. Berk M. Aung P.P. Lavery I.C. Achkar J.P. Li L. et al.Plasma lysophosphatidylcholine levels: potential biomarkers for colorectal cancer.J. Clin. Oncol. 2007; 25: 2696-2701Crossref PubMed Scopus (158) Google Scholar) cancer as well as sepsis (11Drobnik W. Liebisch G. Audebert F.X. Frohlich D. Gluck T. Vogel P. Rothe G. Schmitz G. Plasma ceramide and lysophosphatidylcholine inversely correlate with mortality in sepsis patients.J. Lipid Res. 2003; 44: 754-761Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). A disease-specific species pattern was also observed for CER (11Drobnik W. Liebisch G. Audebert F.X. Frohlich D. Gluck T. Vogel P. Rothe G. Schmitz G. Plasma ceramide and lysophosphatidylcholine inversely correlate with mortality in sepsis patients.J. Lipid Res. 2003; 44: 754-761Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) in sepsis patients. Taken together, lipid class composition as well as the species pattern of lipoprotein fractions may be important for lipoprotein function and may be altered in various disorders. To gain insight into lipid class and species composition of lipoproteins, fractionation is required prior to analysis. Classical lipoprotein isolation techniques by ultracentrifugation (12Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6498) Google Scholar) or preparative free-solution isotachophoresis (13Bottcher A. Schlosser J. Kronenberg F. Dieplinger H. Knipping G. Lackner K.J. Schmitz G. Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions.J. Lipid Res. 2000; 41: 905-915Abstract Full Text Full Text PDF PubMed Google Scholar) are too tedious for large studies and may alter the composition of lipoproteins (14Herbert P.N. Forte T.M. Shulman R.S. La Piana M.J. Gong E.L. Levy R.I. Fredrickson D.S. Nichols A.V. Structural and compositional changes attending the ultracentrifugation of very low density lipoproteins.Prep. Biochem. 1975; 5: 93-129Crossref PubMed Scopus (39) Google Scholar, 15Kunitake S.T. Kane J.P. Factors affecting the integrity of high density lipoproteins in the ultracentrifuge.J. Lipid Res. 1982; 23: 936-940Abstract Full Text PDF PubMed Google Scholar). In contrast, fast performance liquid chromatography (FPLC) offers rapid and reproducible separation of lipoproteins by size (16Ha Y.C. Barter P.J. Rapid separation of plasma lipoproteins by gel permeation chromatography on agarose gel Superose 6B.J. Chromatogr. 1985; 341: 154-159Crossref PubMed Scopus (51) Google Scholar). This technique has been proven to be reproducible and reliable for cholesterol determination in lipoproteins and does not affect lipoprotein composition (17Marz W. Siekmeier R. Scharnagl H. Seiffert U.B. Gross W. Fast lipoprotein chromatography: new method of analysis for plasma lipoproteins.Clin. Chem. 1993; 39: 2276-2281Crossref PubMed Scopus (51) Google Scholar, 18Innis-Whitehouse W. Li X. Brown W.V. Le N.A. An efficient chromatographic system for lipoprotein fractionation using whole plasma.J. Lipid Res. 1998; 39: 679-690Abstract Full Text Full Text PDF PubMed Google Scholar). In order to generate a comprehensive lipid pattern including species information from a small amount of serum, we analyzed FPLC-fractions using well-established methods for quantitative lipid analysis based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) (19Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 20Liebisch G. Drobnik W. Lieser B. Schmitz G. High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.Clin. Chem. 2002; 48: 2217-2224Crossref PubMed Scopus (151) Google Scholar, 21Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (250) Google Scholar–22Liebisch G. Binder M. Schifferer R. Langmann T. Schulz B. Schmitz G. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).Biochim. Biophys. Acta. 2006; 1761: 121-128Crossref PubMed Scopus (333) Google Scholar). Application of this sensitive and fast technique provides detailed cholesterol, glycerophospholipid, and sphingolipid composition of the separated lipoprotein fractions and may help to identify novel biomarkers in disorders of lipid and lipoprotein metabolism. Methanol (HPLC grade) and chloroform (freshly purchased, analytical grade, stabilized with 0.6–1.0 % ethanol) were purchased from Merck (Darmstadt, Germany). Lipid standards for quantitative lipid mass spectrometry were obtained from Avanti Polar Lipids (Alabaster, AL) and Sigma (Deisenhofen, Germany) with purities higher than 99% as described previously (19Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 20Liebisch G. Drobnik W. Lieser B. Schmitz G. High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.Clin. Chem. 2002; 48: 2217-2224Crossref PubMed Scopus (151) Google Scholar, 21Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (250) Google Scholar–22Liebisch G. Binder M. Schifferer R. Langmann T. Schulz B. Schmitz G. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).Biochim. Biophys. Acta. 2006; 1761: 121-128Crossref PubMed Scopus (333) Google Scholar). Ammonium acetate and acetyl chloride of the highest analytical grade available were purchased from Fluka (Buchs, Switzerland). Lipoprotein fractions were prepared from 21 healthy blood donors (Caucasians, 10 female, 11 male with mean age of 28 ± 6). All donors underwent a complete physical examination and laboratory medicine screening to exclude diseases especially related to infectious causes. Donors did not take any medication within 2 weeks before blood drawing. We received informed consent of all donors in written form. Serum lipoprotein levels analyzed by routine diagnostic assays were as follows: VLDL: 16 ± 14 [mg/dl]; LDL: 106 ± 21 [mg/dl]; HDL: 62 ± 12 [mg/dl] (mean ± SD; n = 21). VLDL, LDL, and HDL were isolated from serum of 21 healthy and overnight fasting human blood donors as previously described (18Innis-Whitehouse W. Li X. Brown W.V. Le N.A. An efficient chromatographic system for lipoprotein fractionation using whole plasma.J. Lipid Res. 1998; 39: 679-690Abstract Full Text Full Text PDF PubMed Google Scholar). In brief, a Pharmacia Smart System® FPLC equipped with a Superose 6 PC 3.2/30 column (GE Healthcare Europe GmbH, Munich, Germany) was used with Dulcobecco’s PBS containing 1 mM EDTA as a running buffer. After loading 50 μl serum the system was run with a constant flow of 40 μl/min, and fractionation was started after 18 min with 80 μl per fraction. Fractions 1–20 containing the human serum lipoproteins were used for further analysis on a Cobas Integra 400 (Roche Diagnostic, Penzberg, Germany) to determine cholesterol and triglyceride levels of each fraction and for mass spectrometric analysis as described below. The cholesterol and triglyceride determination assays are standard enzymatic, colorimetric methods, which are also used in routine diagnostics. Ten microliters of each FPLC fraction were mixed separately with 15 μl Optiprep™ (Axis-Shield, Oslo, Norway) and 7-nitrobenz-2-oxa-1,3-diazole (NBD)-CER, dissolved in 0.1 mg/ml ethylene glycol and 10% methanol. (NBD)-CER was shown to stain serum lipoproteins (23Schmitz G. Mollers C. Richter V. Analytical capillary isotachophoresis of human serum lipoproteins.Electrophoresis. 1997; 18: 1807-1813Crossref PubMed Scopus (84) Google Scholar). Afterwards, 10 μl of the mixture of fraction 6–15, containing LDL, were separated on a 3–8% Tris-acetate polyacrylamide gradient gel (Ready Gels; Invitrogen, Karlsruhe, Germany) at 20 mA per gel for 18 h at 4°C. Ten microliters of the mixture of fraction 12–19, containing HDL particles were separated on 4–20% Tris-glycine polyacrylamide gradient gels (Ready Gels; Bio-Rad, Munich, Germany) at 20 mA per gel for 4 h at 4°C. Electrophoresis was carried out in 20 mM Tris/150 mM glycine buffer. PAGE-gels were scanned on a Typhoon fluorescence scanner (GE healthcare, Freiburg, Germany) with an excitation of 488 nm and emission of 520 nm to detect the fluorescence dye bound to the serum lipoproteins. Five microliters of each FPLC fraction were mixed with 15 μl NuPAGE® LDS sample buffer (Invitrogen, Karlsruhe, Germany) and incubated for 10 min at 70°C in the presence of 50 mM DTT. Samples were run on 4–12% Bis-Tris gels (Ready Gels; Invitrogen, Karlsruhe, Germany) with NuPAGE MOPS SDS running buffer (Invitrogen, Karlsruhe, Germany) at 200 V per gel. Proteins separated within the gels were subsequently transferred to a PVDF membrane. Incubation with antibodies against apolipoprotein A-I (apoA-I) and albumin was performed in 1% nonfat dry milk in PBS and 0.1% Tween-20. The immune complexes were detected with an ECL plus (GE Healthcare, Freiburg, Germany). Primary rabbit anti-human apoA-I and anti-human albumin antibodies were purchased from Calbiochem (Darmstadt, Germany) and secondary peroxidase conjugated anti-rabbit antibody from Jackson Immuno Research (Hamburg, Germany). FPLC fractions were extracted according to the method by Bligh and Dyer (24Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar) in the presence of nonnaturally occurring lipid species used as internal standards (PC 14:0/14:0, PC 22:0/22:0, PE 14:0/14:0, PE 20:0/20:0 (di-phytanoyl), LPC 13:0, LPC 19:0, Cer 14:0, Cer 17:0, D7-FC, CE 17:0 and CE 22:0). Lipid species were quantified by ESI-MS/MS using methods validated and described previously (19Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 20Liebisch G. Drobnik W. Lieser B. Schmitz G. High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.Clin. Chem. 2002; 48: 2217-2224Crossref PubMed Scopus (151) Google Scholar, 21Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (250) Google Scholar–22Liebisch G. Binder M. Schifferer R. Langmann T. Schulz B. Schmitz G. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).Biochim. Biophys. Acta. 2006; 1761: 121-128Crossref PubMed Scopus (333) Google Scholar). In brief, samples were analyzed by direct flow injection on a Quattro Ultima triple-quadrupole mass spectrometer (Micromass, Manchester, UK) by direct-flow injection analysis using a HTS PAL autosampler (Zwingen, Switzerland) and an Agilent 1100 binary pump (Waldbronn, Germany) with a solvent mixture of methanol containing 10 mM ammonium acetate and chloroform (3:1, v/v). A flow gradient was performed starting with a flow of 55 μl/min for 6 s followed by 30 μl/min for 1.0 min and an increase to 250 μl/min for another 12 s. A precursor ion scan of m/z 184 specific for phosphocholine containing lipids was used for PC, SM (21Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (250) Google Scholar), and lysophosphatidylcholine (LPC) (20Liebisch G. Drobnik W. Lieser B. Schmitz G. High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.Clin. Chem. 2002; 48: 2217-2224Crossref PubMed Scopus (151) Google Scholar). A neutral loss scan of m/z 141 was used for phosphatidylethanolamine (PE) (25Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (736) Google Scholar), and PE-based plasmalogens (PE-pl) were analyzed according to the principles described by Zemski-Berry (26Zemski Berry K.A. Murphy R.C. Electrospray ionization tandem mass spectrometry of glycerophosphoethanolamine plasmalogen phospholipids.J. Am. Soc. Mass Spectrom. 2004; 15: 1499-1508Crossref PubMed Scopus (224) Google Scholar). In brief, fragment ions of m/z 364, 380, and 382 were used for PE p16:0, p18:1, and p18:0 species, respectively. CER was analyzed similar to a previously described methodology (19Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar) using N-heptadecanoyl-sphingosine as internal standard. Free cholesterol (FC) and CE species were determined after selective acetylation of FC (22Liebisch G. Binder M. Schifferer R. Langmann T. Schulz B. Schmitz G. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).Biochim. Biophys. Acta. 2006; 1761: 121-128Crossref PubMed Scopus (333) Google Scholar). Quantification was achieved by calibration lines generated by addition of naturally occurring lipid species (19Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 20Liebisch G. Drobnik W. Lieser B. Schmitz G. High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.Clin. Chem. 2002; 48: 2217-2224Crossref PubMed Scopus (151) Google Scholar, 21Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (250) Google Scholar, 22Liebisch G. Binder M. Schifferer R. Langmann T. Schulz B. Schmitz G. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).Biochim. Biophys. Acta. 2006; 1761: 121-128Crossref PubMed Scopus (333) Google Scholar) to plasma (extraction of 20 μl 5-fold diluted plasma for single FPLC fractions or 20 μl undiluted plasma for pooled lipoprotein fractions). All lipid classes were quantified with internal standards belonging to the same lipid class, except SM (PC internal standards) and PE-pl (PE internal standards). Calibration lines were generated for the following naturally occurring species: PC 34:1, 36:2, 38:4, 40:0, and PC O 16:0/20:4; SM 34:2, 36:2, 36:1; LPC 16:0, 18:1, 18:0; PE 34:1, 36:2, 38:4, 40:6; and PE p16:0/20:4; Cer 16:0, 18:0, 20:0, 24:1, 24:0; FC, CE 16:0, 18:2, 18:1, 18:0. These calibration lines were also applied for not calibrated species, as follows: concentrations of saturated, monounsaturated, and polyunsaturated species were calculated using the closest related saturated, monounsaturated, and polyunsaturated calibration line slope, respectively. For example PE 36:2 calibration was used for PE 36:1, PE 36:3, and PE 36:4; PE 38:4 calibration was used for PE 38:3, PE 38:5, and so on. Ether-PC species were calibrated using PC O 16:0/20:4 and PE-pl were quantified independent from the length of the ether linked alkyl chain using PE p16:0/20:4. Correction of isotopic overlap of lipid species as well as data analysis was preformed by self programmed Excel macros for all lipid classes according to the principles described previously (21Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (250) Google Scholar). Lipoprotein fractions were isolated from sera of individual normo-lipidemic volunteers by sequential ultracentrifugation as described previously (27Drobnik W. Borsukova H. Bottcher A. Pfeiffer A. Liebisch G. Schutz G.J. Schindler H. Schmitz G. Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.Traffic. 2002; 3: 268-278Crossref PubMed Scopus (141) Google Scholar). Statistical analysis was performed with SPSS© (SPSS Inc., Chicago, IL). We used a Wilcoxon signed-rank test as a nonparametric alternative to a paired Student’s t-test (n = 21 donors). We prepared 20 fractions from 50 μl serum of a healthy blood donor by FPLC-size exclusion chromatography similar to the method of Innis-Whitehouse et al. (18Innis-Whitehouse W. Li X. Brown W.V. Le N.A. An efficient chromatographic system for lipoprotein fractionation using whole plasma.J. Lipid Res. 1998; 39: 679-690Abstract Full Text Full Text PDF PubMed Google Scholar). In order to check whether the major lipoprotein classes were properly separated, total cholesterol (TC) and triglyceride concentrations were analyzed by routine clinical chemistry assays (Fig. 1A). As shown previously (18Innis-Whitehouse W. Li X. Brown W.V. Le N.A. An efficient chromatographic system for lipoprotein fractionation using whole plasma.J. Lipid Res. 1998; 39: 679-690Abstract Full Text Full Text PDF PubMed Google Scholar), three major peaks were found, representing the lipoprotein classes VLDL, LDL, and HDL, respectively. From the same fractions we quantified PC, SM, lysophosphatidylcholine (LPC), CER, PE, PE-pl, FC, and CE including their fatty acid species by previously published tandem mass spectrometric assays (19Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 20Liebisch G. Drobnik W. Lieser B. Schmitz G. High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.Clin. Chem. 2002; 48: 2217-2224Crossref PubMed Scopus (151) Google Scholar, 21Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (250) Google Scholar–22Liebisch G. Binder M. Schifferer R. Langmann T. Schulz B. Schmitz G. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).Biochim. Biophys. Acta. 2006; 1761: 121-128Crossref PubMed Scopus (333) Google Scholar). As expected, most of these lipids revealed a distribution representing the three main lipoprotein classes (Fig. 1B). However, 60% of total LPC were found in fractions 14–17 (Fig. 1B). Because it is known that LPC can bind to albumin (28Thumser A.E. Voysey J.E. Wilton D.C. The binding of lysophospholipids to rat liver fatty acid-binding protein and albumin.Biochem. J. 1994; 301: 801-806Crossref PubMed Scopus (118) Google Scholar), this fraction shift may represent LPC bound albumin. To identify the albumin containing fractions, FPLC fractions were analyzed by nondenaturing PAGE and SDS-PAGE. Corresponding to the main LPC fractions 14 to 17, we found a protein band at a MW of 66 kDa identified as albumin (Fig. 2C). Another question was whether other lipids than LPC coelute with albumin containing fractions. Therefore, we subjected a HDL-deficient serum of a Tangier patient (29Orso E. Broccardo C. Kaminski W.E. Bottcher A. Liebisch G. Drobnik W. Gotz A. Chambenoit O. Diederich W. Langmann T. et al.Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and Abc1-deficient mice.Nat. Genet. 2000; 24: 192-196Crossref PubMed Scopus (431) Google Scholar, 30Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez M. et al.The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1349) Google Scholar) to our method. Consistent with the HDL deficiency, fractions 13 to 17 contained only traces of PC, SM, CER, PE, PE-pl, and cholesterol, but more than 50% of LPC (Fig. 1C). In summary, we could show a clear overlap of albumin and LPC containing fractions, which is in accordance with previous studies (28Thumser A.E. Voysey J.E. Wilton D.C. The binding of lysophospholipids to rat liver fatty acid-binding protein and albumin.Biochem. J. 1994; 301: 801-806Crossref PubMed Scopus (118) Google Scholar, 31Brousseau T. Clavey V. Bard J.M. Fruchart J.C. Sequential ultracentrifugation micromethod for separation of s" @default.
• W1968112386 created "2016-06-24" @default.
• W1968112386 creator A5024189740 @default.
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• W1968112386 creator A5042625397 @default.
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• W1968112386 creator A5078928038 @default.
• W1968112386 date "2009-03-01" @default.
• W1968112386 modified "2023-09-29" @default.
• W1968112386 title "Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry" @default.
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