Measurement of cholesterol of major serum lipoprotein classes by anion-exchange HPLC with perchlorate ion-containing eluent

We have developed a high-performance liquid chromatography (HPLC) method for measurement of cholesterol in the major classes of serum lipoproteins, i.e., HDL, LDL, IDL, VLDL, and chylomicrons. Lipoproteins in serum were separated on a column containing diethylaminoethyl-ligand nonporous polymer-based gel by elution with a step gradient of sodium perchlorate concentration, and detected by post-column reaction with a reagent containing cholesterol esterase and cholesterol oxidase. The within-day assay and between-day assay coefficients of variation for cholesterol concentration in lipoproteins were in the ranges of 0.9–6.4% and 1.1–11.9%, respectively. The correlation coefficients between the values of HDL, LDL, IDL, VLDL, and chylomicron cholesterol measured by the HPLC method and those estimated by an ultracentrifugation method were 0.892, 0.921, 0.840, 0.930, and 0.873, respectively. Values of remnant-like particle cholesterol measured by an immunoseparation technique (Japan Immunoresearch Laboratories, Japan) were significantly correlated with VLDL and chylomicron cholesterol values measured by the HPLC method (r = 0.883 and r = 0.729, respectively).This rapid and accurate HPLC method was successfully applied to the analysis of plasma lipoproteins of patients with hyperlipidemia. We have developed a high-performance liquid chromatography (HPLC) method for measurement of cholesterol in the major classes of serum lipoproteins, i.e., HDL, LDL, IDL, VLDL, and chylomicrons. Lipoproteins in serum were separated on a column containing diethylaminoethyl-ligand nonporous polymer-based gel by elution with a step gradient of sodium perchlorate concentration, and detected by post-column reaction with a reagent containing cholesterol esterase and cholesterol oxidase. The within-day assay and between-day assay coefficients of variation for cholesterol concentration in lipoproteins were in the ranges of 0.9–6.4% and 1.1–11.9%, respectively. The correlation coefficients between the values of HDL, LDL, IDL, VLDL, and chylomicron cholesterol measured by the HPLC method and those estimated by an ultracentrifugation method were 0.892, 0.921, 0.840, 0.930, and 0.873, respectively. Values of remnant-like particle cholesterol measured by an immunoseparation technique (Japan Immunoresearch Laboratories, Japan) were significantly correlated with VLDL and chylomicron cholesterol values measured by the HPLC method (r = 0.883 and r = 0.729, respectively). This rapid and accurate HPLC method was successfully applied to the analysis of plasma lipoproteins of patients with hyperlipidemia. Hyperlipidemia is a risk factor for atherosclerotic events (1Multiple Factor Intervention Trial Research GroupMultiple Risk Factor Intervention Trial. Risk factor changes and mortality results.J. Am. Med. Assoc. 1982; 248: 1465-1477Google Scholar). LDL cholesterol plays a causal role in the development of atherosclerosis, and the guidelines adopted by the National Cholesterol Education Adult Treatment Panel in 1988 (ATP-I) recommended that normal values of LDL cholesterol are <3.36 mmol/l (2Report of the National Cholesterol Education Program Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults. The Expert Panel.Arch. Intern. Med. 1988; 148: 36-69Google Scholar, 3Current status of blood cholesterol measurement in clinical laboratories in the United States: A report from the Laboratory Standardization Panel of the National Cholesterol Education Program. Clin. Chem. 34: 193–201.Google Scholar). 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It is well known that the major classes of human lipoproteins are HDL, LDL, IDL, VLDL, and chylomicrons (6Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1995; 34: 1345-1353Google Scholar, 7Schumaker V.N. Puppione D.L. Sequential flotation ultracentrifugation.Methods Enzymol. 1986; 128: 155-170Google Scholar). Numerous studies have investigated the relationship between IDL level and the risk of CAD (8Tatami R. Mabuchi H. Ueda K. Ueda R. Haba T. Kametani T. Ito S. Koizumi J. Ohta M. Miyamoto S. Nakayama A. Kanaya H. Oiwake H. Genda A. Takeda R. Intermediate-density lipoprotein and cholesterol-rich very low density lipoprotein in angiographically determined coronary artery disease.Circulation. 1981; 64: 1174-1184Google Scholar, 9Peardon M.F. Nestel P.J. Craig I.H. Harper R.W. Lipoprotein predictor of the severity of coronary artery disease in men and women.Circulation. 1985; 71: 881-888Google Scholar, 10Hamsten A. Walldius G. Szamosi A. Dahlen G. Faire U.de Relationship of angiographically defined coronary artery disease to serum lipoproteins and apolipoproteins in young survivors of myocardial infarction.Circulation. 1986; 73: 1097-1110Google Scholar, 11Krauss R.M. Lindgren F.T. Williams P.T. Kelsey S.F. Brensike J. Vranizan K. Detre K.M. Levy R.I. Intermediate-density lipoproteins and progression of coronary artery disease in hypercholesterolaemic men.Lancet. 1987; 2: 62-66Google Scholar, 12Steiner G. Schwartz L. Shumak S. Poapst M. The association of increased levels of intermediate-density lipoproteins with smoking and with coronary artery disease.Circulation. 1987; 75: 124-130Google Scholar). Krauss et al. (11Krauss R.M. Lindgren F.T. Williams P.T. Kelsey S.F. Brensike J. Vranizan K. Detre K.M. Levy R.I. Intermediate-density lipoproteins and progression of coronary artery disease in hypercholesterolaemic men.Lancet. 1987; 2: 62-66Google Scholar) reported that IDL mass and ratios of HDL cholesterol to total cholesterol (TC) or LDL cholesterol were predictors for progression of CAD. Tarami et al. (8Tatami R. Mabuchi H. Ueda K. Ueda R. Haba T. Kametani T. Ito S. Koizumi J. Ohta M. Miyamoto S. Nakayama A. Kanaya H. Oiwake H. Genda A. Takeda R. Intermediate-density lipoprotein and cholesterol-rich very low density lipoprotein in angiographically determined coronary artery disease.Circulation. 1981; 64: 1174-1184Google Scholar) reported that a high IDL cholesterol level was associated with a high frequency of CAD. Various methods for analysis of lipoproteins by ultracentrifugation (6Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1995; 34: 1345-1353Google Scholar, 7Schumaker V.N. Puppione D.L. Sequential flotation ultracentrifugation.Methods Enzymol. 1986; 128: 155-170Google Scholar, 13Caslake M.J. Packard C.J. The use of ultracentrifugation for the separation of lipoproteins.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 509-529Google Scholar), electrophoresis (14Noble R.P. Electrophoretic separation of plasma lipoproteins in agarose gel.J. Lipid Res. 1968; 9: 693-700Google Scholar, 15Frings C.S. Foster L.B. Cohen P.S. Electrophoretic separation of serum lipoproteins in polyacrylamide gel.Clin. Chem. 1971; 17: 111-114Google Scholar, 16Kido T. Kurata H. Matsumoto A. Tobiyama R. Musha T. Hayashi K. Tamai S. Utsunomiya K. Tajima N. Fidge N. Itakura H. Kondo K. Lipoprotein analysis using agarose gel electrophoresis and differential staining of lipids.J. Atheroscler. Thromb. 2001; 8: 7-13Google Scholar), gel-permeation chromatography (17Okazaki M. Sasamoto K. Muramatsu T. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 531-548Google Scholar, 18Sata T. Estrich D.L. Wood P.D.S. Kinsell L.W. Evaluation of gel chromatography for plasma lipoprotein fractionation.J. Lipid Res. 1970; 11: 331-340Google Scholar), and anion-exchange chromatography (19Haginaka J. Yamaguchi Y. Kunitomo M. Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins.Anal. Biochem. 1995; 232: 163-171Google Scholar) have been reported. The cholesterol levels of all the major classes of lipoproteins in serum can be measured by ultracentrifugation, but it takes a long time to perform the analysis (6Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1995; 34: 1345-1353Google Scholar, 7Schumaker V.N. Puppione D.L. Sequential flotation ultracentrifugation.Methods Enzymol. 1986; 128: 155-170Google Scholar, 13Caslake M.J. Packard C.J. The use of ultracentrifugation for the separation of lipoproteins.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 509-529Google Scholar). The other methods have a poor ability to measure IDL cholesterol levels (14Noble R.P. Electrophoretic separation of plasma lipoproteins in agarose gel.J. Lipid Res. 1968; 9: 693-700Google Scholar, 15Frings C.S. Foster L.B. Cohen P.S. Electrophoretic separation of serum lipoproteins in polyacrylamide gel.Clin. Chem. 1971; 17: 111-114Google Scholar, 16Kido T. Kurata H. Matsumoto A. Tobiyama R. Musha T. Hayashi K. Tamai S. Utsunomiya K. Tajima N. Fidge N. Itakura H. Kondo K. Lipoprotein analysis using agarose gel electrophoresis and differential staining of lipids.J. Atheroscler. Thromb. 2001; 8: 7-13Google Scholar, 17Okazaki M. Sasamoto K. Muramatsu T. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 531-548Google Scholar, 18Sata T. Estrich D.L. Wood P.D.S. Kinsell L.W. Evaluation of gel chromatography for plasma lipoprotein fractionation.J. Lipid Res. 1970; 11: 331-340Google Scholar, 19Haginaka J. Yamaguchi Y. Kunitomo M. Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins.Anal. Biochem. 1995; 232: 163-171Google Scholar). It is generally thought that remnant lipoproteins promote atherosclerosis. Remnant lipoproteins are products of partially catabolized chylomicrons and VLDL generated by lipoprotein lipase. Recently, an immunoseparation method was developed in order to determine serum levels of cholesterol of remnant-like particle (RLP) (20Nakajima K. Saito T. Tamura A. Suzuki M. Nakano T. Adachi M. Tanaka A. Tada N. Nakamura H. Campos E. Havel R.J. Cholesterol in remnant-like lipoproteins in human serum using monoclonal anti apo B-100 and anti apo A-I immunoaffinity mixed gels.Clin. Chim. Acta. 1993; 223: 53-71Google Scholar). These RLP fractions consist of chylomicron remnants and a fraction of VLDL enriched in apolipoprotein E (apoE) (21Hirany S. O'Byrne D. Devaraj S. Jialal I. Remnant-like particle-cholesterol concentrations in patients with type 2 diabetes mellitus and end-stage renal disease.Clin. Chem. 2000; 46: 667-672Google Scholar, 22Leary E.T. Wang T. Baker D.J. Cilla D.D. Zhong J. Warnick G.R. Nakajima K. Havel R.J. Evaluation of immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma.Clin. Chem. 1998; 44: 2490-2498Google Scholar, 23Campos E. Nakajima K. Tanaka A. Havel R.J. Properties of an apolipoprotein E-enriched fraction of triglyceride-rich lipoproteins isolated from human blood plasma with a monoclonal antibody to apolipoprotein B-100.J. Lipid Res. 1992; 33: 369-380Google Scholar). RLP cholesterol levels have been found to be high in of patients with CAD Nakajima K. Remnant lipoprotein cholesterol and triglyceride ranges from the Chem. 1998; 44: Scholar, S. G. R. Jialal I. Remnant-like cholesterol levels in patients with or coronary artery J. Med. 1998; Scholar), K. Saito T. Tamura A. Suzuki M. Nakano T. Adachi M. Tanaka A. Tada N. Nakamura H. T. A for the of type by Thromb. Scholar), and H. M. Saito T. of serum remnant-like in patients with Chim. Acta. 1993; Scholar). We have developed a method for lipoprotein analysis by anion-exchange chromatography on a diethylaminoethyl-ligand The use of a sodium perchlorate concentration gradient in the and rapid separation and of HDL, LDL, IDL, VLDL, and chylomicrons in The cholesterol levels of lipoproteins were with RLP cholesterol This high-performance liquid chromatography (HPLC) method was as for the rapid and accurate analysis and of the major lipoprotein classes in The cholesterol reagent for HPLC was the HDL and LDL cholesterol in were determined using HDL, and LDL respectively. RLP cholesterol levels were determined with a Immunoresearch The HPLC was of an anion-exchange a post-column and a The of nonporous polymer-based gel with was in The column was been A and sodium perchlorate were to the We a Japan) for A and are for gradient The was A and were mixed The step gradient for separation of the lipoprotein classes were for for for for for and for it to the assay of The the a of serum the changes in the step gradient by a was The of the column was with a column The from the column was mixed with an cholesterol cholesterol cholesterol and sodium The of the cholesterol reagent was by using a The mixed for in a A was for the The from the was using a with a were by a It was that the concentration of sodium perchlorate in with in the cholesterol reagent serum = TG = LDL cholesterol = and HDL cholesterol = was The cholesterol concentration of lipoprotein in of was by the of the of lipoprotein to the total the levels of were by the of total of lipoprotein of to the total of of the serum with known concentration of from a = TG = LDL cholesterol = and HDL cholesterol = as the and from patients = TG = LDL cholesterol = and HDL cholesterol = = TG = LDL cholesterol = and HDL cholesterol = were for separation of HDL, LDL, IDL, VLDL, and chylomicrons by were to between the by the HPLC method and that by an ultracentrifugation The for the were as = = = TG = = = HDL cholesterol = = = and LDL cholesterol = = = of and patients were for the of lipoprotein cholesterol by the HPLC method and RLP cholesterol The were from blood a The were and of the and TG were mmol/l and respectively. The with diabetes or renal The patients serum levels of mmol/l or TG Sequential ultracentrifugation of serum lipoproteins was by the method reported (6Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1995; 34: 1345-1353Google Scholar, 7Schumaker V.N. Puppione D.L. Sequential flotation ultracentrifugation.Methods Enzymol. 1986; 128: 155-170Google Scholar). The flotation of chylomicrons and VLDL were and in a of sodium = of IDL, LDL, and HDL were as and respectively. Japan) and an were for serum = TG = LDL cholesterol = and HDL cholesterol = was The were with and were the within-day assay and between-day assay the serum was The was HDL, LDL, IDL, VLDL, and chylomicron fractions from the were separated by the ultracentrifugation Cholesterol levels of lipoprotein fraction and the serum were determined using an cholesterol with an chemical The between cholesterol values of lipoprotein measured by the HPLC method and those estimated by an ultracentrifugation method or RLP cholesterol values were in of correlation was for the of between cholesterol values of lipoprotein and RLP cholesterol values of the and those of the were in the of serum from a These were and and were detected and The HDL, LDL, IDL, VLDL, and chylomicron fractions of the serum were by the HPLC method to the the of the lipoprotein The of HDL, LDL, IDL, and VLDL from the serum were and and that the major of and of the serum are HDL, LDL, IDL, and VLDL, respectively. the the lipoprotein of the chylomicron fraction was is as the of chylomicrons in serum is very were in of These were and respectively. The lipoprotein fractions of the were by the HPLC method to the and the of the lipoprotein The of HDL, LDL, IDL, and chylomicrons in the serum of were and the serum of the lipoprotein were and The major of VLDL in the serum from and IDL and VLDL in the serum from 2 were and and that the major of and of the are HDL, LDL, IDL, VLDL, and respectively. the IDL fraction of serum from were found and The of IDL in the serum and the serum of were the of HDL and LDL IDL is the VLDL fraction of from and were found and The was found in the VLDL fraction of the serum a of the serum for the were found between the of lipoprotein and and total and in the of to the of HPLC method applied to a The values of within-day assay and between-day assay coefficients of variation of cholesterol concentration of lipoprotein were and respectively. The was assay and between-day assay values of time were is for assay of was the as that for the LDL, IDL, VLDL, and chylomicrons lipoprotein are and in cholesterol concentration was from the total of of The was the as that for the HDL, LDL, IDL, VLDL, and chylomicrons lipoprotein are and in cholesterol concentration was from the total of in a of The of the between of lipoprotein and in to the high that the cholesterol levels of lipoprotein and total lipoproteins in can be determined by HPLC between the values of HDL, LDL, IDL, VLDL, and chylomicron cholesterol and in measured by the are in The cholesterol concentrations were from the of the lipoprotein was from total of the The and the coefficients of correlation between values of HDL LDL IDL VLDL chylomicron and found by the HPLC method and those estimated by using ultracentrifugation and an chemical were = (r = = (r = = (r = = (r = = (r = and = (r = respectively. The between the of the methods the of HPLC method for of cholesterol levels in HDL, LDL, IDL, VLDL, and total for were from and 2 the of the were and in the and and in the LDL cholesterol levels were patients significantly levels of HDL cholesterol and cholesterol levels of IDL, VLDL, and RLP cholesterol The correlation between the cholesterol levels of the lipoprotein classes by the HPLC method and the RLP cholesterol levels was The correlation coefficients of HDL, LDL, IDL, VLDL, and chylomicrons were and 0.729, Cholesterol concentrations of VLDL and chylomicrons by the HPLC method were significantly correlated with RLP cholesterol and of and = = cholesterol triglyceride cholesterol cholesterol cholesterol cholesterol cholesterol cholesterol are as cholesterol and triglyceride were determined using the HDL, LDL, VLDL, and chylomicron cholesterol were determined by the high-performance liquid chromatography with those of by in a Values are as cholesterol and triglyceride were determined using the HDL, LDL, VLDL, and chylomicron cholesterol were determined by the high-performance liquid chromatography with those of by We that the major classes of lipoproteins LDL, IDL, VLDL, and in serum can be separated by of a anion-exchange HPLC elution with concentration changes of perchlorate The flotation ultracentrifugation method the ability to major classes of lipoproteins of LDL, IDL, and VLDL can be separated by using a flotation ultracentrifugation method M.J. Packard C.J. The use of ultracentrifugation for the separation of lipoproteins.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 509-529Google it takes to the major classes of lipoproteins by flotation ultracentrifugation M.J. Packard C.J. The use of ultracentrifugation for the separation of lipoproteins.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 509-529Google Scholar). HPLC methods for separation of lipoproteins have been reported T. Kurata H. Matsumoto A. Tobiyama R. Musha T. Hayashi K. Tamai S. Utsunomiya K. Tajima N. Fidge N. Itakura H. Kondo K. Lipoprotein analysis using agarose gel electrophoresis and differential staining of lipids.J. Atheroscler. Thromb. 2001; 8: 7-13Google Scholar, 17Okazaki M. Sasamoto K. Muramatsu T. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 531-548Google Scholar, 18Sata T. Estrich D.L. Wood P.D.S. Kinsell L.W. Evaluation of gel chromatography for plasma lipoprotein fractionation.J. Lipid Res. 1970; 11: 331-340Google Scholar, 19Haginaka J. Yamaguchi Y. Kunitomo M. Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins.Anal. Biochem. 1995; 232: 163-171Google Scholar). et al. developed a separation method of plasma lipoproteins using gel permeation with analysis of takes (17Okazaki M. Sasamoto K. Muramatsu T. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 531-548Google Scholar). the separation between HDL and LDL was but the separation between LDL and VLDL was for measurement of the cholesterol levels of lipoprotein (17Okazaki M. Sasamoto K. Muramatsu T. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 531-548Google Scholar). VLDL, IDL, and LDL in plasma from a with in IDL is a major lipoprotein (17Okazaki M. Sasamoto K. Muramatsu T. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. The American Association for Clinical Chemistry, Inc. Press, Washington, D.C.1997: 531-548Google Scholar). et al. reported that HDL, LDL, and VLDL in plasma were separated using an anion-exchange HPLC method with elution (19Haginaka J. Yamaguchi Y. Kunitomo M. Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins.Anal. Biochem. 1995; 232: 163-171Google the separation of IDL in plasma (19Haginaka J. Yamaguchi Y. Kunitomo M. Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins.Anal. Biochem. 1995; 232: 163-171Google Scholar). a reported an HPLC method for serum lipoprotein using a column with Y. H. H. K. Tada N. Analysis method for lipoprotein by high-performance liquid chromatography with column and Biochem. 2002; Scholar). It was that HDL, LDL, IDL, and VLDL in serum were in order from the column with a concentration gradient of and that IDL a in in the LDL and the VLDL Y. H. H. K. Tada N. Analysis method for lipoprotein by high-performance liquid chromatography with column and Biochem. 2002; Scholar). IDL a in elution with a step gradient of concentration were to HDL, LDL, IDL, VLDL, and chylomicrons in by using an anion-exchange column with a step gradient of sodium perchlorate concentration, the serum lipoproteins were separated with a step gradient of sodium or sodium concentration It is known that as and and Y. of and by Scholar). It is that the between the lipoproteins and the gel in the column was by the containing perchlorate a lipoproteins in a serum were separated by using the reported anion-exchange HPLC with a gradient of sodium perchlorate the gradient of sodium HDL LDL IDL and VLDL were separated the containing sodium chylomicrons were from the The HDL and the of LDL and VLDL the of lipoproteins, but to be The analysis of using agarose gel electrophoresis that the chylomicrons were T. Kurata H. Matsumoto A. Tobiyama R. Musha T. Hayashi K. Tamai S. Utsunomiya K. Tajima N. Fidge N. Itakura H. Kondo K. Lipoprotein analysis using agarose gel electrophoresis and differential staining of lipids.J. Atheroscler. Thromb. 2001; 8: 7-13Google Scholar). chylomicrons a the the chylomicrons in were to the gel and by containing a high concentration of perchlorate The chylomicrons were the other lipoproteins to the of the have that LDL, IDL, and VLDL in are D.J. J. M.J. Packard C.J. J. of plasma triglyceride in the of plasma low density lipoprotein of LDL to coronary disease 106: Scholar, Y. M. M. T. M. H. density lipoprotein particle and risk of 2000; 148: Scholar, D. M. M. S. of on very low density lipoprotein density lipoprotein and low density lipoprotein Scholar, E. G. of on the concentration and composition of very low density and low density lipoprotein in 1995; Scholar, E. of human very low density lipoproteins by Lipid Res. Scholar, M.F. A. of VLDL by density gradient Clin. Med. 1995; Scholar). We major and in the IDL fraction of 2 and serum from a of IDL the LDL fraction and the VLDL fraction a the IDL to of LDL and VLDL, and be the the IDL cholesterol concentrations estimated by HPLC method were those measured by ultracentrifugation the VLDL fraction of were detected The chylomicron cholesterol concentrations by HPLC method were to those estimated by the ultracentrifugation This that chylomicrons for the in the VLDL serum lipoproteins were partially separation by ultracentrifugation, in of detected from the VLDL isolated by ultracentrifugation, but to be It been found that the cholesterol levels of VLDL and chylomicrons found by the HPLC method were correlated with RLP cholesterol levels et al. reported that RLP cholesterol was correlated with VLDL cholesterol measured by an ultracentrifugation method with IDL cholesterol E.T. Wang T. Baker D.J. Cilla D.D. Zhong J. Warnick G.R. Nakajima K. Havel R.J. Evaluation of immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma.Clin. Chem. 1998; 44: 2490-2498Google Scholar). are with RLP chylomicron remnants and VLDL enriched in (21Hirany S. O'Byrne D. Devaraj S. Jialal I. Remnant-like particle-cholesterol concentrations in patients with type 2 diabetes mellitus and end-stage renal disease.Clin. Chem. 2000; 46: 667-672Google Scholar, 22Leary E.T. Wang T. Baker D.J. Cilla D.D. Zhong J. Warnick G.R. Nakajima K. Havel R.J. Evaluation of immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma.Clin. Chem. 1998; 44: 2490-2498Google Scholar, 23Campos E. Nakajima K. Tanaka A. Havel R.J. Properties of an apolipoprotein E-enriched fraction of triglyceride-rich lipoproteins isolated from human blood plasma with a monoclonal antibody to apolipoprotein B-100.J. Lipid Res. 1992; 33: 369-380Google Scholar). It was reported that VLDL enriched in a cholesterol level and particle with the values of the VLDL fraction E. of human very low density lipoproteins by Lipid Res. Scholar, M.F. A. of VLDL by density gradient Clin. Med. 1995; Scholar). These are with the correlation between VLDL cholesterol and RLP It is known that chylomicron remnants are products of partially catabolized chylomicrons in have been by lipoprotein and of the of chylomicrons are in chylomicron remnants Havel R.J. to the plasma Enzymol. 1986; 128: Scholar). of the cholesterol of the chylomicron fraction in are in chylomicron the correlation between chylomicron cholesterol and RLP cholesterol but to be that the major classes of lipoproteins in human can be separated by a anion-exchange chromatography elution with an containing and that cholesterol levels in lipoprotein can be We the HPLC method by and and the correlation of values measured by the HPLC method with estimated by a flotation ultracentrifugation The that the method is for rapid and accurate of cholesterol levels of HDL, LDL, IDL, VLDL, and chylomicrons in human of variation coronary artery disease remnant-like particle total cholesterol triglyceride