A simple and precise method for measuring HDL-cholesterol subfractions by a single precipitation followed by homogenous HDL-cholesterol assay

HDL consists of two major subfractions, HDL2 and HDL3. This paper describes a simple method for assaying HDL subspecies by combining a single precipitation with a direct high density lipoprotein-cholesterol (HDL-C) assay. A precipitation reagent (0.06 ml) containing 1,071 U/ml heparin, 500 mmol/l MnCl2, and 12 mg/ml dextran sulfate was added to a serum (0.3 ml). The sample was incubated and centrifuged at 10,000 rpm for 10 min. HDL3-C was measured by a homogenous HDL-C assay in the supernatant, and HDL2-C was estimated by subtracting the HDL3-C from the direct HDL-C. The HDL3-C and HDL2-C values determined by the precipitation method were identical to those determined by ultracentrifugation, and there were excellent correlations between the methods in the measurements of HDL3-C and HDL2-C (r = 0.933 and 0.978, respectively; n = 102). The two methods also proved to be highly correlated in the measurement of apolipoprotein A-I and A-II in HDL subfractions. The HDL-C subfractions determined by ultracentrifugation were more closely associated with the homogenous HDL-C assay than with the total cholesterol assay, especially in the hypertriglyceridemic samples. Our method is far simpler and more precise than the classical dual precipitation method for HDL-C subfractions, and it can be easily performed in a routine chemical laboratory HDL consists of two major subfractions, HDL2 and HDL3. This paper describes a simple method for assaying HDL subspecies by combining a single precipitation with a direct high density lipoprotein-cholesterol (HDL-C) assay. A precipitation reagent (0.06 ml) containing 1,071 U/ml heparin, 500 mmol/l MnCl2, and 12 mg/ml dextran sulfate was added to a serum (0.3 ml). The sample was incubated and centrifuged at 10,000 rpm for 10 min. HDL3-C was measured by a homogenous HDL-C assay in the supernatant, and HDL2-C was estimated by subtracting the HDL3-C from the direct HDL-C. The HDL3-C and HDL2-C values determined by the precipitation method were identical to those determined by ultracentrifugation, and there were excellent correlations between the methods in the measurements of HDL3-C and HDL2-C (r = 0.933 and 0.978, respectively; n = 102). The two methods also proved to be highly correlated in the measurement of apolipoprotein A-I and A-II in HDL subfractions. The HDL-C subfractions determined by ultracentrifugation were more closely associated with the homogenous HDL-C assay than with the total cholesterol assay, especially in the hypertriglyceridemic samples. Our method is far simpler and more precise than the classical dual precipitation method for HDL-C subfractions, and it can be easily performed in a routine chemical laboratory High density lipoprotein-cholesterol (HDL-C) is a negative risk factor for coronary heart disease (CHD), and low HDL-C increases the risk of CHD just as powerfully as high LDL-C. HDL consists of two major subfractions, large buoyant HDL2 (d = 1.063–1.125 g/ml) and small dense HDL3 (d = 1.125–1.210 g/ml). Several lines of evidence suggest that the protective effect of HDL may be better reflected in the concentrations of HDL2-C than in those of total-HDL-C or HDL3-C (1.Miller N.E. Hammett F. Saltissi S. Rao S. Zeller H.van Coltart J. Lewis B. Relation of angiographically defined coronary artery disease to plasma lipoprotein subfractions and apolipoproteins.Br. Med. J. 1981; 282: 1741-1744Crossref PubMed Scopus (399) Google Scholar, 2.Calabresi L. Franceschini G. Sirtori M. Gianfranceschi G. Werba P. Sirtori C.R. Influence of serum triglycerides on the HDL pattern in normal subjects and patients with coronary artery disease.Atherosclerosis. 1990; 84: 41-48Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 3.Syvanne M. Ahola M. Lahdenpera S. Kahri J. Kuusi T. Virtanen K.S. Taskinen M.R. High density lipoprotein subfractions in non-insulin-dependent diabetes mellitus and coronary artery disease.J. Lipid Res. 1995; 36: 573-582Abstract Full Text PDF PubMed Google Scholar). Other studies, meanwhile, provide no support for this (4.Kempen H.J. van Gent C.M. Buytenhek R. Buis B. Association of cholesterol concentrations in low-density lipoprotein, high-density lipoprotein, and high-density lipoprotein subfractions, and of apolipoproteins AI and AII, with coronary stenosis and left ventricular function.J. Lab. Clin. Med. 1987; 109: 19-26PubMed Google Scholar, 5.Smuts C.M. Weich H.F. Weight M.J. Faber M. Kruger M. Lombard C.J. Benade A.J. Free cholesterol concentrations in the high-density lipoprotein subfraction-3 as a risk indicator in patients with angiographically documented coronary artery disease.Coron. Artery Dis. 1994; 5: 331-338Crossref PubMed Scopus (27) Google Scholar, 6.Lamarche B. Moorjani S. Cantin B. Dagenais G.R. Lupien P.J. Despres J.P. Associations of HDL2 and HDL3 subfractions with ischemic heart disease in men. Prospective results from the Quebec Cardiovascular Study.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1098-1105Crossref PubMed Scopus (161) Google Scholar). It remains to be determined whether a shift in the distribution of HDL particles confers a greater benefit than an increase in total HDL alone. Which of the two is the more powerful negative risk factor for CHD events remains open to debate. Although studies have yet to determine which of the two is the more powerful negative risk factor for CHD events, a simple and reliable assay system for measuring the HDL subfractions will have to be developed to determine the clinical significance of the HDL species. Research laboratories use various ultracentrifugation techniques to determine HDL2 and HDL3 in serum and plasma (7.Scanu A. Studies on the conformation of human serum high-density lipoproteins HDL2 and HDL3.Proc. Natl. Acad. Sci. 1965; 54: 1699-1705Crossref PubMed Scopus (24) Google Scholar, 8.Chapman M.J. Goldstein S. Lagrange D. Laplaud P.M. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum.J. Lipid Res. 1981; 22: 339-358Abstract Full Text PDF PubMed Google Scholar, 9.Patsch J.R. Sailer S. Kostner G. Sandhofer F. Holasek A. Braunsteiner H. Separation of the main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation.J. Lipid Res. 1974; 15: 356-366Abstract Full Text PDF PubMed Google Scholar). Yet the specialized skills, time, and expensive equipment required for these techniques are disadvantageous for the processing of the large numbers of samples used in epidemiological studies. Several groups have already developed methods for measuring HDL subfractions by precipitation instead of ultracentrifugation (10.Gidez L.I. Miller G.J. Burstein M. Slagle S. Eder H.A. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.J. Lipid Res. 1982; 23: 1206-1223Abstract Full Text PDF PubMed Google Scholar, 11.Kostner G.M. Molinari E. Pichler P. Evaluation of a new HDL2/HDL3 quantitation method based on precipitation with polyethylene glycol.Clin. Chim. Acta. 1985; 148: 139-147Crossref PubMed Scopus (97) Google Scholar, 12.Whitaker C.F. Srinivasan S.R. Berenson G.S. Simplified methods for measuring cholesterol concentrations of high-density lipoprotein subclasses in serum compared.Clin. Chem. 1986; 32: 1274-1278Crossref PubMed Scopus (11) Google Scholar, 13.Patsch W. Brown S.A. Morrisett J.D. Gotto Jr., A.M. Patsch J.R. A dual-precipitation method evaluated for measurement of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 in human plasma.Clin. Chem. 1989; 35: 265-270Crossref PubMed Scopus (86) Google Scholar, 14.Dias V.C. Parsons H.G. Boyd N.D. Keane P. Dual-precipitation method evaluated for determination of high-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations.Clin. Chem. 1988; 34: 2322-2327Crossref PubMed Scopus (31) Google Scholar). All of these methods, however, require dual-step precipitation: VLDL and LDL fractions are precipitated with heparin-manganese (Mn), whereupon HDL2 is precipitated with dextran sulfate (DS) in the supernatant from the heparin-Mn precipitation. Dual precipitation requires two steps with the centrifuge, which prolongs the total assay time and introduces complicated steps that may compromise the accuracy of the values. Dual precipitation is also very unsuitable for the accurate measurement of HDL subfractions in severely hypertriglyceridemic samples containing chylomicrons (15.Bachorik P.S. Albers J.J. Precipitation methods for quantification of lipoproteins.Methods Enzymol. 1986; 127: 78-100Crossref Scopus (259) Google Scholar). By modifying the dual precipitation method originally described by Gidez et al. (10.Gidez L.I. Miller G.J. Burstein M. Slagle S. Eder H.A. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.J. Lipid Res. 1982; 23: 1206-1223Abstract Full Text PDF PubMed Google Scholar), our group developed a single precipitation procedure for selectively separating HDL3 from other lipoproteins with a heparin/Mn/DS mixture. In the present study, we used this single precipitation procedure and then measured cholesterol in a heparin/Mn/DS supernatant by direct HDL-C homogenous assay. Previously, we reported a successful attempt to measure small dense LDL-C by a combined method of heparin/magnesium precipitation followed by direct LDL-C assay (16.Hirano T. Ito Y. Saegusa H. Yoshino G. A novel and simple method for quantification of small, dense LDL.J. Lipid Res. 2003; 44: 2193-2201Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Gleaning hints from our previous study, we used direct HDL-C instead of total cholesterol (TC) assay for the measurement of cholesterol in the heparin/Mn/DS supernatant. The direct HDL-C assay is expected to eliminate the contamination of cholesterol in apolipoprotein B (apoB)-containing lipoproteins and to yield more accurate values, especially in the measurement of severely hypertriglyceridemic samples. The brief total assay time of <1 h ensures that the method can be applied easily in routine clinical laboratories. Blood was sampled in a fasting state from healthy subjects and patients, some of them dyslipidemic, during visits to Showa University Hospital and affiliated hospitals. Written informed consent was obtained from all subjects, and the study was approved by the local ethics committee. Table 1 lists the serum lipid, apoA-I, and apoA-II levels in the enrolled subjects (n = 102). Data on the TG, apoA-I, and apoA-II levels were available for 90 of the samples. The HDL-C levels ranged widely from 9 to 118 mg/dl, because a patient with Tangier disease was included in the subjects. There were also two chylomicronemic samples from subjects with severe hypertriglyceridemia (1,214 and 1,259 mg/dl). Serum was isolated within 2 h after blood sampling and stored at −80°C until analysis. The precipitation and ultracentrifugation were performed on the same day, the latter in accordance with the method described earlier (17.Okada M. Matsui H. Ito Y. Fujiwara A. Direct measurement of HDL cholesterol: method eliminating apolipoprotein E-rich particles.J. Clin. Lab. Anal. 2001; 15: 223-229Crossref PubMed Scopus (23) Google Scholar). HDL2 (d = 1.063–1.125 g/ml) and HDL3 (d = 1.125–1.210 g/ml) were separated by adding sodium bromide to sequentially adjust the sera to different densities and then performing ultracentrifugation at 453,000 g for 8 h at 16°C in a Himac centrifuge with a PR80A rotor (Hitachi, Tokyo, Japan). Cholesterol concentrations in the isolated HDL2 and HDL3 were measured by TC assay.TABLE 1.Serum lipid and apoA-I and apoA-II levels in enrolled subjectsValueTCHDL-CTGApoA-IApoA-IIMean (SD)210 (51)62 (23)212 (295)146 (37)32 (8)Range99–3758–12826–168315–2326–57ApoA-I, apolipoprotein A-I; HDL-C, high density lipoprotein-cholesterol; TC, total cholesterol; TG, triglyceride. n = 102 for TC and HDL-C; n = 90 for TG, apoA-I, and apoA-II. Open table in a new tab ApoA-I, apolipoprotein A-I; HDL-C, high density lipoprotein-cholesterol; TC, total cholesterol; TG, triglyceride. n = 102 for TC and HDL-C; n = 90 for TG, apoA-I, and apoA-II. The HDL-C was determined directly in the serum using a commercially available test kit (HDL-EX; Denka Seiken, Co., Tokyo, Japan) with a special surfactant (the homogenous method), based on a principle fully described previously (17.Okada M. Matsui H. Ito Y. Fujiwara A. Direct measurement of HDL cholesterol: method eliminating apolipoprotein E-rich particles.J. Clin. Lab. Anal. 2001; 15: 223-229Crossref PubMed Scopus (23) Google Scholar). The HDL-C levels determined by this method correlated excellently with those determined by ultracentrifugation (d = 1.063–1.210 g/ml; r = 0.99, n = 200) and those determined using another commercially available kit by the direct method (Determina HDL-C; Kyowa Medics, Tokyo, Japan) (r = 0.96, n = 16) (18.Okamoto Y. Tanaka S. Nakano H. Direct measurement of HDL cholesterol preferable to precipitation method.Clin. Chem. 1995; 41: 1784Crossref PubMed Scopus (15) Google Scholar). The within-run imprecision (coefficient of variation) was 1.25%, and the between-assay coefficient of variation was 0.79%. No significant interference was observed when bilirubin, to at a of to or was incubated during the measurement of HDL-C. The dual-step precipitation was performed to the procedure described by and Albers (15.Bachorik P.S. Albers J.J. Precipitation methods for quantification of lipoproteins.Methods Enzymol. 1986; 127: 78-100Crossref Scopus (259) Google Scholar), based on the procedure by Gidez et al. (10.Gidez L.I. Miller G.J. Burstein M. Slagle S. Eder H.A. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.J. Lipid Res. 1982; 23: 1206-1223Abstract Full Text PDF PubMed Google Scholar). total HDL-C by a combined of mg/ml and was added to of The concentrations of the were mmol/l and mg/ml of at the was centrifuged at g for 1 h at of the supernatant were for the assay of the HDL-C and precipitation of the The HDL2 was precipitated by in and adding a of supernatant. The of was at the was centrifuged at 10,000 rpm for at The measured for total HDL-C was by and that for HDL3 was by to for by the HDL3-C was measured by the direct HDL-C homogenous assay instead of the TC assay, for with the single precipitation method described The procedure was performed in a single by adding heparin/Mn/DS reagent to the lipoproteins and with various concentrations of in the of the studies that 12 mg/ml was for the precipitation of HDL2 in this single precipitation The reagent of and The precipitation reagent (0.06 ml) was added to of left at for and centrifuged at 10,000 rpm for 10 at The concentrations of heparin, MnCl2, and were mg/ml mmol/l and of the supernatant was for HDL3-C HDL-C in the supernatant was determined by homogenous HDL-C assay (HDL-EX; Denka The measured for total HDL3-C was by to for by the The for HDL2-C was as the between the total HDL-C determined in the serum by and were in at of two using two different reagent The and of variation were for sample for and for reagent imprecision was by in two and of variation were for the and for the No significant reagent variation or variation was No significant in the HDL2-C and HDL3-C levels were in serum stored at −80°C for at we to use the serum for the study, the results of using plasma instead of the serum were the The of severe hypertriglyceridemia on this assay were also was obtained from a patient with lipoprotein T. M. A. M. T. T. No evidence of in a patient for the in the lipoprotein 2001; Full Text Full Text PDF PubMed Scopus (31) Google by or was added to normal serum and of this sample was with of the precipitation HDL3-C in the supernatant was measured by direct HDL-C assay or TC assay. and TC were measured by laboratory Serum apoA-I and apoA-II were determined by an assay Co., Tokyo, Japan). was measured by a commercially available test kit were performed with Tokyo, Japan). between were by analysis. The was used to the between two methods for the measurement of the same was at The dual-step and precipitation methods for the of HDL subfractions were The concentrations for the dual-step and methods were the determined to be for the precipitation for HDL2 in the study by Gidez et al. (10.Gidez L.I. Miller G.J. Burstein M. Slagle S. Eder H.A. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.J. Lipid Res. 1982; 23: 1206-1223Abstract Full Text PDF PubMed Google Scholar). The HDL3-C and HDL2-C values determined by the dual and single precipitation methods correlated excellently with those determined by ultracentrifugation the for HDL3-C and HDL2-C determined by precipitation were and more The values determined by the two precipitation methods were also correlated with other (r = and for HDL3-C and respectively; in the measurement of HDL3-C by a precipitation we to determine the in the heparin/Mn/DS for the of HDL3 from 12 mg/ml = was the of the between the precipitation and ultracentrifugation for HDL3-C was very to and the was to = The was than that used by Gidez et al. (10.Gidez L.I. Miller G.J. Burstein M. Slagle S. Eder H.A. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.J. Lipid Res. 1982; 23: 1206-1223Abstract Full Text PDF PubMed Google for dual precipitation The was at a low and more at concentrations were to determine the between the single precipitation method and ultracentrifugation for HDL-C subfractions in more samples (n = The HDL3-C values determined by single precipitation with heparin/Mn/DS followed by homogenous HDL-C assay were excellently correlated with the values determined by ultracentrifugation (r = The was and the was mg/dl). A was observed for HDL2-C = r = in the with the apoA-I and apoA-II levels in HDL subfractions determined by the single precipitation method were correlated with those determined by for and were and (n = between ultracentrifugation and the single precipitation for the measurement of apolipoprotein A-I and apoA-II levels in HDL subfractions in 90 Table 2 HDL3-C and HDL2-C levels determined by the ultracentrifugation and the single precipitation method with direct HDL-C assay or TC assay. The HDL2-C was the HDL3-C as reported in subjects H. T. high-density lipoprotein cholesterol subfractions, and in Full Text PDF PubMed Scopus Google Scholar, Y. M. F. Y. A. J. Tanaka H. M. of on the high-density lipoprotein cholesterol in healthy Full Text PDF PubMed Scopus Google Scholar). The HDL3-C determined by ultracentrifugation and precipitation HDL assay was and the between the two methods was <1 mg/dl, the was greater when HDL3-C was measured by the TC assay mg/dl). The HDL2-C determined by ultracentrifugation and the precipitation HDL-C assay was also and the between the two methods was mg/dl, the greater when HDL3-C was measured by TC assay mg/dl). the between the ultracentrifugation and single precipitation methods using TC or direct HDL-C assay for the determination of The values determined by the TC were correlated with those determined by ultracentrifugation (r = the between the ultracentrifugation and precipitation TC assay a between the two were from samples with severe hypertriglyceridemia between and precipitation with direct HDL-C or TC assay for determination of HDL2-C and HDL3-C are (n = Open table in a new tab are (n = the of the serum on the between ultracentrifugation and the precipitation method with HDL-C or TC assay. A was observed in the HDL3-C values measured by TC assay in the samples with severe No significant was however, in the measurements by the precipitation HDL-C assay. A in the supernatant after the precipitation of the chylomicronemic to yield HDL3-C values by direct HDL-C assay, however, by the supernatant the a The HDL3-C values, on the other when the TC assay was used for the chylomicronemic samples the direct HDL-C assay was to the TC assay, we the effect of chylomicrons on HDL3-C measurement by the precipitation The of chylomicrons T. M. A. M. T. T. No evidence of in a patient for the in the lipoprotein 2001; Full Text Full Text PDF PubMed Scopus (31) Google to serum triglycerides from to The HDL3-C determined by the direct HDL-C assay from to when the chylomicrons were In the HDL3-C determined by the TC assay from to determine the effect of on the precipitation we measured HDL-C subfractions in a sample with a high of mg/dl). The HDL3-C and HDL2-C levels determined by ultracentrifugation were identical to the levels determined by the precipitation method and in this a simple and precise assay for measuring HDL-C subfractions by a single precipitation with a heparin/Mn/DS followed by a direct HDL-C assay. By measuring the total HDL-C directly in serum with a homogenous assay, we can determine the total HDL-C the A single precipitation procedure the total assay time and may precipitation is widely used for separating HDL3 from HDL2 also for the precipitation of lipoproteins (15.Bachorik P.S. Albers J.J. Precipitation methods for quantification of lipoproteins.Methods Enzymol. 1986; 127: 78-100Crossref Scopus (259) Google Scholar). we that it be to a of and for the precipitation of HDL2 and lipoproteins the HDL3 in the supernatant. our to test this by a heparin/Mn/DS in the same concentrations used by Gidez et al. (10.Gidez L.I. Miller G.J. Burstein M. Slagle S. Eder H.A. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.J. Lipid Res. 1982; 23: 1206-1223Abstract Full Text PDF PubMed Google Scholar). we that a single precipitation was for measuring HDL3-C an to that of the classical dual-step precipitation. also however, that the for HDL3-C and HDL2-C were and more in the two precipitation This that the precipitation of HDL2 was and required by of the we that 12 mg/ml was the for the precipitation. a at a low and a more at concentrations we that at high concentrations some of the HDL3 the various methods for may be the closely with the ultracentrifugation for the determination of HDL2-C and HDL3-C (r = and The apoA-I and apoA-II levels determined in HDL subfractions by the precipitation method were also correlated with those determined by This the accuracy of our single precipitation method for the HDL et al. H. Evaluation of the dual-precipitation method for determination of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 in Chem. 1986; 32: PubMed Scopus Google the precipitation method by the procedure of Gidez et al. (10.Gidez L.I. Miller G.J. Burstein M. Slagle S. Eder H.A. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.J. Lipid Res. 1982; 23: 1206-1223Abstract Full Text PDF PubMed Google with density gradient ultracentrifugation, that mg/ml was required to HDL2-C and HDL3-C values with also observed that as the concentrations some HDL3 to the precipitation of the et al. V.C. Parsons H.G. Boyd N.D. Keane P. Dual-precipitation method evaluated for determination of high-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations.Clin. Chem. 1988; 34: 2322-2327Crossref PubMed Scopus (31) Google used mg/ml to HDL3 from HDL2 in a total HDL previously isolated by Our however, to HDL2 from HDL3 using concentrations than the other The of heparin/Mn/DS is to require a than the supernatant for the accurate precipitation of HDL2 from HDL3. and Pichler G.M. Molinari E. Pichler P. Evaluation of a new HDL2/HDL3 quantitation method based on precipitation with polyethylene glycol.Clin. Chim. Acta. 1985; 148: 139-147Crossref PubMed Scopus (97) Google reported a single precipitation for HDL subfractions with polyethylene and R. Precipitation with polyethylene and ultracentrifugation for high-density lipoprotein subclasses HDL2 and Chem. PubMed Scopus Google Scholar), on the other a high coefficient between ultracentrifugation and the polyethylene method (r = for HDL2-C and r = for et al. A. J. P. with precipitation methods in the determination of and J. Clin. Lab. 1987; PubMed Scopus Google the of of HDL subfractions with polyethylene precipitation Our group applied direct HDL-C measurement instead of TC assay for the determination of The direct HDL-C assay by ultracentrifugation HDL-C values than the TC assay. The homogenous HDL-C assay also the to measure HDL-C the serum (17.Okada M. Matsui H. Ito Y. Fujiwara A. Direct measurement of HDL cholesterol: method eliminating apolipoprotein E-rich particles.J. Clin. Lab. Anal. 2001; 15: 223-229Crossref PubMed Scopus (23) Google Scholar). In severe hypertriglyceridemic HDL3-C values determined by ultracentrifugation were from those determined by TC assay to those determined by homogenous HDL-C assay. The HDL-C assay also when we added to the that the TC assay cholesterol in lipoproteins it is that contamination by in the supernatant the TC assay to In the single precipitation and direct HDL-C assay is a simple and precise procedure for the measurement of HDL-C subfractions and that for chylomicronemic samples. The of our single precipitation method is than that for the ultracentrifugation A homogenous HDL-C assay as is to an which the assay Our method is to be for the of different HDL subfractions in and clinical studies.

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