Enzymatic assessment of cholesterol on electrophoresis gels for estimating HDL size distribution and plasma concentrations of HDL subclasses
2010; Elsevier BV; Volume: 51; Issue: 6 Linguagem: Inglês
10.1194/jlr.d004358
ISSN1539-7262
AutoresPaola Toledo-Ibelles, Cynthia García-Sánchez, Nydia Ávila-Vazzini, Elizabeth Carreón‐Torres, Carlos Posadas‐Romero, Gilberto Vargas‐Alarcón, Óscar Pérez‐Méndez,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoThe aim of this study was to develop an enzymatic cholesterol staining method to determine HDL subclasses in a polyacrylamide gradient gel electrophoresis, which further allows staining by protein in the same electrophoresis lane. HDLs from 120 healthy individuals were separated through nondenaturing PAGE. HDLs were stained for cholesterol using an enzymatic semisolid mixture. Once the gels were unstained, they were stained again for proteins with Coomassie blue. The proportions of HDL subclasses were determined by densitometry. HDL subclasses were transformed to concentrations using as reference HDL-cholesterol plasma levels. This method is comparable in linearity and reproducibility to Coomassie blue staining, although it provides quantitative data. As expected, HDL size distribution shifted toward larger particles when determined by cholesterol as compared with protein. With this method, we observed different proportions of HDL subclasses between men and women as compared with Coomassie blue staining. We described a method to determine HDL size distribution by enzymatic cholesterol staining on polyacrylamide gels. The method allows the quantification of the cholesterol plasma concentration of each HDL subclass with the possibility to further stain the protein in the same sample. The combination of HDL staining by cholesterol and protein on electrophoresis gels provides information that may have clinical relevance. The aim of this study was to develop an enzymatic cholesterol staining method to determine HDL subclasses in a polyacrylamide gradient gel electrophoresis, which further allows staining by protein in the same electrophoresis lane. HDLs from 120 healthy individuals were separated through nondenaturing PAGE. HDLs were stained for cholesterol using an enzymatic semisolid mixture. Once the gels were unstained, they were stained again for proteins with Coomassie blue. The proportions of HDL subclasses were determined by densitometry. HDL subclasses were transformed to concentrations using as reference HDL-cholesterol plasma levels. This method is comparable in linearity and reproducibility to Coomassie blue staining, although it provides quantitative data. As expected, HDL size distribution shifted toward larger particles when determined by cholesterol as compared with protein. With this method, we observed different proportions of HDL subclasses between men and women as compared with Coomassie blue staining. We described a method to determine HDL size distribution by enzymatic cholesterol staining on polyacrylamide gels. The method allows the quantification of the cholesterol plasma concentration of each HDL subclass with the possibility to further stain the protein in the same sample. The combination of HDL staining by cholesterol and protein on electrophoresis gels provides information that may have clinical relevance. apolipoprotein body mass index coronary heart disease cholesterol-to-protein ratio coefficient of variation high-density lipoprotein-cholesterol low-density lipoprotein-cholesterol thiazolyl blue teratozolium bromide phenazine methosulphate Tris-borate buffer total cholesterol triglycerides Several epidemiological studies have demonstrated a negative correlation between HDL-cholesterol (HDL-C) and the development of coronary heart disease (CHD) (1Assmann G. Schulte H. von Eckardstein A. Huang Y. High-density lipoprotein cholesterol as a predictor of coronary heart disease risk: the PROCAM experience and pathophysiological implications for reverse cholesterol transport.Atherosclerosis. 1996; 124: S11-S20Abstract Full Text PDF PubMed Scopus (628) Google Scholar). The causal relationship between plasma HDL-C concentration and CHD has been explained by the role played by these lipoproteins in reverse cholesterol transport, as well as by the potentially anti-atherogenic properties of HDL, such as their anti-inflammatory, anti-oxidative, anti-aggregatory, anti-coagulant, and pro-fibrinolytic effects [for review see Ref. (2Feig J.E. Shamir R. Fisher E.A. Atheroprotective effects of HDL: beyond reverse cholesterol transport.Curr. Drug Targets. 2008; 9: 196-203Crossref PubMed Scopus (74) Google Scholar)].HDLs comprise a heterogeneous group of lipoproteins that may be classified by decreasing size in HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c, as assessed by nondenaturing PAGE in conjunction with automated densitometry (3Blanche P.J. Gong E.L. Forte T.M. Nichols A.V. Characterization of human high-density lipoproteins by gradient gel electrophoresis.Biochim. Biophys. Acta. 1981; 665: 408-419Crossref PubMed Scopus (443) Google Scholar, 4Huesca-Gómez C. Carreón-Torres E. Nepomuceno-Mejía T. Sánchez-Solorio M. Galicia-Hidalgo M. Mejía A.M. Montaño L.F. Franco M. Posadas-Romero C. Pérez-Méndez O. Contribution of cholesteryl ester transfer protein and lecithin:cholesterol acyltranferase to HDL size distribution.Endocr. Res. 2004; 30: 403-415Crossref PubMed Scopus (26) Google Scholar). However, HDLs have been classified by other approaches, such as selective precipitation (5Gidez 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, 6Lamarche B. Moorjani S. Cantin B. 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 (158) Google Scholar), ultracentrifugation (7Kulkarni K.R. Cholesterol profile measurement by vertical auto profile method.Clin. Lab. Med. 2006; 26: 787-802Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), NMR (8Otvos J.D. Jeyarajah E.J. Bennett D.W. Quantification of plasma lipoproteins by proton nuclear magnetic resonance spectroscopy.Clin. Chem. 1991; 37: 377-386Crossref PubMed Scopus (189) Google Scholar), electronic microscopy (9Elkhalil L. Majd Z. Bakir R. Perez-Mendez O. Castro G. Poulain P. Lacroix B. Duhal N. Fruchart J.C. Luc G. Fish-eye disease: structural and in vivo metabolic abnormalities of high-density lipoproteins.Metabolism. 1997; 46: 474-483Abstract Full Text PDF PubMed Scopus (32) Google Scholar), and two-dimensional electrophoresis (10Asztalos B.F. Lefevre M. Foster T.A. Tulley R. Windhauser M. Wong L. Roheim P.S. Normolipidemic subjects with low HDL cholesterol levels have altered HDL subpopulations.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1885-1893Crossref PubMed Scopus (37) Google Scholar). All these strategies recognize the existence of HDL particles of different sizes or densities that might have different properties against atherosclerosis development.Nevertheless, it is still a controversial issue as to which HDL fraction confers better cardiovascular protection; it has been postulated that the large HDL fraction is the most atheroprotective, because CHD patients have lower levels of these particles than controls, as assessed by selective precipitation or NMR (6Lamarche B. Moorjani S. Cantin B. 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 (158) Google Scholar, 11Harchaoui K. Arsenault B.J. Franssen R. Després J.P. Hovingh G.K. Stroes E.S. Otvos J.D. Wareham N.J. Kastelein J.J. Khaw K.T. et al.High-density lipoprotein particle size and concentration and coronary risk.Ann. Intern. Med. 2009; 150: 84-93Crossref PubMed Scopus (193) Google Scholar). In contrast, small HDL particles are the best acceptors of cholesterol from peripheral tissues (12Castro G.R. Fielding C.J. Early incorporation of cell-derived cholesterol into pre-beta-migrating high density lipoprotein.Biochemistry. 1988; 12: 25-29Crossref Scopus (562) Google Scholar) and also have better antioxidant properties than large HDL (13Kontush A. Chantepie S. Chapman M.J. Small dense HDL particles exert potent protection of atherogenic LDL against oxidative stress.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1881-1888Crossref PubMed Scopus (342) Google Scholar, 14Deakin S. Leviev I. Gomaraschi M. Calabresi L. Franceschini G. James R.W. Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high affinity, saturable, desorption mechanism.J. Biol. Chem. 2002; 277: 4301-4308Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Moreover, thiazolidinediones as well as fibrates both antiatherogenic drugs that increase HDL-cholesterol plasma levels, shift HDL size distribution toward small HDL particles (15Carreón-Torres E. Juárez-Meavepeña M. Cardoso-Saldaña G. Gómez C.H. Franco M. Fievet C. Luc G. Juárez-Oropeza M.A. Pérez-Méndez O. Pioglitazone increases the fractional catabolic and production rates of high-density lipoproteins apo AI in the New Zealand White Rabbit.Atherosclerosis. 2005; 181: 233-240Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 16Huesca C. Luc G. Duhal N. Pérez-Méndez O. Ciprofibrate increases synthesis and catabolism of HDL apo AI and AII in patients with hypertriglyceridemia.Atherosclerosis. 2004; 5: 64Google Scholar). In addition, some subjects with severe hypoalphalipoproteinemia who do not develop CHD have a high proportion of small HDL (9Elkhalil L. Majd Z. Bakir R. Perez-Mendez O. Castro G. Poulain P. Lacroix B. Duhal N. Fruchart J.C. Luc G. Fish-eye disease: structural and in vivo metabolic abnormalities of high-density lipoproteins.Metabolism. 1997; 46: 474-483Abstract Full Text PDF PubMed Scopus (32) Google Scholar), suggesting an atheroprotective role of these particles.The existing methods for the estimation of HDL subclasses involve the determination of at least one of their components. NMR, vertical autoprofile-II (VAP-II), as well as selective precipitation are based on HDL lipid content for estimating subclasses (5Gidez 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, 8Otvos J.D. Jeyarajah E.J. Bennett D.W. Quantification of plasma lipoproteins by proton nuclear magnetic resonance spectroscopy.Clin. Chem. 1991; 37: 377-386Crossref PubMed Scopus (189) Google Scholar, 17Warnick G.R. McNamara J.R. Boggess C.N. Clendenen F. Williams P.T. Landolt C.C. Polyacrylamide gradient gel electrophoresis of lipoprotein subclasses.Clin. Lab. Med. 2006; 26: 803-846Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Kulkarni K.R. Marcovina S.M. Krauss R.M. Garber D.W. Glasscock A.M. Segrest J.P. Quantification of HDL2 and HDL3 cholesterol by the Vertical Auto Profile-II (VAP-II) methodology.J. Lipid Res. 1997; 38: 2353-2364Abstract Full Text PDF PubMed Google Scholar), whereas protein is the HDL component determined in other methods, such as two-dimensional electrophoresis (10Asztalos B.F. Lefevre M. Foster T.A. Tulley R. Windhauser M. Wong L. Roheim P.S. Normolipidemic subjects with low HDL cholesterol levels have altered HDL subpopulations.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1885-1893Crossref PubMed Scopus (37) Google Scholar) and PAGE in native conditions (17Warnick G.R. McNamara J.R. Boggess C.N. Clendenen F. Williams P.T. Landolt C.C. Polyacrylamide gradient gel electrophoresis of lipoprotein subclasses.Clin. Lab. Med. 2006; 26: 803-846Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Because small HDL particles are protein rich and lipid poor, whereas large particles are the contrary, the relative proportion of HDL subclasses is dependent on the component determined for the quantification. The wide variety of methods used for determining HDL subclasses may explain, at least in part, the apparent controversy concerning which is the most antiatherogenic fraction of HDL. Therefore, the aim of this study was to develop an enzymatic cholesterol staining method to determine HDL subclasses in a polyacrylamide gradient electrophoresis gel, which further allows staining by protein in the same electrophoresis lane. In the first step, the enzymatic procedure specifically stains cholesterol on the gel. In the second step, HDL proteins are stained, and in both cases, the HDL subclasses distribution is determined by optical densitometry. This new method has two main advantages: it could be quantitative, and the HDL subclasses can be estimated also by protein for every sample in the same electrophoresis lane.MATERIALS AND METHODSMaterialsCholesterol esterase, cholesterol oxidase, and peroxidase were purchased from MP Biomedicals (Selem, OH). Sodium cholate, Triton 100×, thiazolyl blue teratozolium bromide (MTT), phenazine methosulfate (PMS), carboxymethylcellulose 5-cholesten-3-one, cholesteryl palmitate, and cholesterol were from Sigma-Aldrich (St. Louis, MO). Noble agar was purchased from Becton Dickinson (Franklin Lakes, NJ).Study subjectsOne hundred and twenty individuals between 18 and 85 years of age were recruited in the department of Endocrinology and the Outpatients service of the National Institute of Cardiology, Mexico. Volunteers without personal or family history of type 1 or 2 diabetes, pancreatitis, high blood pressure, angina pectoris, and CHD were accepted. All subjects had a fasting glucose of <110 mg/dl, a body mass index (BMI) of < 32 kg/m2, total cholesterol (TC) of < 220 mg/dl, triglycerides (TGs) of < 200 mg/dl, smoked fewer than five cigarettes per day, and had normal hepatic, thyroid, and renal functions, as assessed by routine laboratory analyses. All the participants gave their informed consent to take part in the study that was approved by the Ethics Committee of the National Institute of Cardiology "Ignacio Chávez", Mexico.Laboratory assessmentAll patients were instructed to avoid strenuous exercise and to eat a light dinner the day before blood drawings were performed. Blood samples were obtained in EDTA tubes after 12 h overnight fasting from an antecubital vein after subjects had been seated for 15 min. Samples were centrifuged at 4°C, plasma was separated and analyzed or frozen at −80°C until analysis. For lipoprotein isolation and plasma lipid profile (TC, TGs, HDL-C, and LDL-C), samples were processed within 2 h after collection. Plasma glucose, TC, and TGs were determined by commercially available enzymatic methods. The phosphotungstic acid-Mg2+ precipitation procedure of apolipoprotein (apo) B-containing lipoproteins was used before quantifying HDL-C. Quality control of lipid measurements was assessed through standardization to the Center for Disease Control and Prevention (Atlanta, GA). LDL-C was estimated using the Friedewald equation modified by De Long (19DeLong D.M. DeLong E.R. Wood P.D. Lippel K. Rifkind B.M. A comparison of methods for the estimation of plasma low- and very low density lipoprotein cholesterol. The Lipid Research Clinics Prevalence Study.JAMA. 1986; 256: 2372-2377Crossref PubMed Scopus (317) Google Scholar). Serum levels of all lipids were determined within 48 h after drawing blood samples.Isolation of HDLHDLs were separated by ultracentrifugation in a Beckman optima TLX table centrifuge at 110,000 rpm in 3.2 ml polycarbonate tubes as described previously (20Huesca-Gómez C. Franco M. Luc G. Montaño L.F. Massó F. Posadas-Romero C. Pérez-Méndez O. Chronic hypothyroidism induces abnormal structure of high-density lipoproteins and impaired kinetics of apolipoprotein A-I in the rat.Metabolism. 2002; 51: 443-450Abstract Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, total apo B-containing lipoproteins (density < 1.063 mg/dl) were obtained after 2.16 h, whereas total HDL (1.063 < density < 1.21 g/ml) took 2.5 h. Under these conditions, 80 to 85% of total plasma apo A-I was recovered from the HDL fraction without apo B-contamination. HDLs were dialyzed against 0.09 M Tris/0.08 M boric acid/3 mM EDTA (TBE) buffer, pH 8.4.Enzymatic staining of cholesterol on polyacrylamide gelHDLs were separated by their hydrodynamic diameter in an 8 × 10 × 0.15 cm nondenaturing 3–30% gradient polyacrylamide gel electrophoresis, using TBE during 24 h at 170 V as previously described (3Blanche P.J. Gong E.L. Forte T.M. Nichols A.V. Characterization of human high-density lipoproteins by gradient gel electrophoresis.Biochim. Biophys. Acta. 1981; 665: 408-419Crossref PubMed Scopus (443) Google Scholar, 4Huesca-Gómez C. Carreón-Torres E. Nepomuceno-Mejía T. Sánchez-Solorio M. Galicia-Hidalgo M. Mejía A.M. Montaño L.F. Franco M. Posadas-Romero C. Pérez-Méndez O. Contribution of cholesteryl ester transfer protein and lecithin:cholesterol acyltranferase to HDL size distribution.Endocr. Res. 2004; 30: 403-415Crossref PubMed Scopus (26) Google Scholar, 17Warnick G.R. McNamara J.R. Boggess C.N. Clendenen F. Williams P.T. Landolt C.C. Polyacrylamide gradient gel electrophoresis of lipoprotein subclasses.Clin. Lab. Med. 2006; 26: 803-846Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Twenty five micrograms of HDL protein sample, corresponding approximately to 10 µg of cholesterol, were deposited per well.Gels were stained for cholesterol using an enzymatic mixture of cholesterol esterase, cholesterol oxidase, and peroxidase at a final concentration of 0.075 U/ml, 0.05 U/ml, and 0.25 U/ml, respectively, in a 150 mM NaCl, 8.6 mM Na2HPO4, 1.4 mM NaH2PO4 buffer (PBS), pH 7.4. The reaction mixture also included 3 mM sodium cholate, 0.1% Triton 100×, 0.4 mM MTT, and 0.6 mM PMS. In a first series of experiments, we directly submerged the electrophoresis gels in the reaction mixture. In order to enhance the cholesterol staining, we further added Noble agar at 1.0% or carboxymethylcellulose at 1.4% as viscosant agents to the reaction mixture. For the Noble agar, 100 mg were heated in 10 ml the buffer reaction until dissolution, cooled to about 40°C, and then the enzymes were added. The final concentration of enzymes was as described above. Electrophoresis gels were kept in contact with the reaction mixture containing the viscosant agent during 1 h at 37°C in the dark. At the end of the incubation time, the reaction mixture was removed and the gels were gently washed in PBS to eliminate any remaining residue of agar or carboxymethylcellulose. Electrophoresis gels were then scanned in a GS-670 BioRad densitometer (scan 1), distained with methanol:acetic acid:water 5:2:13, and further restained for proteins with Coomassie R-250. Afterwards, gels were again scanned (scan 2). The relative proportions of each HDL subclass determined by protein were estimated by optical densitometry analysis of scan 2, using as reference globular proteins (thyroglobulin, 17 nm; ferritin, 12.2 nm; lactate deshydrogenase, 8.2 nm; and albumin, 7.1 nm; high-molecular weight calibration kit, Amersham Pharmacia Biotech, Buckimghamshire, UK) that were run in the same gel (3Blanche P.J. Gong E.L. Forte T.M. Nichols A.V. Characterization of human high-density lipoproteins by gradient gel electrophoresis.Biochim. Biophys. Acta. 1981; 665: 408-419Crossref PubMed Scopus (443) Google Scholar, 4Huesca-Gómez C. Carreón-Torres E. Nepomuceno-Mejía T. Sánchez-Solorio M. Galicia-Hidalgo M. Mejía A.M. Montaño L.F. Franco M. Posadas-Romero C. Pérez-Méndez O. Contribution of cholesteryl ester transfer protein and lecithin:cholesterol acyltranferase to HDL size distribution.Endocr. Res. 2004; 30: 403-415Crossref PubMed Scopus (26) Google Scholar, 17Warnick G.R. McNamara J.R. Boggess C.N. Clendenen F. Williams P.T. Landolt C.C. Polyacrylamide gradient gel electrophoresis of lipoprotein subclasses.Clin. Lab. Med. 2006; 26: 803-846Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Relative proportion of each HDL subclass is expressed as the percentage of the total HDL area under the curve, integrated from 7.94 to 13.59 nm.The relative proportion of HDL subclasses by their cholesterol content was determined in scan 1 using the migration distances of the reference globular proteins obtained from the scan 2. Considering that the area under the curve in the densitogram represents 100% of the cholesterol in the HDL, the cholesterol plasma concentration of each HDL subclass was estimated as follows: HDLn-C = (% HDLn determined by cholesterol × HDL-C)/100 where n represents the HDL subclass, and the HDL-C is the HDL cholesterol plasma concentration. For classification of the HDL subclasses, we considered the following size intervals: HDL3c, 7.94–8.45 nm; HDL3b, 8.45–8.98 nm; HDL3a, 8.98–9.94 nm; HDL2a, 9.94–10.58 nm; and HDL2b, 10.58–13.59 nm (4Huesca-Gómez C. Carreón-Torres E. Nepomuceno-Mejía T. Sánchez-Solorio M. Galicia-Hidalgo M. Mejía A.M. Montaño L.F. Franco M. Posadas-Romero C. Pérez-Méndez O. Contribution of cholesteryl ester transfer protein and lecithin:cholesterol acyltranferase to HDL size distribution.Endocr. Res. 2004; 30: 403-415Crossref PubMed Scopus (26) Google Scholar).Analysis of the enzymatic reaction homogeneityIn order to determine whether the enzymatic reaction is homogeneous along the polyacrylamide gradient gel, we used HDL labeled with [3H]cholesterol as we have previously described (4Huesca-Gómez C. Carreón-Torres E. Nepomuceno-Mejía T. Sánchez-Solorio M. Galicia-Hidalgo M. Mejía A.M. Montaño L.F. Franco M. Posadas-Romero C. Pérez-Méndez O. Contribution of cholesteryl ester transfer protein and lecithin:cholesterol acyltranferase to HDL size distribution.Endocr. Res. 2004; 30: 403-415Crossref PubMed Scopus (26) Google Scholar). By this procedure, 90% of the radioactive cholesterol within the HDL was esterified, whereas the remaining 10% remained as free cholesterol. Radiolabeled HDLs were then separated by electrophoresis as described above; as a consequence, the radioactive label was distributed along the whole sample in the electrophoresis lane. After the migration time, we monitored the conversion of cholesteryl esters and free cholesterol to cholestenone in the sections of the gel corresponding to the HDL subclasses. For this purpose, polyacrylamide gels were stained for cholesterol using the enzymatic mixture and scanned as described above. The sections corresponding to the different HDL subclasses were cut, lipids were extracted with chloroform:methanol 2:1, the organic solvent was evaporated under nitrogen stream, and the sample was resuspended in 75 µL of toluene. Cholestenone, cholesteryl esters, and free cholesterol were separated by thin layer chromatography in silica gel plates (Sigma Aldrich) using petroleum ether:ethylic ether:acetic acid 90:10:5 as mobile phase. Spots were identified using cold 5-cholesten-3-one (Rf=0.07), cholesteryl palmitate (Rf=0.71) and cholesterol (Rf=0.20) as standards, scratched from the plate, and counted for radioactivity in a liquid scintillation analyzer (TRI-CARB 2200CA, Packard). The rate of conversion of cholesterol to cholestenone was estimated by dividing the counted radioactivity of the cholestenone spot by the addition of the radioactivity corresponding to cholestenone, cholesteryl esters, and free cholesterol. The results were expressed as % of conversion.Statistical analysisCentral tendency and dispersion measurements were estimated by conventional methods. Comparisons between multiple groups were assessed by ANOVA test. Normal distribution of the variables was evaluated by the Kolmogorov-Smirnoff test. The significance of the differences among parameters between men and women was tested by the Student's t-test for normally distributed variables. TGs values were logarithmically transformed for parametric statistical analysis. Comparisons of nonnormally distributed variables were performed by Mann-Whitney U and Wilcoxon tests for independent groups and paired variables, respectively. Partial correlations adjusted by age and gender were performed and statistical significance was set at P < 0.05. Unless otherwise indicated, values are expressed as mean ± SD for variables with normal distribution and as median and interquartile interval for nonnormally distributed variables. Statistical analysis was performed using SPSS V 11 software.RESULTSFor the assessment of the optimal viscosant agent and to test the homogeneity of the enzymatic reaction along the gel, we obtained a pool of plasma from five volunteers. HDLs were isolated from this pool by ultracentrifugation and further separated on the basis of their hydrodynamic diameter by nondenaturating electrophoresis in a 3–30% PAGE, as indicated in the Methods section. The enzymatic method for the assessment of the HDL subclasses required a gel-phase reagent to enhance the intensity of the cholesterol staining, because a part of the formed products were washed away by the buffer and the mechanical agitation needed during incubation in an aqueous solution (Fig. 1). Both carboxymethylcellulose and Noble agar enhanced the intensity of the staining signal as compared with the enzymatic mixture in buffer without viscosant agent as it can be observed in the corresponding densitograms (Fig. 1). We finally chose carboxymethylcellulose at 1.4%, because the staining intensity is better than that obtained with Noble agar. Moreover, carboxymethylcellulose is soluble in the buffer at room temperature, avoiding heating-cooling processes that are necessary for agar solubilization. The best cholesterol staining with the lower background was obtained between 60 to 75 min of incubation (Supplementary Fig. I). This new procedure to stain cholesterol is linear for total HDL between 5 and 30 µg of protein deposited by well in the electrophoresis gel (Fig. 2). For HDL subfractions, the method is linear from 5 to 25 µg of protein for HDL 2b, 2a, and 3a, from 5 to 30 µg of protein for HDL3b, and up to 40 µg of protein for HDL3c (Fig. 2).Fig. 2Linearity tests for enzymatic staining of cholesterol on electrophoresis gels. Total HDL (HDLt) were run in polyacrylamide 3-30% gradient gel electrophoresis, stained for cholesterol (black triangles) and further for protein (black circles). Plots represent area under the curve (AUC) obtained by densitometric analysis for HDLt and HDL subclasses (for details, see Methods section) versus protein deposited by well. For comparison purposes, protein AUC ranges were adjusted.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To rule out the possible influence of polyacrylamide gradient on the enzymatic reaction, i.e., the lower the polyacrylamide concentration, the higher conversion of cholesterol to cholestenone, we determined the % of conversion of substrates to products during 1 h using HDL labeled with [3H]cholesterol as described in the Methods section. The mean percentage of cholesterol conversion to cholestenone was 58.9 ± 7.9% for the whole HDL (n = 5). Concerning the HDL subclasses, the mean percentage of conversion was 55.7 ± 6.1, 51.4 ± 5.1, 49.0 ± 8.6, 52.2 ± 12.8, and 56.0 ± 5.2% for HDL2b, 2a, 3a, 3b, and 3c, respectively; there was not any significant difference among these values (ANOVA test P = 0.606).For the assessment of the intra- and inter-assay coefficient, we obtained a pool of plasma from five healthy volunteers. HDLs were isolated and separated on the basis of their hydrodynamic diameter by nondenaturating electrophoresis in a 3–30% PAGE. Cholesterol and protein were sequentially stained, densitograms were obtained, and analyzed as mentioned above. Samples were run in duplicate and the reported values are the mean of the two determinations. Under these conditions, four samples and the corresponding duplicate of the reference globular proteins were run per each electrophoresis gel. The intra-assay coefficient of variation (CV) of the method was 6.9% as assessed after 32 repetitions of the same sample in eight independent gels. The mean inter-assay CV was 4.3% as determined with the analysis of the same sample in 25 independent electrophoresis gels.Once the conditions for this new method were established, we determined HDL subclasses distribution in 120 subjects without clinical history of CHD. Table 1 shows the lipid profile and clinical characteristics of the subjects included in the study. For comparison purposes, data of women and men were analyzed separately.TABLE 1.Demographic data and lipid profile of the individuals included in the studyAll SubjectsMenWomenpaStudent's t-test or Mann-Whitney U for variables with normal or nonnormal distribution variables, respectively.n1206060Age (years)33.4 ± 16.029.2 ± 12.036.5 ± 17.90.012BMI25.1 ± 4.825.2 ± 4.425.0 ± 4.20.844SBP111.6 ± 16.1113.4 ± 14.6110.1 ± 16.90.320DBP71.7 ± 11.671.1 ± 11.172.2 ± 13.70.659Triglycerides (mg/dl)108 [71–147]102.0 [70.4–134.0]109.5 [71.7–170.5]0.193Total cholesterol (mg/dl)169.2 ± 42.9157.4 ± 35.6178.5 ± 46.10.009LDL-cholesterol (mg/dl)99.8 ± 34.495.1 ± 29.7103.4 ± 37.50.205HDL-cholesterol (mg/dl)47.3 ± 14.542.9 ± 13.150.6 ± 14.80.006Data represent mean ± SD for normally distributed variables or median [interquartile interval] for nonnormally distributed variables.a Student's t-test or Mann-Whitney U for variables with normal or nonnormal distribution variables, respectively. Open table in a new tab Concerning the analysis of HDL subclasses, the relative proportion of HDL2a and HDL3c determined by cholesterol did not have a normal distribution. In contrast, the relative proportions of all HDL subclasses determined by protein were normally distributed (Table 2). In agreement with previous observations (7Kulkarni K.R. Cholesterol profile measurement by vertical auto profile method.Clin. Lab. M
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