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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2670-2676

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2670.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Distribution of ApoA-I–Containing HDL Subpopulations in Patients With Coronary Heart Disease

Bela F. Asztalos; Paul S. Roheim; Richard L. Milani; Michael Lefevre; Judith R. McNamara; Katalin V. Horvath; Ernst J. Schaefer

From the Lipid Metabolism Laboratory, Jean Mayer USDA Human Nutrition Center on Aging at Tufts University, and the Division of Endocrinology, Metabolism, Diabetes, and Molecular Medicine (B.F.A., J.R.M., K.V.H., E.J.S.), New England Medical Center, Boston, Mass; the Physiology Department (P.S.R.), Louisiana State University Medical Center, New Orleans; the Division of Cardiology (R.L.M.), Alton Ochsner Hospital, New Orleans, La; and Pennington Biomedical Research Center (M.L.), Baton Rouge, La.

Correspondence to Bela F. Asztalos, PhD, JM-USDA/HNRC at Tufts University, Lipid Metabolism Laboratory, 711 Washington St, Boston, MA 02111. E-mail belaasztalos{at}yahoo.com


*    Abstract
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Abstract—High density lipoproteins (HDLs) and their subspecies play a role in the development of coronary heart disease (CHD). HDL subpopulations were measured by 2-dimensional nondenaturing gel electrophoresis in 79 male control subjects and 76 male CHD patients to test the hypothesis that greater differences in apolipoprotein (apo)A-I–containing HDL subpopulations would exist between these 2 groups than for traditional lipid levels. In CHD subjects, HDL cholesterol (HDL-C) was lower (-14%, P<0.001), whereas total cholesterol and the low density lipoprotein cholesterol/HDL-C ratio were higher (9% [P<0.05] and 21% [P<0.01], respectively) compared with control levels. No significant differences were found for low density lipoprotein cholesterol, triglyceride, and apoA-I levels. In CHD subjects, there were significantly (P<0.001) lower concentrations of the large lipoprotein (Lp)A-I {alpha}1 (-35%), pre-{alpha}1 (-50%), pre-{alpha}2 (-33%), and pre-{alpha}3 (-31%) subpopulations, whereas the concentrations of the small LpA-I/A-II {alpha}3 particles were significantly (P<0.001) higher (20%). Because {alpha}1 was decreased more than HDL-C and plasma apoA-I concentrations in CHD subjects, the ratios of HDL-C to {alpha}1 and of apoA-I to {alpha}1 were significantly (P<0.001) higher by 36% and 57%, respectively, compared with control values. Subjects with low HDL-C levels (<=35 mg/dL) have different distributions of apoA-I–containing HDL subpopulations than do subjects with normal HDL-C levels (>35 mg/dL). Therefore, we stratified participants according to HDL-C concentrations into low and normal groups. The differences in lipid levels between controls and HDL-C–matched cases substantially decreased; however, the significant differences in HDL subspecies remained. Our research findings support the concept that compared with control subjects, CHD patients not only have HDL deficiency but also have a major rearrangement in the HDL subpopulations with significantly lower {alpha}1 and pre-{alpha}1–3 (LpA-I) and significantly higher {alpha}3 (LpA-I/A-II) particles.


Key Words: HDL subpopulations • coronary heart disease • lipids • lipoproteins • apolipoproteins


*    Introduction
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Coronary heart disease (CHD) remains the leading cause of death and disability in the United States. Approximately 14 million people have CHD, and 1.5 million individuals experience a myocardial infarction annually, leading to {approx}500 000 deaths per year.1 Atherosclerosis is a multifactorial disease affected by lifestyle and genetic factors.2 Independent risk factors for CHD as defined by the National Cholesterol Education Program (NCEP) Adult Treatment Panel II include the following: age, sex, hypertension, smoking, diabetes, family history of premature CHD, elevated plasma levels of LDL cholesterol (LDL-C >=160 mg/dL), and low levels of HDL (HDL-C <35 mg/dL).3 4 5 6 7 8 9 10 The NCEP panel classified an HDL-C level >=60 mg/dL (>1.55 mmol/L) as protective against the development of CHD.3 Many prospective epidemiological studies have indicated that a decreased HDL-C level is a significant independent risk factor for heart disease.5 6 7 9 11 12

HDL is found in human plasma at a density of 1.063 to 1.21 g/mL and contains {approx}50% protein, 25% phospholipid, 20% cholesterol (mainly esterified), and 5% triglycerides by weight. The 2 major protein constituents are apoA-I and apoA-II. A variety of methods, including analytical ultracentrifugation, differential precipitation, immunoaffinity chromatography, and nondenaturing 1D and 2D gel electrophoresis, have been developed to separate HDL into different subclasses. These HDL subpopulations differ in apolipoprotein and lipid composition, as well as in size and charge, and probably have different physiological functions.13 14 15 16

Separation of HDL subclasses on the basis of charge and size by 2D nondenaturing gel electrophoresis was developed in the late 1980s14 and later was adopted by others.16 17 18 By use of this method, the majority of apoA-I in plasma has {alpha} mobility. These HDL particles have been classified as {alpha}1, {alpha}2, and {alpha}3, with sizes of 11.2, 9.51, and 7.12 nm, respectively.18 Small amounts of HDL particles containing apoA-I with pre-ß mobility (pre-ß1 and pre-ß2) have been detected as well. We have standardized the 2D nondenaturing gel electrophoresis methodology and have reported 12 apoA-I–containing HDL subpopulations, including 4 new subpopulations with pre-{alpha} mobility containing only apoA-I.18 19 ApoA-II is present in {alpha}2 and {alpha}3 HDL subpopulations.19 Subjects with low HDL-C levels have significant decreases in the large LpA-I HDL particles, {alpha}1 and pre-{alpha}1.19

The precise function and the metabolic nature of the apoA-I–containing and/or other apolipoprotein-containing HDL subfractions remain to be elucidated. There are a number of epidemiological studies on the relationship between CHD and HDL subspecies with conflicting results. Most investigators report an overall reduction in HDL-C and apoA-I in CHD subjects compared with control subjects.20 Depending on the methodology used, however, some scientists report significantly lower levels of HDL2 or LpA-I in CHD subjects,21 22 23 24 25 and others report reductions in HDL3, in HDL2 and HDL3, or in LpA-I and LpA-I/A-II.23 26 27 28 29 30 Using immunoaffinity chromatography and nondenaturing gel electrophoresis, Cheung et al31 reported significant differences in the size of HDL particles between CHD patients and control individuals. They found that the presence of CHD was more strongly associated with HDL particle size distribution than with a low HDL-C level. The discrepancy between studies may be due to sample size and/or subject selection. Alternatively, the difference in results between studies may be explained by the fact that HDL2, HDL3, LpA-I, and LpA-I/A-II are heterogeneous fractions and contain a variety of different HDL subspecies. These issues require resolution with larger sample sizes and with more complete characterization of HDL subspecies.

Our goal was to determine and compare HDL subpopulations, as defined by 2D nondenaturing gel electrophoresis, in healthy male controls and male subjects with CHD. The present study provides additional information about the potential role of HDL subpopulations in the risk for CHD.


*    Methods
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Study Population
Fasting plasma samples were obtained from 79 healthy male subjects, aged 30 to 82 years. Participants were selected to have total plasma cholesterol and LDL-C levels between the 10th and 90th percentile, HDL-C levels >25 mg/dL or below the 95th percentile, and triglycerides below the 95th percentile. Lipid cutoff points were selected to eliminate subjects with lipid disorders. Exclusion criteria included the presence of cardiovascular, renal, hepatic, endocrine, gastrointestinal, or other systemic disease, body mass index >32 kg/m2, and hypertension. During screening visits, physical examinations, blood chemistry, lipid profiles, and urinalyses were performed.

Fasting plasma samples were also obtained from 76 male patients, aged 33 to 81 years, with established CHD, as documented by history of prior myocardial infarction, coronary artery bypass grafting, percutaneous transluminal coronary angioplasty, documented disease on coronary angiography (>50% stenosis in >=1 coronary artery), or the presence of angina pectoris with a positive stress test. Exclusion criteria included triglyceride values >400 mg/dL, body mass index >35 kg/m2, secondary causes of hyperlipoproteinemia, diabetes, any systemic disease interfering with lipoprotein metabolism, lipid-lowering medications, and/or myocardial infarction, and/or coronary artery bypass graft within the last 12 weeks.

Lipid and Lipoprotein Analysis
Fasting blood samples were collected into tubes containing 1.5 g/L EDTA and centrifuged at 3000g for 20 minutes at 4°C to obtain plasma. Aliquots of each plasma sample were stored at -80°C or in liquid nitrogen until use. Total cholesterol, HDL-C, and triglycerides were determined by use of automated enzymatic assays. HDL-C was determined after precipitation of the non-HDL fraction by the dextran sulfate method.32 LDL-C was calculated by use of the Friedewald equation.33 ApoA-I was measured in plasma by automated immunoturbidometric assay (Wako Inc). Coefficients of variation for all assays between and within runs were <10%.

HDL Subpopulation Analysis
Separation of HDL subpopulations was carried out similarly as previously described in detail.18 19 Plasma samples stored at -80°C were placed into liquid nitrogen for {approx}30 minutes before thawing at 37°C to avoid any alterations in the physicochemical composition of the particles. The effects of storage and thawing conditions were carefully investigated (Table 1Down). No significant differences in the percent distribution of the apoA-I–containing HDL subpopulations were detected (at 95% CI) when fresh plasma and stored plasma samples (up to 1 year) were compared.


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  		Table 1. ApoA-I–Containing HDL Subpopulations in Fresh and Stored Plasma Samples

In the first dimension, we separated HDL according to charge into pre-ß, {alpha}, and pre-{alpha} mobility particles. Four microliters of plasma was applied to 0.7% LE SeaKem agarose gels (3 mm thick) and electrophoresed in a vertical slab gel electrophoresis unit (Pharmacia GE 2/4 recirculating apparatus) in 25 mmol/L Tris-tricine buffer at pH 8.6 (10°C) at 250 V until the albumin (the blue band) reached 3.5 cm. Gel strips were cut out, placed, and sealed (with 65°C agarose) on the top of the nondenaturing 3% to 34% concave gradient polyacrylamide gels, followed by electrophoresis in the second dimension. Gels (3 mm thick) were prepared in a Hoefer gel-caster (SE-600). Electrophoresis was carried out in a Hoefer SE-600 electrophoresis apparatus (Amersham Pharmacia Biotech) for 24 hours at 250 V in buffer containing 90 mmol/L Tris, 80 mmol/L boric acid, and 2.5 mmol/L EDTA, pH 8.3. 125I-labeled molecular weight standard proteins (Pharmacia High Molecular Weight Standard) were applied in the middle of each polyacrylamide gel. Electrotransfer onto nitrocellulose membranes (BA-S83, 0.2 µm, Schleicher & Schuell) was carried out at 30 V for 24 hours at 10°C. Posttransfer membrane operations included fixing with 0.3% glutaraldehyde in PBS for 10 minutes, rinsing with Tris-buffered PBS (PBST), blocking with 5% dry milk for 10 minutes, and incubating with monospecific polyclonal goat anti-human apoA-I sera for 6 hours. Unbound antibody was removed by washing 3 times for 3 minutes each with PBST, followed by incubation with 125I-labeled rabbit anti-goat Fa(b)2 IgG fragment overnight. Membranes then were washed 3 times for 3 minutes each with PBST, dried, and placed in cassettes for overnight exposure. Quantification was carried out in a PhosphorImager with the use of an ImageQuant software package (Molecular Dynamics). After the peaks were determined by integrating the designated areas,18 each HDL subpopulation was delineated. The program automatically measured the volume of each area and the percent distribution of each of the encircled HDL subpopulations. Data were expressed as pixels linearly correlated with the disintegrations per minute of the 125I bound to the antigen-antibody complex.34 Absolute concentrations (in milligrams apoA-I per deciliter plasma) were calculated by multiplying the plasma total apoA-I concentration (milligrams per deciliter) by the percentile value of each subpopulation.

For further analysis, nonnormally distributed data were transformed by use of the natural logarithm to approach a gaussian distribution. To test the hypothesis of no difference in mean levels of given variables between categories, ANOVA was used with the Stat Most statistical software package.


*    Results
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Information about age, concentrations of plasma lipids and apoA-I, and the ratio of LDL-C to HDL-C for control subjects (n=79) and CHD patients (n=76) is presented in Table 2Down. The CHD group was significantly (P<0.001) older (8.4 years) than the control group. Other significant differences between the control and CHD groups were as follows: HDL-C (-14%, P<0.001), total cholesterol (9%, P<0.05), and the ratio of LDL-C to HDL-C (23%, P<0.01). Concentrations of LDL-C and triglycerides were higher in the CHD group by 5% and 18%, respectively, but the differences were not significant, whereas apoA-I concentrations were similar in the 2 groups. When the apoA-I–containing HDL subpopulations were compared between the 2 groups (Table 3Down), we found significantly lower concentrations of {alpha}1 (-35%), pre-{alpha}1 (-50%), pre-{alpha}2 (-33%), and pre-{alpha}3 (-31%, P<0.001) in CHD patients. In contrast, the {alpha}3 HDL subpopulation was significantly higher (20%, P<0.001) in the case patients, whereas differences in {alpha}2, pre-ß1, and pre-ß2 were not significantly different between the 2 groups. We calculated the ratios of apoA-I to {alpha}1 and of HDL-C to {alpha}1 and found that both ratios were significantly (P<0.001) higher in the CHD group (by 57% and 36%, respectively) than in the control group.


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  		Table 2. Concentrations of Lipids, ApoA-I, and Differences Between Control and CHD Subjects


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  		Table 3. Concentrations of ApoA-I–Containing HDL Subpopulations and Differences Between Control and CHD Subjects

To test the hypothesis that HDL-C concentration has an influence on the values of the above-measured parameters, both groups were further separated according to HDL-C level into low (HDL-C <=35 mg/dL) and normal (HDL-C >35 mg/dL) groups. There were 24 control (30.4%) and 37 CHD (48.7%) subjects in the low HDL-C group and 55 control (69.6%) and 39 CHD (51.3%) subjects in the normal HDL-C group. In the low HDL-C group, CHD subjects had significantly higher total cholesterol (20%, P<0.05) and significantly lower HDL-C (-6%, P<0.05) concentrations compared with HDL-C–matched control subjects (Table 4Down). LDL-C, triglyceride, and apoA-I levels were higher by 13%, 15%, and 5%, respectively, but these differences were not significant. In the normal HDL-C group, only the average HDL-C concentration was significantly different (-9%, P<0.05) in CHD patients compared with control subjects. The ratios of LDL-C to HDL-C were higher in CHD patients in the low (21%) and normal (9%) HDL-C groups, but these differences were not significant (Table 4Down).


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  		Table 4. Comparison of Plasma Lipid Values After Subjects Were Stratified Into Low (<=35 mg/dL) and Normal (>35 mg/dL) HDL-C Groups

When the apoA-I–containing HDL subpopulations were compared between control and CHD subjects, very similar patterns were found in low and normal HDL-C groups (Table 5Down), and the differences resembled those obtained when all CHD patients were compared with all control individuals. In CHD patients with low HDL-C levels compared with control subjects, {alpha}1, pre-{alpha}1, pre-{alpha}2, and pre-{alpha}3 subpopulations were significantly lower (P<0.001), by -30%, -48%, -33%, and -32%, respectively, whereas pre-ß1 and {alpha}3 were significantly higher by 40% (P<0.05) and 21% (P<0.01), respectively (Table 5Down). Levels of pre-ß2 (-7%) and {alpha}2 (8%) were not significantly different between cases and controls with low HDL-C levels. In patients with normal HDL-C levels, the {alpha}1, pre-{alpha}1, pre-{alpha}2, and pre-{alpha}3 subpopulations were also significantly (P<0.001) lower by -29%, -44%, -29%, and -29%, respectively, whereas {alpha}3 was significantly higher by 21% (P<0.001), and the concentrations of pre-ß1, pre-ß2, and {alpha}2 were not significantly different when CHD cases were compared with the appropriate controls. The ratios of apoA-I to {alpha}1 and of HDL-C to {alpha}1 were significantly higher in CHD patients in the low and normal HDL-C groups by 57%, 37%, and 44%, 33%, respectively, compared with ratios in the appropriate control groups.


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  		Table 5. Comparison of ApoA-I–Containing HDL Subpopulations After Subjects Were Stratified Into Low (<35 mg/dL) and Normal (>35 mg/dL) HDL-C Groups

The {alpha}1 and {alpha}2 HDL subpopulations were strongly correlated with HDL-C in control subjects (r=0.756 and r=0.714, respectively) and CHD subjects (r=0.774 and r=0.575, respectively). Concentration of the {alpha}3 subpopulation did not correlate with HDL-C. In addition, a very strong positive correlation was found between the {alpha}1 and {alpha}2 subpopulations and apoA-I levels in control subjects (r=0.832 and r=0.852, respectively) and CHD subjects (r=0.600 and r=0.798, respectively). The concentration of the {alpha}3 subpopulation was weakly correlated with apoA-I values in both groups. In control and CHD groups, the {alpha}1 subpopulation correlated negatively with the plasma triglyceride level (r=-0.476 and r=-0.517, respectively), whereas the {alpha}3 subpopulation correlated positively with the plasma triglyceride level (r=0.380 and r=0.230, respectively). No correlation was found between the {alpha}-mobility HDL subpopulations and either total cholesterol or LDL-C. In Figure 1Down, we present the regression lines and the 95% CIs around the regression lines for the various {alpha}-mobility subpopulations versus HDL-C and plasma total apoA-I for control subjects superimposed with the x-y scatterplot of the same parameters for CHD subjects. The majority of data points for CHD subjects of {alpha}1 versus HDL-C (66%) and apoA-I (86%) were below the 95% CIs calculated for control individuals. The majority of data points on CHD subjects with regard to {alpha}3 versus HDL-C (67%) and apoA-I (78%) were above the 95% CIs calculated for control individuals. Data points of cases on {alpha}2 subpopulations versus HDL-C (42%) and {alpha}2 versus apoA-I (38%) also showed a trend for values above the 95% CIs of control subjects.



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  		Figure 1. Up Plots of {alpha}-migrating HDL subpopulations vs HDL-C (left) and plasma total apoA-I concentration (right). Solid line represents estimated regression lines for control individuals (n=79). Dotted lines indicate 95% CIs around these regression lines. Symbols (open triangles) represent plots of same parameters for CHD subjects.

Because earlier studies on the relation of HDL subspecies and CHD used ultracentrifugally separated subfractions, we compared the 2 methodologies. Ultracentrifugally separated HDL2 (density [d]<1.125) and HDL3 (d=1.125 to 1.24) were subjected to 2D nondenaturing gel electrophoresis (Figure 2Down). The majority of HDL2 was found in the {alpha}1 and pre-{alpha}1 positions, whereas HDL3 was a composite of the smaller {alpha}2, {alpha}3, pre-{alpha}2, pre-{alpha}3, and pre-ß1 subpopulations. All of the pre-ß2 and some apoA-I from the other particles were found in the d>1.21 g/mL fraction in accordance with our previous observation.18 Because it is known that ß-blockers can affect lipoproteins, we examined HDL subspecies in CHD patients on and off ß-blockers and saw no significant effects.



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  		Figure 2. Up ApoA-I–containing HDL subpopulations of normolipidemic healthy male subject. HDL subfractions were separated by ultracentrifuge (Beckman L8-M, at 40 000 rpm for 48 hours with an SW40 rotor, HDL2 d<1.125 and HDL3 d=1.125 to 1.24) before analysis by nondenaturing 2D gel electrophoresis, immunoblot, and image analysis as described in Methods.


*    Discussion
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 *Discussion
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The inverse association between the incidence of CHD and HDL has been known for decades.7 8 16 20 21 22 23 24 25 26 27 28 29 30 31 35 36 37 38 HDL is heterogeneous, containing multiple subpopulations differing in size, density, flotation rate, lipid and apolipoprotein composition, and functional properties.19 39 40 41 42 In the last 2 decades, many laboratories have tried to assess whether any of these subpopulations have a stronger relationship with risk for CHD than others.20 21 22 23 24 25 26 27 28 29 30 31 35 36 37 Despite conflicting data, as Silverman et al43 reviewed in 1993 and Montali et al30 summarized in 1994, many investigators believe that measuring HDL subfractions may provide additional information about atherosclerotic risk as well as about the underlying physiological mechanism responsible for that risk.

In the present study, we investigated the relationship between plasma lipid levels and the apoA-I–containing HDL subpopulations, separated by nondenaturing 2D gel electrophoresis, and CHD. Data generated in the present study support earlier observations that a low HDL-C level, even in the presence of a normal LDL-C concentration, is a potential risk factor for CHD43 44 (Table 2Up). Our results indicate that in the CHD group, the lower concentration of HDL-C (-14%) rather than the higher LDL-C level (5%) was mainly responsible for the significantly increased ratio of LDL-C to HDL-C (23%).

We have previously published that subjects with low HDL-C levels (<=35 mg/dL) have different HDL subpopulation profiles than do subjects with normal levels of HDL-C (>35 mg/dL).19 Therefore, we further partitioned both groups into low and normal HDL-C subgroups. Comparing the HDL-C–matched CHD and control groups, we found 20% (P<0.05) higher total cholesterol levels in the low HDL-C CHD subjects versus only 3% higher total cholesterol values in CHD subjects in the normal HDL-C group (Table 4Up). Therefore, CHD subjects in the low HDL-C group were responsible for the significantly higher total cholesterol in the combined CHD group compared with the combined control group. Similar to total cholesterol, differences in the concentrations of LDL-C and triglycerides and the ratio of LDL-C to HDL-C were greater in low HDL-C cases than in cases with normal HDL-C levels compared with the appropriate control groups. Because the ratios of LDL-C to HDL are dependent on HDL-C concentrations, this ratio gives more precise information about risk for CHD when CHD patients are compared with HDL-C–matched control individuals. In contrast, HDL-C {alpha}1 ratio did not show this discriminative difference between groups with different HDL-C levels and was significantly lower in CHD cases in the low and normal HDL-C groups.

As we discussed earlier, the major HDL constituents, apoA-I and HDL-C, did not differ strikingly between control and CHD subjects. However, a major reorganization of the apoA-I–containing HDL subpopulations was observed in CHD patients compared with control individuals (Tables 3Up and 5Up). The {alpha}1 and all of the pre-{alpha} subpopulations were significantly lower, whereas the {alpha}3 subpopulation was significantly higher in CHD subjects compared with control subjects. The differences in the concentrations of the {alpha}2 and the pre-ß mobility subpopulations were not significant between the 2 groups. It has to be noted that there were significantly higher pre-ß1 HDL subpopulations in CHD subjects than in control subjects when percent distribution was calculated. These data suggest that in CHD subjects the lipid-poor pre-ß1 maturation into larger discoidal HDL particles (probably {alpha}1) is impaired. Because {approx}85% of apoA-I is in {alpha} mobility particles, our focus was on differences in these particles. Previously, we have reported that neither the {alpha}1 nor the pre–{alpha}-mobility HDL subpopulations contain apoA-II; therefore, they are LpA-I particles.19 LpA-I particles appear to play a pivotal role in reverse cholesterol transport and in the development of CHD.23 Our finding of decreased large LpA-I particles, including {alpha}1 and pre-{alpha}1, in CHD subjects versus control subjects supports recently published observations of Decossin et al.45

We have also demonstrated that subjects with low (<=35 mg/dL) and normal (>35 mg/dL) HDL-C levels have very different distributions of apoA-I–containing HDL subpopulations. Therefore, we examined whether the differences in the HDL subpopulation profiles between control and CHD subjects are only reflections of the differences in HDL-C levels. The subgroup analyses proved that the differences in HDL subpopulation profile between the control and CHD groups are still significant after the HDL-C level is taken into account. The {alpha}1 subpopulation is decreased by 41% in the low HDL-C groups compared with the normal HDL-C groups and is further decreased by about another 30% in CHD individuals compared with HDL-C–matched control subjects (Table 5Up).

Our results indicate that despite the decrease in apoA-I and HDL-C, the extensively decreased {alpha}1 (-35%) resulted in increased ratios of apoA-I to {alpha}1 (57%) and HDL-C to {alpha}1 (36%) in CHD individuals (Table 2Up). The differences in these ratios were still significant between controls and cases after subgrouping individuals into low and normal HDL-C groups (Table 5Up). The lower {alpha}1 and higher {alpha}3 concentrations in CHD subjects were also a characteristic for the group and independent of HDL-C level (Figure 1Up). These results indicate that some patients have CHD caused by factors that do not alter the lipid and HDL subpopulation profile. However, the majority of normolipidemic CHD patients have an altered distribution of HDL subpopulations.

On the basis of our results, we may be able to explain the discrepancies among laboratories on the possible role of HDL subclasses in the development of CHD. By our 2D analysis, the majority of HDL2 is in the {alpha}1 and pre-{alpha}1 positions (Figure 2Up). Earlier, we demonstrated that {alpha}1 and pre-{alpha}1 consist of LpA-I particles. Data generated by ultracentrifugation, immunoaffinity chromatography, or nondenaturing gel electrophoresis indicate lower levels of these particles in CHD individuals compared with control subjects.21 22 23 31 These findings are understandable, inasmuch as {alpha}1 is a homogeneous subpopulation and shows a very strong positive correlation with HDL-C and apoA-I concentrations, which are usually lower in CHD subjects. However, the picture is more complicated for HDL3, because it consists of many different HDL subspecies (mainly {alpha}2 and {alpha}3) of varying sizes and apolipoprotein composition (Figure 2Up). The concentration of these particles is differentially altered ({alpha}2 decreases, whereas {alpha}3 increases) in CHD individuals compared with control subjects.

Data generated in the present study on normolipidemic subjects support our hypothesis that the apoA-I–containing HDL subpopulation profile, obtained by 2D nondenaturing gel electrophoresis, is significantly different between control and CHD individuals. These differences were not influenced significantly by HDL-C or apoA-I concentrations. Our research findings support the concept that compared with control subjects, CHD patients not only have HDL deficiency but also have a major rearrangement in the HDL subpopulations with significantly lower {alpha}1 and pre-{alpha}1–3 (LpA-I) and significantly higher {alpha}3 (LpA-I/A-II) particles.


*    Acknowledgments
 
This research was supported by a grant from Parke Davis, Morris Plain, NJ, and by the National Institutes of Health/National Heart, Lung, and Blood Institute (HL-56160). The authors wish to thank Dana Bass for her excellent help in collecting medical histories on CHD patients.

Received April 26, 2000; accepted September 21, 2000.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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