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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1885-1893

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1885-1893.)
© 1997 American Heart Association, Inc.

Articles

Normolipidemic Subjects With Low HDL Cholesterol Levels Have Altered HDL Subpopulations

Bela F. Asztalos; Michael Lefevre; Theda A. Foster; Richard Tulley; Marlene Windhauser; Laurence Wong; ; Paul S. Roheim

From the Division of Lipoprotein Metabolism and Pathophysiology, Department of Physiology, Louisiana State University Medical Center, New Orleans (B.F.A., T.A.F., L.W., P.S.R.), and the Division of Diet and Heart Disease, Pennington Biomedical Research Center, Baton Rouge (M.L., R.T., M.W.), La.

Correspondence to Paul S. Roheim, MD, Louisiana State University Medical Center, Department of Physiology, 1542 Tulane Ave, New Orleans, LA 70112. E-mail dbarra{at}nomvs.lsumc.edu


*    Abstract
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 *Abstract
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Abstract Epidemiological studies have established that plasma concentration of HDL is inversely correlated with the risk of coronary heart disease, even in the absence of increased LDL cholesterol levels. We postulate that specific HDL subpopulations may be responsible for antiatherogenic properties of HDL. HDL subpopulations were quantitated by two-dimensional gel electrophoresis in 79 normolipidemic healthy male subjects. To eliminate the influence of diet, volunteers consumed an average American diet for 6 weeks. After the diet period, subjects were stratified according to their HDL cholesterol (HDL-C) levels to low HDL-C <0.91 mmol/L (<35 mg/dL), medium >0.91<1.30 mmol/L (>35 <50 mg/dL), and high >=1.30 mmol/L (>=50 mg/dL) groups. Plasma triglycerides and insulin levels were in the normal range, but subjects with low HDL-C levels had higher concentrations of plasma triglycerides and insulin than subjects with medium or high HDL-C concentrations. The absolute concentration (mg/dL) of apoA-I in the largest {alpha}-migrating HDL subpopulation ({alpha}1) was (P<.01) lower in the low HDL-C subjects compared with the medium and high HDL-C groups. The relative concentration (percent distribution) of apoA-I was decreased (P<.01) in {alpha}1 and increased (P<.01) in {alpha}3 subpopulations. A positive correlation between HDL-C and {alpha}1 (P<.001) and a negative correlation between HDL-C and {alpha}3 were observed. The inverse correlation of apoA-I distribution (relative concentration) between {alpha}1 and {alpha}3 suggests an interconversion of {alpha}1 and {alpha}3 subpopulations, possibly by cholesteryl ester transfer protein. Pre-ß subpopulations showed an inverse trend with HDL-C, while the pre-{alpha} subpopulation behaved similarly to the {alpha}-migrating subpopulation. Colocalization of apoA-I and apoA-II particles in the different HDL subpopulations demonstrated that {alpha}1, pre-ß1, and pre-ß2 subpopulations are apoA-I–only particles rather than apoA-I:A-II particles.


Key Words: apoA-I–containing HDL subpopulations • LpA-I • LpA-I:A-II particles • cholesterol • diet


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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The inverse relationship between HDL and risk of coronary heart disease (CHD) has been established for several decades.1 Low plasma levels of HDL are associated with increased risk of CHD, even in the absence of elevated LDL concentrations.2 3 4 5 Plasma concentration of HDL is influenced by several factors, including gender, race, diet, and lifestyle.6 7 8

HDLs are heterogeneous, containing several subpopulations differing in size,9 hydrated density, lipid and apoprotein composition, and functional properties.10 11 Subclasses of HDL can be separated by ultracentrifugation,10 11 gel filtration chromatography,12 polyanion precipitation,13 one-9 and two-dimensional14 15 16 polyacrylamide gel electrophoresis, or by immunoaffinity chromatography.17 18

Although most data have been obtained through subfractionation of HDL subpopulations by ultracentrifugation, this procedure produces artifacts resulting in loss of pre-ß1 and pre-ß2 particles and an overall change in the distribution of {alpha} and pre-{alpha} particles.15 Additional artifacts can be produced by in vitro remodeling of HDL subpopulations by factors present in plasma,11 requiring appropriate methods for sample collection and preservation. Association of HDL subpopulations with CHD has been widely investigated, but conclusions are equivocal.18 19 20 21 22 Separation of HDL subpopulations based on apoprotein composition17 18 22 suggests that a decrease of apoA-I–only particles is associated with an increase in the risk of CHD. Using a 2DE system, it is possible to separate and quantify HDL subpopulations reproducibly and precisely.15 These well-defined apoA-I–containing subpopulations differ in charge (pre-ß, {alpha}, and pre-{alpha}) and size.

It has been postulated that HDL subpopulations may directly influence the atherogenic process.23 Using immunoaffinity chromatography and nondenaturing polyacrylamide gel electrophoresis, Cheung et al24 reported significant differences in the size of HDL in patients with CHD compared with healthy subjects. They found that the presence of CHD was more strongly associated with HDL particle size distribution than with low HDL-C levels.

Considerable variation is reported in HDL-C levels among normolipidemic subjects,25 but no information is available on the distribution of HDL subpopulations at different HDL-C levels in normolipidemic subjects. The scarcity of information is more evident in groups whose dietary intake is controlled. In this study using 2DE, we compared the distribution of apoA-I–containing HDL subpopulations in normolipidemic male subjects consuming an average American diet for 6 weeks with low (<0.91 mmol/L, <35 mg/dL), medium (>0.91<1.30 mmol/L, >35 <50 mg/dL), and high (>=1.30 mmol/L, >=50 mg/dL) HDL-C concentrations.


*    Methods
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 *Methods
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Subjects
Seventy-nine male subjects (ages 22 to 65 years) were recruited to participate in a study examining the relationship between diet and risk factors for CHD. Participants were selected to have total plasma cholesterol and LDL cholesterol between the 10th and 90th percentile. HDL-C above 0.65 mmol/L (25 mg/dL) or below the 95th percentile and triglycerides below the 95th percentile were adjusted for age and race as determined by the NHANES II data.26 The lipid cutoff points were selected to eliminate subjects with lipid disorders.

Exclusion criteria included the presence of (1) renal, hepatic, cardiovascular, endocrine, gastrointestinal, or other systemic disease; (2) body mass index greater than 32; (3) hypertension; (4) history of drug or alcohol abuse; and (5) data obtained from medical questionnaires, psychological evaluations, blood chemistry and lipid profiles, and urinalyses and physical examination during screening visits.

Diet
Subjects consumed an AAD designed to provide 38% calories as fat (Table 1Down).


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  		Table 1. Target Values for AAD1

Study Design
A 6-week dietary period was chosen to enable stabilization of the lipoprotein end points.27 Participants were provided with all foods during this time. On weekdays, the study protocol required subjects to consume breakfast and dinner at the Pennington Biomedical Research Center dining facility. Weekday packaged lunches were distributed at breakfast, and evening snacks were distributed at dinner. Weekend meals were packaged and distributed at the Friday dinner. Subjects were allowed the option of a "free choice meal" for Saturday dinner; approximately 25% of Saturday dinners were "free choice." Since the Pennington Biomedical Research Center provided 95% of caloric intake, we assumed that this free meal would not substantially influence variables measured in this study. Furthermore, performing measurements at the end of the sixth week, when dietary influences were stabilized, minimized the potential short-term effects of this "free choice meal." Participants were allowed a limited choice of items such as alcoholic drinks and sugar-free beverages. Subjects recorded symptoms of illness, medication use, selected food items, and any deviations in food diaries. Dietary energy adjustments were made as needed to maintain weight. Meals were prepared at four energy levels (2200, 2600, 3000, and 3400 kcal/d). One hundred kcal unit foods that matched the composition of the diet were used for energy adjustments. Subjects started on the energy level most closely matching their estimated energy requirement. Body weight (without shoes, jackets, or heavy sweaters) was measured twice weekly. If a subject's weight differed from the initial level by more than 1 kg, the subject was switched to another energy level or the number of unit foods was changed until the weight returned to within 1 kg of the initial value.

Processing of Blood
At the end of the 6-week dietary period, fasting blood samples for lipid and apolipoprotein analysis were collected in tubes containing 1.2 g/L EDTA and placed on ice immediately after collection. Plasma was isolated by centrifugation at 30 000g min. Multiples (0.5 to 1.0 mL) of each sample were stored in cryovials at -80°C until samples were analyzed for lipids and apoproteins.

For 2DE, blood samples were collected and placed on ice with the following additives in final concentrations: 1.2 g/L EDTA, 0.1 g/L sodium azide, 80 mg/L gentamicin sulfate, and 10 KU/L kallikrein inactivator (aprotinin). Plasma was separated immediately by centrifugation at 5°C. Additional preservatives were added to the plasma in 1 mmol/L final concentration: PMSF in DMSO, benzamidine in DMSO, and N'-ethylmaleimide (dissolved just before use because it is stable in water only for about 1 hour) in water.

After separation, plasma was frozen and stored in liquid nitrogen until samples were processed by 2DE. Four microliters of plasma was applied and run on agarose in the first dimension followed by electrophoresis into a 3% to 35% nondenaturing concave gradient polyacrylamide gel at 280 V for 24 hours at 10°C. In this 2DE system, the agarose electrophoresis differs from the one generally used in that the agarose contains no albumin. This modification provides a clearer separation of {alpha} and pre-{alpha} particles. Electrophoretic transfer, fixing, blocking, and immunolocalization were performed as described previously.15 Bound radioactivity was quantified by PhoshorImager analysis (Molecular Dynamics).28 Monospecific polyclonal anti–apoA-I and –apoA-II antibodies used for these studies were produced in goats in our laboratory as previously described.29 For secondary antibody, F(ab')2 fragments of anti-goat gamma globulins were used (Zymed). Subpopulations were characterized by: (1) charge (pre-ß, {alpha}, and pre-{alpha}) based on their relative mobility with respect to albumin and (2) size determined from molecular weight standards run simultaneously in the same gel. With this system, we were able to separate and quantify apoA-I–containing subpopulations. Interassay and intra-assay coefficients of variation for different subpopulations varied between 3% ({alpha} subpopulations) and 15% (pre-ß subpopulations). Criteria for designation of HDL subfractions of {alpha}1, {alpha}2, and {alpha}315 are based on integration of {alpha}-migrating HDL in the two-dimensional system. Three distinct peaks are observed, providing a basis for the designation of {alpha}1, {alpha}2, {alpha}3 (Fig 1Down). Quantification was calculated based on valley-to-valley area under the scanned curves.



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  		Figure 1. Typical two-dimensional separation of apoA-I–containing subpopulations for subjects with different HDL cholesterol levels: low (<0.91 mmol/L, <35 mg/dL; a); medium (>0.91<1.30 mmol/L, >35<50 mg/dL; b); and high (>=1.30 mmol/L, >=50 mg/dL; c) HDL-C. Plasma was electrophoresed in the first dimension in agarose followed by application of the agarose strip to the top of a nondenaturing 3% to 35% polyacrylamide gel and subsequently electrophoresed. On the left side of each gel, a Pharmacia high-molecular-weight standard was applied. Asterisks indicate the position of human serum albumin. The horizontal inset on top represents apoA-I distribution on a duplicate agarose strip. Curves under the pictures represent a scan of the {alpha}-migrating region on 2DE.15

Chemical Methods
Serum total cholesterol, HDL-C, and triglycerides were determined using enzymatic assays on a Beckman Synchron CX5 automated chemistry analyzer. HDL-C was determined after precipitation of the non-HDL fractions by dextran sulfate (50 000 molecular weight; DMA) following the protocol of Warnick et al.30 Assayed controls provided by DMA were used to verify accuracy. VLDL was calculated using the Friedewald equation. The interassay coefficient of variation was less than 2.0%.

ApoA-I and apoB were measured on a Beckman Array analyzer employing reagents supplied by the manufacturer. The interassay coefficients of variation for assays were less than 7%.

Statistical Analysis
Initially, descriptive statistics, scatterplots, and Pearson correlation coefficients summarized data. For analysis, nonnormally distributed data were transformed using the natural logarithm to approach a Gaussian distribution. ANOVA tested the hypothesis of no difference in mean levels of given variables among three HDL-C categories. Post hoc (a posteriori) comparisons, appropriate only when ANOVA is statistically significant,31 32 33 evaluated all pairwise differences among three HDL-C subgroups by Duncan's multiple range test.


*    Results
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 up arrowAbstract
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 up arrowMethods
 *Results
down arrowDiscussion
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The objective of this study was to compare the distribution of HDL subpopulations in low, medium, and high HDL-C subjects consuming the AAD for 6 weeks. Subjects with <0.91 mmol/L (<35 mg/dL) HDL-C were designated as low HDL-C, while subjects between >0.91<1.30 mmol/L (>35 <50 mg/dL) and >=1.30 mmol/L (>=50 mg/dL) HDL-C were defined as medium and high HDL-C individuals, respectively.34

Table 2Down presents screening values for lipids, insulin, and selected anthropometric variables by the three HDL-C categories and all categories combined. Plasma total and LDL cholesterol concentrations were similar in all three groups, whereas triglycerides and insulin levels were the highest in the low HDL-C group. Table 3Down presents lipid values obtained after consuming the AAD for 6 weeks. In general, the overall values were very similar to the values obtained at screening.


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  		Table 2. Screening Values


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  		Table 3. Lipid Values on AAD

After 6 weeks on the AAD, lipid values were stabilized27 so that the distribution of HDL subpopulations would be more representative for each HDL-C group. On the AAD, the average HDL-C values were 0.82, 1.09, and 1.46 mmol/L (31.6, 42.3, and 56.5 mg/dL) in the low, medium, and high HDL-C groups, respectively (Table 3Up). No significant differences in total cholesterol or LDL cholesterol were noted among the three groups, but triglyceride and VLDL cholesterol concentrations were higher (33% and 72%) in the low HDL-C group than the medium and high HDL-C groups, respectively. It should be noted that in this sample of normolipidemic subjects, triglyceride concentration was inversely proportional to HDL-C concentration. ApoA-I concentrations paralleled those of HDL-C. Low, medium, and high HDL-C groups had mean apoA-I concentrations of 108.4, 124.7, and 138.4 mg/dL, respectively, and were significantly different from one another. ApoB concentration was the lowest in the high HDL-C, high apoA-I individuals. The low HDL-C group had a significantly lower HDL-C/apoA-I ratio compared with the medium and high HDL-C groups, suggesting a decrease in cholesterol-rich HDL particles.

Using 2DE, the apoA-I–containing HDL subpopulations were compared in these three HDL-C groups. Fig 1Up shows a typical distribution of HDL subpopulations for subjects with low (A), medium (B), and high (C) HDL-C. It is apparent that low HDL-C subjects had a considerable decrease in the largest HDL subpopulation ({alpha}1).

In this study, we distinguished between absolute concentrations of apoA-I (mg/dL apoA-I in the subpopulations) and relative concentrations (percent distribution of apoA-I among the subpopulations). The absolute apoA-I concentration in a subpopulation is a function of plasma apoA-I concentration and its percent distribution among the subpopulations.

ApoA-I concentrations in the different HDL subpopulations were compared in the three HDL-C groups (Fig 2ADown). Absolute concentrations of apoA-I in the {alpha}1 subpopulation was the lowest in the low HDL-C group and highest in the high HDL-C group. The concentration of apoA-I in {alpha}2 behaved similarly to {alpha}1, but the differences were smaller between the low and the high HDL-C groups. Absolute concentrations of apoA-I in {alpha}3 was the lowest in the high HDL-C group. Absolute concentrations of pre-{alpha} subpopulations followed the patterns of {alpha} subpopulations.



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  		Figure 2. Distribution of apoA-I–containing HDL subpopulations in subjects with low (<0.91 mmol/L, <35 mg/dL), medium (>0.91<1.30 mmol/L, >35<50 mg/dL), and high (>=1.30 mmol/L, >=50 mg/dL) HDL cholesterol concentrations. a, Absolute concentration (mg/dL) of apoA-I. b, Relative concentration (apoA-I percent distribution) of the subpopulations. All values represent mean±SE; means with different letters are statistically different (a, b, c: P<.05; d, e, f: P<.01).*Data were ln transformed for analysis.

When relative concentrations of apoA-I in HDL subpopulations were compared (Fig 2BUp), {alpha}1 had the lowest apoA-I concentrations in the low HDL-C group and the highest in the high HDL-C group. In contrast to our observations indicating a difference in absolute concentration of {alpha}2 among HDL-C groups (P<.05), relative concentrations of apoA-I (percent distribution) did not differ among HDL-C categories. Relative concentration of apoA-I in {alpha}3 was the highest in the low HDL-C group and the lowest in the high HDL-C group (P<.01) in contrast to absolute apoA-I concentrations where no difference was found among HDL-C groups (P>.05). Pre-{alpha} subpopulations in general followed the patterns of the {alpha} subpopulations. Percent distribution of pre-ß2a and pre-ß2c in the low HDL-C group exceeded that in the high HDL-C group. The pre-ß2b was the highest in the low HDL-C group compared with medium and high HDL-C categories. Although no significant differences were noted among HDL-C groups in the percent distribution of pre-ß1 (P=.0792, F=2.62, ANOVA), a lower mean level was observed in the high HDL-C category.

To better evaluate the interrelationship among the three major {alpha}-migrating HDL subpopulations, HDL-C concentrations were correlated with both the absolute and relative concentrations of apoA-I in {alpha}1, {alpha}2, and {alpha}3 subpopulations (Fig 3Down). It is apparent that HDL-C is highly correlated with apoA-I concentrations in the {alpha}1 subpopulation in both absolute (Fig 3aDown) and relative concentrations (Fig 3bDown). In the {alpha}2 subpopulation, HDL-C correlated well with the absolute concentrations (Fig 3cDown) but was not statistically different from zero with respect to relative concentrations (Fig 3dDown) of apoA-I. In the {alpha}3 subpopulation, HDL-C did not correlate (P>.05) with absolute apoA-I concentration (Fig 3eDown) but showed a significant negative correlation with the relative concentration of apoA-I (Fig 3fDown).



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  		Figure 3. Correlation of {alpha}-migrating subpopulations of all subjects with HDL cholesterol concentrations: absolute and relative concentration of {alpha}1 (a and b); absolute and relative concentration of {alpha}2 (c and d); absolute and relative concentration of {alpha}3 (e and f).

Since it appears that changes in relative distributions of apoA-I can be explained by the interconversion of {alpha}1 and {alpha}3, we determined the correlation among the relative concentrations of apoA-I in {alpha}1, {alpha}2, and {alpha}3 (Fig 4Down). A negative correlation was observed in the relative concentrations (Fig 4aDown) of {alpha}1 and {alpha}3, which suggests that in vivo redistribution of apoA-I may be responsible for decreased apoA-I levels in {alpha}1 and increased levels in {alpha}3 subpopulations. While the correlation between {alpha}1 and {alpha}3 is very strong, those between {alpha}1 and {alpha}2 and between {alpha}2 and {alpha}3 are not different from zero (P>.05; Fig 4bDown and 4cDown).



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  		Figure 4. Correlation of relative concentrations (percent distribution) of {alpha}-migrating apoA-I–containing subpopulations of all subjects. a, Correlation between {alpha}1 and {alpha}3. b, Correlation between {alpha}1 and {alpha}2. c, Correlation between {alpha}2 and {alpha}3.

Correlations were also calculated between pre-ß1, pre-ß2, and {alpha}1. In both cases, the r value was less than 0.26 and there was no statistical significance. It should be noted that the absolute as well as relative concentrations of {alpha}1 apoA-I were lower in the low HDL-C group than in the medium and high HDL-C groups (Fig 2aUp and 2bUp).

Low HDL-C subjects have increased risk of CHD and a decrease in LpA-I–only concentrations.18 35 A major difference in the {alpha}1 subpopulations was found among the low HDL-C and the medium and high HDL-C subjects; therefore, it was important to establish the nature of this particle and determine whether the {alpha}1 is an LpA-I–only or an LpA-I:A-II particle. The distribution of apoA-I and apoA-II in the HDL subpopulations were examined in two different ways. After 2DE, apoA-I and apoA-II were immunolocalized separately on individual membranes and superimposed to evaluate colocalization. It was found that the {alpha}1 particle did not contain apoA-II; thus, it is an apoA-I–only particle. Similar results were obtained when the membranes were immunolocalized first for apoA-II followed by immunolocalization of apoA-I on the same membrane (Fig 5Down). It was also shown that both pre-ß1 and pre-ß2 subpopulations are apoA-I–only particles and do not contain apoA-II. ApoA-I and apoA-II colocalized in the {alpha}2 and {alpha}3 subpopulations. These results should not be considered to imply that {alpha}2 and {alpha}3 subpopulations are only apoA-I:apoA-II particles. It is likely that apoA-I–only particles may also be present in the same area.



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  		Figure 5. Colocalization of apoA-I and apoA-II–containing subpopulations. This colocalization is from an individual with medium HDL-C (45 mg/dL). a, Two-dimensional separation of apoA-II–containing subpopulations. b, Two-dimensional separation of apoA-I–containing subpopulations. c, Colocalization of apoA-II subpopulations with apoA-I by superimposing the apoA-I distribution on the distribution of apoA-II subpopulations. Asterisks 1, 2, and 3 are reference points used for colocalization of superimposed membranes: 1, macroglobulin; 2, BSA; 3 endogenous serum albumin. The different subpopulations are indicated in b. Subjects irrespective of HDL-C levels provided similar results.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
We compared the lipid values obtained at screening with values 6 weeks after the consumption of the AAD. The values at screening and after 6 weeks on the AAD showed no difference. These data suggest that the diet of subjects before the study was similar to the AAD consumed during the study. However, it should be noted that 23% of the subjects shifted within HDL-C categories; considering the categories at screening, six subjects moved from low to medium and seven subjects from medium to low HDL-C groups. From the high HDL-C categories, three subjects shifted to medium, while one subject moved to the high HDL-C group. Despite these changes of subjects among HDL-C categories, the lipid compositions were similar at screening and after AAD, suggesting characteristic lipid composition for each category.

We also compared the screening insulin and triglyceride levels in subjects with low, medium, and high HDL-C (Table 2Up). Insulin and TG levels segregated on the basis of HDL-C concentration (ie, low HDL-C subjects had higher insulin and plasma triglyceride concentrations). However, according to the screening criteria for this study, subjects were normolipidemic with triglyceride concentrations within normal limits.25 These data suggest possible insulin resistance among low HDL-C individuals.

Considerable variation is found in HDL-C levels in normolipidemic subjects.25 Our experimental design assumed that controlled consumption of the AAD for 6 weeks stabilized plasma lipid values and decreased or eliminated variations of diet-induced changes.27 36 There is no information available on the distribution of HDL subpopulation of healthy normolipidemic subjects consuming a standard well-controlled diet for a defined period of time. The objective of this study was to compare HDL subpopulations in normolipidemic individuals with different HDL-C levels. Table 3Up shows the comparison of lipid values in the different HDL-C groups at the end of 6 weeks on an AAD. An increase (33% and 72%) in plasma triglycerides was found in the low HDL-C subjects compared with the medium and high HDL-C groups, respectively.

Two-dimensional electrophoresis was used for the quantification of apoA-I–containing HDL subpopulations. This method established that HDL subpopulations can be separated accurately and reproducibly.15 In this study, special attention was paid to prevent in vitro remodeling of HDL subpopulations by using the appropriate preservation at the time of blood sampling.

When apoA-I levels in HDL subpopulations were compared among different HDL-C groups, we observed that the {alpha}1 subpopulation was the lowest in the low HDL-C group, in both absolute and relative concentrations of apoA-I. These findings are also supported by the decreased HDL-C/apoA-I ratios (Table 3Up) in low HDL-C groups, suggesting a decrease in cholesterol-rich HDL particles. The {alpha}2 subpopulation was significantly different in HDL-C groups in absolute concentrations but not in the relative concentrations (percent distributions of apoA-I). In contrast to {alpha}2 subpopulations, there was no significant difference in the absolute concentrations of apoA-I in {alpha}3, but the relative concentrations (percent distributions) of apoA-I were significantly lower in the medium and high HDL-C groups.

Mechanisms for these differences may be partially explained by differences in CETP activities. Increased CETP activity has been demonstrated in subjects with low HDL-C concentration.37 We did not measure CETP mass or CETP activity; however, we should consider that low HDL-C subjects had increased TG concentrations, which favors enhanced cholesterol ester transfer despite similar CETP mass.38 Increased CETP activity may be responsible for the decreased relative concentration of apoA-I in {alpha}1 subpopulations and the increase in relative concentrations of {alpha}3. When CETP in conjunction with hepatic lipase activity transfers cholesterol ester to LDL and VLDL from large HDL particles, a decrease in the large ({alpha}1) and an increase in the small ({alpha}3) HDL particles takes place.39 40 If increased CETP activity results in a redistribution of apoA-I from {alpha}1 to {alpha}3, then we should be able to demonstrate a correlation between the relative concentration of {alpha}1 and {alpha}3 (Fig 4aUp) but no correlation with {alpha}2 and {alpha}3 (Fig 4bUp and 4cUp). Indeed, we observed a strong negative correlation between the relative concentrations (percent distributions) of {alpha}1 and {alpha}3 (Fig 4aUp) but no correlation (P>.05) in relative concentrations between either {alpha}1 and {alpha}2 or {alpha}2 and {alpha}3 subpopulations (Fig 4bUp and 4cUp). Increased CETP activity is consistent with the observation of an increased fractional catabolic rate of apoA-I41 42 and may explain decreased HDL-C concentrations. It is possible that in addition to increased CETP activity, a decrease in phospholipid transfer protein activity may also be responsible for the changes in HDL subpopulations. It has been shown that phospholipid transfer protein activity favors the formation of larger-sized HDL particles.43 The differences in subpopulation mass may not necessarily be the result of interconversion, but rather there may be differences in the metabolism of subpopulations in the three HDL-C populations.

Low levels of HDL-C concentrations have been attributed to changes in lipoprotein lipase and hepatic triglyceride lipase activities; decreased lipoprotein lipase/ hepatic triglyceride lipase ratio was observed in low HDL-C subjects.37 41 This decreased ratio will result in an increased fractional catabolic rate of an apoA-I–only particle.41 This is consistent with our finding that the {alpha}1, an apoA-I–only particle, is decreased in low HDL-C subjects.44

HDL can be separated into LpA-I–only and LpA-I:A-II particles.18 22 35 Low HDL-C subjects have low levels of LpA-I–only lipoproteins. It has been shown that the incidence of CHD is associated with a decrease of LpA-I–only lipoproteins.22 Therefore, it was important to determine whether the {alpha}1 subpopulation is an LpA-I–only particle. We colocalized apoA-I and apoA-II (Fig 5Up) and found that apoA-II did not colocalize with {alpha}1; thus, {alpha}1 HDL subpopulation is an LpA-I–only particle. These data are similar to data reported previously.14 We have also shown that both pre-ß1 and pre-ß2 subpopulations are LpA-I–only particles, in agreement with Hennessy et al.45 Colocalization of apoA-I and apoA-II was observed in {alpha}2 and {alpha}3 subpopulations. Lack of colocalization can be proof of the existence of a separate apoA-I–only or an apoA-II–only particle, but colocalization does not necessarily mean that apoA-I and apoA-II are on the same particle.

These studies were conducted on normolipidemic male subjects. Since HDL-C levels in females are considerably higher,46 it would be important to establish what HDL-C level would result in an alteration of HDL-C subpopulations in females that is characteristic of low HDL-C subjects in males. Postmenopausal women have an increased incidence of CHD47 48 ; therefore, it also would be important to follow changes in HDL subpopulations in this segment of the population.

It has been shown that cholesterol efflux correlates well with HDL-C concentrations.49 It is possible that altered HDL subpopulations observed in low HDL-C subjects influence susceptibility to CHD by decreased reverse cholesterol transport. We hypothesize that a decreased {alpha}1 subpopulation may be partially responsible or is a marker for susceptibility to development of CHD. The finding that {alpha}1 is an apoA-I–only particle and that it is decreased in the low HDL-C group is consistent with the observation that decreased LpA-I is associated with CHD.22

The Framingham studies have demonstrated that CHD preferentially occurs in subjects with low HDL-C.3 We do not know whether changes in HDL subpopulations in the low HDL-C group are causally related to increased susceptibility to atherosclerosis or whether it is an epiphenomenon. It is also possible that the change in HDL subpopulations is an indicator that the subjects are prone to CHD. In the future, it will be important to establish the association of HDL-C and changes in HDL subpopulations in the development of CHD.


*    Selected Abbreviations and Acronyms
 
AAD = average American diet
apo = apolipoprotein
CETP = cholesteryl ester transfer protein
CHD = coronary heart disease
2DE = two-dimensional nondenaturing polyacrylamide gel electrophoresis
HDL-C = HDL cholesterol


*    Acknowledgments
 
This work was supported by a grant from the Dairy Management, Inc. and the National Institutes of Health/National Heart, Lung, and Blood Institute (HL-56160 and HL-25596). The authors wish to thank Dr Howard Eder for his suggestions, Katalin Horvath and Colleen Tierney for their excellent technical help, and Debbie Plaisance and Marci Crosby for their editorial assistance and manuscript preparation.

Received May 5, 1997; accepted August 4, 1997.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
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 *References
 

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