Bela F. Asztalos

High-Density Lipoprotein Subpopulation Profile and Coronary Heart Disease Prevalence in Male Participants of the Framingham Offspring Study

Objective—High-density lipoprotein (HDL) is a heterogeneous lipoprotein class and there is no consensus on the value of HDL subspecies in coronary heart disease (CHD) risk assessment. We tested the hypothesis whether specific HDL subpopulations are significantly associated with CHD-prevalence. Methods and Results—ApoA-I concentrations (mg/dL) in HDL subpopulations were quantitatively determined by native 2d gel electrophoresis, immunoblotting, and image analysis in male participants in the Framingham Offspring Study (FOS). CHD cases (n=169) had higher pre&bgr;-1 and &agr;-3 particle and lower &agr;-1, pre&agr;-3, and pre&agr;-1 particle levels than either all (n=1277) or HDL cholesterol-matched (n=358) controls. &agr;-1 and pre&agr;-3 levels had an inverse association, whereas &agr;-3 and pre&agr;-1 particle levels had a positive association with CHD prevalence after adjusting the data for established CHD risk factors. Standardized logit coefficients indicated that &agr;-1 HDL was most significantly associated with CHD prevalence. Moreover, each mg/dL increase in &agr;-1 particle level decreased odds of CHD by 26% (P<0.0001), whereas each mg/dL increase in HDL cholesterol decreased odds of CHD by 2% in a model including all established CHD risk factors. Conclusions—Specific HDL subpopulations were positively correlated, whereas others were inversely correlated with CHD prevalence in male subject in the FOS, indicating that the various HDL particles might have different roles in the cause of CHD.

The Ability to Promote Efflux Via ABCA1 Determines the Capacity of Serum Specimens With Similar High-Density Lipoprotein Cholesterol to Remove Cholesterol From Macrophages

Objective—We measured efflux from macrophages to apolipoprotein B-depleted serum from 263 specimens and found instances in which serum having similar high-density lipoprotein cholesterol (HDL-C) differed in their efflux capacity. Thus, we wanted to elucidate why efflux capacity could be independent of total HDL-C or apolipoprotein A-I (apoA-I). Methods and Results—To understand why sera with similar HDL-C or apoA-I could differ in total efflux capacity, we assessed their ability to promote efflux via the pathways expressed in cAMP-treated J774 macrophages. Briefly, macrophages were preincubated with probucol to block ABCA1, with BLT-1 to block SR-BI, and with both inhibitors to measure residual efflux. ABCG1 efflux was measured with transfected BHK-1 cells. We used apolipoprotein B-depleted serum from specimens with similar HDL-C values at the 25th and 75th percentiles. Specimens in each group were classified as having high or low efflux based on total efflux being above or below the group average. We found that independently of HDL-C, sera with higher efflux capacity had a significant increase in ABCA1-mediated efflux, which was significantly correlated to the concentration of pre&bgr;-1 HDL. The same result was obtained when these sera were similarly analyzed based on similar apoA-I. Conclusion—Sera with similar HDL-C or apoA-I differ in their ability to promote macrophage efflux because of differences in the concentration of pre&bgr;-1 HDL.

Value of High-Density Lipoprotein (HDL) Subpopulations in Predicting Recurrent Cardiovascular Events in the Veterans Affairs HDL Intervention Trial

Objective—To test the hypothesis whether determination of high-density lipoprotein (HDL) subpopulations provides more power to predict recurrent cardiovascular disease (CVD) events (nonfatal myocardial infarction, coronary heart disease death, and stroke) than traditional risk factors in the Veterans Affairs HDL Intervention Trial (VA-HIT). Methods and Results—Apolipoprotein A-I (apoA-I)–containing HDL subpopulations were quantitatively determined by nondenaturing 2D gel electrophoresis. Hazard ratios of recurrent CVD events were calculated by comparing VA-HIT subjects with (n=398) and without (n=1097) such events. Subjects with new CVD events had significantly lower HDL-C, apoA-I, and large cholesterol-rich HDL particle (α-1, α-2, pre–α-1, and pre–α-2) levels, significantly higher triglyceride, and small poorly lipidated HDL particle (pre–β-1 and α-3) levels than subjects without such events. Multivariate analyses indicated that α-1 and α-2 particle levels were significant negative risk factors, whereas α-3 level was a significant positive risk factor for new CVD events. Pre–β-1 level was a significant risk factor for new CVD events only in univariate analysis. A forward selection model indicated that α-1 was the most significant risk factor for recurrent CVD events among HDL particles. Conclusions—An altered HDL subpopulation profile marked with low α-1 and α-2 levels and a high α-3 level in coronary heart disease patients indicated an elevated risk for new CVD events. Moreover, α-1 and α-2 levels were superior to HDL-C levels in risk assessment in patients with low HDL-C in VA-HIT.

Effects of Cholesteryl Ester Transfer Protein Inhibition on High-Density Lipoprotein Subspecies, Apolipoprotein A-I Metabolism, and Fecal Sterol Excretion

Objective—Pharmacological inhibition of the cholesteryl ester transfer protein (CETP) in humans increases high-density lipoprotein (HDL) cholesterol (HDL-C) levels; however, its effects on apolipoprotein A-I (apoA-I) containing HDL subspecies, apoA-I turnover, and markers of reverse cholesterol transport are unknown. The present study was designed to address these issues. Methods and Results—Nineteen subjects, 9 of whom were taking 20 mg of atorvastatin for hypercholesterolemia, received placebo for 4 weeks, followed by the CETP inhibitor torcetrapib (120 mg QD) for 4 weeks. In 6 subjects from the nonatorvastatin cohort, the everyday regimen was followed by a 4-week period of torcetrapib (120 mg BID). At the end of each phase, subjects underwent a primed-constant infusion of (5,5,5-2H3)-l-leucine to determine the kinetics of HDL apoA-I. The lipid data in this study have been reported previously. Relative to placebo, 120 mg daily torcetrapib increased the amount of apoA-I in &agr;1-migrating HDL in the atorvastatin (136%; P<0.001) and nonatorvastatin (153%; P<0.01) cohorts, whereas an increase of 382% (P<0.01) was observed in the 120 mg twice daily group. HDL apoA-I pool size increased by 8±15% in the atorvastatin cohort (P=0.16) and by 16±7% (P<0.0001) and 34±8% (P<0.0001) in the nonatorvastatin 120 mg QD and BID cohorts, respectively. These changes were attributable to reductions in HDL apoA-I fractional catabolic rate (FCR), with torcetrapib reducing HDL apoA-I FCR by 7% (P=0.10) in the atorvastatin cohort, by 8% (P<0.001) in the nonatorvastatin 120 mg QD cohort, and by 21% (P<0.01) in the nonatorvastatin 120 mg BID cohort. Torcetrapib did not affect HDL apoA-I production rate. In addition, torcetrapib did not significantly change serum markers of cholesterol or bile acid synthesis or fecal sterol excretion. Conclusions—These data indicate that partial inhibition of CETP via torcetrapib in patients with low HDL-C: (1) normalizes apoA-I levels within &agr;1-migrating HDL, (2) increases plasma concentrations of HDL apoA-I by delaying apoA-I catabolism, and (3) does not significantly influence fecal sterol excretion.

Two-dimensional electrophoresis of plasma lipoproteins: Recognition of new apo A-I-containing subpopulations

Two-dimensional electrophoresis has been used to resolve 12 distinct apo A-I-containing high-density lipoprotein (HDL) subpopulations in human plasma. The subpopulations were quantitated by 125I-labeled, monospecific antibody and phosphor-imaging. Modification and standardization of the agarose electrophoresis (first dimension) enabled us to recognize new HDL subpopulations. Lipoprotein mobilities in agarose were expressed relative to the mobility of the samples endogenous albumin. We demonstrated the presence of lipoproteins with mobilities faster than and similar to albumin, as well as subpopulations with mobilities slower than albumin. We refer to these as pre alpha, alpha and pre beta, respectively. Lipoprotein molecular sizes were determined with a non-denaturing polyacrylamide gradient gel electrophoresis (PAGE) (2% to 36%) in the second dimension. Internal standard of 125I-labeled proteins of known molecular size was run simultaneously in each gel permitting accurate size determination. We have demonstrated that ultracentrifugally-isolated lipoproteins are different from the native apo A-I-containing subpopulations. The major difference observed was the loss of pre beta 1 and pre beta 2 particles from the d < 1.21 g/ml fractions to the d > 1.21 g/ml fractions. Possible physiologic and pathologic implications of these findings are also discussed.

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

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 &agr;1 (−35%), pre-&agr;1 (−50%), pre-&agr;2 (−33%), and pre-&agr;3 (−31%) subpopulations, whereas the concentrations of the small LpA-I/A-II &agr;3 particles were significantly (P <0.001) higher (20%). Because &agr;1 was decreased more than HDL-C and plasma apoA-I concentrations in CHD subjects, the ratios of HDL-C to &agr;1 and of apoA-I to &agr;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 &agr;1 and pre-&agr;1–3 (LpA-I) and significantly higher &agr;3 (LpA-I/A-II) particles.

Metabolic and functional relevance of HDL subspecies.

Purpose of review Our purpose is to review recent findings highlighting the metabolic and functional diversity of HDL subspecies. Recent findings HDL heterogeneity – both structural and functional – is the main focus of this review. Recent work indicates that the metabolism and functionality of HDL particles differ greatly among HDL subspecies. With the introduction of new and improved methodology (e.g., proteomics), new aspects of the structural complexity and functionality of HDL have been revealed. It has been confirmed that HDL functions – including, but not limited to decreasing inflammation, apoptosis, macrophage adhesion to the endothelium and insulin resistance – are due to HDLs ability to remove cholesterol from cells (reverse cholesterol transport). A new level of HDL complexity has recently been revealed by investigating the lipid composition of HDL with gas chromatography, gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry. There are about 100 different HDL-associated proteins; however, there are many more lipid species potentially associated with HDL particles. Summary The most important recent findings disclose that HDL is more complex than previously thought. HDL subclasses differ in physical–chemical properties, protein and lipid composition, metabolism, physiological functions and pathophysiological significance. The staggering complexity of HDL demands significantly more investigation before we can truly begin to understand HDL metabolism and function in humans.

Protection From Retinopathy and Other Complications in Patients With Type 1 Diabetes of Extreme Duration: The Joslin 50-Year Medalist Study

OBJECTIVE To assess complication prevalence and identify protective factors in patients with diabetes duration of ≥50 years. Characterization of a complication-free subgroup in this cohort would suggest that some individuals are protected from diabetes complications and allow identification of endogenous protective factors. RESEARCH DESIGN AND METHODS Cross-sectional, observational study of 351 U.S. residents who have survived with type 1 diabetes for ≥50 years (Medalists). Retinopathy, nephropathy, neuropathy, and cardiovascular disease were assessed in relation to HbA1c, lipids, and advanced glycation end products (AGEs). Retrospective chart review provided longitudinal ophthalmic data for a subgroup. RESULTS A high proportion of Medalists remain free from proliferative diabetic retinopathy (PDR) (42.6%), nephropathy (86.9%), neuropathy (39.4%), or cardiovascular disease (51.5%). Current and longitudinal (the past 15 years) glycemic control were unrelated to complications. Subjects with high plasma carboxyethyl-lysine and pentosidine were 7.2-fold more likely to have any complication. Of Medalists without PDR, 96% with no retinopathy progression over the first 17 years of follow-up did not experience retinopathy worsening thereafter. CONCLUSIONS The Medalist population is likely enriched for protective factors against complications. These factors might prove useful to the general population with diabetes if they can be used to induce protection against long-term complications. Specific AGE combinations were strongly associated with complications, indicating a link between AGE formation or processing with development of diabetic vasculopathy.

Extended-Release Niacin Alters the Metabolism of Plasma Apolipoprotein (Apo) A-I and ApoB-Containing Lipoproteins

Objectives—Extended-release niacin effectively lowers plasma TG levels and raises plasma high-density lipoprotein (HDL) cholesterol levels, but the mechanisms responsible for these effects are unclear. Methods and Results—We examined the effects of extended-release niacin (2 g/d) and extended-release niacin (2 g/d) plus lovastatin (40 mg/d), relative to placebo, on the kinetics of apolipoprotein (apo) A-I and apoA-II in HDL, apoB-100 in TG-rich lipoproteins (TRL), intermediate-density lipoproteins (IDL) and low-density lipoproteins (LDL), and apoB-48 in TRL in 5 men with combined hyperlipidemia. Niacin significantly increased HDL cholesterol and apoA-I concentrations, associated with a significant increase in apoA-I production rate (PR) and no change in fractional catabolic rate (FCR). Plasma TRL apoB-100 levels were significantly lowered by niacin, accompanied by a trend toward an increase in FCR and no change in PR. Niacin treatment significantly increased TRL apoB-48 FCR but had no effect on apoB-48 PR. No effects of niacin on concentrations or kinetic parameters of IDL and LDL apoB-100 and HDL apoA-II were noted. The addition of lovastatin to niacin promoted a lowering in LDL apoB-100 attributable to increased LDL apoB-100 FCR. Conclusion—Niacin treatment was associated with significant increases in HDL apoA-I concentrations and production, as well as enhanced clearance of TRL apoB-100 and apoB-48.

Role of LCAT in HDL remodeling : investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (−77% and −13%), and LCAT activities (−99% and −39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated preβ-1 and α-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with α-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, preβ-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with preβ-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of preβ-1 into α-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.