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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1184-1188.)
© 1996 American Heart Association, Inc.

Articles

Apolipoprotein E Polymorphism and LDL Size in a Biethnic Population

Steven M. Haffner; Michael P. Stern; Heikki Miettinen; David Robbins; Barbara V. Howard

the Division of Clinical Epidemiology, Department of Medicine, University of Texas Health Science Center at San Antonio (S.M.H., M.P.S., H.M.), and Medlantic Research Institute, Washington, DC (D.R., B.V.H.).

Correspondence to Steven Haffner, MD, Division of Clinical Epidemiology, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7873.

	Abstract

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Polymorphisms in the apolipoprotein E (apoE) phenotype (especially E₄) are associated with increased cardiovascular risk and particularly with increased concentrations of LDL cholesterol. Little is known, however, about whether alterations in LDL size are associated with the apoE₄ phenotype. LDL size was determined by gradient gel electrophoresis, and apoE phenotype was determined by isoelectric focus in 337 nondiabetic subjects from the San Antonio Heart Study, a population-based study of diabetes and cardiovascular risk factors in Mexican Americans and non-Hispanic whites. ApoE₄ was associated not only with increases in LDL cholesterol concentrations but also with decreased LDL size. After adjustment for age, sex, body mass index, waist-to-hip ratio, triglyceride, HDL cholesterol, fasting insulin, and diabetic status, the apoE phenotype remained significantly related to LDL size (Å) in both men (apoE₂₃, 260.0; apoE₃₃, 256.3; and apoE₃₄, 252.6; P=.01) and women (apoE₂₃, 261.7; apoE₃₃, 257.9; and apoE₃₄, 256.7, P=.045). Variations in apoE phenotype are associated not only with changes in the absolute concentration of LDL cholesterol but also with changes in its composition. These changes are only partly explained by associations of apoE with insulin, triglyceride, and HDL cholesterol.

Key Words: apolipoprotein E • LDL size • LDL cholesterol • triglyceride

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Variation in apoE phenotype may account for a considerable part of the variation in prevalence of CHD.^{1 2 3} ApoE may participate in the receptor-mediated clearance of cholesterol.^{1 2} In the Framingham study, both male and female subjects with the phenotype E₄ had an increased risk of CHD that was greater than that for any other genetic lipid abnormality.⁴ The European Atherosclerosis Research Study, a multicenter group of 14 centers in 11 countries, concluded that polymorphism in apoE strongly contributes to the development of CHD.⁵ The gene that encodes apoE lies on chromosome 19, and its three common alleles, ₂, ₃, and ₄ code for the isoforms apoE₂, apoE₃, and apoE₄, respectively.^{6 7} The frequencies of these isoforms vary from population to population, but apoE₃ always shows the highest frequency (50%) and apoE₂ the lowest frequency (15%).⁸ Population studies have shown that compared with the isoform apoE₃, the isoform apoE₂ is associated with low and the isoform apoE₄ with high levels of plasma LDL cholesterol.^{2 3 4 5} In a recent analysis, subjects with allele E₂ had increased triglyceride levels relative to subjects with the allele E₃.⁹ Similarly, compared with diabetic patients carrying the isoform E₃, the cardiovascular risk profile seems better in diabetic patients carrying apoE phenotype E₂ and worse in those carrying the isoform apoE₄.^{10 11 12}

Considerable heterogeneity exists in the size and density of LDL particles.^{13 14} A preponderance of small, dense LDL particles is associated with increased CHD, although this relationship may not be independent of triglyceride levels.^{15 16 17} Austin et al¹⁶ found that most individuals can be assigned to one of two LDL subclass patterns (A or B). Relatively few subjects have an intermediate pattern (I). Small, dense LDL (type B) is associated with diabetes, hyperinsulinemia, higher triglyceride, lower concentrations of HDL cholesterol, and male sex.^{18 19 20 21 22 23 24 25} LDL heterogeneity may also have a genetic basis. In two family studies, the LDL subclass pattern was found to have a strong genetic determinant.^{26 27} Nishina et al²⁸ have provided evidence for linkage between the proposed gene for pattern B and the LDL receptor locus located on the short arm of chromosome 19.

Few data are available relating to the effect of apoE phenotypes on LDL size. Schaefer et al²⁹ have reported higher LDL score (which represents smaller, denser LDL) in subjects with apoE₄. This trend was significant in men but not in women. In contrast, Zhao et al³⁰ did not observe a relationship between apoE phenotype and LDL size in men. We have previously shown an association of apoE with insulin concentrations in the San Antonio Heart Study,³¹ and Despres et al³² have shown associations of triglycerides with insulin in certain apoE phenotypes, making an association between apoE and LDL composition at least plausible. In this report, we examine associations between apoE and LDL compositions and whether these associations are further associated with alterations in triglyceride and insulin concentrations.

	Methods

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The San Antonio Heart Study is a population-based study of diabetes and cardiovascular risk factors in 3302 Mexican Americans and 1877 non-Hispanic whites, 25 to 64 years of age at entry, who were enrolled in two phases (phase I: 1979 to 1982 and phase II: 1984 to 1988). Detailed descriptions of these surveys have appeared elsewhere.^{33 34} Ethnic assignment (Mexican American versus non-Hispanic white) was based on an algorithm published previously,³⁵ considering concordance (Spanish versus non-Spanish) between father's surname and mother's maiden name, birthplace of both parents, respondent's preferred ethnic identity when it indicated a unique national original, and the stated ethnic background of all four grandparents.

An 8-year follow-up of the phase I cohort has been completed, with 80.8% (n=1685) of the surviving subjects having been reexamined according to procedures identical to those used at the baseline examination. The follow-up to the phase II cohort is currently in progress. The subjects in this report (n=337) are from the first two census tracts of the phase II follow-up, a middle-income neighborhood (50% Mexican American and 50% non-Hispanic white) and a low-income neighborhood (100% Mexican American).

For each individual, blood samples were obtained after a 12-hour fast and 2 hours after administration of a 75-g oral glucose equivalent load (Orangedex, Custom Laboratories). Glucose was measured by a glucose oxidase method. Insulin was measured by a solid-phase radioimmunoassay (Diagnostic Products Corporation).³⁴ Serum cholesterol was determined by the cholesterol oxidase method, using an Abbott VP autoanalyzer,³⁶ and triglyceride level was measured after hydrolysis of the glyceride, using an enzymatic method for glycerol determination.³⁷ HDL, LDL, and VLDL were measured by the procedures used by the Lipid Research Clinics.³⁸ Lipoprotein concentrations are expressed in terms of their cholesterol concentrations. VLDL was removed by ultracentrifugation in those specimens having a total serum triglyceride concentration >300 mg/dL. In specimens with triglyceride <300 mg/dL, VLDL cholesterol was estimated as triglyceride divided by 5.³⁸ LDL cholesterol was estimated as total cholesterol minus the sum of HDL and VLDL cholesterol. We measured HDL cholesterol after precipitation of ß lipoproteins by the dextran sulfate method.³⁹ Diabetes was diagnosed by World Health Organization criteria.⁴⁰ Subjects who were under treatment with either oral antidiabetic agents or insulin were also considered to have diabetes regardless of their plasma glucose levels. Subjects with diabetes were excluded from the present report.

The anthropometric measurements, waist and hip circumferences, weight, and height were made using standard procedures.⁴¹ BMI was calculated as weight (in kilograms) divided by height (in meters) squared. The WHR was used as an index of upper body adiposity.

The apoE phenotypes were determined from small amounts (10 µL) of whole fasting plasma by using isoelectric focusing followed by immunoblotting.⁴² Briefly, polyacrylamide gels were prepared on 200x260-mm glass plates. The cathode and anode solutions were 1.0 mmol/L NaOH and 1.0 mmol/L H₃PO₄, respectively. LKB 2197 power supply and LKB 2117 Multiphor II electrophoresis units (LKB) were used for electrofocusing at 2000 V. Fifteen-minute prefocusing was carried out before applying the samples. A 4x10-mm piece of Whatman 3MM chromatography paper (sample wick) was dipped into each sample tube and, after blotting, was placed on the gel 5 mm from the cathode (80 samples per plate). Initial focusing was carried out for 30 minutes, with voltage increasing from 1000 to 1600 V. The sample wicks were removed after 30 minutes, and subsequent focusing was conducted for an additional 1.5 hours. After electrophoresis, the protein was transferred by simple diffusion using a 0.45-µm (pore size) nitrocellulose membrane (BA-585; Schleicher & Schuell). The immunoblotting was completed as described as Kataoka et al.⁴²

LDL size and subclass assessment were determined on fasting plasma samples by the method of Krauss and Burke¹⁴ in the laboratory of the Medlantic Research Institute, Washington, DC. Fasting plasma samples for determination of LDL size were stored at -70°C (without thawing) until the analyses for LDL size were done an average of 5 months later. Gradient gels were obtained from Isolab, Inc. Mean particle sizes were calibrated using LDL subfractions whose molecular diameter had been determined by analytical ultracentrifugation (courtesy of Dr R. Krauss, Donner Laboratories, Berkeley, Calif).

Subjects were classified into three groups on the basis of size and shape of the major gradient peak. A major peak of >257 Å, with skewing toward smaller particles, was classified as pattern A. Individuals with a predominant peak of <253.5 Å, with skewing toward larger particles, were classified as pattern B. If the size of the predominant peak was between 253.5 Å and 257 Å, subjects were classified as pattern I unless the size was very close to the cut points and the peak had the shape of the A or B pattern. The mean sizes (Å) of the A, I, and B peaks were 264.7±0.3, 255.3±0.4, and 244.5±0.4, respectively. Quality control pools were done on each run. The interassay coefficient of variation for eight control pools (240 Å to 263 Å) ranged from 1.8% to 3.6% measured over a period of 8 months.

Statistical techniques included analysis of variance, ² test, Mantel Haenszel ², and multiple logistic regression analyses (SAS, Proc GLM).⁴³ The ² test was used to assess whether Mexican Americans and non-Hispanic whites were in Hardy Weinberg equilibrium for the apoE gene. Allele frequencies were calculated by the gene-counting technique. We confirmed that LDL size was normally distributed by using normal probability plots and skewness and kurtosis. The skewness of the size of the LDL predominant peaks in the San Antonio Heart Study population was -0.16, and its kurtosis was -0.71, also suggesting an approximately normal distribution. Since both triglyceride and insulin concentrations were markedly skewed to the right, these variables were transformed using natural logarithms, and the results were then back transformed into their natural units for presentation in the tables. The effect of apoE phenotypes on LDL size was assessed by analyses of variance. Interactions between LDL size and other variables (apoE phenotype, ethnicity, diabetic status, obesity, etc) were also examined by analysis of variance. There were no statistically significant interactions (P>.20). Multiple logistic regression analyses were used to assess the effect of apoE phenotype on LDL subclass pattern (B versus A) after adjustment for other covariates. (Subjects with pattern I were excluded from these analyses.) No significant interactions (at P>.10) between apoE and other independent variables (age, ethnicity, etc) were found.

	Results

Top Abstract Introduction Methods Results Discussion References

 
The distribution of apoE phenotypes is shown in Table 1. Both Mexican Americans (P=.615) and non-Hispanic whites (P=.0374) were in Hardy Weinberg equilibrium. The estimated allele frequencies in Mexican Americans were E₂, 0.0238; E₃, 0.9048; and E₄, 0.0714 and in non-Hispanic whites were E₂, 0.0906; E₃, 0.7874; and E₄, 0.1220. Because of the small numbers of subgroups with the phenotypes E₂₂ (n=0), E₂₄ (n=3), and E₄₄ (n=5), we excluded these subjects from the remainder of the report to simplify the data presentation. Results with and without the eight subjects with rare phenotypes were very similar.

View this table: [in this window] [in a new window]   Table 1. Distribution of ApoE Phenotype by Sex and Ethnicity

The distribution of major apoE phenotypes did not differ by ethnicity (Table 1). The relationship of LDL size to apoE phenotype is shown in the Figure. In all four ethnicity/gender groups, LDL size was decreased in subjects with apoE₃₄; these results were significant only in non-Hispanic white women (P=.033) and Mexican American men (P=.001). We next tested whether the relation of apoE phenotype to LDL size differed by ethnicity; the apoExethnicity interaction was not statistically significant in either men (P=.206) or women (P=.605). After adjustment for ethnicity, apoE phenotype was significantly related to LDL size in both men (P=.002) and women (P=.605). In the rest of this report, we pool the ethnic groups and adjust for ethnicity.

View larger version (23K): [in this window] [in a new window]   Figure 1. Relationship of LDL size (Å) to apoE phenotype in Mexican American (MA) women (top left), non-Hispanic white (NHW) women (top right), MA men (bottom left), and NHW men (bottom right).

Table 2 shows the distribution of metabolic and clinical characteristics by sex. LDL size (Å) was 256.1±0.8 in men and 258.1±0.3 (P=.038). As expected, the distribution of apoE phenotypes did not differ by sex.

View this table: [in this window] [in a new window]   Table 2. Clinical Characteristics of the Study Subjects by Gender

Table 3 shows the distribution of cardiovascular risk factors by apoE status in men. LDL size (Å) differed significantly by apoE phenotype (E₂₃, 261.0; E₃₃, 256.6; and E₃₄, 251.1, P=.002). ApoE phenotype was also significantly related to HDL, LDL, and total cholesterol. The proportion of subjects with LDL subclass pattern was also significantly higher in subjects with apoE₃₄ phenotype. In women (Table 4), apoE phenotype was also significantly related to LDL subclass pattern and LDL size, as well as to the absolute concentration of LDL cholesterol.

View this table: [in this window] [in a new window]   Table 3. Mean Values of Metabolic Characteristics by ApoE Phenotype in Men

View this table: [in this window] [in a new window]   Table 4. Mean Values of Metabolic Characteristics by ApoE Phenotype in Women

After adjustment for age, ethnicity, BMI, WHR, triglyceride, HDL cholesterol, and fasting insulin (by multiple linear regression), LDL size (Å) continued to be significantly lower in subjects with apoE₃₄ in both men (E₂₃, 260.0; E₃₃, 256.3; and E₃₄, 252.5, P=.015) and women (E₂₃, 261.7; E₃₃, 257.9; and E₃₄, 256.7, P=.045). The risk of LDL subclass pattern B (in multiple logistic regression after similar adjustments) was also related to apoE phenotype (OR_34/23=16.15, P=.003; OR_33/23=3.63, P=.042; and OR_34/33=4.45, P=.012) in men. In women, the association of LDL subclass pattern B (in multiple logistic regression) was also significant (OR_34/23=12.0; OR_33/23=1.70; P=.247; and OR_34/33=7.06, P=.025).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have shown in this report that the apoE₃₄ phenotype is not only associated with higher LDL cholesterol concentrations but also with a preponderance of small, dense LDL particles. This was true in all major subgroups studied, including men, women, Mexican Americans and non-Hispanic white subjects. In a previous report from the Framingham study²⁹ a preponderance of smaller, denser LDL in subjects with apoE₄ was observed in men but not in women. However, in a second study in men, apoE phenotype was not significantly related to LDL size.³⁰

We have previously shown that apoE₄₃ was associated with increased triglyceride and insulin concentrations in normoglycemic subjects in the San Antonio Heart Study.³¹ In this report, triglyceride levels tended to be lower in some subgroups with apoE₃₂. In a previous meta-analysis, apoE₂₂ and E₂₃ were associated with increased triglyceride concentrations.⁶ Previous studies have indicated that dyslipidemia, hyperinsulinemia, and insulin resistance are strongly associated with smaller, denser LDL.^{18 19 20 21 22 23 24 25} Adjustment for these factors slightly reduced the differences in LDL size by apoE phenotype; however, these differences remained statistically significant.

Subjects with the apoE₄ phenotype have markedly increased rates of CHD.^{1 2 3 4 5 44} Much of the variation in CHD with different apoE phenotypes may be due to differences in lipoproteins, especially LDL cholesterol. However, it is likely that apoE may be related to cardiovascular risk through other pathways as well. In the Pathobiological Determinants of Atherosclerosis in Youth study, apoE was strongly related to the degree of atherosclerosis at autopsy in 720 young males.⁴⁵ However, adjustment for cholesterol levels did not appreciably change the association of apoE with variations in atherosclerotic lesion in that study.⁴⁵ We have also shown associations of apoE with insulin concentrations in the San Antonio Heart Study.³¹

A number of pathways may be responsible for the elevated LDL cholesterol levels in subjects with apoE₄. In these subjects, catabolism of LDL is decreased because of lower activity of the LDL receptor.⁴⁶ Subjects who are homogenous for apoE₂ may have low LDL levels because of upregulation of LDL receptors due to lower availability of intracellular cholesterol associated with decreased uptake of chylomicron or VLDL remnants, as well as increased availability of hepatic LDL receptors due to the lower competition by the VLDL or chylomicron remnants.^{46 47}

The precise mechanism by which apoE phenotype might affect the composition or LDL size is not well understood but may involve a number of related mechanisms. ApoE isoforms affect the catabolism of VLDL and LDL remnants by the apoB/E and apoE receptors in the liver,^{47 48 49} conversion of VLDL and IDL to LDL,^{46 50} cholesterol absorption,^{51 52} and LDL catabolism by the liver.⁵⁰ Increased cholesterol absorption and increased uptake of VLDL remnants by the liver in subjects with apoE₃₄ phenotype will lead to the downregulation of hepatic apoB/E receptor. On the other hand, lower cholesterol absorption and a decreased uptake of VLDL remnants in subjects with apoE₂₃ phenotype will lead to the upregulation of hepatic apoB/E receptor. However, since apoB- and apoE₂₃-containing particles will be removed slowly because of their poor recognition by the hepatic apoE/B receptor, subjects with apoE₂₃ phenotype will accumulate larger LDL particles, as has been found in the present study.

Variations in apoE phenotype may explain a considerable part of the variation in occurrence of CHD. While previous studies have concentrated on the association of apoE₄ with increased LDL cholesterol levels, data from the San Antonio Heart Study show an association of the apoE₄ polymorphism with atherogenic changes in LDL size as well.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BMI = body mass index CHD = coronary heart disease OR = odds ratio WHR = waist-to-hip ratio

	Acknowledgments

 
This grant was supported by grants from the National Heart, Lung, and Blood Institute (R01 HL24799 and R37 HL36820). We thank Dr R. Krauss of Donner Laboratories, Berkeley, Calif, for performing the analytical ultracentrifugation.

	Footnotes

 
Presented in part at the International Atherosclerosis Society meeting in Montreal, Canada, October, 1994.

Received July 31, 1995; revision received March 20, 1996;

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