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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1040.)
© 1996 American Heart Association, Inc.

Articles

Compositional Differences of LDL Particles in Normal Subjects With LDL Subclass Phenotype A and LDL Subclass Phenotype B

Warren H. Capell; Alberto Zambon; Melissa A. Austin; John D. Brunzell; John E. Hokanson

the Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (W.H.C., A.Z., J.D.B., J.E.H.), and the Department of Epidemiology, School of Public Health and Community Medicine (M.A.A., J.E.H), University of Washington, Seattle.

Correspondence to John E. Hokanson, MPH, PhC, Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, RG-26, University of Washington, Seattle, WA 98195.

Abstract

A predominance of small LDL particles (subclass phenotype B), as determined by gradient-gel electrophoresis is found among patients with myocardial infarction. Despite physical differences in phenotype A and B particles, differences in lipid composition of particles in these phenotypes have yet to be reported in an unselected population of males and females. The present study used lipid/apoB ratios to analyze the amount of lipid per LDL particle, isolated by density-gradient ultracentrifugation, in 70 healthy subjects. Relative to apoB, the LDL particles from phenotype B subjects were found to contain less free cholesterol (0.391±0.05 versus 0.465±0.05; mean±SD; P<.001), phospholipid (1.26±0.2 versus 1.43±0.2; P<.001), and cholesteryl ester (1.97±0.1 versus 2.11±0.2; P<.001) than particles from phenotype A subjects. The amount of triglyceride per LDL particle did not differ between the two phenotypes (0.410±0.1 versus 0.406±0.1; P=NS) despite higher plasma triglyceride levels in the phenotype B subjects. LDL size and buoyancy were positively correlated with particle free cholesterol, phospholipid, and cholesteryl ester but not with particle triglyceride. These data suggest that the physical properties of LDL from subjects with phenotype A and B reflect their lipid composition. The compositional differences between LDL particles of the two phenotypes may provide new insight into the increased risk of myocardial infarction in subjects with small, dense LDL.

Key Words: triglyceride • apoB • gradient-gel electrophoresis • density-gradient ultracentrifugation • LDL subclass phenotype B

Low density lipoprotein has long been recognized as a heterogeneous population of particles on the basis of their physical properties.^{1 2 3 4 5 6 7} Studies of LDL chemical composition have traditionally analyzed multiple-density subfractions of LDL. These studies have reported a trend toward decreased percent cholesterol (free and esterified) and PL with increasing density of LDL.^{3 8 9 10} Trends in percent TG in LDL density fractions were less consistent. Attempts to estimate the actual LDL particle composition on the basis of calculations from the hydrodynamic properties of LDL and its lipid constituents have also suggested similar trends in the lipid content of LDL.^{3 9}

Individual subjects can be characterized into distinct, genetically influenced LDL subclass phenotypes based on the size distribution of LDL particles as determined by gradient-gel electrophoresis.¹¹ Individuals with a predominance of large, buoyant LDL particles are identified as phenotype A, while individuals with a predominance of small, dense LDL particles are identified as phenotype B. Approximately 15% of the population have an intermediate phenotype (phenotype I), often characterized by a broad distribution of particles of intermediate size.

Phenotype B subjects, and other subjects with small, dense LDL, have been shown to be at increased risk for myocardial infarction in both cross-sectional^{10 12 13 14 15 16 17} and longitudinal¹⁸ studies. In addition, a predominance of small LDL is associated with insulin resistance^{19 20 21} and non–insulin-dependent diabetes mellitus.^{19 22 23 24} These risks, however, may not be independent of other lipids, in particular, high levels of plasma TG and low levels of HDL cholesterol.²⁵ Although the mechanism or mechanisms underlying the increased risk of coronary disease in phenotype B subjects remain to be established,²⁶ LDL particle composition may play an important role. Compositional analysis of LDL in this clinically important group of individuals has shown a decrease in the carbohydrate content of LDL in phenotype B subjects relative to phenotype A subjects.²⁷ In addition, in familial combined hyperlipidemia, a select population with small LDL, LDL particles are cholesterol and PL depleted.²⁸ Despite the abundance of data on LDL composition, differences in lipid composition of LDL particles in these two LDL subclass phenotypes from an unselected population have yet to be reported.

Given the clinical significance of LDL subclass phenotype B and the high prevalence in the general population,^{11 29} we investigated the compositional differences of LDL particles in normal phenotype A and B subjects prior to the potential development of coronary disease. Since there is only one apoB molecule per LDL particle,³⁰ the lipid/apoB ratio (wt/wt) provides a measure of the lipid composition of individual particles in a sample of LDL. In this analysis, we used this approach to compare the CE, FC, PL, and TG content of LDL particles in a group of phenotype A and B subjects.

Methods

Study Subjects
Seventy-three subjects with no known lipid disorders and taking no lipid-altering medications, including ß-blockers and estrogens, were recruited through advertisements at the University of Washington for participation in these studies. All subjects gave informed consent and were paid for their participation. Three subjects were excluded from the present analysis: one who was diagnosed as having type III hyperlipidemia and two with small amounts of LDL cholesterol (37 and 78 mg/dL) who did not produce detectable LDL gradient-gel electrophoresis scans. The resulting sample size was 70 subjects (see Table 1). Nine phenotype I subjects (seven men and two women) were excluded from the comparison of LDL composition between discrete phenotypes, rendering a sample size of 61. The phenotype I subjects were included in linear regression analyses of LDL size and buoyancy as continuous variables.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Dichotomized Study Sample

Blood Collection
Subjects were instructed to fast for 12 hours prior to donating blood. Samples were collected in 0.1% EDTA and centrifuged immediately at 4°C. The plasma obtained was stored at 4°C, for no longer than 7 days, until lipid analyses were performed.

Nondenaturing Gradient-Gel Electrophoresis
Gradient-gel electrophoresis was used to estimate the particle diameter of the major LDL peak and to determine LDL subclass phenotypes, as previously described.^{4 13} Briefly, 6 µL of plasma from each subject was diluted 4:1 with a 50% sucrose/0.01% bromophenol blue tracking solution and electrophoresed at 10°C in 2% to 16% polyacrylamide gradient gels (Pharmacia) at 125 V for 24 hours, using a tris(hydroxymethyl)aminomethane (0.09 mol/L)/boric acid (0.08 mol/L)/Na₂EDTA (0.003 mol/L) buffer at pH 8.3. The gels were stained with oil red O and scanned at 490 nm, producing a graphical representation of the size distribution of lipoprotein particles. The standard lane was stained with Coomassie blue R250, scanned, and a quadratic curve constructed based on the migration distances of four standards of known diameter: ferritin (12.2 nm), thyroglobulin (17.0 nm), thyroglobulin dimer (23.6 nm, Pharmacia), and carboxylated latex beads (38.0 nm, Dow Chemical). The diameters of particles in the major LDL peak were estimated from this standard curve. Peak particle diameter was reproducible, with a CV of 2.5% from 87 consecutive gels.³¹

LDL subclass phenotype A, B, or I was assigned to each subject by two independent, blinded observers (J.E.H. and A.Z.) according to previously published criteria, which included both measurement of major peak particle diameter and assessment of the distribution skewing due to minor peaks.²⁵ Twenty-seven subjects (39%) were identified as phenotype A, having a major LDL diameter peak of >255 Å, with skewing toward the smaller particles. Thirty-four subjects (49%) were assigned the B phenotype, having major LDL diameter peaks of 255 Å, with skewing toward the larger-diameter sizes. Nine subjects (13%) displaying either one broad LDL peak in the 255- to 260-Å range or a bimodal distribution were designated phenotype I.

DGUC
Nonequilibrium DGUC was used to isolate LDL from plasma for particle lipid analysis and to calculate the peak Rf and density distribution of lipoprotein lipids.³² This technique is a variation of the single vertical spin method by Chung et al,³³ in which the resolution of apoB-containing particles is optimized. Briefly, 2 mL of plasma was adjusted to 5 mL of 1.08 g/mL solution with aqueous KBr (d=1.21 g/mL) and NaCl (d=1.006 g/mL). This solution was then pipetted under 12 mL of 1.006 g/mL NaCl solution in a Sorvall TV-865B tube (Du Pont) to form a discontinuous salt gradient. The plasma was centrifuged against this gradient at 65 000 rpm for 90 minutes at 10°C. The tube was then drained from the bottom at a flow rate of 1.7 mL/min into 38 fractions. Fig 1 shows the cholesterol distribution of lipoprotein classes isolated by standard sequential ultracentrifugation³⁴ and subjected to this DGUC. LDL is restricted to fractions 7 through 18. Of measured LDL isolated by this method, HDL and IDL contamination represent 1.8% and 2.6%, respectively, in the study population.

View larger version (21K): [in this window] [in a new window]   Figure 1. DGUC of lipoproteins previously isolated by sequential equilibrium ultracentrifugation. Values in each DGUC fraction represent percent cholesterol within the lipoprotein density class being evaluated. It should be noted that these values do not represent percentages of total plasma cholesterol; for example, the overlap of HDL with the LDL range (fractions 7 through 18) is only 1.8% of LDL in whole plasma.

Cholesterol was assayed (see below) in each fraction and plotted against fraction number for each subject, producing a DGUC lipoprotein cholesterol profile. To normalize for variations in the distribution of total plasma cholesterol, the percent of cholesterol in each fraction was calculated. Rf, used to estimate relative LDL buoyancy, was calculated for each subject by dividing the fraction number containing the LDL peak by the total number of fractions collected. Rf values were highly reproducible, with a CV of 0.2% by replicate analysis.³¹ The DGUC profile for each subject was used to identify the fractions that contained LDL (fractions 7 through 18). These fractions were then pooled for each subject for subsequent lipid analysis.³⁵

Lipid and Apolipoprotein Analysis
Lipids and apolipoproteins were analyzed by the Northwest Lipid Research Laboratory. Control samples were routinely included in each assay. CV values are based on a minimum of 50 control samples.

VLDL was isolated using standard preparative ultracentrifugation,³⁶ and HDL was separated from LDL by using dextran sulfate.³⁷ Total cholesterol was measured by enzymatic methods (cholesterol esterase from Boehringer Mannheim; cholesterol oxidase from Calbiochem-Behring; horseradish peroxidase from Sigma Chemical Company) in plasma, VLDL, LDL, HDL, and the pooled LDL fractions isolated by DGUC.³⁸ The CV of the cholesterol assay was 1.2%. TG was determined in plasma and in the pooled LDL fractions with an enzymatic kit (Sigma). The CV for the TG assay was 2.1%. FC assays on pooled DGUC fractions were analogous to that of total cholesterol but without cholesterol esterase. The CV for the FC assay was 3.3%. PLs cleaved by phospholipase D were assayed on pooled DGUC fractions by enzymatic methods, using phosphatidylcholine as the standard (phospholipase D and choline oxidase from Fermco Biochemics, Inc).³⁸ The CV for the PL assay was 1.9%. CE values were calculated by multiplying the difference between total cholesterol and FC by 1.67.

ApoB was measured in plasma and in the pooled DGUC LDL fractions by immunonephelometry (Behring Diagnostic), using a polyclonal antibody, as previously described.³⁹ The CV for the apoB assay was 3.0%. To verify this method between the different phenotypes, the apoB immunoassay was compared with the protein assay of Lowry et al,⁴⁰ using established methods and BSA as the standard, in isolated LDL from phenotype A and phenotype B subjects (10 and 7 samples, respectively). The apoB immunoassay measured 87.1±4.3% of the Lowry protein in LDL of phenotype A versus 83.7±1.3% of the Lowry protein in LDL of phenotype B (P>.25). LDL particle lipid composition was calculated as lipid/apoB (wt/wt) ratio.

Statistical Analysis
The DGUC difference profile comparing phenotype B and phenotype A groups was constructed by plotting the difference in mean percent cholesterol and 95% CI⁴¹ for each fraction.

Two-tailed Student's t tests were performed for comparing phenotype A and B subjects, excluding phenotype I subjects. For correlation analyses, Pearson's correlation coefficients were generated for the entire study population (N=70). The level of statistical significance used in this study was P<.05.

Results

General characteristics of phenotype A and B subjects are summarized in Table 1. There were no significant differences in age or body mass index between phenotype A and B subjects. Phenotype B subjects had a higher mean value of total plasma TG than phenotype A subjects (102±54 versus 51±20 mg/dL; mean±SD; P<.001). Plasma TG ranged from 27 to 102 mg/dL in phenotype A subjects and from 36 to 244 mg/dL in phenotype B subjects. Eight of the phenotype B subjects had plasma TG levels >140 mg/dL; two of these were >200 mg/dL. Phenotype B subjects also had higher mean values than phenotype A subjects of VLDL cholesterol (20±11 versus 10±4 mg/dL; P<.001), LDL cholesterol (112±31 versus 95±23 mg/dL; P<.05), and apoB (100±24 versus 77±17 mg/dL; P<.001). HDL cholesterol, on the other hand, was lower in the phenotype B group (48±9 versus 64±13 mg/dL; P<.001). These differences in HDL persisted when men and women were analyzed separately (data not shown).

LDL physical and chemical properties were compared between males and females, with males having smaller and more dense LDL. However, within each phenotype there were no differences in LDL size, buoyancy, or lipid composition of LDL between sexes. Therefore, males and females within each phenotype were combined for comparisons of LDL composition.

Mean DGUC lipoprotein cholesterol profiles for phenotype A and B subjects, presented in Fig 2A and 2B, respectively, illustrate the plasma lipoprotein cholesterol distributions in the two phenotypes. Distinct differences in lipoprotein distribution were evident when the difference in mean percent cholesterol between phenotype B and A subjects was plotted (Fig 2C). Consistent with the lipoprotein measurements in Table 1, the phenotype B group had significantly less HDL cholesterol (fractions 1 through 6) and more VLDL cholesterol (fractions 30 through 38) than the phenotype A subjects. A redistribution of particles by buoyancy was noted in the LDL region (fractions 7 through 18), with the phenotype B subjects having a lower proportion of their cholesterol in a more buoyant LDL subclass and a greater proportion in the form of dense LDL. As expected, a direct comparison of the physical properties of LDL in the dichotomized population (Table 1) revealed that the particle diameter of the major LDL subclass was smaller in phenotype B subjects (249±5 versus 268±7 Å; P<.001) and that LDL particles were less buoyant in phenotype B subjects (Rf=0.298±0.023 versus 0.328±0.012; P<.001).

View larger version (18K): [in this window] [in a new window]   Figure 2. DGUC lipoprotein cholesterol profiles for 27 phenotype A (A) and 34 phenotype B (B) subjects. Mean percent cholesterol and 95% CI are shown for each fraction. Note that the major peak of cholesterol falls within the LDL range for both phenotype A and B subjects. The DGUC difference profile (difference in mean percent cholesterol and 95% CI of the difference) between phenotype B and A subjects is also shown (C). Standard lipoprotein fractions isolated by sequential ultracentrifugation are as follows: HDL, (1.063<d<1.21 g/mL) in fractions 1 through 6; LDL, (1.019<d<1.063 g/mL) in fractions 7 through 18; IDL, (1.006<d<1.019 g/mL) in fractions 19 through 29; and VLDL, (d<1.006 g/mL) in fractions 30 through 38, as in Fig 1.

A comparison of the chemical composition of the pooled LDL in phenotype A and B subjects (Table 2), not controlled for the number of particles in the LDL, indicated a greater concentration of apoB (90±19 versus 75±17 mg/dL, P<.01), TG (36±8 versus 29±5 mg/dL, P<.001), and CE (176±33 versus 156±32 mg/dL, P<.05) in the LDL of phenotype B subjects. The concentrations of FC (35±8 versus 34±7 mg/dL, P=.71) and PL (111±19 versus 104±17 mg/dL, P=.14) in LDL did not differ between the two phenotypes.

View this table: [in this window] [in a new window]   Table 2. Concentrations of LDL Compositional Components (mg/dL) and Fractional Composition of Pooled LDL

Analysis of percent composition of LDL produced quite different trends (Table 2). Percent apoB (20±1.3% versus 19±1.5%, P<.001) was significantly greater in the LDL of phenotype B subjects. Percent FC (8±0.8% versus 9±0.5%, P<.001) and percent PL (25±1.5% versus 26±1.5%, P<.001) were both lower in the LDL of phenotype B subjects, while percent TG (8±1.7% versus 7±1.3%, P=.095) and percent CE (39±1.5% versus 39±1.6%, P=.704) in the LDL of phenotype A and B subjects did not differ.

Comparing actual LDL particle composition, by controlling for the number of particles in LDL with lipid/apoB ratios, revealed statistically significant differences between the two phenotypes (Table 3, Fig 3). LDL from phenotype B subjects contained significantly less FC per particle relative to apoB (0.391±0.046 versus 0.465±0.045, P<.001), less PL per particle (1.26±0.16 versus 1.43±0.21, P<.001), and less CE per particle (1.97±0.12 versus 2.11±0.18, P<.001) than LDL from phenotype A subjects. Interestingly, the TG content per LDL particle did not differ between the two phenotypes (0.410±0.10 versus 0.406±0.10) despite higher plasma TG levels in the phenotype B subjects.

View this table: [in this window] [in a new window]   Table 3. Comparison of Lipid/ApoB Ratios (wt/wt) in LDL Particles of Phenotype A and Phenotype B Subjects

View larger version (12K): [in this window] [in a new window]   Figure 3. Mean lipid/apo B ratios (wt/wt) of the four major lipid components of LDL in phenotype A () and phenotype B (•) subjects. Error bars represent 95% CIs. The differences in lipid/apoB ratios between phenotype A and B subjects were significant for particle FC/apoB, PL/apoB, and CE/apoB (P<.001).

To determine whether these per-particle chemical differences account for the observed physical differences in the LDL particles, correlation analyses were performed, with LDL peak particle size and buoyancy as continuous variables (Table 4). LDL peak particle size was found to be positively correlated with the per-particle amounts of FC (r=.639, P<.001), PL (r=.369, P<.01), and CE (r=.406, P=.001). There was no statistically significant relationship between LDL size and TG per particle (r=-.100, P=.42). Correlations between LDL peak buoyancy and the per-particle lipid components displayed a similar but stronger relationship (FC, r=.679; PL, r=.455; CE, r=.497; P<.001 for all). LDL TG per particle was not related to LDL buoyancy (r=-.066, P=.60).

View this table: [in this window] [in a new window]   Table 4. Pearson Correlation Coefficients Between Physical and Chemical Properties of LDL Particles

Plasma TG level was associated with several LDL characteristics, but the patterns of correlations differed between phenotype A and B subjects. Among phenotype B subjects, plasma TG was correlated negatively with LDL size (r=-.511, P<.01), LDL FC/apoB (r=-.864, P<.001), and LDL CE/apoB (r=-.407, P<.05); plasma TG and LDL PL/apoB were not correlated (r=-.187, P=.29). However, plasma TG did correlate positively with LDL TG/apoB among phenotype B subjects (r=.467, P<.01). This correlation does not appear to be driven solely by outliers with very high TG levels, since, with respect to plasma TG, the difference in mean TG/apoB between the upper quartile and lower three quartiles of phenotype B subjects was only marginally significant (P=.07). Among the phenotype A subjects, plasma TG was correlated only with LDL FC/apoB (r=-.512, P<.01), and not with LDL size (r=-.243, P=.22), LDL PL/apoB (r=-.179, P=.37), LDL TG/apoB (r=-.09, P=.66), or LDL CE/apoB (r=.009, P=.96).

Discussion

The main objective of the present study was to analyze the compositional differences between LDL particles of unselected, healthy subjects dichotomized into two distinct phenotypes by gradient-gel electrophoresis, LDL subclass phenotypes A and B, which are relevant to clinical disease. Using mass ratios of lipid components to the fixed apoB content per LDL particle, we have demonstrated that LDL from subjects with phenotype B contains significantly less FC, PL, and CE per particle than that from phenotype A subjects. We have also shown that the amount of LDL TG per particle in these two phenotypes does not differ.

Previous studies of LDL composition have analyzed multiple density fractions between 1.019 and 1.063 g/mL. These studies have reported overall trends of decreasing lipid content as LDL density increases, with variable trends in the TG content. We have also obtained results that support these previously described trends of decreasing FC, PL, and CE per particle when LDL size and buoyancy are analyzed as continuous variables.

The association between small LDL and high plasma TGs is well documented both in cross-sectional^{13 25 42 43 44 45 46} and longitudinal²⁹ studies. Our data agree with these findings, in that phenotype B subjects had significantly higher plasma TG levels than phenotype A subjects. Deckelbaum et al^{47 48} have suggested that TG enrichment of LDL occurs with increasing plasma TG levels due to accelerated cholesteryl ester transfer protein–mediated transfer of VLDL TG into LDL. Consistent with this model, there was a positive correlation between plasma TG and LDL TG content among phenotype B subjects. However, when the LDL TG was adjusted for plasma TG concentration, the LDL TG was similar in the phenotype A and B subjects (P=.36).

Our results also illustrate the importance of controlling for particle number in comparing the composition of LDL. In measuring the concentrations of LDL components, the total LDL pool of phenotype B subjects is enriched in TG and CE relative to the LDL of phenotype A subjects (Table 2). However, these differences are the result of a greater number of LDL particles present in LDL of phenotype B subjects, as evidenced by the greater concentration of apoB in LDL. In fact, LDL particle composition is depleted in CE in phenotype B subjects (Table 3). In addition, comparing percent composition of LDL (for example, CE) does not provide a meaningful comparison of the lipid content in LDL. There is no difference in the percent composition of LDL CE between phenotype B and phenotype A subjects (Table 2), but the absolute concentration of LDL CE is higher (Table 2) and the per-particle composition of LDL CE lower in phenotype B subjects. Therefore, to compare actual particle composition, one must measure concentration and control for differing particle numbers, such as is accomplished by LDL lipid/apoB ratios (Table 3).

This study indicates that males as a group have smaller and more dense LDL than do females. However, within each phenotype, there is no difference in the composition or physical properties of LDL between males and females. The differences in LDL between males and females can be fully accounted for by the previously documented differences in distribution of LDL subclass phenotypes between males and females.^{11 45 46}

Though the subjects in our study were all healthy, the compositional differences we have shown in the LDL of phenotype B subjects resemble those reported in other clinically important disease groups. Patients with coronary artery disease,¹⁰ familial combined hyperlipidemia,²⁸ and non–insulin-dependent diabetes mellitus²³ who have small, dense LDL were found to have similar trends of lipid-depleted LDL. Therefore, healthy subjects displaying LDL subclass phenotype B have differences in LDL composition that could lead to increased risk of atherosclerosis and subsequent heart disease.

There are several hypotheses to explain the increased atherogenicity of LDL particles in phenotype B subjects, including the increased number of apoB-containing particles,⁴⁹ differences in glycosylation of LDL particles,²⁷ and altered conformation and receptor affinity of the apoB molecule.⁵⁰ One explanation with which our data are consistent, is that phenotype B is associated with an atherogenic lipid profile^{13 25 43} that may be directly responsible for the increased risk of myocardial infarction. The phenotype B subjects in our study had higher plasma VLDL cholesterol and TG, along with lower plasma HDL cholesterol, than phenotype A subjects. Under this hypothesis, phenotype B could be viewed as a marker for a high-risk lipid profile.

Another hypothesis, which proposes an active role of small LDL particles in accelerating atherosclerosis, comes from observations that LDL subfractions containing small, dense particles are more susceptible to in vitro oxidation.^{51 52 53} With increasing density of LDL subfractions, the increased susceptibility to oxidation bears an inverse relationship to FC concentrations in these subfractions.⁵² Our results indicate that the decreased size and buoyancy of LDL particles in phenotype B subjects are most strongly associated with a decrease in the amount of FC, with differences in per-particle FC accounting for 41% and 46% of the variation in LDL size and buoyancy, respectively. Coresh et al¹⁰ have also shown similar results using percent FC in LDL. Thus, the increased risk of atherosclerosis in phenotype B subjects may result in part from a greater susceptibility to oxidation of the LDL depleted in FC.

In summary, we have described the use of lipid component/apoB ratios in analyzing the composition of LDL particles isolated by DGUC from normal subjects. Using these ratios, we have found that LDL particles from phenotype B subjects contain less FC, PL, and CE than particles from phenotype A subjects. LDL particle FC, PL, and CE were all positively correlated to LDL size and buoyancy. These results suggest that differences in the chemical composition underlie the physical differences in LDL particles of phenotype A and B individuals and may relate to an increased risk of atherosclerosis among phenotype B subjects.

Selected Abbreviations and Acronyms

CE = cholesteryl ester CI = confidence interval CV = coefficient of variation DGUC = density-gradient ultracentrifugation FC = free cholesterol PL = phospholipid Rf = relative flotation rate TG = triglyceride

Acknowledgments

This study was supported in part by National Institutes of Health Program Project grant HL-30086. Dr Zambon was a recipient of a CNR (National Research Committee) of Italy grant, bando n. A.1. 94.00474.04. Dr Capell completed these studies as a medical student to fulfill the research requirement at the University of Washington School of Medicine. This work was performed during Dr Austin's tenure as an Established Investigator of the American Heart Association. The authors wish to thank Dr S. Marcovina of the Northwest Lipid Research Laboratory, Steve Hashimoto, Alem Nicodimos, Chris Casazza, and Jennifer Watanabe for their technical assistance.

Footnotes

Presented in part at the 6th European Symposium on Metabolism, Padova, Italy, May 24-26, 1993, and previously published (New York, NY: Excerpta Medica; 1993:197-200). Also presented in part at the 39th Western Clinical Research Meeting, Carmel, Calif, February 9-12, 1994, and published in abstract form (Clin Res. l994;42:86A).

Received May 1, 1995; revision received February 7, 1996; References

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