Patricia J. Blanche

Enhanced oxidative susceptibility and reduced antioxidant content of metabolic precursors of small, dense low-density lipoproteins

PURPOSE Elevated plasma concentrations of low-density lipoproteins (LDL) increase risk for coronary heart disease. However, lipoprotein profiles rich in small, dense LDL particles confer greater risk than those that mainly consist of large, buoyant LDL. This may be due, in part, to the greater oxidative susceptibility of small, dense LDL. In the current studies, we tested whether differences in the oxidative behavior of buoyant and dense LDL arise from differences in their immediate metabolic precursors, intermediate-density lipoproteins. SUBJECTS AND METHODS We compared the properties of intermediate-density lipoproteins and buoyant and dense LDL subfractions in 9 subjects with the large, buoyant LDL phenotype versus 6 with the small, dense LDL phenotype. Oxidative susceptibility was evaluated based on conjugated diene formation and parinaric acid oxidation induced by copper. Antioxidants (ubiquinol-10 and alpha-tocopherol) were measured by high-performance liquid chromatography. RESULTS Oxidative susceptibility was increased and antioxidant concentrations were decreased with increasing lipoprotein density (intermediate intermediate-density lipoproteins to buoyant LDL to dense LDL). Intermediate-density lipoproteins from subjects with the small, dense LDL phenotype had a greater oxidative susceptibility (by the parinaric acid test) and lower antioxidant concentrations than corresponding particles from subjects with the large, buoyant LDL phenotype. CONCLUSIONS Differences in oxidative susceptibility between large, buoyant and small, dense LDL particles are apparent in their lipoprotein precursors. These results suggest that lipoprotein oxidative susceptibility may be metabolically programmed and that intermediate-density lipoproteins may contribute to the increased risk associated with the small, dense LDL phenotype.

Direct Determination of Lipoprotein Particle Sizes and Concentrations by Ion Mobility Analysis

BACKGROUND Current methods for measuring the concentrations of lipoprotein particles and their distributions in particle subpopulations are not standardized. We describe here and validate a new gas-phase differential electrophoretic macromolecular mobility-based method (ion mobility, or IM) for direct quantification of lipoprotein particles, from small, dense HDL to large, buoyant, very-low-density lipoprotein (VLDL). METHODS After an ultracentrifugation step to remove albumin, we determined the size and concentrations of lipoprotein particles in serum samples using IM. Scan time is 2 min and covers a particle range of 17.2-540.0 A. After scanning, data are pooled by totaling the particle number across a predetermined size range that corresponds to particular lipoprotein subclasses. IM results were correlated with those of standard methods for cholesterol and apolipoprotein analysis. RESULTS Intra- and interassay coefficients of variation for LDL particle size were <1.0%. The intra- and interassay variation for LDL and HDL particle subfraction measurements was <20%. IM-measured non-HDL correlated well with apolipoprotein B (r = 0.92). CONCLUSIONS The IM method provides accurate, reproducible, direct determination of size and concentration for a broad range of lipoprotein particles. Use of this methodology in studies of patients with cardiovascular disease and other pathologic states will permit testing of its clinical utility for risk assessment and management of these conditions.

Detection and quantitation of LDL subfractions

A number of procedures are available for analysis and quantitation of the multiple discrete subclasses of LDL in human plasma. In view of the differing relationships of these subclasses to coronary artery disease risk, LDL subclass analysis augments information provided by standard measurements of total LDL cholesterol.

Smallest LDL Particles Are Most Strongly Related to Coronary Disease Progression in Men

Objective—LDLs include particle subclasses that have different mobilities on polyacrylamide gradient gels: LDL-I (27.2 to 28.5 nm), LDL-IIa (26.5 to 27.2 nm), LDL-IIb (25.6 to 26.5 nm), LDL-IIIa (24.7 to 25.6 nm), LDL-IIIb (24.2 to 24.7 nm), LDL-IVa (23.3 to 24.2 nm), and LDL-IVb (22.0 to 23.3 nm in diameter). We hypothesized that the association between smaller LDL particles and coronary artery disease (CAD) risk might involve specific LDL subclasses. Methods and Results—Average 4-year onstudy lipoprotein measurements were compared with annualized rates of stenosis change from baseline to 4 years in 117 men with CAD. The percentages of total LDL and HDL occurring within individual subclasses were measured by gradient gel electrophoresis. Annual rate of stenosis change was related concordantly to onstudy averages of total cholesterol (P =0.04), triglycerides (P =0.05), VLDL mass (P =0.03), total/HDL cholesterol ratio (P =0.04), LDL-IVb (P =0.01), and HDL3a (P =0.02) and inversely to HDL2-mass (P =0.02) and HDL2b (P =0.03). The average annual rate in stenosis change was 6-fold more rapid in the fourth quartile of LDL-IVb (≥5.2%) than in the first quartile (<2.5%, P =0.03). Stepwise multiple regression analysis showed that LDL-IVb was the single best predictor of stenosis change. Conclusions—LDL-IVb was the single best lipoprotein predictor of increased stenosis, an unexpected result, given that LDL-IVb represents only a minor fraction of total LDL.

Alterations in low-density lipoprotein and high-density lipoprotein subclasses among Hispanic women with polycystic ovary syndrome: influence of insulin and genetic factors

OBJECTIVE To examine the influence of hyperandrogenism on low-density lipoprotein (LDL) and high-density lipoprotein (HDL) subclass levels as well as lipoprotein (a) levels in hyperandrogenic women compared with a control group. DESIGN Case-control study. SETTING University-based outpatient clinic. PATIENT(S) Sixteen Hispanic women with polycystic ovary syndrome were compared with 21 controls matched for age, weight, and ethnicity. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Fasting serum levels of testosterone, insulin, and lipoproteins. RESULT(S) Compared with controls, women with polycystic ovary syndrome had significantly lower levels of apolipoprotein A-I (95+/-28 mg/dL versus 144+/-42 mg/dL) and HDL2a (30.9%+/-4.4% versus 36.6%+/-5.4%) but significantly higher levels of HDL3c (5.1%+/-2.2% versus 2.4%+/-1.5%). There were no statistically significant differences in LDL subclasses between groups, but there was a high incidence (54%) of the atherogenic lipoprotein phenotype B in this Hispanic population. As a group, Hispanic women with the abnormal B phenotype had significantly higher levels of insulin, HDL, HDL2b, and triglycerides. CONCLUSION(S) Hyperandrogenemia may have an adverse effect on serum lipoproteins through effects on HDL subclasses. Hispanic women may have a higher incidence of the atherogenic lipoprotein phenotype B, which may increase their risk for atherosclerosis.

Behavioral Versus Genetic Correlates of Lipoproteins and Adiposity in Identical Twins Discordant for Exercise

Background—Lipoprotein and weight differences between vigorously active and sedentary monozygotic (MZ) twins were used to (1) estimate the effects of training while controlling for genotype and (2) estimate genetic concordance (ie, similarity) in the presence of divergent lifestyles. Methods and Results—Thirty-five pairs of MZ twins (25 male, 10 female) were recruited nationally who were discordant for vigorous exercise (running distances differed by ≥40 km in male and ≥32 km in female twins). The active twins ran an average (mean±SD) of 63.0±20.4 km/wk, whereas the mostly sedentary twins averaged 7.0±13.5 km/wk. The active twins had significantly lower body mass index (difference±SE, −2.12±0.57 kg/m2, P=0.0007) and significantly higher HDL cholesterol (0.14±0.04 mmol/L, P=0.004), HDL2 (2.71±1.04 U, P=0.01), and apolipoprotein (apo) A-I (0.10±0.03 g/L, P=0.004). Despite the difference in lifestyle, when adjusted for sex, the correlations between the discordant MZ twin pairs were significant (P<0.01) for HDL cholesterol (r=0.69), apoA-I (r=0.58), and HDL2 (r=0.67). There was no significant MZ twin correlation for body mass index (r=0.17). None of the active twins having an overweight twin were themselves overweight. Conclusions—Behavior (vigorous exercise) may reduce genetic influences on body mass index. In contrast, genetics (or shared environment) substantially influences HDL cholesterol and HDL subclasses, even in the presence of extreme behavioral differences. There may be greater individual control over moderate degrees of obesity, whereas low HDL cholesterol may be largely predetermined and less effectively treated by vigorous exercise.

Overexpression of Human Hepatic Lipase and ApoE in Transgenic Rabbits Attenuates Response to Dietary Cholesterol and Alters Lipoprotein Subclass Distributions

The effect of the expression of human hepatic lipase (HL) or human apoE on plasma lipoproteins in transgenic rabbits in response to dietary cholesterol was compared with the response of nontransgenic control rabbits. Supplementation of a chow diet with 0.3% cholesterol and 3.0% soybean oil for 10 weeks resulted in markedly increased levels of plasma cholesterol and VLDL and IDL in control rabbits as expected. Expression of either HL or apoE reduced plasma cholesterol response by 75% and 60%, respectively. The HL transgenic rabbits had substantial reductions in medium and small VLDL and IDL fractions but not in larger VLDL. LDL levels were also reduced, with a shift from larger, more buoyant to smaller, denser particles. In contrast, apoE transgenic rabbits had a marked reduction in the levels of large VLDLs, with a selective accumulation of IDLs and large buoyant LDLs. Combined expression of apoE and HL led to dramatic reductions of total cholesterol (85% versus controls) and of total VLDL+IDL+LDL (87% versus controls). HDL subclasses were remodeled by the expression of either transgene and accompanied by a decrease in HDL cholesterol compared with controls. HL expression reduced all subclasses except for HDL2b and HDL2a, and expression of apoE reduced large HDL1 and HDL2b. Extreme HDL reductions (92% versus controls) were observed in the combined HL+apoE transgenic rabbits. These results demonstrate that human HL and apoE have complementary and synergistic functions in plasma cholesterol and lipoprotein metabolism.

Effects on Lipoprotein Subclasses of Combined Expression of Human Hepatic Lipase and Human apoB in Transgenic Rabbits

Objective—The effects of combined expression of human hepatic lipase (HL) and human apolipoprotein B (apoB) on low-density lipoprotein (LDL) subclasses were examined in rabbits, a species naturally deficient in HL activity. Methods and Results—In apoB-transgenic rabbit plasma, >80% of the protein was found in the 1.006- to 1.050-g/mL fraction. Gradient gel electrophoresis (GGE) of this fraction revealed two distinct species, designated large and small LDL. A denser fraction (d=1.050 to 1.063 g/mL) contained small LDL as well as another discrete LDL subspecies, designated very small LDL. Expression of HL resulted in reductions in protein concentrations in the 1.006- to 1.050-g/mL density-gradient subfractions containing large (6.5±4.1 versus 32.6±12.0 mg/dL, P <0.005) and small LDL (59.6±17.4 versus 204.3±50.3 mg/dL, P <0.002). A concomitant small but not significant increase in protein concentration in the denser LDL fraction (48.0±28.2 versus 44.6±18.2 mg/dL) was due primarily to an increase in very small LDL (25.9±3.1 versus 9.6±5.4% of total LDL GGE densitometric area, P <0.002). Conclusion—These findings support a direct role for HL in regulating total plasma LDL concentrations as well as in the production of smaller, denser LDL from larger, more buoyant precursors.

Characterization of complexes of egg yolk phosphatidylcholine and apolipoprotein A-II prepared in the absence and presence of sodium cholate

Complexes of apolipoprotein A-II and egg yolk phosphatidylcholine were prepared in mixtures of different composition in the absence and presence of sodium cholate. By gradient gel electrophoresis, complex preparations were polydisperse and particle size distributions were influenced by the composition of the reconstitution mixture. Complexes generally exhibited a discoidal morphology by electron microscopy, but showed increased formation of vesicular complexes at elevated levels of egg yolk PC in the mixtures. By chemical crosslinking, complexes formed in the absence of cholate were shown to consist primarily of discoidal species with three apolipoprotein A-II molecules per particle in the mixtures investigated; complexes formed in the presence of cholate included species ranging from three to five apolipoprotein A-II per particle. The number of apolipoprotein A-II per particle and the sizes of the complexes, prepared in cholate, increased with increase of egg yolk PC in the reconstitution mixture. Relative to the particle size distribution of discoidal complexes formed in the absence of cholate, those prepared in cholate showed a distribution shifted to larger particle sizes. Complexes of similar particle size distribution formed in the presence or absence of cholate showed similar physical-chemical properties. Discoidal complexes with the same number of apolipoprotein A-II per particle but of different size and composition were observed, suggesting the possibility of some conformational adaptation of apolipoprotein A-II leading to stabilization of egg yolk PC bilayers of different diameter. Properties of particle size distributions of discoidal complexes prepared in cholate of apolipoprotein A-II and egg yolk PC were compared with those of complexes of apolipoprotein A-I previously reported (Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) Biochim. Biophys. Acta 750, 353-364).

Influence of ApoE Content on Receptor Binding of Large, Buoyant LDL in Subjects With Different LDL Subclass Phenotypes

We investigated the influence of apolipoprotein (apo) E-containing particles on LDL receptor binding of large, buoyant LDL subfractions (LDL I) from subjects with predominantly large (phenotype A) and small (phenotype B) LDL particles. Direct binding by human fibroblast LDL receptors was tested at 4 degrees C before and after removal of apoE-containing particles by immunoaffinity chromatography. The binding affinity of total LDL I in phenotype B was greater than that in phenotype A (Kd of 1.83+/-0.3 and 3.43+/-0.9 nmol/L, respectively, P<.05). LDL I from phenotype B subjects had a higher apoE to apoB molar ratio than did that from phenotype A (0.16+/-0.04 versus 0.06+/-0.02, P<.05). Nondenaturing gradient gel electrophoresis of apoE-containing LDL I isolated by immunoaffinity chromatography revealed a substantially larger peak particle diameter than in apoE-free LDL I, and comparison of LDL I composition before and after immunoaffinity chromatography suggested an increase in triglyceride content of apoE-containing particles. After removal of these particles, there was a greater than twofold reduction in LDL receptor affinity of phenotype B LDL (Kd of 1.83+/-0.3 to 3.76+/-0.6, P<.01), whereas in phenotype A no change was observed (Kd of 3.43+/-0.9 to 3.57+/-0.4, respectively). The receptor affinity of apoE-free LDL I from phenotype A and B subjects did not differ. These findings confirm that large, buoyant LDL particles from phenotype B subjects have a higher LDL receptor affinity than does LDL I from phenotype A subjects and suggest that this difference is due to an increased content of large, triglyceride-enriched, apoE-containing lipoproteins. It is possible that the accumulation of these particles reflects abnormalities in the metabolism of remnant lipoproteins that contribute to atherosclerosis risk in phenotype B subjects.