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.

Increased plasma concentrations of lipoprotein(a) during a low-fat, high-carbohydrate diet are associated with increased plasma concentrations of apolipoprotein C-III bound to apolipoprotein B-containing lipoproteins.

BACKGROUND: Low-fat, high-carbohydrate (LFHC) diets have been shown to increase plasma concentrations of lipoprotein(a) [Lp(a)] and of triacylglycerol- rich lipoproteins (TRLs). OBJECTIVE: We tested whether increases in plasma Lp(a) induced by an LFHC diet are related to changes in TRLs. DESIGN: Healthy men (study 1; n = 140) consumed for 4 wk each a high-fat, low-carbohydrate diet (HFLC; 40% fat, 45% carbohydrate) and an LFHC diet (20% fat, 65% carbohydrate). Plasma lipids; lipoproteins; apolipoprotein (apo) B, A-I, and C-III; and Lp(a) were measured at the end of each diet. In a second group of men following a similar dietary protocol (study 2; n = 33), we isolated apo(a)-containing particles by immunoaffinity chromatography and determined the concentrations of apo C-III in ultracentrifugally isolated subfractions of apo B-containing lipoproteins. RESULTS: In study 1, plasma concentrations of Lp(a) (P < 0.001), triacylglycerol (P < 0.001), apo B (P < 0.005), apo C-III (P < 0.005), and apo C-III in apo B-containing lipoproteins (non-HDL apo C-III) (P < 0.001) were significantly higher with the LFHC diet than with the HFLC diet. Stepwise multiple linear regression analysis showed that the association of changes in Lp(a) with changes in non-HDL apo C-III was independent of changes in body mass index, apo B, LDL cholesterol, and HDL cholesterol. Plasma lipid and lipoprotein changes were similar in study 2, and we found that both total apo C-III and the apo C-III content of apo(a)-containing particles were increased in a TRL fraction consisting predominantly of large VLDL particles [TRL-apo(a)]. CONCLUSIONS: The increase in plasma Lp(a) with an LFHC diet is significantly associated with an increase in non-HDL apo C-III. Enrichment of TRL-apo(a) with apo C-III may contribute to this dietary effect on Lp(a) concentrations.

Separate effects of reduced carbohydrate intake and weight loss on atherogenic dyslipidemia.

BACKGROUND: Low-carbohydrate diets have been used to manage obesity and its metabolic consequences. OBJECTIVE: The objective was to study the effects of moderate carbohydrate restriction on atherogenic dyslipidemia before and after weight loss and in conjunction with a low or high dietary saturated fat intake. DESIGN: After 1 wk of consuming a basal diet, 178 men with a mean body mass index (in kg/m(2)) of 29.2 +/- 2.0 were randomly assigned to consume diets with carbohydrate contents of 54% (basal diet), 39%, or 26% of energy and with a low saturated fat content (7-9% of energy); a fourth group consumed a diet with 26% of energy as carbohydrate and 15% as saturated fat. After 3 wk, the mean weight loss (5.12 +/- 1.83 kg) was induced in all diet groups by a reduction of approximately 1000 kcal/d for 5 wk followed by 4 wk of weight stabilization. RESULTS: The 26%-carbohydrate, low-saturated-fat diet reduced triacylglycerol, apolipoprotein B, small LDL mass, and total:HDL cholesterol and increased LDL peak diameter. These changes were significantly different from those with the 54%-carbohydrate diet. After subsequent weight loss, the changes in all these variables were significantly greater and the reduction in LDL cholesterol was significantly greater with the 54%-carbohydrate diet than with the 26%-carbohydrate diet. With the 26%-carbohydrate diet, lipoprotein changes with the higher saturated fat intakes were not significantly different from those with the lower saturated fat intakes, except for LDL cholesterol, which decreased less with the higher saturated fat intake because of an increase in mass of large LDL. CONCLUSIONS: Moderate carbohydrate restriction and weight loss provide equivalent but nonadditive approaches to improving atherogenic dyslipidemia. Moreover, beneficial lipid changes resulting from a reduced carbohydrate intake were not significant after weight loss.

Phenotypic predictors of response to simvastatin therapy among African-Americans and Caucasians: the Cholesterol and Pharmacogenetics (CAP) Study.

Although statins are effective lipid-lowering agents, the phenotypic and demographic predictors of such lowering have been less well examined. We enrolled 944 African-American and white men and women who completed an open-label, 6-week pharmacogenetics trial of 40 mg of simvastatin. The phenotypic and demographic variables were examined as predictors of the change in lipids and lipoproteins using linear regression analysis. On average, treatment with simvastatin lowered low-density lipoprotein (LDL) cholesterol by 54 mg/dl and increased high-density lipoprotein (HDL) cholesterol by 2 mg/dl. Compared with African-Americans, whites had a 3-mg/dl greater LDL reduction and a 1-mg/dl higher HDL elevation, independent of other variables, including baseline lipoprotein levels (p <0.01). Multivariate analyses revealed moderate subgroup differences, with older participants having a larger decrease in LDL cholesterol and apolipoprotein B levels compared with younger participants (p <0.001), women having larger increases in HDL than men (p <0.01), nonsmokers having larger decreases in LDL and triglyceride levels compared with smokers (p <0.05), those with hypertension having smaller decreases in apolipoprotein B than those without hypertension (p <0.05), and those with a larger waist circumference having a diminished lowering of triglycerides in response to treatment with simvastatin (p <0.01). In conclusion, treatment with simvastatin produced favorable lipid and lipoprotein changes among all participants. The magnitude of the lipid and lipoprotein responses, however, differed among participants according to a number of phenotypic and demographic characteristics.

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 > or =40 km in male and > or =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.

Concordant lipoprotein and weight responses to dietary fat change in identical twins with divergent exercise levels 1.

BACKGROUND: Individuals vary greatly in their lipoprotein responses to low-fat diets, with some of this variation being attributable to genes. OBJECTIVE: The purpose was to test the extent to which individual lipoprotein responses to diet can be attributed to genes in the presence of divergent exercise levels. DESIGN: Twenty-eight pairs of male monozygotic twins (one twin mostly sedentary, the other running an average of 50 km/wk more than the sedentary twin) went from a 6-wk 40%-fat diet to a 6-wk 20%-fat diet in a crossover design. The diets reduced fat primarily by reducing saturated and polyunsaturated fat (both from 14% to 4%) while increasing carbohydrate intake from 45% to 65%. RESULTS: Despite the twins' differences in physical activity, the dietary manipulation produced significantly correlated changes (P < 0.05) in the twins' total cholesterol (r = 0.56); LDL cholesterol (r = 0.70); large, buoyant LDL [Svedberg flotation rate (S(f)) 7-12; r = 0.52]; apolipoprotein A-I (r = 0.49); lipoprotein(a) (r = 0.49); electrophoresis measurements of LDL-I (LDLs between 26 and 28.5 nm in diameter; r = 0.48), LDL-IIB (25.2-24.6 nm; r = 0.54), and LDL-IV (22-24.1 nm; r = 0.50); and body weight (r = 0.41). Replacing fats with carbohydrates significantly decreased the size and ultracentrifuge flotation rate of the major LDL and the LDL mass concentrations of large, buoyant LDL; LDL-I; HDL cholesterol; and apolipoprotein A-I and significantly increased concentrations of LDL-IIIA (24.7-25.5 nm) and lipoprotein(a). CONCLUSIONS: Even in the presence of extreme differences in exercise, genes significantly affect changes in LDL, apolipoprotein A-I, lipoprotein(a), and body weight when dietary fats are replaced with carbohydrates.

Role of CYP27A in cholesterol and bile acid metabolism.

The CYP27A gene encodes a mitochondrial cytochrome P450 enzyme, sterol 27-hydroxylase, that is expressed in many different tissues and plays an important role in cholesterol and bile acid metabolism. In humans, CYP27A deficiency leads to cerebrotendinous xanthomatosis. To gain insight into the roles of CYP27A in the regulation of cholesterol and bile acid metabolism, cyp27A gene knockout heterozygous, homozygous, and wild-type littermate mice were studied. In contrast to homozygotes, heterozygotes had increased body weight and were mildly hypercholesterolemic, with increased numbers of lipoprotein particles in the low density lipoprotein size range. Cyp7A expression was not increased in heterozygotes but was in homozygotes, suggesting that parts of the homozygous phenotype are secondary to increased cyp7A expression and activity. Homozygotes exhibited pronounced hepatomegaly and dysregulation in hepatic cholesterol, bile acid, and fatty acid metabolism. Hepatic cholesterol synthesis and synthesis of bile acid intermediates were increased; however, side chain cleavage was impaired, leading to decreased bile salt concentrations in gallbladder bile. Expression of Na-taurocholate cotransporting polypeptide, the major sinusoidal bile salt transporter, was increased, and that of bile salt export pump, the major canalicular bile salt transporter, was decreased. Gender played a modifying role in the homozygous response to cyp27A deficiency, with females being generally more severely affected. Thus, both cyp27A genotype and gender affected the regulation of hepatic bile acid, cholesterol, and fatty acid metabolism.

Plasma clearance of human low-density lipoprotein in human apolipoprotein B transgenic mice is related to particle diameter.

To test for intrinsic differences in metabolic properties of low-density lipoprotein (LDL) as a function of particle size, we examined the kinetic behavior of 6 human LDL fractions ranging in size from 251 to 265 A injected intravenously into human apolipoprotein (apo) B transgenic mice. A multicompartmental model was formulated and fitted to the data by standard nonlinear regression using the Simulation, Analysis and Modeling (SAAM II) program. Smaller sized LDL particles (251 to 257 A) demonstrated a significantly slower fractional catabolic rate (FCR) (0.050 +/- 0.045 h(-1)) compared with particles of larger size (262 to 265 A) (0.134 +/- -0.015 h(-1), P <.03), and there was a significant correlation between FCR and the peak LDL diameter of the injected fractions (R(2) =.71, P <.034). The sum of the equilibration parameters, k(2,1) and k(1,2), for smaller LDL (0.255 h(-1) and 0.105 h(-1), respectively) was significantly smaller than that for larger LDL (0.277 h(-1) and 0.248 h(-1), respectively; P <.01), indicative of slower intravascular-extravascular exchange for smaller LDL. Therefore in this mouse model, smaller LDL particles are cleared more slowly from plasma than larger LDL and are exchanged more slowly with the extravascular space. This might be due to compositional or structural features of smaller LDL that lead to retarded clearance.

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). CONCLUSIONS: 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.

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 HDL(3a) (P=0.02) and inversely to HDL(2)-mass (P=0.02) and HDL(2b) (P=0.03). The average annual rate in stenosis change was 6-fold more rapid in the fourth quartile of LDL-IVb (>or=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.

ApoC-III content of apoB-containing lipoproteins is associated with binding to the vascular proteoglycan biglycan.

Retention of apolipoprotein (apo)B and apoE-containing lipoproteins by extracellular vascular proteoglycans is critical in atherogenesis. Moreover, high circulating apoC-III levels are associated with increased atherosclerosis risk. To test whether apoC-III content of apoB-containing lipoproteins affects their ability to bind to the vascular proteoglycan biglycan, we evaluated the impact of apoC-III on the interaction of [(35)S]SO(4)-biglycan derived from cultured arterial smooth muscle cells with lipoproteins obtained from individuals across a spectrum of lipid concentrations. The extent of biglycan binding correlated positively with apoC-III levels within VLDL (r = 0.78, P < 0.01), IDL (r = 0.67, P < 0.01), and LDL (r = 0.52, P < 0.05). Moreover, the biglycan binding of VLDL, IDL, and LDL was reduced after depletion of apoC-III-containing lipoprotein particles in plasma by anti-apoC-III immunoaffinity chromatography. Since apoC-III does not bind biglycan directly, enhanced biglycan binding may result from a conformational change associated with increased apo C-III content by which apoB and/or apoE become more accessible to proteoglycans. This may be an intrinsic property of lipoproteins, since exogenous apoC-III enrichment of LDL and VLDL did not increase binding. ApoC-III content may thus be a marker for lipoproteins characterized as having an increased ability to bind proteoglycans.

Metabolic effects of nandrolone decanoate and resistance training in men with HIV.

Thirty human immunodeficiency virus (HIV)-infected men were randomized to a high dose of nandrolone decanoate weekly (group 1) or nandrolone plus resistance training (group 2) for 12 wk. For the two groups, nandrolone had no significant effects on total cholesterol, LDL cholesterol, LDL phenotype, or fasting triglycerides, although triglycerides decreased by 66 +/- 124 mg/dl for the entire population (P = 0.01). Group 2 subjects had a favorable increase of 5.2 +/- 7.7A in LDL particle size (P = 0.03), whereas there was no change in group 1. Lipoprotein(a) decreased by 7.3 +/- 6.8 mg/dl for group 1 (P = 0.002) and by 6.9 +/- 8.1 for group 2 (P = 0.013). However, HDL cholesterol decreased by 8.7 +/- 7.4 mg/dl for group 1 (P < 0.001) and by 10.6 +/- 5.9 for group 2 (P < 0.001). Percentages of HDL(2b) (9.7-12 nm) and HDL(2a) (8.8-9.7 nm) subfractions decreased similarly for the two groups, whereas HDL(3a) (8.2-8.8 nm) and HDL(3b) (7.8-8.2 nm) increased in the groups during study therapy (P < or = 0.02 for all comparisons). There was no evidence of a decreased insulin sensitivity in either group, whereas fasting glucose, fasting insulin, and homeostasis model assessment improved in group 2 (P < 0.05). These metabolic effects were favorable (other than for HDL), but changes were generally transient (except for HDL in group 2), with measurements returning to baseline 2 mo after the interventions were completed.

Analysis and quantitation of biotinylated apoB-containing lipoproteins with streptavidin-Cy3.

Non denaturing gradient gel electrophoresis (GGE) is commonly used to analyze the size distribution of lipoprotein particles. Its relatively low sensitivity and linear dynamic range limit use of GGE to quantify protein content of lipoproteins. We demonstrate a new high sensitivity method for analysis and quantitation of biotinylated apolipoprotein B (apoB)-containing lipoproteins using a fluorescent streptavidin-Cy3 conjugate and non covalent preelectrophoretic binding. Forty-four lipoprotein subfractions spanning the VLDL and LDL particle spectrum subfractions (11 each from four human subjects) were prepared by density gradient ultracentrifugation. An aliquot of each sample was biotinylated and GGE was performed. Gels also were stained for lipid with Oil Red O (32 samples) and for protein with Coomassie Brilliant Blue (30 samples). There was a significant relationship between the Cy3 fluorescent label area under the curve and the mass of apoB (P < 0.02-0.004) and total cholesterol (P < 0.03-0.004). Particle diameters of each absorbence/fluorescent peak were comparable between Oil-Red O and streptavidin-Cy3 treated biotinylated lipoproteins (+/-3.54 A, P = 0.3). Biotinylation and prestaining of lipoprotein particle with streptavidin-Cy3 provides a new fluorescence-based method for detection and quantitative analysis of lipoprotein subspecies by gradient gel electrophoresis.

Properties of triglyceride-rich and cholesterol-rich lipoproteins in the remnant-like particle fraction of human blood plasma.

An immunoassay procedure that quantifies remnant-like particle (RLP) cholesterol in human blood plasma has shown considerable promise as a clinically applicable risk marker for atherosclerotic disease. The lipoproteins included in this assay include not only certain TG-rich lipoproteins [all particles containing apolipoprotein B-48 (apoB-48) and a fraction of those containing apoB-100] but also a very small proportion of plasma cholesterol-rich lipoproteins. The TG-rich lipoprotein component of RLP has been partially characterized, but relatively little is known about the component cholesterol-rich lipoproteins. We have further characterized the properties of the TG-rich component that is included in RLP in which about 25% of the particles contain apoB-48 and the remainder apoB-100. We show that the cholesterol-rich component is comprised mainly of beta-migrating LDLs that contain predominantly apoB-100. ApoE found in the LDL fraction of RLP resides on pre-beta lipoproteins that lack apoA-I as well as apoB. The TG-rich component of RLP is responsible for increased RLP-cholesterol concentrations associated with hypertriglyceridemia. By contrast, the cholesterol-rich component is a major contributor to plasma RLP-cholesterol in individuals with low plasma TG. Our results suggest that particle heterogeneity in the RLP fraction is likely to affect the ability of RLP-cholesterol concentration to predict atherosclerotic risk. RLP-cholesterol concentrations in individuals with low plasma TG may not have the same clinical significance as they do in those with hypertriglyceridemia.

Altered activities of anti-atherogenic enzymes LCAT, paraoxonase, and platelet-activating factor acetylhydrolase in atherosclerosis-susceptible mice.

We examined whether the putative anti-atherogenic enzymes LCAT, paraoxonase (PON), and platelet-activating factor acetylhydrolase (PAF-AH) are impaired in 8 week old atherosclerosis susceptible apolipoprotein E (apoE)(-/-) and LDL receptor (LDLr)(-/-) mice and whether plasma concentrations of bioactive oxidized phospholipids accumulate in plasma. ApoE(-/-) mice had reduced (28%) LCAT activity and elevated lysophosphatidylcholine and bioactive oxidized phospholipids (1-palmitoyl-2-oxovaleryl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine) compared with controls on the chow diet. Elevated oxidized phospholipids and reduced LCAT activity may, in part, contribute to spontaneous lesions in these mice on a chow diet. A Western diet decreased LCAT activity further (50% of controls) and PON activity was decreased 38%. The LDLr(-/-) mice showed normal LCAT activity on chow diet and little accumulation of oxidized phospholipids. On a Western diet, LDLr(-/-) mice had reduced LCAT activity (21%), but no change in PON activity. All genotypes had reduced PAF-AH activity on the Western diet. ApoE(-/-) and LDLr(-/-) mice, but not controls, had elevated plasma bioactive oxidized phospholipids on the Western diet. We conclude that impairment of LCAT activity and accumulation of oxidized phospholipids are part of an early atherogenic phenotype in these models.