Nondenaturing Polyacrylamide Gradient Gel Research Articles

An Automatic Fast High Resolution HDL Subfractioning Assay Using Microfluidic Electrophoretic System

Objectives: Studies in recent decade demonstrated the importance of HDL subfractionation in precision medicine. It is notable that not all HDL subfractions are “good” lipoprotein. The lack of a reliable technique that can precisely identify each particle of HDL subfractions remains the major obstacle in clinical application. Based on the principle of microfluidic electrophoresis, we have developed an automatic system (MFE) that meets the need of clinical applications. The objectives of this paper is to test the system for its application, precision, and also the normal range in healthy population using one of major subfraction, HDL-2b, as a target. Methods: The MFE was compared with traditional non-denaturing polyacrylamide gradient gel electrophoresis (GGE). Intra-chip, inter-chip, inter-instrument, inter-lab, and inter-operator precisions were all tested by using the sample in various conditions. An instruction was provided with the instrument and HDL subfractioning kit. Data were analyzed with SPSS for correlation of variation, regressions, etc. Results: The new system exhibited a linear correlation with the traditional GGE system. The intra-chip %CVs, inter-chip %CVs, inter-instrument %CVs of HDL-2b were less than 10%, which was the set acceptance criteria. With 243 samples from healthy individuals demonstrated that the mean value of HDL-2b% is about 25% in both male and female. Conclusions: Our data demonstrated that the MFE system is a fast, automatic, and reliable system that can obtain the percentage of HDL-2b with high precision

Lipoproteins and cholesterol homeostasis in paediatric nephrotic syndrome patients.

IntroductionThe aim of this study was to investigate lipoprotein particle distributions and the likelihood of achieving cholesterol homeostasis in the remission phase of nephrotic syndrome (NS) in paediatric patients. We hypothesized that lipoprotein particle distributions moved toward less atherogenic profile and that cholesterol homeostasis was achieved.Materials and methodsThirty-three children, 2 to 9 years old with NS were recruited. Blood sampling took place both in the acute phase and during remission. Serum low-density lipoprotein particles (LDL) and high-density lipoprotein particles (HDL) were separated using non-denaturing polyacrylamide gradient gel (3-31%) electrophoresis. Serum non-cholesterols sterols (NCSs), desmosterol, lathosterol, 7-dehydrocholesterol (7-DHC), campesterol and β-sitosterol were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).ResultsAll patients had desirable serum HDL cholesterol concentrations during remission. The dominant lipoprotein diameters and LDL subclass distribution did not change significantly during follow-up. In contrast, HDL lipoprotein particle distribution shifted towards larger particles. The absolute concentration of desmosterol was significantly lower during remission (P = 0.023). β-sitosterol concentration markedly increased during remission (P = 0.005). Desmosterol/β-sitosterol (P < 0.001) and 7-DHC/β-sitosterol (P = 0.005) ratios significantly declined during disease remission.ConclusionsFavourable changes in the serum lipid profiles, HDL particle subclass distribution and cholesterol metabolism in paediatric patients with NS during remission took place. For the first time, we found that cholesterol homeostasis changed in favour of increased cholesterol absorption during disease remission. Nevertheless, complete cholesterol homeostasis was not achieved during disease remission.

The Effect of Biochanin A as PPAR γ agonist on LDL Particles Diameter and Type 2 Diabetic Dyslipidemia

Background and Aims: Small dense low-density lipoproteins (sd-LDL) particles are smaller and heavier than typical LDL ones. They can penetrate into the endothelium of coronary arteries more easily because of their small size. Diabetes mellitus is accompanied by dyslipidemia such as increasing concentration of plasma very low density lipoprotein and sd-LDL. Peroxisome proliferator activated receptor γ (PPARγ ) can decrease the level of sd-LDL in plasma. Biochanin A (BCA), a natural compound, is a PPARγ agonist. The present study was designed to investigate the effect of BCA on sd-LDL-Clolesterol level in diabetic animals.&#x0D; Materials and Methods: Adult male rats (Wistar strain) were used as the animal models in this study. Animals were made diabetic by single intraperitoneal injection of Streptozotocin- Nicotinamide and then treated by 1 and 5 mg/kg of BCA for 28 days. Body weight and fasting blood glucose were also tested before and at the end of treatment. Furthermore, the size of LDL particles were measured by nondenaturing polyacrylamide gradient gel electrophoresis assay.&#x0D; Results: Results of the present study indicated that BCA administration at dose of 5mg/kg decreased fasting blood glucose level and increased body weight and diameter of LDL particles in diabetic animals significantly.&#x0D; Conclusions: BCA seems to be an appropriate agent in diabetes mellitus, because it improves the diabetic dyslipidemia, which is the most important complication in diabetic patients.

Molecular basis for the protective effects of low-density lipoprotein receptor-related protein 1 (LRP1)-derived peptides against LDL aggregation

Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly1127-Cys1140 exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly1127-Cys1140 (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA2)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA2-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA2. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima.

Transcoronary gradients of HDL-associated MicroRNAs in unstable coronary artery disease

AimsMicroRNAs (miRNAs) are transported on high-density lipoproteins (HDLs) and HDL-associated miRNAs are involved in intercellular communication. We explored HDL-associated miRNAs concentration gradients across the coronary circulation in stable and unstable coronary artery disease patients and whether changes in the transcoronary gradient were associated with changes in HDL composition and size. MethodsAcute coronary syndrome (ACS, n=17) patients, those with stable coronary artery disease (stable CAD, n=19) and control subjects without CAD (n=6) were studied. HDLs were isolated from plasma obtained from the coronary sinus (CS), aortic root (arterial blood) and right atrium (venous blood). HDL-associated miRNAs (miR-16, miR-20a, miR-92a, miR-126, miR-222 and miR-223) were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. HDL composition was measured immunoturbidometrically or enzymatically. ResultsA concentration gradient across the coronary circulation was observed for all the HDL-associated miRNAs. In ACS patients, there was a significant inverse transcoronary gradient for HDL-associated miR-16, miR-92a and miR-223 (p<0.05) compared to patients with stable CAD. Changes in HDL-miRNA transcoronary gradients were not associated with changes in HDL composition or size. ConclusionHDLs are depleted of miR-16, miR-92a and miR-223 during the transcoronary passage in patients with ACS compared to patients with stable CAD.

Low-density-lipoprotein particle size predicts a poor outcome in patients with atherothrombotic stroke.

Background and PurposeLow-density lipoprotein (LDL) particle size is considered to be one of the more important cardiovascular risk factors, and small LDL particles are known to have atherogenic potential. The aim of this study was to determine whether LDL particle size is associated with stroke severity and functional outcome in patients with atherothrombotic stroke.MethodsBetween January 2009 and May 2011, 248 patients with first-episode cerebral infarction who were admitted to our hospital within 7 days after symptom onset were prospectively enrolled. LDL particle size was measured using the nondenaturing polyacrylamide gradient gel electrophoresis assay. Stroke severity was assessed by applying the National Institutes of Health Stroke Scale (NIHSS) at admission. Functional outcome was investigated at 3 months after the index stroke using the modified Rankin Scale (mRS), and poor functional outcome was defined as an mRS score of ≥3.ResultsThe LDL particle size in the 248 patients was 25.9±0.9 nm (mean±SD). LDL particle size was inversely correlated with the degree of cerebral artery stenosis (p=0.010). Multinomial multivariate logistic analysis revealed that after adjustment for age, sex, and variables with p<0.1 in univariate analysis, LDL particle size was independently and inversely associated with stroke severity (NIHSS score ≥5; reference, NIHSS score 0-2; odds ratio=0.38, p=0.028) and poor functional outcome (odds ratio=0.44, p=0.038).ConclusionsThe results of this study demonstrate that small LDL particles are independently correlated with stroke outcomes. LDL particle size is thus a potential biomarker for the prognosis of atherothrombotic stroke.

Bariatric surgery in morbidly obese patients improves the atherogenic qualitative properties of the plasma lipoproteins

ObjectiveThe purpose of this study was to evaluate the effect of weight loss induced in morbidly obese subjects by Roux-en-Y gastric bypass bariatric surgery on the atherogenic features of their plasma lipoproteins. MethodsTwenty-one morbidly obese subjects undergoing bariatric surgery were followed up for up to 1 year after surgery. Plasma and lipoproteins were assayed for chemical composition and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity. Lipoprotein size was assessed by non-denaturing polyacrylamide gradient gel electrophoresis, and oxidised LDL by ELISA. Liver samples were assayed for mRNA abundance of oxidative markers. ResultsLipid profile analysis revealed a reduction in the plasma concentrations of cholesterol and triglycerides, which were mainly associated with a significant reduction in the plasma concentration of circulating apoB-containing lipoproteins rather than with changes in their relative chemical composition. All patients displayed a pattern A phenotype of LDL subfractions and a relative increase in the antiatherogenic plasma HDL-2 subfraction (>2-fold; P < 0.001). The switch towards predominantly larger HDL particles was due to an increase in their relative cholesteryl ester content. Excess weight loss also led to a significant decrease in the plasma concentration of oxidised LDL (∼−25%; P < 0.01) and in the total Lp-PLA2 activity. Interestingly, the decrease in plasma Lp-PLA2 was mainly attributed to a decrease in the apoB-containing lipoprotein-bound Lp-PLA2. ConclusionOur data indicate that the weight loss induced by bariatric surgery ameliorates the atherogenicity of plasma lipoproteins by reducing the apoB-containing Lp-PLA2 activity and oxidised LDL, as well as increasing the HDL-2 subfraction.

Changes in lipoprotein(a), oxidized phospholipids, and LDL subclasses with a low-fat high-carbohydrate diet

Low-fat diets have been shown to increase plasma concentrations of lipoprotein(a) [Lp(a)], a preferential lipoprotein carrier of oxidized phospholipids (OxPLs) in plasma, as well as small dense LDL particles. We sought to determine whether increases in plasma Lp(a) induced by a low-fat high-carbohydrate (LFHC) diet are related to changes in OxPL and LDL subclasses. We studied 63 healthy subjects after 4 weeks of consuming, in random order, a high-fat low-carbohydrate (HFLC) diet and a LFHC diet. Plasma concentrations of Lp(a) (P < 0.01), OxPL/apolipoprotein (apo)B (P < 0.005), and OxPL-apo(a) (P < 0.05) were significantly higher on the LFHC diet compared with the HFLC diet whereas LDL peak particle size was significantly smaller (P < 0.0001). Diet-induced changes in Lp(a) were strongly correlated with changes in OxPL/apoB (P < 0.0001). The increases in plasma Lp(a) levels after the LFHC diet were also correlated with decreases in medium LDL particles (P < 0.01) and increases in very small LDL particles (P < 0.05). These results demonstrate that induction of increased levels of Lp(a) by an LFHC diet is associated with increases in OxPLs and with changes in LDL subclass distribution that may reflect altered metabolism of Lp(a) particles.

Comparative effects of rosiglitazone and pioglitazone on fasting and postprandial low-density lipoprotein size and subclasses in patients with Type 2 diabetes

Objective: To assess the effects of pioglitazone and rosiglitazone on fasting and postprandial low-density lipoprotein (LDL) size and subclasses in patients with Type 2 diabetes. Research design and methods: Nine Type 2 diabetic patients (age 61 ± 10 years, body mass index 30 ± 5 kg/m2, glycosylated hemoglobin [HbA1c] 7.5 ± 0.5%) were randomized in a crossover trial to rosiglitazone 4 mg b.i.d. or pioglitazone 45 mg/day for 12 weeks with an 8-week wash-out period. LDL size and subclasses were determined by non-denaturing polyacrylamide gradient gel electrophoresis. A standardized breakfast was served and variables were assessed after 3 and 6 h. Results: HbA1c, insulin sensitivity (as assessed by the homeostasis model assessment) and LDL size and subclasses did not differ before treatments. Rosiglitazone and pioglitazone resulted in a similar improvement in HbA1c and insulin sensitivity. Fasting total cholesterol increased more after rosiglitazone compared with pioglitazone (p = 0.04), whereas triglycerides decreased after pioglitazone and increased after rosiglitazone (p = 0.004). Fasting LDL size similarly increased after both treatments, mainly due to increased LDL-I particles. Pioglitazone resulted in a more prominent LDL-IIA subfraction compared with rosiglitazone (p = 0.03). After 3 h, pioglitazone lead to increased LDL-IIB (p = 0.01) and decreased LDL-IVB (p = 0.05), however, after 6 h no significant changes were found. Conclusions: Pioglitazone was more effective than rosiglitazone in increasing larger LDL concentrations (in both fasting and postprandial status) as well as in reducing levels of atherogenic small, dense particles (in postprandial status only). Whether or not these findings differentially affect the atherogenic process and the clinical endpoints such as cardiovascular events remains to be determined.

Gender-related effect of apo E polymorphism on lipoprotein particle sizes in the middle-aged subjects

Objectives We determined the frequencies of apolipoprotein E (apo E) alleles and examined the effect of apo E polymorphism on lipoprotein particle sizes in Serbian healthy, middle-aged individuals. Design and methods We performed apo E phenotype by immunobloting method in 183 men and 143 women (mean years: 56.3 ± 10.60 and 54.9 ± 10.31, respectively). Plasma lipid and apolipoprotein levels were measured by routine laboratory methods. LDL and HDL particle sizes were determined by nondenaturing polyacrylamide gradient (3–31%) gel electrophoresis. Results The apo E allele frequencies were ε2—4.9%, ε3—86.5%, and ε4—8.6%. Men with ε4 allele had lower HDL-C and Apo AI concentrations than ε3 men. The ε2 allele men had the smallest LDL particles, highest percent of subjects with LDL phenotype B and highest TG/HDL-C ratio. Women with ε2 allele had lowest concentration of apo B. The ε4 allele women had smallest HDL particles and highest percent of the subjects with small-sized HDL phenotype. Conclusions This study showed gender-related effect of apo E polymorphism on lipoprotein particle size. In men, possession of the ε2 allele is associated with small LDL particles, whereas in women, ε4 allele is associated with small HDL particles. Differences in gender-related influence of apo E polymorphism on LDL and HDL particle sizes could be clinically useful in strategy for reduction of coronary disease risk in middle-aged men and women.

Post-prandial alterations in LDL size and subclasses in patients with growth hormone deficiency

ObjectiveSeveral studies have suggested that lipoproteins generated during the post-prandial phase are highly atherogenic, with modifications in low-density lipoproteins (LDL) size and density. In the present study we assessed post-prandial variations in LDL size and subclasses in patients with growth hormone deficiency (GHD). DesignWe studied in 12 hypopituitary patients with GHD and 10 healthy control subjects matched for gender, age and body mass index (BMI) post-prandial variations after a standardized meal consisting of 35% fat, 45% carbohydrate and 20% of protein (Clinutren Mix®, Nestlé) and containing calories corresponding to 1/3 of estimated basal metabolic rate. Blood samples were collected at baseline and after 2 and 4h to measure plasma lipids and LDL size and subclasses by nondenaturing polyacrylamide gradient gel electrophoresis. ResultsAt baseline patients had similar plasma lipids than controls, with the exception of higher triglycerides (1.2±0.8 vs. 0.7±0.4mmol/L, p=.0024). Baseline LDL size was similar between the two groups and LDL subclass analysis revealed a small increase in LDL-IIIA (p=.0046). During post-prandial phase no significant differences were found in LDL size and subclasses in patients vs. controls with the sole exception of increased levels of LDL-IVB after 2h (p=.0295) and LDL-IIIB after 4h (p=.0478). ConclusionsIt is, therefore, unlikely that a post-prandial variation in levels of small, dense LDL may significantly contribute to the atherogenic potential in hypopituitary patients with GHD.

Comparison of a dietary portfolio diet of cholesterol-lowering foods and a statin on LDL particle size phenotype in hypercholesterolaemic participants

The effect of diet v. statins on LDL particle size as a risk factor for CVD has not been examined. We compared, in the same subjects, the impact of a dietary portfolio of cholesterol-lowering foods and a statin on LDL size electrophoretic characteristics. Thirty-four hyperlipidaemic subjects completed three 1-month treatments as outpatients in random order: a very-low saturated fat diet (control); the same diet with 20 mg lovastatin; a dietary portfolio high in plant sterols (1 g/4200 kJ), soya proteins (21.4 g/4200 kJ), soluble fibres (9.8 g/4200 kJ) and almonds (14 g/4200 kJ). LDL electrophoretic characteristics were measured by non-denaturing polyacrylamide gradient gel electrophoresis of fasting plasma at 0, 2 and 4 weeks of each treatment. The reductions in plasma LDL-cholesterol levels with the dietary portfolio and with statins were comparable and were largely attributable to reductions in the estimated concentration of cholesterol within the smallest subclass of LDL (portfolio - 0.69 (se 0.10) mmol/l, statin - 0.99 (se 0.10) mmol/l). These were significantly greater (P < 0.01) than changes observed after the control diet ( - 0.17 (se 0.08) mmol/l). Finally, baseline C-reactive protein levels were a significant predictor of the LDL size responsiveness to the dietary portfolio but not to the other treatments. The dietary portfolio, like the statin treatment, had only minor effects on several features of the LDL size phenotype, but the pronounced reduction in cholesterol levels within the small LDL fraction may provide additional cardiovascular benefit over the traditional low-fat diet of National Cholesterol Education Program Step II.

Abstract 4161: Cholesterol Levels in Different LDL Subclasses and CHD Incidence in Apparently Healthy Men and Women: The EPIC-Norfolk Prospective Population Study

Background: Measurement of cholesterol levels in small LDL particles represents a novel metabolic marker that might add to the coronary risk prediction attributed to LDL cholesterol and LDL particle size taken separately. We examined, in a nested case-control study performed in the prospective EPIC-Norfolk cohort, the association of LDL cholesterol in small LDL particles to future CHD in apparently healthy men and women aged 45 to 79 years who developed fatal or nonfatal CHD over 10-year follow-up. Methods and results: Cases (n=1035) were matched to 1920 controls who remained free of CHD, by age, gender and enrollment time. Electrophoretic characteristics of LDL particles were measured using 2–16% nondenaturing polyacrylamide gradient gel electrophoresis. Cholesterol levels in small LDL particles (&lt;255 Å) were higher in cases than in controls (1.26 ± 0.87 vs. 1.07 ± 0.78 mmol/l, P&lt;0.001) Participants in the top tertile of cholesterol concentrations in small LDL particles were characterized by the highest waist circumference, by the highest triglyceride, C-reactive protein and apolipoprotein B levels and by the lowest HDL cholesterol concentrations. These participants had an unadjusted odds ratio (OR) for future CHD of 1.72 (95% CI, 1.42 to 2.09; P&lt;0.001) which remained significant after adjustment for classical risk factors such as age, smoking, diabetes, body mass index, systolic blood pressure and LDL cholesterol concentrations (OR, 1.43; 95% CI, 1.16 to 1.76; P=0.001). However, after further adjustments for triglyceride and HDL cholesterol levels, the association between cholesterol in small LDL particles and CHD was no longer significant (OR, 1.18; 95% CI, 0.95 to 1.48; NS). Conclusions: Cholesterol concentrations in different LDL subclasses show different relationships with CHD incidence in a European cohort representative of a contemporary Western population.

Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (+/-SD) 42.1 +/- 9.5 years, body mass index 29.8 +/- 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means +/- SD, 1.22 +/- 0.49 mg/kg/day vs. 1.15 +/- 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 +/- 0.13 pool/day vs. 0.61 +/- 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 +/- 0.12 mg/kg/day) and a 60% lower FCR (0.21 +/- 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL <255 A (r = 0.72, P = 0.002). These results suggest that all apoC-III isoforms, especially the predominant CIII1 and CIII2 isoforms, contribute to hypertriglyceridemia and that apoC-III2 may play a significant role in the expression of the small, dense LDL phenotype.

Association of heterozygous familial hypercholesterolemia with smaller HDL particle size

Small, dense HDL particles have been associated with factors known to increase the risk of cardiovascular disease, such as obesity, hypertriglyceridemia, small dense LDL particles, decreased HDL-cholesterol levels and increased apoA-I fractional catabolic rate from plasma. In order to assess the potential contribution of HDL particle size to atherosclerosis in heterozygous familial hypercholesterolemia (FH), we examined the electrophoretic characteristics of HDL particles in a large cohort of well defined FH heterozygotes and controls. A total of 259 FH heterozygotes and 208 controls participated in the study. FH subjects were carriers of one of the nine French Canadian mutations in the LDL receptor gene. All subjects were apoE3 homozygotes. HDL particles were characterized by non-denaturing polyacrylamide gradient gel electrophoresis following a 6-week lipid-lowering drug-free baseline period. The integrated HDL size was significantly smaller in the FH group compared to controls (FH = 87.3 ± 5.2 Å versus controls = 91.6 ± 4.9 Å, P < 0.0001). In each groups, men had smaller HDL particles than women. Multiple regression linear analyses showed that the FH/Control status accounted for 20.3% of the variance in the integrated HDL size. These results suggest that the FH/control status was independently associated with variations in HDL particle size and that these variations could contribute to the development of premature atherosclerosis in these patients.

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.

Small high density lipoprotein subclasses: some of their physico-chemical properties and stability in solution.

Small high density lipoproteins (SHDL) contribute to the protection from atherosclerosis, but detailed information about their properties is not available yet. We isolated four of the smallest HDL subclasses that contain apoA-I alone, the small lipoprotein A-I (SLpAI), by their separation on gradient polyacrylamide gel followed by electroelution. Their physico-chemical properties were calculated from their displacement in non-denaturing gradient polyacrylamide gel under the effect of electrical potential. The properties are: Stokes' radii 2.96-3.56 nm; molecular masses 42-70 kDa; net negative charge 7.2-13.5; surface charge densities 3139-4069 -esu.cm(-2); surface potentials 10.6-15.7 -mV; coefficients of friction 5.74-6.90 x 10(-8) g.s(-1); and diffusion coefficients 5.76-6.94 x 10(-7) cm(2).s(-1). We found that these particles were of low stability as they underwent molecular transformation into larger particles on storage. The estimated dimensions of these particles do not support ellipsoidal shape, therefore, the most probable shape is spherical; consequently, their hydrated characteristics were estimated. We conclude that these particles have high values of negative surface charge and diffusion coefficients, and are of low stability. Their small Stokes' radii were similar to each other and they are spherical and highly hydrated.

Screening for Dysbetalipoproteinemia by Plasma Cholesterol and Apolipoprotein B Concentrations

Dysbetalipoproteinemia (type III hyperlipidemia) is a highly atherogenic mixed hyperlipidemia characterized by the accumulation of remnants of triglyceride-rich lipoproteins (chylomicrons and VLDL) (1). The binding of these remnants to hepatic lipoprotein receptors is mediated by apolipoprotein E (apoE). At the apoE gene locus, there are three common alleles: e2, e3, and e4 (2). ApoE2 binds poorly to hepatic lipoprotein receptors, leading to impaired remnant clearance. Consequently, remnants become enriched with cholesterol and migrate abnormally on electrophoresis. More than 90% of dysbetalipoproteinemic patients are homozygous for apoE2, but only a minority (1 in 20) of apoE2 homozygotes will be overtly hyperlipidemic. Remnant accumulation sufficient to cause hyperlipidemia usually occurs only when a second metabolic hit increases lipoprotein production (e.g., diabetes) or further decreases remnant clearance (e.g., hypothyroidism) (3). Dysbetalipoproteinemia is highly atherogenic but responds well to lifestyle changes and lipid-modifying medications. Genetic counseling is important, particularly in areas where there is a high local prevalence of autosomal dominant apoE mutations (4)(5). Dysbetalipoproteinemia is often suspected when both the total cholesterol (TC) and triglyceride (TG) concentrations are increased and the TC/TG molar ratio approximates 2:1. Clinically, pathognomonic palmar crease xanthomata are found in only ∼20% of patients (4)(6)(7). There are no simple diagnostic tests for dysbetalipoproteinemia. Diagnostic tests are based either on the demonstration of remnant accumulation or characterization of apoE. Electrophoretic techniques include serum agarose gel electrophoresis, but a broad β band is found in less than one half of patients (8)(9). Ultracentrifugation is required to demonstrate β-migrating VLDL. Nondenaturing polyacrylamide gradient gel electrophoresis is an effective screening technique (9). Cholesterol-enriched VLDL is diagnostic of dysbetalipoproteinemia (10)(11)(12), but sample preparation requires ultracentrifugation, which is not available in routine diagnostic laboratories. ApoE phenotyping requires isoelectric focusing, and genotyping is …

Physiological importance of SR-BI in the in vivo metabolism of human HDL and LDL in male and female mice

The physiological role of murine scavenger receptor class B type I (SR-BI) was evaluated by in vivo clearances of human HDL3 and LDL in normal and SR-BI knockout (KO) mice. In normal mice, cholesteryl esters (CEs) were removed faster than proteins, indicating a selective uptake process from both HDL3 and LDL. SR-BI KO mice showed 80% losses of HDL-CE selective uptake and the complete loss of LDL-CE selective uptake in the first phase of clearance. However, the second phase was characterized by an acceleration of CE disappearance in SR-BI KO mice. Thus, SR-BI is the only murine receptor mediating HDL-CE selective uptake, whereas a SR-BI-independent pathway specific to LDL can rescue SR-BI deficiency. The analysis of LDL recovered 3 h after injection in mice from different genotypes revealed that LDLs are significantly depleted in CE (reduction from 19% to 50% of the CE/protein ratios). A smaller LDL size in comparison with that of noninjected LDL was also detectable but was more evident for LDL recovered from normal mice. All LDL preparations migrate faster than noninjected LDL on agarose-barbital gels. Thus, both SR-BI-dependent and -independent pathways lead to substantial changes in LDL.

Rapid, simple laser-light-scattering method for HDL particle sizing in whole plasma.

HDL particles are composed of an outer layer of phospholipids and free cholesterol surrounding a hydrophobic core that consists primarily of cholesterol esters and small amounts of triglycerides (1). Apolipoprotein A-I (apo A-I) accounts for ∼60% of the protein content of HDL. Other apolipoprotein species found in HDL particles include apo A-II, apo A-IV, apo CI, apo CIII, and apo E. Several subfractions of HDL have been identified on the basis of density, electrophoretic mobility, particle size, and apolipoprotein composition (2). Differences in particle size are ascribed mainly to the number of apolipoprotein molecules on the particle surface and the amounts of cholesterol esters in the core (1)(2). Furthermore, there is growing evidence suggesting that most of the cardioprotective properties of HDL are associated with the HDL2 fraction (larger particles) rather than the HDL3 fraction (smaller particles) in patients with coronary artery disease (3)(4), in postmenopause women (5), in diabetes (6), and in patients with familial hypercholesterolemia (9). Several methods, such as sequential ultracentrifugation, chemical precipitation, immunoaffinity chromatography, and nondenaturing polyacrylamide gradient gel electrophoresis, have been used to separate HDL subfractions (8). The sizes of the subfractions have been estimated by nondenaturing polyacrylamide gradient gel electrophoresis (9) and, more recently, by nuclear magnetic resonance spectroscopy (10). In general, these procedures are either laborious or expensive. Laser-light scattering (LLS) has been used in the measurement of LDL particle sizes after isolation of this fraction by ultracentrifugation (11). There is great similarity between the data obtained with this approach and those obtained with the nondenaturing polyacrylamide gradient gel electrophoresis method ( P <0.0001; r = 0.78). To date, however, LLS has not been used to perform HDL sizing. HDL sizing by LLS can be performed after chemical precipitation of the apo B-containing lipoprotein (8). This approach …