Zonal Ultracentrifugation Research Articles

High-density Lipoprotein-cholesterol Subfractions and Coronary Artery Calcium: The ELSA-Brasil Study

Although elevated high-density lipoprotein cholesterol (HDL-C) is considered protective against atherosclerotic cardiovascular disease, no causal relationship has been demonstrated. HDL-C comprises a group of different subfractions that might have different effects on atherosclerosis. Our objective was to investigate the association between HDL-C subfractions with the coronary artery calcium (CAC) score. We included 3,674 (49.8±8.3years, 54% women) participants from the ELSA-Brasil study who had no prior history of CVD and were not currently using lipid-lowering medications. We measured the fasting lipoprotein cholesterol fractions (in mmol/l) by a zonal ultracentrifugation method (VAP). We analyzed the independent predictive values of total HDL-C, HDL2-C, and HDL3-C subfractions and in the HDL2-C/HDL3-C ratio using linear regression to predict Ln(CAC+1) and logistic regression to predict the presence of CAC. Overall 912 (24.8%) of the participants had CAC>0, and 294 (7.7%) had CAC>100. The mean total HDL-C, HDL2-C, and HDL3-C were: 1.42±0.37, 0.38±0.17 and 1.03±0.21mmol/l, respectively. Individuals with CAC>0 had lower levels of total HDL-C as well as of each subfraction (p<0.001). When adjusted for age, gender, smoking, hypertension, alcohol use, physical activity, and LDL-C, we observed an inverse association between HDL-C and its subfractions and CAC (p<0.05). However, by adding triglycerides in the adjustment, neither total HDL-C nor its subfractions remained independently associated with the presence or extent of CAC. In this cross-sectional analysis, neither the total HDL-C nor its subfractions (HDL2-C and HDL3-C, as well as HDL2-C/HDL3-C ratio) measured by VAP are independently associated with the presence or extent of coronary calcification.

Abstract P198: Diabetes Alters the Association Between High-density Lipoprotein Subfractions and Carotid Intima-media Thickness: The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)

Background: Although HDL-C is inversely associated with cardiovascular risk, no causal relationship has been demonstrated. Since HDL comprises a group of heterogeneous subfractions, they might have different effects on atherosclerosis, and its effects may be modified by the presence of diabetes mellitus. Hypothesis: HDL-C subfractions exert different effects on subclinical carotid atherosclerosis, and diabetes modifies these effects. Methods: We evaluated 3,930 individuals between 35 and 74 years (45.6% men) without previous cardiovascular disease not using lipid-lowering drugs. HDL 2 -C and HDL 3 -C were obtained by zonal ultracentrifugation. The relationships between each HDL-C subfraction and cIMT were analyzed by multiple linear regression models. Results: The mean cIMT was 0.796±0.195 mm, the mean total HDL-C was 55.1±14.4 mg/dl, HDL 2 -C was 14.9±6.7, and HDL 3 was 40.1±8.4 mg/dl. Total HDL-C and subfractions, as well as HDL 2 -C/HDL 3 -C ratio were negatively associated with cIMT after adjustment for race/ethnicity, age, and sex (all p&lt;0.001) and further for smoking status, alcohol use, physical activity, LDL-C, systolic blood pressure, waist circumference, fasting serum glucose, body mass index, triglycerides and antihypertensive use (HDL-C: p = 0.003, HDL 2 -C: p = 0.01; HDL 3 -C: p = 0.003; HDL 2 -C/HDL 3 -C ratio: p = 0.02). When stratified by diabetes status, both HDL 2 -C (p=0.03) and HDL 2 -C/HDL 3 -C ratio (p= 0.01) showed a negative association with cIMT in people with diabetes (n=531) after adjusting for confounding variables. These associations were not observed in individuals without diabetes (p = 0.11 and p = 0.30, respectively). Conclusion: HDL 2 -C and HDL 3 -C subfractions, as well as the HDL 2 /HDL 3 -C ratio, are inversely associated with cIMT after adjustment for traditional risk factors. This association of HDL 2 -C/HDL 3 -C ratio and HDL 2 -C is modified by the presence of diabetes, where it is more pronounced.

Relationship between lipoprotein subfraction cholesterol and residual risk for cardiovascular outcomes: A post hoc analysis of the AIM-HIGH trial

The AIM-HIGH (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides and Impact on Global Health Outcomes) trial failed to demonstrate incremental clinical benefit of extended-release niacin (ERN) in 3414 statin-treated patients with established cardiovascular (CV) disease who had low baseline levels of high-density lipoprotein cholesterol (HDL-C) as compared to placebo. A previous secondary analysis suggested that ERN provided outcome benefits in ERN-treated patients with high triglycerides (TGs; >200mg/dL) and very low HDL-C (<32mg/dL) at baseline. The current analysis sought to ascertain how changes in TG-enriched lipoproteins and HDL subfractions impact residual risk in the comparator treatment arms. We evaluated the relationship between niacin treatment, lipoproteins and their subfractions, and CV outcomes in a non-prespecified, post hoc analysis of the AIM-HIGH trial. Lipoprotein subfraction analysis was performed with zonal ultracentrifugation in 2457 AIM-HIGH participants at baseline and 1year of treatment. Hazard ratios were estimated using Cox proportional hazards models for relationships between lipoproteins and the composite primary endpoint of CV death, myocardial infarction, acute coronary syndrome, ischemic stroke, or symptom-driven revascularization. Analyses were performed for the entire cohort and in participants with TGs > 200mg/dL and HDL-C < 32mg/dL. Apoprotein B-containing lipoproteins and their subfractions decreased significantly in both treatment arms but decreased more with ERN treatment. HDL-C and its subfractions increased significantly in both treatment groups, but more so in patients treated with ERN. For the entire study population, neither apoB- nor apoA1-containing lipoprotein subfractions predicted risk at baseline or at 1year of follow-up. In the high TG and low HDL-C subgroup treated with placebo, changes at 1year in HDL2-C, total cholesterol/HDL2-C, and non-HDL-C/HDL2-C may be associated with increased CV events, whereas in the ERN treatment arm, changes at 1year in very low-density lipoprotein cholesterol and very low-density lipoprotein subfractions, total remnant lipoproteins, and various risk ratios may be associated with increased CV events, while HDL2-C may be associated with reduced risk. We provide hypothesis-generating findings that ERN may confer benefit in patients with coronary heart disease who have high TGs and low HDL by reducing serum levels of remnant lipoprotein cholesterol and increasing HDL2-C.

Separation of double-wall carbon nanotubes by electronic type and diameter.

We introduce a new procedure for the efficient isolation and subsequent separation of double-wall carbon nanotubes (DWCNTs). A simplified, rate zonal ultracentrifugation (RZU) process is first applied to obtain samples of highly-enriched DWCNTs from a raw carbon nanotube material that has both single- and double-wall carbon nanotubes. Using this purified DWCNT suspension, we demonstrate for the first time that DWCNTs can be further processed using aqueous two-phase extraction (ATPE) for sequential separation by electronic structure and diameter. Additionally, we introduce analytical ultracentrifugation (AUC) as a new method for DWCNT characterization to assess DWCNT purity in separated samples. Results from AUC analysis are utilized to compare two DWCNT separation schemes. We find that RZU processing followed by sequential bandgap and diameter sorting via ATPE provides samples of highest DWCNT enrichment, whereas single-step redox sorting of the same raw material through ATPE yields SWCNT/DWCNT mixtures of similar diameter and electronic character. The presented methods offer significant advancement in DWCNT processing and separation while also providing a promising alternative for DWCNT sample analysis.

Immunological and biochemical characterizations of coxsackievirus A6 and A10 viral particles

Childhood exanthema caused by different serotypes of coxsackievirus (CV-A) and enterovirus A71 (EV-A71) has become a serious global health problem; it is commonly known as hand, foot, and mouth disease (HFMD). Current EV-A71 vaccine clinical trials have demonstrated that human antibody responses generated by EV-A71 vaccinations do not cross-neutralize coxsackievirus A16 (CV-A16). An effective multivalent HFMD vaccine is urgently needed. From molecular epidemiological studies in Southeast Asia, CV-A6 and CV-A10 are commonly found in HFMD outbreaks. In this study, CV-A6 and CV-A10 were individually cultured in rhabdomyosarcoma (RD) cells grown in medium containing serum, harvested and concentrated. In viral downstream purification, two viral fractions were separated by sucrose gradient zonal ultracentrifugation and detected using a SDS-PAGE analysis and a virus infectivity assay. These two viral fractions were formalin-inactivated, and only the infectious particle fraction was found to be capable of inducing CV-A serotype-specific neutralizing antibody responses in animal immunogenicity studies. These mouse and rabbit antisera also failed to cross-neutralize EV-A71 and CV-A16 infections. Only a combination of formalin-inactivated EV-A71, CV-A6, CV-A10 and CV-A16 multivalent vaccine candidates elicited cross-neutralizing antibody responses in both mouse and rabbit immunogenicity studies. The current results certainly provide important information for multivalent HFMD vaccine development.

Efficient chromatographic reduction of ovalbumin for egg-based influenza virus purification

Vaccination is the most effective prevention strategy to avoid influenza infection and for protection of large populations. The vast majority of influenza vaccines are still produced with allantoic fluid from fertilized chicken eggs. The presence of ovalbumin, which can constitute over 60% of the total protein content in allantoic fluid, can result in severe allergies. Consequently, efficient reduction of ovalbumin is critical during egg based vaccine manufacturing. Here we present Capto Core 700, a novel core bead chromatographic flow through mode resin for removal of ovalbumin and compare it to sucrose zonal gradient ultracentrifugation, which is the industry standard for egg-based vaccine production. The results demonstrate that core bead chromatography is fully comparable to zonal centrifugation in removing ovalbumin to meet regulatory requirements. Furthermore, the scalability and the shorter process times of this method have the potential to significantly improve the productivity and economy for industrial production compared to zonal centrifugation.

Immunological and biochemical characterization of coxsackie virus A16 viral particles.

BackgroundCoxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.Principal FindingIn this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >106 the tissue culture's infectious dose (TCID50) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10−5 was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24–28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35–38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176–190.ConclusionThese results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

BackgroundEnterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.Principal FindingIn this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10−5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225).ConclusionThese results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.

Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient

Ultracentrifugation in sucrose density gradient remains the most commonly used technique for hRSV purification. However, the high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus leading to loss of infectivity. To overcome these limitations, an alternative purification technique was developed using iodixanol as gradient medium, incorporating MgSO4 as a stabilizing agent and EDTA to disaggregate the virus prior to infectivity assay. Virus particles were banded at the 20–36% interface after purification of polyethylene glycol-concentrated viruses by rate zonal ultracentrifugation on a 20–52% discontinuous iodixanol gradient. The presence of the virus was confirmed by viral fusion glycoprotein content using ELISA. After further purification by buoyant density ultracentrifugation on a 20–52% continuous gradient, the virus was recovered in the region of density 1.15–1.19g/ml and this was confirmed by the coincidence of the infectivity titre, viral genome and fusion glycoprotein peaks. Analysis of recovery rates showed that the use of iodixanol increased the virus yield up to 69%. Iodixanol was also found to be non-toxic to HeLa cells used in infectivity assay, eliminating the need of its downstream removal by dialysis.

Complexation of integral membrane proteins by phosphorylcholine-based amphipols

Amphiphilic macromolecules, known as amphipols, have emerged as promising candidates to replace conventional detergents for handling integral membrane proteins in water due to the enhanced stability of protein/amphipol complexes as compared to protein/detergent complexes. The limited portfolio of amphipols currently available prompted us to develop amphipols bearing phosphorylcholine-based units (PC). Unlike carboxylated polymers, PC-amphipols remain soluble in aqueous media under conditions of low pH, high salt concentration, or in the presence of divalent ions. The solubilizing properties of four PC-amphipols were assessed in the case of two membrane proteins, cytochrome b 6 f and bacteriorhodopsin. The protein/PC-amphipol complexes had a low dispersity in size, as determined by rate zonal ultracentrifugation. Short PC-amphipols (< M>≈ 22 kDa) of low dispersity in length, containing ∼ 30 mol% octyl side groups, ∼ 35 mol% PC-groups, and ∼ 35 mol% isopropyl side groups, appeared best suited to form stable complexes, preserving the native state of BR over periods of several days. BR/PC-amphipol complexes remained soluble in aqueous media at pH ≥ 5, as well as in the presence of 1 M NaCl or 12 mM calcium ions. Results from isothermal titration calorimetry indicated that the energetics of the conversion of BR/detergent complexes into BR/amphipol complexes are similar for PC-amphipols and carboxylated amphiphols.

Purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation

Sucrose equilibrium density ultracentrifugation remains the most widely used technique for retrovirus purification. However, purified virus preparations obtained by this routine method usually contain considerable amounts of contaminating cell membrane vesicles. In addition, sucrose solutions are highly viscous and hyperosmotic which jeopardizes the integrity and functionality of the retrovirus particle. In order to overcome these limitations, an alternative purification technique using rate zonal ultracentrifugation and iodixanol as gradient medium was developed. Recombinant retrovirus particles were produced by 293-GPG packaging cells grown in suspension in the presence of 10% FBS. Concentrated supernatants were purified by rate zonal sedimentation on a 10–30% continuous iodixanol gradient. Virus particles were recovered intact and active from the central fractions of the gradient. By using this strategy, high levels of purification were achieved, with no evident contamination with cell membrane vesicles as indicated by subtilisin treatment studies. The level of purity of the retrovirus preparation is over 95% as shown by SDS-PAGE analysis and size-exclusion chromatography. Purified particles appear homogenous in size and morphology according to negative stain electron microscopy. In addition, large amounts of defective retrovirus particles produced by 293-GPG packaging cells can be separated from functional retrovirus particles using this purification strategy.

Partial fractionation of wheat starch amylose and amylopectin using zonal ultracentrifugation

A novel technique for partial separation of amylose and amylopectin using zonal ultracentrifugation is introduced, supported by analytical ultracentrifuge measurements. Starch was first solubilised in 90% DMSO and 5% sucrose and then layered onto a gradient forming solution of 10–30% sucrose in 90% DMSO, with a cushion made up of 36% caesium chloride and 25% sucrose in water. Solutions were then run in a preparative ultracentrifuge at a rotor speed of 14000 rpm for approximately 25 min. The results for wheat starch showed that this method produced highly purified amylopectin in both soluble and highly aggregated forms, and at least partial purification of the amylose fractions.

Omacor in familial combined hyperlipidemia: effects on lipids and low density lipoprotein subclasses

Elevations of plasma cholesterol and/or triglycerides, and the prevalence of small, dense LDL particles remarkably increase coronary risk in patients with familial combined hyperlipidemia (FCHL). A total of 14 FCHL patients were studied, to investigate the ability of Omacor, a drug containing the n-3 fatty acids eicosapentaenoic and docosahexaenoic acid (EPA and DHA), to favorably correct plasma lipid/lipoprotein levels and LDL particle distribution. The patients received four capsules daily of Omacor (providing 3.4 g EPA+DHA per day) or placebo for 8 weeks in a randomized, double-blind, cross-over study. Omacor significantly lowered plasma triglycerides and VLDL-cholesterol levels, by 27 and 18%, respectively. Total cholesterol did not change but LDL-cholesterol and apolipoprotein B (apoB) concentrations increased by 21 and 6%. As expected, LDL particles were small (diameter=24.9±0.3 nm) and apoB-rich (LDL-cholesterol/apoB ratio=1.27±0.26) in the selected subjects. After Omacor treatment LDL became enriched in cholesterol (LDL-cholesterol/apoB ratio=1.40±0.17), mainly cholesteryl esters, indicating accumulation in plasma of more buoyant and core enriched LDL particles. Indeed, the separation of LDL subclasses by rate zonal ultracentrifugation showed an increase of the plasma concentration of IDL and of the more buoyant, fast floating LDL-1 and LDL-2 subclasses after Omacor, with a parallel decrease in the concentration of the denser, slow floating LDL-3 subclass. However, the average LDL size did not change after Omacor (25.0±0.3 nm). The resistance of the small LDL pattern to drug-induced modifications implies that a maximal lipid-lowering effect must be achieved to reduce coronary risk in FCHL patients.

LDL particle size: an important drug target?

Low density lipoprotein (LDL) is the main carrier of plasma cholesterol and a major component of atherosclerotic plaque [1]. Lowering LDL cholesterol reduces coronary events and mortality from coronary artery disease (CAD) [2–4], however, the relation between LDL cholesterol concentration and (CAD) is complex. Many patients with CAD have plasma LDL cholesterol concentrations in the normal rangefor the general population [5]. Thus, it could be that coronary risk goes beyond LDL cholesterol concentration to the characteristics of the LDL particles themselves. The purpose of this communication is to address the issue of whether LDL particle size and density influences its atherogenecity and how this might be modified by drug therapy.

O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2 Species Formed Early during Biosynthesis Are Insoluble and Contain Nonreducible Intermolecular Bonds

The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857-18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L. -A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.

Effects of pancreas transplantation on distribution and composition of plasma lipoproteins

In type I (insulin-dependent) diabetic patients, peripheral hyperinsulinemia due to subcutaneous insulin treatment is associated with increased high-density lipoprotein (HDL) cholesterol, and also with an altered surface composition of HDL. Pancreas grafts also release insulin into the systemic rather than into the portal venous system, giving rise to pronounced peripheral hyperinsulinemia. We hypothesized that if peripheral hyperinsulinemia is responsible for high HDL cholesterol and/or altered surface composition of HDL in diabetic subjects, similar changes in the lipid profile should be present in pancreas-kidney transplant recipients (PKT-R). Using zonal ultracentrifugation, we isolated HDL 2, HDL 3, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) from fasting plasma of 14 type I diabetic PKT-R, eight nondiabetic kidney transplant recipients (KT-R), and 14 healthy control subjects and determined the level and composition of the above lipoproteins. HDL 2 cholesterol was increased in PKT-R as compared with KT-R and healthy controls (both P < .05), whereas HDL 3 cholesterol was unchanged. However, an altered lipoprotein surface composition was evident in PKT-R: HDL 2, HDL 3, and LDL were enriched in unesterified cholesterol ([UC] PKT-R v KT-R, P = .13, P < .005, and P < .05, respectively; PKT-R v controls, all P < .005); HDL 2 was enriched in phospholipids; and LDL was depleted of phospholipid. KT-R, in contrast, showed no changes in lipoprotein surface composition but a substantial triglyceride enrichment of HDL 2 as compared with PKT-R and healthy controls (both P < .05). LDL size as determined by gradient gel electrophoresis was increased in PKT-R compared with controls ( P < .005). The plasma concentration of cholesteryl ester (CE) transfer protein (CETP), involved also in phospholipid transfer, was increased in both transplant groups compared with healthy controls (both P < .05). Insulin concentrations in fasting plasma were directly related to CETP levels and to the weight-percentage of UC in HDL 3, and inversely to the weight-percentage of phospholipids in LDL (all P < .05). We explain the increase in HDL 2 cholesterol and LDL size in PKT-R by their high lipoprotein lipase (LPL) activity conferring an excellent capacity to clear chylomicron triglycerides. Effective handling of postprandial triglycerides, high HDL 2 cholesterol, and predominance of LDL pattern A, respectively, are established indicators of a low risk of atherosclerosis. However, it is presently unclear what effects the compositional changes on the surface of HDL and LDL may have on cardiovascular risk in clinically stable PKT-R.

Slowß-migrating lipoprotein: An atherogenic subclass of low density lipoproteins

Objective: To clarify the possibility that midband Lp in LDL fractions might act as an atherogenic lipoprotein in their interaction with macrophages. Design and Methods: Low density lipoproteins (LDL) isolated by zonal ultracentrifugation from midband lipoprotein-positive serum in type IIb hyperlipidemics were subjected to polyacrylamide gel disc electrophoresis. Results: A part of midband lipoprotein was observed between preβ- and β-band, in addition to the main β-band. We named this midband lipoprotein “slowβ-migrating Lp (slowβ-Lp).” The larger LDL subtraction from midband-lipoprotein positive serum on Sepharose 2B column chromatography contained much slowβ-Lp, named slowβ-Lp-rich LDL. The smaller LDL subfraction contained a little slowβ-Lp, named slowβ-Lp-poor LDL. Slowβ-Lp-rich LDL had similar composition to the control LDL except for apolipoprotein E. The uptake of [ 3H]cholesteryl linoleate-labeled slowβ-Lp-rich LDL by J774 macrophages was higher than that of control LDL. The cholesterol ester content of J774 macrophages incubated with slowβ-Lp-rich LDL increased significantly compared with slowβ-Lp-poor LDL, β-VLDL, and control LDL. Conclusion: These results suggest that slowβ-Lp- in type IIb might generate foam cells from macrophages in atherosclerotic lesions.

Effects of molecular crowding on protein self-association: a potential source of error in sedimentation coefficients obtained by zonal ultracentrifugation in a sucrose gradient.

Theoretical and experimental studies have illustrated a potential source of error in sedimentation coefficients obtained by sucrose density gradient centrifugation of proteins undergoing reversible self-association. The error stems from the excluded volume (molecular crowding) effect of the sucrose on the activity coefficients of monomeric and polymeric states. The consequent displacement of the equilibrium position in favor of polymeric state(s) is a function of sucrose concentration, and can therefore result in failure to detect the equilibrium coexistence of monomer if 5% sucrose suffices to displace the equilibrium completely toward dimer. In less extreme situations, it may result in the evaluation of an average sedimentation coefficient whose magnitude is a function of sucrose concentration and hence of the distance migrated into the sucrose gradient. These features are illustrated by the results of computer-simulated sedimentation of reversibly dimerizing systems in a sucrose gradient, and by conventional sedimentation velocity experiments on yeast enolase.

Levels and physicochemical properties of lipoprotein subclasses in moderate hypertriglyceridemia.

Moderate hypertriglyceridemia is associated with several abnormalities of the plasma lipoprotein particles and it may be a risk factor for atherosclerotic vascular diseases. Plasma lipid, lipoprotein and apolipoprotein levels, as well as lipoprotein composition and physical properties, were examined by ultracentrifugation in a zonal rotor and by gradient gel electrophoresis in 14 patients with moderate hypertriglyceridemia (plasma triglycerides 4.00 +/- 0.32 mmol/l, mean +/- S.D.) and in 14 control subjects. Based on zonal ultracentrifugation hypertriglyceridemic patients have higher levels of cholesterol in all VLDL subclasses (Sf > 200, 100-200, 60-100 and 20-60), in IDL and in small and dense LDL. Both HDL2 and HDL3 cholesterol levels are reduced. The LDL flotation rate is inversely related to plasma triglyceride levels, thus indicating that the higher the plasma triglycerides the smaller and/or denser the LDL are. The triglyceride percent content of LDL2 and HDL3 is increased, while that of esterified cholesterol is reduced in hypertriglyceridemic patients. Gradient gel electrophoresis shows that the LDL peak size is lower (25.2 +/- 0.5 nm, mean +/- S.D.) in hypertriglyceridemic than in control subjects (27.1 +/- 0.4 nm; P < 0.0001). Considering both hypertriglyceridemic and control subjects the LDL peak effluent volume from the zonal rotor (which reflects the LDL flotation rate) is inversely related to the LDL peak size determined by gradient gel electrophoresis (r = -0.71; P = 0.0006) and the plasma triglyceride levels are related to LDL peak effluent volume (r = 0.74; P = 0.0002) and to LDL peak size (r = -0.95; P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein(a) is present in the triglyceride-rich fraction in type IV hypertriglyceridemia

This study was designed to quantify the levels of apo(a) and assess their distribution among lipoprotein fractions in type IV hypertriglyceridemia. Plasma density < 1.006 g/mL fraction (VLDL) was obtained by preparative and zonal ultracentrifugation. Apo(a) was detected by immunoblotting with anti-apo(a) antibodies of agarose gel electrophoresed lipoproteins. Apo(a) was consistently found in VLDL in 30 hypertriglyceridemic subjects, but not in 10 normolipidemic or 10 hypercholesterolemic subjects with normal triglyceride levels. Apo(a)B particle concentrations were then measured using a selective 'sandwich' ELISA with anti-apo(a) as the capture antibody and anti-apoB as the detecting antibody. The mean apo(a)B level of VLDL in six hypertriglyceridemic patients was 30% (range 1.3-50%) of the total plasma concentration. Apo(a)-containing particles of both plasma, d < 1.006 g/mL and d > 1.006 g/mL, had the same pre-beta mobility on agarose gel electrophoresis and a similar apparent molecular weight on non-denaturing 2-16% gradient polyacrylamide gel electrophoresis. Fractionation of plasma by zonal ultracentrifugation confirmed the presence of a heterogeneous distribution of apo(a) in hypertriglyceridemic subjects. Apo(a) was present throughout the density range from VLDL to Lp(a). After normalization of plasma triglyceride levels with dietary fish oil, apo(a) was no longer detected in VLDL suggesting that detection of apo(a) in the plasma, d < 1.006 g/mL, density fraction is related to type IV hypertriglyceridemia.