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PubMed1979

Cholesterol in high-density lipoproteins: a comparison between dextran sulfate-magnesium chloride precipitation and preparative ultracentrifugation.

The cholesterol in high density lipoproteins (HDL) has been determined in 140 serum samples after dextran sulfate-magnesium chloride precipitation of the apolipoprotein B-containing lipoproteins. The values correlated well with simultaneously measured values from preparative ultracentrifugation (slope 0.98, r = 0.93). Furthermore, there was a significant correlation in 60 specimen between the dextran sulfate-MgCl2 and heparin-MnCl2-precipitation method.

Chemical Precipitation↗

PubMed2005

Modeling analytical ultracentrifugation experiments with an adaptive space-time finite element solution of the Lamm equation.

Analytical ultracentrifugation experiments can be accurately modeled with the Lamm equation to obtain sedimentation and diffusion coefficients of the solute. Existing finite element methods for such models can cause artifactual oscillations in the solution close to the endpoints of the concentration gradient, or fail altogether, especially for cases where somega(2)/D is large. Such failures can currently only be overcome by an increase in the density of the grid points throughout the solution at the expense of increased computational costs. In this article, we present a robust, highly accurate and computationally efficient solution of the Lamm equation based on an adaptive space-time finite element method (ASTFEM). Compared to the widely used finite element method by Claverie and the moving hat method by Schuck, our ASTFEM method is not only more accurate but also free from the oscillation around the cell bottom for any somega(2)/D without any increase in computational effort. This method is especially superior for cases where large molecules are sedimented at faster rotor speeds, during which sedimentation resolution is highest. We describe the derivation and grid generation for the ASTFEM method, and present a quantitative comparison between this method and the existing solutions.

Algorithms↗

PubMed2006

Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation.

Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The sedimentation coefficient distribution c(s) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the sedimentation coefficient consistent with the traditional s approximately M(2/3) power law. It provides a high hydrodynamic resolution, where diffusional broadening of the sedimentation boundaries is deconvoluted from the sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of sedimentation coefficient and frictional ratio, c(s,f(r)), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c(M,R(S)), and sedimentation coefficient-molar mass distributions c(s,M). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.

Algorithms↗

PubMed1985

Electron microscopy after direct ultracentrifugation.

A direct ultracentrifugation technique was used in the preparation of skin lesion specimens for examination by electron microscopy. The concentration factor of centrifuged specimens was estimated to be in excess of 1,000-fold compared to conventional adsorption techniques. This resulted in an increase of over 300% in the detection rate of herpesviruses and poxviruses from skin lesion specimens.

Herpesviridae Infections↗

PubMed1984

Behavior of hyaluronic acid from gingival epithelium and connective tissue on the analytical ultracentrifuge.

The molecular weights of hyaluronic acid (HA) isolated from separated specimens of human gingival epithelium and connective tissue as well as standard hyaluronic acid preparations have been estimated. The values were determined following substitution of sedimentation values into a previously determined empirical relationship between the reciprocal of the sedimentation coefficient at zero concentration (S-1)0 and molecular weights estimated by sedimentation-diffusion (MsD). The values of (S-1)0 for connective tissue and standard low-molecular weight HA preparations obtained by linear regression of all points indicated molecular weights (MsD) of 340,000 and 205,000 respectively. However, epithelial and standard high-molecular weight HA behaved differently during ultracentrifugation generating a curvilinear relationship between s-1 and concentration. Nevertheless linear extrapolation of a line of best fit of the very lowest concentrations (those which approached zero concentration) provided molecular weight estimates of 860,000 and 2,500,000 respectively. Moreover, similar treatment of s-1 values derived from the previously published data of Laurent, Ryan and Pietruszkiewicz has validated the use of linear regression of s-1 at the lower concentrations alone, to calculate high-molecular weight HA. The curvilinear relationship for s-1 throughout the whole concentration range (0.15-2.3 mg/ml) has been regarded only as a qualitative indication that the HA samples are of relatively high-molecular weight, while a straight line through such data points implies a qualitatively lower molecular weight for HA.

Connective Tissue↗

PubMed1979

Isolation of stable aggregates of IgG by zonal ultracentrifugation in sucrose gradients containing albumin.

Soluble heat aggregates of [125I]IgG (A-IgG) were prepared and separated by gel filtration or by zonal ultracentrifugation, and fractions containing different size aggregates were then analyzed by reultracentrifugation. A-IgG formed single narrow peaks with constant S rates when isolated, stored and analyzed in solution containing 0.5% serum albumin. Thus, stable homogeneous aggregates of IgG of known size can be simply prepared and should prove useful both as a model for immune complexes of specified sizes and as a standard for immune complex assays.

Animals↗

PubMedDate not supplied

Characterization of chick serum lipoproteins isolated by density gradient ultracentrifugation.

Serum lipoproteins from 12h fasted male chicks (15-day-old) were separated into 20 fractions by isopycnic density gradient ultracentrifugation. A new procedure was described by collecting the different fractions from the bottom of tube instead of by aspiration from the meniscus of each tube. Analyses of chemical composition of serum lipoproteins have permitted to reevaluate the density limits of major classes: VHDL, d greater than 1.132 g/ml; HDL, d 1.132-1.084 g/ml; LDL, d 1.084-1.038; IDL, d 1.038-1.022; and VLDL d less than 1.022. HDL fractions clearly predominated (approx. 77% of total lipoproteins) while IDL and VLDL were present at low percentage. LDL was the fraction richest in cholesterol; triacylglycerol content clearly increased from HDL to VLDL, while protein content decreased. All the chemical components of chick serum lipoproteins were accumulated in HDL, although triacylglycerol was relatively distributed in all the lipoprotein classes.

Animals↗

PubMed1976

Determination of physical properties of bovine serum lipoproteins by analytical ultracentrifugation.

Pure samples of low density lipoprotein-one (d 1.019 to 1.039 g/ml), low density lipoprotein-two (d 1.039 to 1.060 g/ml), and high density lipoprotein (d 1.060 to 1.210 g/ml) were isolated from bovine serum and their properties studied in the analytical ultracentrifuge. Approach-to-equilibrium experiments indicated that the lipoprotein classes were homogeneous. Molecular weights of the lipoproteins given by this method (low density lipoprotein-one, 1.14 x 10(6); low density lipoprotein-two, 2.37 x 10(6); high density lipoprotein, .576 x 10(6)) agreed well with those obtained by a high-speed-equilibrium method (1.03 x 10(6), 2.15 x 10(6), and .567 x 10(6)). Linear plots of flotation rate (corrected for viscosity) against the density of the medium were obtained in sedimentation velocity experiments and on extrapolation gave values for the hydrated density of 1.032 g/ml for low density lipoprotein-one, 1.044 g/ml for low density lipoprotein-two, and 1.071 g/ml for high density lipoprotein. The density of the material which would have to be added to high density lipoprotein to give it the physical properties of low density lipoprotein one was less than 1 g/ml, which suggested that it was predominantly lipid.

Animals↗

PubMed1975

A model for ultracentrifugal quantification of (35-S) bromosulphthalein-binding to plasma proteins in the presence of radioimpurities.

Sloutions of (35S)bromosulphthalein ((35S)BSP) in heparinized canine plasma, in the proportions established in vivo after injecting BSP intravenously to test liver function, were ultracentrifugated at 226,000 g for 24 hr at 5 degrees. Protein-free supernatant was replaced by Krebs-Ringer buffer (pH 7.40), the protein sediment resuspended, and the mixture recentrifugated. That process was repeated several times, and the radio-activity of each resulting supernatant was measured. Since (35-S)BSP could not be adequately purified, supernatant radioactivities reflected both (35-S)BSP and radioimpurity. Therfore, a model was derived that (i) interpreted the rapid decrease in supernatant radioactivities of initial centrifugations and the gradual fall therafter; and (ii) allowed us to determine picomoles of non-protein-bound (35-S)BSP. Results indicated that only 0.053% (SD .0013%) of BSP in our system was not protein-bound.

Animals↗

PubMed1993

Characterization of lipoprotein fractions isolated from plasma of male Wistar rats by gradient ultracentrifugation.

Both Wistar and Sprague-Dawley rat strains have been used for experimental studies on lipoprotein metabolism, although the lipoprotein characteristics of the former strain are less well known than those of the latter. We have defined more precise conditions for separating by density gradient ultracentrifugation the different lipoprotein classes from plasma of young male Wistar rats. Present results confirm that Wistar rats, like other rat strains, have negligible amounts of low density lipoproteins and levels of very low density lipoproteins much lower than high density lipoproteins. The differences between rat strains appeared in the profile of lipoprotein denser than 1.08 g/ml. In fact, we showed Wistar rats have considerable amounts of high density lipoprotein-3 subfraction with a density range between 1.17 and 1.21 g/ml, a mean diameter of 4.9 nm, and a low cholesterol content. In analogy with the Sprague-Dawley strain, Wistar rats had a small proportion of high density lipoprotein-1 subfraction (about 17% of total lipoproteins) and, at variance, in Wistar rats, we did not observe the presence of the so-called very high density lipoprotein fraction described previously in the Sprague-Dawley strain.

Animals↗

PubMed1999

Ratio of remnant-like particle-cholesterol to serum total triglycerides is an effective alternative to ultracentrifugal and electrophoretic methods in the diagnosis of familial type III hyperlipoproteinemia.

BACKGROUND: Familial type III hyperlipoproteinemia (HLP) is characterized by the presence of beta-migrating VLDL (beta-VLDL) and increased risk of cardiovascular disease. Assessment of plasma beta-VLDL is achieved by measuring the ratio of VLDL-cholesterol (VLDL-C) to total plasma triglycerides (TGs) or by detecting beta-VLDL in total VLDL. The objective of this study was to compare the clinical utility of the ratio of remnant-like particle-cholesterol (RLP-C) to total TGs with that of the current methods for diagnosing type III HLP. METHODS: Detection of beta-VLDL by electrophoresis of VLDL was used to define type III HLP. Twenty-eight patients with type III HLP and 43 subjects lacking beta-VLDL were investigated. Fasting TG concentrations were >2.26 mmol/L in all subjects. Subjects were separated into three groups: group 1, serum total cholesterol </=5.18 mmol/L (n = 11); group 2, total cholesterol >5.18 mmol/L and TGs between 2.26 and 9.04 mmol/L (n = 51); and group 3, TGs >9.04 mmol/L (n = 9). RESULTS: In group 2, a RLP-C-to-total TG molar ratio >/=0.23 (>/=0.10 when using mg/dL) and a VLDL-C-to-total TG molar ratio >/=0.69 (>/=0.30 when using mg/dL) correctly classified 94% and 90% of the subjects, respectively. The utility of the RLP-C-to-total TG ratio in diagnosing type III HLP decreased in patients in the other two groups. CONCLUSION: When used in an appropriate target population, the RLP-C-to-total TG ratio is a convenient and effective alternative to ultracentrifugal and electrophoretic methods for diagnosing type III HLP.

Apolipoproteins↗

PubMed2001

Evolution of methods for measurement of HDL-cholesterol: from ultracentrifugation to homogeneous assays.

BACKGROUND: Adoption of automated homogeneous assays for HDL-cholesterol (HDL-C) is increasing, driven by the need of clinical laboratories to cope with increasing workloads while containing costs. However, performance characteristics of homogeneous assays often differ in important aspects from those of the earlier precipitation methods. This review provides an overview of the new generation of homogeneous assays for HDL-C within the historical context of the evolution of methods and the efforts to standardize measurements of the lipoproteins. APPROACH: This is a narrative review based on method evaluations conducted in the laboratories of the authors as well as on relevant publications, especially comparative evaluation studies, from the literature. Publications considered here have been collected by the authors over the past 30 years of involvement as methods for HDL-C made the transition from their early use in lipid research laboratories to clinical laboratories and the recent emergence of homogeneous assays. CONTENT: The presentation includes descriptions of methodologies, including homogeneous, precipitation, electrophoresis, and ultracentrifugation assays. Reference methods and recommended approaches for assessing accuracy are described. Accuracy and imprecision are summarized in the context of the National Cholesterol Education Program (NCEP) standards for analytical performance. The effects of interfering substances and preanalytical sources of variation are presented. SUMMARY: Homogeneous assays have been shown to be reasonably well suited for use in routine clinical laboratories, generally meeting the NCEP criteria for precision, accuracy, and total error. However, discrepant results compared with the reference methods have been observed with some of the assays, and the sources of discrepancies are not well characterized. Some homogeneous reagents have not been thoroughly evaluated. At least three of the reagents have experienced successive adjustments in formulation; hence, the reagents may not yet be fully optimized. For these reasons, the homogeneous assays cannot be confidently recommended for use in long-term clinical trials and other research applications without thorough validation.

Chemical Precipitation↗

PubMed1992

Analysis of cholesterol in all lipoprotein classes by single vertical ultracentrifugation of fingerstick blood and controlled-dispersion flow analysis.

This new, highly sensitive analytical system, based on controlled dispersion of the flowing sample, gives a rapid, continuous, and direct analysis for cholesterol in all lipoprotein classes, separated by single vertical-spin density-gradient ultracentrifugation. In this Vertical Auto Profile-II fingerstick system, designated VAP-IIfs, a narrow-bore Teflon coil serves as the reactor with no segmentation of the analytical stream by air bubbles, in contrast to the Technicon AutoAnalyzer used in the VAP-I method. Concentrations of high-, low-, intermediate-, and very-low-density lipoprotein cholesterol and lipoprotein(a) cholesterol are determined by decomposing the spectrophotometric absorbance curve for the continuous analysis of the centrifuged sample, with use of software developed in this laboratory. Total cholesterol is determined from the total area under the absorbance curve. For assaying total cholesterol, the CV between aliquots within a rotor ranged from 1.35% to 3.15%; the CV between rotors was 2.45%. Because only 18 microL of sample is required, VAP-IIfs can be readily adapted to analysis for lipoprotein cholesterol profiles in capillary blood samples. Total cholesterol values by VAP-IIfs for fingerstick and venous samples from 23 subjects agreed well: slope = 1.01 (SD 0.03), intercept = -21 (SD 51) mg/L, Sy/x = 50 mg/L, and r = 0.992. Results by VAP-IIfs also correlated highly with results for duplicate samples analyzed at the Northwest Lipid Research Laboratories.

Cholesterol↗

PubMed1991

[Assessment of the composition and structure of covalent complexes of superoxide dismutase with aldehyde dextran by analytical ultracentrifugation].

Superoxide dismutase was covalently coupled wih aldehyde dextran, a polymeric carrier of molecular mass of 70 kDa. Modification produced an increase in the enzyme thermostability. Modified preparations retained a high specific activity. The composition of the thus obtained conjugates was analyzed by the ultracentrifugation and diffusion methods. The protein induced the destruction of aldehyde dextran, the enzyme being modified by its fragments. The presence of aldehyde dextran excess in the incubation medium promoted superoxide dismutase dissociation into individual subunits. At the enzyme/carrier ratio of 1:02 modification occurred as covalent coupling of the biocatalyst subunits and its one-point modification.

Dextrans↗

PubMed1976

Plasma high-density lipoprotein cholesterol concentrations determined after removal of other lipoproteins by heparin/manganese precipitation or by ultracentrifugation.

The widely used heparin/MnCl2 precipitation procedure for determination of plasma high-density lipoprotein cholesterol has been re-examined in light of recent reports that isolated preparations of the lipoprotein are only partly precipitated under the test conditions. In the present study, the procedure as applied to plasma tolerated rather wide variations in heparin and MnCl2 concentrations without significant effects on the assayed values in several plasma pools tested. The procedure was further tested on 129 individual samples by comparison with an ultracentrifugal method in which high-density lipoprotein-cholesterol is assumed to be represented by the cholesterol content of the plasma fraction of relative (to water) density greater than 1.063. Our results indicate that high-density lipoprotein is not precipitated under the test conditions when applied to unfractionated plasma.

Cholesterol↗

PubMed1991

The formation of actin oligomers studied by analytical ultracentrifugation.

The small oligomers formed from Mg-G-actin under favorable conditions were studied by sedimentation velocity ultracentrifugation. The critical concentration of actin at pH 7.8 in the presence of 100 microM MgCl2 and 200 microM ATP was 12.5 +/- 2.8 microM. Under these conditions, about 15% of 7.5 microM Mg-actin was converted to oligomers of subunit size four to eight in 5 h at 20 degrees C. In 100 microM MgCl2 and no free ATP, the critical concentration was about 6.5 microM, and about 22% of 7.5 microM Mg-actin was converted to dimers in 80 min. There were no detectable higher oligomers or F-actin present in either case. As determined by the analysis of ATP hydrolysis, most, if not all, of the oligomer subunits contained ATP. When 28.5 microM actin was polymerized to steady state in 100 microM MgCl2 and 200 microM ATP, about 50% of the actin was present as F-actin, consistent with the critical concentration (approximately 12.5 microM), about 50% as oligomers as large as seven subunits, and only about 5% as monomers. When solutions containing oligomers were diluted the oligomers dissociated. Alternatively, when the MgCl2 concentration was raised to 1 mM, the solutions containing oligomers polymerized more rapidly than monomeric Mg-G-actin and to the same final steady state. These data are entirely consistent with the condensation-elongation model for helical polymerization proposed by Oosawa and Kasai (Oosawa, F., and Kasai, M. (1962) J. Mol. Biol. 4, 10-21) according to which, under certain conditions, substantial amounts of short linear and helical oligomers should be formed below the critical concentration and linear oligomers should coexist with monomers and F-actin at steady state.

Actins↗

PubMed1978

Polymorphism of apolipoprotein E. I. Methodological aspects and diagnosis of hyperlipoproteinemia type III without ultracentrifugation.

Two methods for phenotyping apolipoprotein E are compared. One is based on preparation of VLDL by conventional ultracentrifugation, whereas the other uses heparin/Mg precipitation of VLDG. In principle, the same results were obtained by both methods. However, the group limits for the three different phenotypes Apo E-N, Apo E-ND and Apo E-D were slightly different by the two methods. Phenotype Apo E-D - the phenotype characterizing type III dyslipoproteinemia - is clearly definable by both methods. Hence the precipitation-I.E.F. method for Apo E phenotyping provides a simple tool for genetic and population genetic studies and also for the routine diagnosis of hyperlipoproteinemia type III, based on the only specific marker known for this disease.

Apolipoproteins↗

PubMed1979

Lipoprotein abnormalities in cholestasis. I. Electrophoretic and ultracentrifugal analyses.

The alterations of lipid composition in sera of patients with liver diseases, particularly intrahepatic cholestasis and biliary obstruction, were studied by ultracentrifugation and polyacrylamide-gel disc-electrophoresis of lipoproteins and apoproteins. The elevation of serum cholesterol in intrahepatic cholestasis was greater than in biliary obstruction. The appearance of lipoprotein X in obstructive disease accounted for most of the increased cholesterol. The level of non-lipoprotein X cholesterol in intrahepatic cholestasis was significantly elevated, this being in part ascribed to the appearance of a new class of cholestatic lipoprotein, Slow-migrating HDL. The electrophoretic pattern of lipoprotein in cholestasis was generally characterized by a decrease in alpha band intensity and, in some types of cholestasis, by the appearance of Slow-migrating HDL. In addition, other abnormal lipoproteins exhibiting the characteristics of triglyceride-rich LDL (LP-Y), LP-X-like HDL and LDL-like HDL were found in some cases of intrahepatic cholestasis and biliary obstruction.

Apolipoproteins↗