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PubMed1990

High-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations determined in serum of newborns, infants, children, adolescents, and adults by use of a micromethod for combined precipitation ultracentrifugation.

Cholesterol (C) concentrations in the two major subfractions of high-density lipoproteins (HDL2-C and HDL3-C) in sera from both sexes, ages ranging from newborns to adults, were measured by use of a micromethod for combined precipitation-ultracentrifugation. Sera were obtained from 91 boys, 68 girls, 15 healthy men, and 14 women. The HDL2-C concentration was higher in women than in men; the HDL3-C concentration was similar in these two groups. This sex-related difference, generally seen in adults, was found to begin at ages 11-15 y. The value of HDL2-C in females increased with age in a stepwise manner, whereas that in males increased up to ages 6-10 y but tended to decline thereafter. The HDL3-C concentration was higher in the adults than in the children. This micromethod for separating operationally defined HDL subfractions is of value for lipoprotein research in children.

Adolescent↗

PubMed1986

Detection of serum proteins by high-pressure gel-permeation chromatography with low-angle laser light scattering, compared with analytical ultracentrifugation.

Human sera were subjected to analytical ultracentrifugation and "high-pressure" gel-permeation chromatography on a system of combined TSK Gel G5000 PW and G3000 SW columns. The chromatographic method produced remarkably superior resolution of the proteins, especially those exceeding 100 000 Da. We calculated the molecular masses of eluted fractions on the basis of their detection by low-angle laser light scattering and their differential refractive index. We discuss the results in relation to the clinical data.

Blood Proteins↗

PubMed1978

Characterization of microtubule protein oligomers by analytical ultracentrifugation.

Samples of microtubule protein prepared by repeated cycles of assembly-disassembly were examined at low temperatures by sedimentation velocity ultracentrifugation. Sedimenting boundaries corresponding to the 6 S tubulin subunit as well as to two oligomeric species with s020,w values of 18.6 S and 30.6 S were observed. The 30 S to 6 S mass ratio varied with total protein concentration, suggesting that a concentration-dependent equilibrium exists between 6 S and oligomeric species of tubulin. A study of the effects of pH on the mass distribution among the species demonstrated that the 6 S species was favored at low pH values (5.8 to 6.5), the 30 S oligomer was favored at moderate pH values (6.5 to 7.4), and the 18 S oligomer was formed in increasing proportions at higher pH values (7.4 to 8.2). Incubation of purified microtubule protein solutions with increasing concentrations of NaCl initially favored conversion of the 30 S oligomer to the 18 S species with further increases in salt concentration resulting in the dissolution of both the 30 S and 18 S oligomers. The depolymerizing effects of high salt concentrations were substantially reversible, providing further evidence for 6 S-oligomer equilibria. The manipulation of the solution variables of pH and ionic strength in a systematic fashion led to the construction of a "phase diagram" for the microtubule protein species which provided a relatively complete description of the mass distribution among the 6 S, 18 S, and 30 S species over a range of physiological and near-physiological solution conditions.

Animals↗

PubMed1987

Characterization of high density lipoprotein subspecies: structural studies by single vertical spin ultracentrifugation and immunoaffinity chromatography.

Affinity columns containing anti-apolipoprotein A-I or A-II were used to fractionate plasma into subpopulations of lipoprotein particles containing: a) apoA-I [Lp(A-I)], b) apoA-I and A-II [Lp(A-I with A-II)], and c) apoA-I but no A-II [Lp(A-I without A-II)]. Single vertical spin and electron microscopy analyses of these HDL subpopulations demonstrated that acid elution from the affinity columns caused no detectable change in size and density of the three subpopulation particles. Analysis by nondenaturing gradient gel electrophoresis of the three subpopulations found in four normal subjects identified nine HDL subspecies, designated [1] through [9] in order of increasing size; [3-7] were the major subspecies. Lp(A-I with A-II) is composed primarily of subspecies [3],[5], and [6], and may contain some subspecies [2] and [7], while Lp(A-I without A-II) represents mainly [4] and [7] and the minor subspecies [1],[2],[8], and [9]. HDL subspecies [4],[5], and [6] are found in the standard sequential flotation density cuts for both HDL3 and HDL2, which illustrates the limitations of the latter terminology. Using single vertical spin ultracentrifugation, HDL fractions were located and isolated for physical and chemical analyses, including immunoassay for apoA-I, A-II, and C-II. The distribution of the Lp(A-I without A-II) particles corresponded closely to the apoC-II distribution. An apoA-I-rich, cholesteryl ester- and phospholipid-poor subspecies was identified in the dense HDL fractions. HDL subspecies [7] was found to contain at least three separate subspecies designated [7a], [7b], and [7c]. Based on these and previously published results (Brouillette, C. G., et al. 1984. Biochemistry. 23: 359-367), we propose that the HDL subspecies adjacent in size generally differ by the association/lack of association of a hinge-like domain of amphipathic helixes in a single molecule of apoA-I.

Adult↗

PubMed1977

[Isolation of flagellin antigens of salmonellae by the method of ultracentrifugation and their use for preparation of erythrocytic H-diagnostic agents].

Flagellar preparations were obtained from 6 salmonellae strains by differential ultracentrifugation; they were characterized by morphological, immunochemical, and serological tests. The results of investigations showed that the preparations possessed high serological activity; somatic antigen admixture was insignificant. Flagellins extracted from the flagellae were used to prepare erythrocytic H-diagnostic agents. The results of their use in the examination of sera of healthy persons, and of those suffering from typhoid fever and salmonellosis indicated the expediency of using passive H-hemagglutination for diagnostic purposes.

Antigens, Bacterial↗

PubMed1988

Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation.

The molecular basis of the heterogeneity of plasma low density lipoproteins (LDL, d 1.024-1.050 g/ml) was evaluated in 40 normolipidemic male subjects following fractionation by isopycnic density gradient ultracentrifugation into eight major subspecies. The mass profile of our subjects' LDL uniformly displayed single symmetric or asymmetric peaks as a function of density; the peak occurred most frequently (20 subjects) in subfraction 7 (d 1.0297-1.0327 g/ml). Several physicochemical properties (hydrodynamic behavior, electrophoretic mobility, chemical composition, size and particle heterogeneity, and apolipoprotein heterogeneity) of the LDL subfractions were examined. Hydrodynamic analyses revealed unimodal distributions and distinct peak Sf degree rates in individual subfractions. Such behavior correlated well with particle size and heterogeneity data, in which LDL subspecies were typically resolved as unique narrow bands by gradient gel electrophoresis. Subspecies with average densities of 1.024 to 1.0409 g/ml ranged from 229 to 214 A in particle diameter. LDL protein content increased in parallel with density while the proportion of triglyceride diminished; cholesteryl esters predominated, accounting for approximately 40% or more by weight. Distinct differences in net electric charge were demonstrated by electrophoresis in agarose gel, the subspecies with average density of 1.0314 g/ml displaying the lowest net negative charge. ApoB-100 was the major apoprotein in all subspecies, and constituted the unique protein component over the density interval 1.0271-1.0393 g/ml. ApoE and apo[a] were detected at densities less than 1.0271 and greater than 1.0393 g/ml. While apoE was evenly distributed within these two regions, representing up to 2% of apoLDL, the distribution of apo[a] was skewed towards the denser region, in which it amounted to 3-7% of apoLDL. ApoC-III was detectable as a trace component at densities greater than 1.0358 g/ml. Calculation of the number of molecules of each chemical component per LDL subspecies showed the presence of one copy of apoB-100 per particle, in association with decreasing amounts of cholesteryl ester, free cholesterol, and phospholipid. These data indicate that a similar overall molecular organization and structure is maintained in a unimodal distribution of LDL particle subspecies over the density range approximately 1.02 to 1.05 g/ml. In sum, our data may be interpreted to suggest that microheterogeneity in the physicochemical properties of human LDL subspecies reflects dissimilarities in their origins, intravascular metabolism, tissular fate, and possibly in their atherogenicity.

Adult↗

PubMed1985

[Studies on normal and pathological human cartilage proteoglycans with metrizamide density gradient ultracentrifugation].

In order to study human articular cartilage proteoglycans (PG) in aging and pathological states, PG extracts of normal cartilage, osteoarthritic cartilage, chondrosarcoma and osteochondroma were subjected to the density gradient ultracentrifugation in 23% metrizamide-6M urea system. Normal cartilages have chondroitin sulfate rich PG and keratan sulfate rich PG. The latter has a higher buoyant density and increases markedly with aging, but is missing in all of the pathological cartilages tested which have juvenile profiles corresponding to the normal cartilage of a child under 10 years of age. No differences in the chain lengths of glycosaminoglycans were observed in any of the cartilages tested, but marked differences were observed in the aggregation PG and hyaluronate in the pathological cartilages. The molecular sizes of hyaluronate are probably small in normal adults but large in the pathological state. Determinations of hexose and uronate distribution in the density gradient characterize easily the differences in aging and pathological cartilages. This system provided more information than the CsCl-guanidine HCl system.

Adolescent↗

PubMed1975

[Obtaining purified preparations of individual Shigella newcastle K- and O-antigens by using an ultracentrifugation method].

A modified method of obtaining the purified K- and O-antigens of Sh. newcastle with the use of ultracentrifugation is described. Serologically individual K- and O-antigens were obtained and their chemical composition was studied. Polysaccharide of the K-antigen consisted of the galacturonic acid, galactose, glucose, rhamnose xylose and hexosamine; it contained no heptoses. The O-antigen contained galactose, glucose, mannose, rhamnose, heptose and hexosamine; in difference to K-antigen was equal to 150000--160000. Thus, the K- and the O-antigen differed not only in the electrophoretic and diffuse mobility and immunological specificity, but also in the chemical composition.

Antigens, Bacterial↗

PubMedDate not supplied

Gel filtration through Sephadex G25 of virus fluorescent compound systems. Comparison with ultracentrifugation results.

A stable interaction could be made evident between influenza A(H1N1) or Sendai virus and two fluorescent naphthalic acid derivatives solubilized in DMSO. The virus-compound interaction was studied spectrophotometrically and spectrofluorometrically, at the wavelengths characteristic of virus protein and of the two compounds, in samples subjected to gel filtration or ultracentrifugation. The percentages of virus-bound compound obtained by the two methods were similar in the case of the vinyl-sulfonic derivative (affecting virus structure to a lesser extent) and considerably different in that of the isocyano derivative (causing important structural damage).

Chromatography, Gel↗

PubMed1983

Use of short analytical ultracentrifugation runs for the isopycnic determination of foot-and-mouth disease virus concentration and density in autoformed caesium chloride gradients.

Short analytical ultracentrifugation runs of less than six hours with autoformed CsCl density gradients were used for routine FMDV density and concentration analysis. The values obtained after short runs were slightly higher for concentration and lower for density than after classical 22 h runs. These differences between the 6 and the 22 h values were related to virus strain and did not seem to result of the incomplete stabilisation of the CsCl gradient, but of the shorter virion/CsCl contact period.

Aphthovirus↗

PubMed1983

[Separation of high density lipoproteins (HDL) using zonal ultracentrifugation in patients with apolipoprotein AI Milano variant].

Investigations of HDL with zonal ultracentrifugation were carried out in three patients with apolipoprotein AIMilano. HDL in patients with AIMilano apolipoprotein are characterized by the practically total absence of HDL2. The HDL3 peak is broader than normal. In spite of the marked reduction or practically total absence of HDL2, which is believed to be a protective factor against atherosclerosis, our patients show no signs of clinical atherosclerosis.

Adult↗

PubMed1984

Sedimentation analysis of von Willebrand and factor VIIIC protein using partition cells in the analytical ultracentrifuge.

Sedimentation analysis of factor VIII complex was performed in the analytical ultracentrifuge using partition cells. This method allowed for the calculation of three different sedimentation coefficients from each run: one based on ristocetin agglutination activity for von Willebrand protein, SWF; one based on coagulant activity for factor VIIIC, SVIIIC; and one based on the schlieren or adsorption data for protein concentration, Sconc. In most cases, there was no agreement between the three values calculated from the same run, indicating a heterogeneous system. The calculated functional sedimentation coefficients give values that require the molecules to be highly asymmetric to be consistent with a glycoprotein of high molecular weight, which is in agreement with results observed in electron microscope studies. The dissociation of VIIIC into a smaller form can be demonstrated by this method. Determination of the three sedimentation coefficients in a series of fractions from gel filtration indicates a uniform size for the VIIIC activity but not for the WF activity. These observations are in agreement with the concept of a copolymer between WF and VIIIC and also with the concept of separate polymers for the two activities.

Antigens↗

PubMed1984

Simplified screening for immune complexes by laser nephelometry of ultracentrifuged serum.

Serum samples were drawn from 14 healthy volunteers after an overnight fast and again after a meal. Each sample was divided into two aliquots. One aliquot was centrifuged at 20,000 rpm for 20 min, and a portion from the middle of the tube was removed by puncturing the tube's side. All samples were then added to cuvettes containing 0.14 M NaCl alone or 1, 2, 3, or 4% polyethylene glycol in 0.15 M KCl. The light scattering of each tube was measured. The centrifuged samples scattered 58% less light than the uncentrifuged controls. The range of values obtained with the centrifuged samples was also smaller as reflected in the standard deviation of the means obtained at each polyethylene glycol concentration. The coefficient of variation for replicate samples ranged from 0 to 16.6% (mean 5.8%), even when samples were processed differently. Storage at -70 degrees C significantly increased light scattering compared to baseline values. Serum samples were drawn from 44 patients with diseases that have been associated with immune complexes. Nephelometry selectively identified some groups of patients as abnormal. We conclude that preparative ultracentrifugation removes much of the background light scattering of serum samples, including that due to lipid, under conditions that do not sediment out immune complexes. Because it measures a physical property of immune complexes, this assay may provide information that is not available from biological receptor-based assays.

Adult↗

PubMed1977

[Immunoelectrophoretic analysis of mink sera lipoproteins fractionated by preparative ultracentrifugation].

Using the method of preparative ultracentrifugation, four lipoprotein fractions were obtained differing in hydrate density. By means of immunoelectrophoretic analysis of the fractions, it is found that mink sera (except chilomicrons) contains at least five lipoproteins; two of them, lipoproteins 2 and 3, bind the major bulk of lipids. Lipoprotein 1 is of very low density. Lipoprotein 2 mostly belongs to the fraction with density less than 1.100 g/ml, although antigenically identical molecules with higher density were identified. Lipoprotein 3 is much more heterogeneous antigenically, electrophoretically and in its density (from 1.006 to 1.210 g/ml and higher). This protein seems to contain several structurally similar, though not identical, apoproteins. Lipoproteins 4 and 5 are very dense (more than 1.210 g/ml) and are weakly stained with Sudan black. These data, together with the results obtained in our previous studies, indicate that lipoproteins 2 and 4 are marked with genetically alloantygenic determinants.

Animals↗

PubMed1984

Evaluation of heat-sealed ultracentrifuge tubes in the isolation of plasma lipoproteins.

An initial step in the measurement of the concentration of lipids in plasma lipoproteins by the Lipid Research Centers' protocol is the separation of very low density lipoproteins from low density and high density lipoproteins by ultracentrifugation. We compared the performance of heat-sealed centrifuge tubes to the conventionally capped centrifuge tubes required by the protocol in terms of the separation and recovery of cholesterol and triglyceride. No significant differences in either parameter were found, however the heat-sealed tube required much less time to prepare and eliminated the need for periodic replacement of relatively expensive cap parts. We recommend the use of heat-sealed tubes for the routine separation of plasma lipoproteins.

Hot Temperature↗

PubMedDate not supplied

[Chemoluminescence of blood serum lipoproteins isolated by means of ultracentrifugation and precipitation with heparin and calcium].

Apo-B containing lipoproteins obtained after precipitation with heparin and calcium as well as very low (LPVLD) and low density lipoproteins (LPLD) produced using preparative scale ultracentrifugation had identical chemoluminescent properties. However, LPVLD are involved only slightly in total chemoluminescence of apo-B containing lipoproteins due to their low concentration in blood serum. In blood serum there is the factor having the quenching effect on the chemoluminescence and also exhibiting the antioxidant activity. Subfraction 3 of high density lipoproteins from patients with atherosclerosis differed from the analogous subfraction of donors by its chemoluminescent properties.

Calcium↗

PubMed1980

[Determination of the high density lipoprotein (HDL) cholesterol in blood serum. Comparison of 3 methods: ultracentrifugation and precipitations (author's transl)].

Three methods of assaying the high density lipoprotein cholesterol were applied simultaneously to a hundred of human sera: ultracentrifugation, precipitation by concavalin A and phosphotungstic acid. The obtained results were compared by statistical means: it was shown that the three procedures were equivalent. Some of their analytical features are discussed.

Chemical Precipitation↗

PubMed1995

Direct determination of lipoprotein(a) cholesterol by ultracentrifugation and agarose gel electrophoresis with enzymatic staining for cholesterol.

Lipoprotein(a) [(Lp(a)], a low-density lipoprotein (LDL)-like particle, contains in addition to LDL a specific protein component, apolipoprotein(a) [apo(a)]. Conventionally, Lp(a) has been measured by immunological methods that distinguish between Lp(a) and LDL by dealing with apo(a) as an antigen. We describe a new method to determine Lp(a) on the basis of its cholesterol content. Very-low-density lipoproteins were removed from serum by preparative ultracentrifugation at a density of 1.006 kg/L. The infranate was subjected to agarose gel electrophoresis to separate Lp(a) and LDL. Lp(a) cholesterol was then determined by direct enzymatic staining for cholesterol. On electrophoresis of the > 1.006 kg/L (bottom) fraction, Lp(a) migrates to the pre-beta position, regardless of the genetic apo(a) isoform. The interassay CVs of Lp(a) cholesterol determinations ranged from 6.9% to 11.5%, and the results correlated well with the Lp(a) concentrations measured by immunonephelometry (r = 0.937). There was an inverse relation between the molecular mass of the genetically determined apo(a) isoforms and Lp(a) cholesterol concentrations. Patients with angiographically proven coronary artery disease (CAD) had significantly more Lp(a) cholesterol than healthy controls did. The ratio of Lp(a) cholesterol to immunologically determined Lp(a) tended to be lower in CAD patients, suggesting that Lp(a) particles contained less cholesterol than apo(a). In addition, the new method allows determination of LDL cholesterol without contamination by Lp(a).

Adult↗