Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ULTRACENTRIFUGATION”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1995

Very fast ultracentrifugation of serum lipoproteins: influence on lipoprotein separation and composition.

A very short run time and small sample volumes in the separation of lipoproteins by preparative ultracentrifugation are needed for several investigations. Recently, a very fast sequential separation method was described that needs only 100 min for one run in a centrifugal field of 625,000 x g. We studied the influence of centrifugal fields of this dimension on lipoprotein separation and lipoprotein particle integrity using a Beckman Optima TLX ultracentrifuge with a TLA-120.2 rotor. Rotor speed (120/90/60/30.10(3) rev./min) and run time (100 min/3 h/6.7 h/27 h) were selected in such a way that the product of centrifugal field and run time remained constant. The first conditions correspond to the very fast ultracentrifugation (VFU) procedure with a centrifugal field of 625,000 x g. Thirty different plasma samples covering a wide range of lipid and protein concentrations were separated in the course of two centrifugal runs at densities of 1.006 and 1.063 kg/l which yielded very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and the subnatant of low-density lipoproteins, including high-density lipoproteins (HDL) and concomitant sedimented plasma proteins. The major lipid components of the lipoproteins, triacylglycerols, free and esterified cholesterol, phospholipids and the apolipoproteins B and A-I, were estimated considering the masses of the tube contents after a slicing procedure. Measurements of lipids and proteins showed a very good recovery of better than 94% and 91%, respectively, and precision-within-series (coefficient of variation) of better than 4.2% and 6.5%, respectively. The effects of the rotor speed on the lipoprotein structure appeared to be weak. With increasing rotor speed, VLDL and LDL lipid constituents principally tended to decrease, whereas they increased in the subnatant of the LDL-run. The mean lipoprotein mass composition, considering the mass percentage of each measured particle constituent, did not show significant alterations. Total protein decreased in VLDL and in LDL and increased in the subnatant of the LDL-run. As checked by an enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein effects were due to nearly complete disappearance of contaminating plasma proteins, especially albumin as the major contamination of VLDL and LDL. The apolipoproteins (apo) B-100, A-I, E and C-I to C-III remained nearly unaffected. The main advantages of VFU were the very short run time (cumulative flotation time is 3.4 h) and the elemination of albumin without repeated runs. The procedure was suitable for the assessment of lipid and protein constituents in lipoproteins from very small plasma samples (500 microliters).

Adult↗
PubMed1994

Very-fast ultracentrifugation of human plasma lipoproteins: influence of the centrifugal field on lipoprotein composition.

A short run-time in separation of lipoproteins by preparative ultracentrifugation is desirable for several reasons. Recently, a method was described that needs only 100 min for one run in a centrifugal field of 625,000 x g. It is assumed that lipoprotein separation depends on the rotor speed but this has not been systematically studied in centrifugal fields of this order. We performed such a study. Rotor speeds of 120, 90, 60 and 30 x 10(3) rev./min and run-times of 100 min, 3 h, 6.7 h and 27 h were selected in such a way that the product of centrifugal field and run-time remained constant. The first conditions correspond to the 'very fast ultracentrifugation' (VFU) procedure with a centrifugal field of 625,000 x g. The Optima tabletop ultracentrifuge, the rotor TLA-120.2 and thick wall open tubes for 1 ml were used. Thirteen different plasma samples covering a wide range of cholesterol and triglyceride concentrations were separated into VLDL, LDL and HDL in the course of two centrifugal runs at densities of 1.006 and 1.063. The constituents of the lipoproteins were calculated considering the mass of the tube contents after slicing. Recoveries of cholesterol, triglycerides and protein were 97%, 98% and 90%, respectively. The influence of the rotor speed on the apparent composition of the lipoproteins was small. With increasing rotor speed, VLDL-cholesterol became higher (by 14%, P < 0.001), VLDL-triglyceride became lower (by 6%, P < 0.012), LDL-cholesterol became lower (by 9%, P < 0.000). The effects on LDL-triglyceride and on HDL-cholesterol and HDL-triglyceride, did not reach statistical significance. Protein in VLDL and in LDL decreased and increased in 'HDL' (the subnatant of the LDL run). As checked by SDS-PAGE the protein effects were due to complete disappearance of albumin from VLDL and LDL while the apolipoproteins B-100, E and C-I to C-III remained unaffected. It is concluded that the main advantages of VFU are the short run-time and the disappearance of albumin from VLDL and LDL. The other compositional changes need to be further investigated.

Apolipoproteins↗
PubMed1985

Selective losses of large immune complexes during density gradient ultracentrifugation and an approach for prevention of these losses.

Substantial amounts (12.3-40.0%) of model immune complexes became nonspecifically adsorbed to centrifuge tubes during sucrose density gradient ultracentrifugation, and the adsorbed complexes were therefore unavailable for subsequent detection by the C1q solid-phase assay. The adsorption was greater for heavier immune complexes; thus detection of large-latticed complexes was impaired more than detection of small-latticed complexes. Loss of complexes could be prevented by incorporation of 0.05% polyoxyethylene (20) sorbitan monolaurate (Tween 20) into sucrose density gradient solutions and precoating tubes with gelatin. Tween 20 did not alter the immune complex lattice and did not prevent detection of immune complexes by the C1q solid-phase assay. Similar selective losses of immune complexes occurred when serum specimens from 2 patients with circulating immune complexes were analyzed by sucrose density gradient ultracentrifugation; the nonspecific adsorption of serum immune complexes which occurred during ultracentrifugation could be prevented by precoating centrifuge tubes with gelatin and incorporating 0.05% Tween 20 into sucrose gradients.

Adsorption↗
PubMed1991

Purification of measles virus by affinity chromatography and by ultracentrifugation: a comparative study.

The purification of viruses by ultracentrifugation in regions with limited resources is often hampered by problems of instrument maintenance. Viral antigens must therefore be imported, which results in delays in obtaining epidemiological data. To address this problem, we undertook the development of purification procedures by affinity chromatography using measles virus (strain Y15) and monkey IgG (anti-measles) coupled covalently via their carbohydrate residues to glass spheres as a model system. The measles virus prepared in this way was compared with measles virus purified by ultracentrifugation using three criteria: the degree of purification, yield and the sensitivity in HA and ELISA. The purification of the antigen by ultracentrifugation (AgU) was undertaken using 10(9) infected cells, and by affinity chromatography (AgAc) using 10(7) infected cells. The total concentration of proteins before purification was 5000 mg for AgU and 11.3 mg for AgAc. After purification by AgU 21.5 mg protein was obtained which was assessed by specific hemagglutinating units (HAU/mg protein) and found to be 179 for AgU and 372-744 for AgAc. Based on total HAU, the yield by centrifugation was 2.4%, whereas the affinity chromatography produced essentially 100%. By ELISA, using reference sera to calibrate the antigens, maximum OD was obtained with 0.5 microgram/well of AgAc and with 2 micrograms/well of AgU. From the relatively small amount of starting material, good yields were obtained with increased specific activity economy in coating by ELISA. These results favour the adoption of affinity chromatography for purifying viral antigens.

Animals↗
PubMed1991

Comparison of gel permeation chromatography, density gradient ultracentrifugation and precipitation methods for quantitation of very-low-, low- and high-density lipoprotein cholesterol.

Human VLDL, LDL and HDL (very-low-, low-, and high-density lipoproteins) were isolated from plasma by gel permeation chromatography with one pre-ultracentrifugation step. The column effluent was monitored at 280 nm. The cholesterol content of the fractions correlated well with fractions from sequential ultracentrifugation (VLDL, r = 0.839; LDL, r = 0.924; HDL, r = 0.766) or precipitation (LDL, r = 0.975; HDL, r = 0.972) methods. The average triglyceride, phospholipid and protein compositions of the separated lipoprotein fractions were close to those of the ultracentrifugally isolated fractions reported previously. Apolipoproteins A1 and B were determined from fractions to confirm the right distribution between different lipoproteins.

Adult↗
PubMed2004

Fluorescence detection for the XLI analytical ultracentrifuge.

Analytical ultracentrifugation (AUC) provides first-principle hydrodynamic and thermodynamic information concerning the size, shape and interactions of macromolecules. The fundamental measurement needed in AUC is the macromolecular concentration as a function of radial position and time. Currently, the Beckman Coulter XLI analytical ultracentrifuge may be equipped with absorbance and refractive detectors, which provide complementary concentration determinations. For detecting trace quantities of materials, fluorescence detection offers unique advantages over either absorbance or interference detection. A prototype fluorescence detector for the XLI analytical ultracentrifuge has been developed and its characteristics determined. An Ar(+) laser provides a continuous 488-nm excitation beam. Radial resolution is achieved by scanning the focused beam along a radial axis. Detection of the fluorescence signal uses a co-axial, front-face optical configuration to reduce inaccuracies in the concentration caused by inner filter effects. A high-speed A/D data acquisition system allows the fluorescence intensity to be monitored continuously and at a sufficiently high angular resolution so that at any radial position the intensities from all of the samples may be acquired at each revolution. The fluorescence detector is capable of detecting concentrations as low as 300 pM for fluorescein-like labels. The radial resolution of the fluorescence detector is comparable to that of the absorbance system. Both sedimentation velocity and sedimentation equilibrium measurements may be made with the fluorescence detector. Results are presented comparing data acquired using the fluorescence with those acquired using the absorbance detector.

Attention↗
PubMed2001

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation.

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M, 21,000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.

Chromatography, Gel↗
PubMed1978

Plasma lipoprotein separations by zonal ultracentrifugation.

Procedures for the separation of plasma lipoprotein classes and subclasses by zonal ultracentrifugation are described. The main density classes, very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL), in plasma can be separated in a single run for 20 hours. For the isolation of VLDL-LDL a centrifugation time of only 90 minutes is needed. Separations can be performed on plasma volumes varying from 10 to 400 ml in the Ti-14 rotor used; VLDL can in this way be isolated from 400 ml plasma in 30 minutes. The advantages and disadvantages of zonal ultracentrifugation in comparison with the commonly employed differential ultracentrifugation for separation of lipoproteins are discussed.

Centrifugation, Zonal↗
PubMedDate not supplied

Double pre-beta lipoprotein. Isolation and characterization of the two population of very low density lipoproteins by zonal ultracentrifugation.

Two electrophoretic populations of very low density lipoprotein are frequently observed in both normal and hyperlipidemic human sera. This gives the appearance of two pre-beta bands, called the "double pre-beta lipoprotein phenomenon." The slow and fast bands forming the double pre-beta lipoprotein were isolated by ultracentrifugation in the zonal rotor under rate flotation conditions. On the basis of the effluent position, the slow band showed a lower flotation coefficient than the fast one. The lipid and apoprotein composition of the two very low density lipoprotein populations isolated by zonal ultracentrifugation was in close agreement with those obtained by preparative agarose gel electrophoresis. The slow and fast pre-beta fractions had the same triglyceride fatty acid composition. Both fractions contained only higher molecular weight apo B-100, while the slow fraction was relatively enriched in apo E and apo C-III. Since the slow pre-beta fraction showed the typical properties of the remnant particle, studying this fraction could clarify the possible relationship between remnant lipoproteins and atherogenesis. Zonal rotor ultracentrifugation may be useful to characterize remnant particles in normal and hyperlipidemic subjects.

Apoproteins↗
PubMed1989

Comparison of microscale ultracentrifugation and equilibrium dialysis for the determination of the unbound fraction of theophylline in human serum.

A microscale ultracentrifugation procedure for the measurement of free theophylline in serum is described and evaluated by comparison with an equilibrium dialysis method. Provided an ultracentrifuge is available, micro-ultracentrifugation is a simple, cost-effective technique for the routine determination of free drug fractions.

Costs and Cost Analysis↗
PubMed1995

The preparation of tissue-type Plasminogen Activator (t-PA) containing liposomes: entrapment efficiency and ultracentrifugation damage.

UNLABELLED: In this study, a method was developed for the efficient entrapment of active tissue-type Plasminogen Activator (t-PA) into liposomes. Experimental conditions were varied to optimize t-PA entrapment: different buffer solutions were used (pH 4 and 7.5), the effect of the incubation concentrations of phospholipid (PL) and t-PA was monitored and the influence of liposome-size was examined. Furthermore, the effect of ultracentrifugation on t-PA containing liposomes was determined in the presence and absence of Tween 80. t-PA entrapment strongly depended on experimental conditions and ranged from 30 up to 90%. Almost quantitative+ (90%) entrapment (entrapment percentage defined as absolute entrapment (IU t-PA/mumol PL) divided by total incubation ratio (IU t-PA/mumol PL), times 100%) was obtained in Hepes buffer pH 7.5, devoid of arginine, with low ionic strength. Ultracentrifugation, used for removal of non-entrapped t-PA, was shown to have a damaging effect on the liposomes (especially in the presence of 0.05% Tween 80), leading to t-PA loss. However, because acceptable alternatives were not available, ultracentrifugation was used during this study. Therefore, the encapsulation-percentage values shown in this study are in fact underestimates for the true entrapment of t-PA. IN CONCLUSION: almost quantitative t-PA entrapment in liposomes can be achieved by selecting the proper milieu and inducing a strong interaction between t-PA and bilayer.

Buffers↗
PubMed2001

Paraformaldehyde protects of hepatitis C virus particles during ultracentrifugation.

Divergent buoyant densities of hepatitis C virus (HCV) have been reported. If the destruction of HCV particles occurs during the ultracentrifugation process to separate fractions with different densities, an accurate evaluation of the HCV buoyant density is difficult. To examine this concern, changes were examined in HCV RNA titer of each density fraction after paraformaldehyde fixation of virus particles in the sera of 9 patients with chronic HCV infection. Serum was treated with 4% paraformaldehyde, and the density fractions were then separated by ultracentrifugation. The HCV RNA titer of each fraction was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay, and the results were compared with those obtained from the serum without paraformaldehyde fixation. After fixation, the HCV RNA titer was significantly increased in the 1.11-1.14 g/mL fraction (P=0.0018), and decreased in the 1.14-1.17 and 1.17-1.20 g/mL fractions (P=0.0457 and 0.0003, respectively). Using immunogold electron microscopy, it was found that morphologically destroyed HCV particles are present mainly in the 1.17 g/mL fraction of paraformaldehyde-untreated samples, whereas the intact HCV virion particles are present in the 1.12 and 1.14 g/mL fractions of the paraformaldehyde-treated samples. These results suggest that the destruction of HCV virions occurs during the ultracentrifugation process and that paraformaldehyde treatment protects from destruction. It was also considered that the accurate buoyant density of the HCV virion is 1.11-1.14 g/mL. This study describes a useful method for the purification of HCV virions, and provides new insights for elucidating the physicochemical properties of HCV particles.

Adult↗
PubMed2003

Characterization of triglyceride rich lipoproteins with very light density by ultracentrifugation and agarose gel electrophoresis using triglyceride- and cholesterol-staining.

Hypertriglyceridemia is an independent risk factor for atherosclerosis. This risk is most likely due to accumulation of circulating triglyceride rich lipoproteins with heterogeneous particles. The identification and characterization of these triglyceride rich lipoproteins is important to detect abnormality of triglyceride metabolism. In the present study, we developed a new method that combines ultracentrifugation and agarose gel electrophoresis with triglyceride- and cholesterol-staining. We investigated 40 subjects with hypertriglyceridemia. Triglyceride rich lipoproteins with very light density were recovered in the aqueous fraction after ultracentrifugation (17,000 x g, 15 min). The lipoproteins recovered in the aqueous fraction contained chylomicrons, if present, their remnants, and light-VLDL (d <1.000 g/ml) containing apoB-100, but not normal VLDL (d <1.006 g/ml) and IDL. Triglyceride rich lipoproteins in the aqueous fraction were characterized by electrophoresis patterns of triglyceride- and cholesterol-staining. Forty patients with hypertriglyceridemia were separated into 8 groups according to their electrophoretic patterns. In lipoproteins recovered in the aqueous fraction from each group, the triglyceride level was correlated with the respective cholesterol level. In summary, a system using ultracentrifugation and agarose gel electrophoresis with triglyceride- and cholesterol-staining is useful for characterization of triglyceride rich lipoproteins and their remnants.

Cholesterol↗
PubMed1977

Turbidimetric ultracentrifugation. Application to the study of human serum very low density lipoprotein distributions.

In this communication it is shown that the sedimentation coefficient distribution may be accurately measured for very large particles using turbidimetric techniques and the ultraviolet-scanning analytical ultracentrifuge. A principal advantage is that turbidity is a function of the product of concentration and molecular weight; thus, large particles may be observed even when present in very small amounts. We propose to call this method of analysis "turbidimetric ultracentrifugation." We have used turbidimetric ultracentrifugation ot determine the sedimentation coefficient distribution for a sample of human serum very low density lipoproteins. This distribution is compared to that found with conventional schlieren techniques with good agreement.

Humans↗
PubMed1977

Routine titration of foot-and-mouth disease virus suspensions by analytical ultracentrifugation 2nd communication: sedimentation equilibrium method.

A routine method for the determination of the virus concentration in FMD virus cultures and vaccines was developed. This method was based on sedimentation equilibrium in the analytical ultraviolet scanning ultracentrifuge. The virus suspension was first clarified. The virions were then sedimented in a preparative ultracentrifuge. The resuspended virions were diluted in a Cesium chloride solution and brought to equilibrium in the density gradient generated in the analytical ultracentrifuge. The optical density of the virus band was measured by the UV scanning system. A calculation procedure was developed to compute the density at the limits and at the maximum of the virus band. The virus concentration expressed as weight, was calculated for the original virus suspension.

Aphthovirus↗
PubMed1988

Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations.

Two populations of apolipoprotein (apo) A-I-containing lipoprotein particles are found in high density lipoproteins (HDL): those that also contain apo A-II[Lp(A-I w A-II)] and those that do not [Lp(A-I w/o A-II)]. Lp(A-I w/o A-II) comprised two distinct particle sizes with mean hydrates Stokes diameter of 10.5 nm for Lp(A-I w/o A-II)1 and 8.5 nm for Lp(A-I w/o A-II)2. To study the effect of ultracentrifugation on these particles, Lp(A-I w/o A-II) and Lp(A-I w A-II) were isolated from the plasma and the ultracentrifugal HDL (d 1.063-1.21 g/ml fractions) of five normolipidemic and three hyperlipidemic subjects. The size subpopulations of these particles were studied by gradient polyacrylamide gel electrophoresis. Several consistent differences were detected between plasma Lp(A-I w/o A-II) and HDL Lp(A-I w/o A-II). First, in all subjects, the relative proportion of Lp(A-I w/o A-II)1 to Lp(A-I w/o A-II)2 isolated from HDL was reduced. Second, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 were considerably reduced in HDL. Third, a distinct population of particles with approximate Stokes diameter of 7.1 nm usually absent in plasma was detected in HDL Lp(A-I w/o A-II). Little difference in subpopulation distribution was detected between Lp(A-I w A-II) isolated from the plasma and HDL of the same subject. When plasma Lp(A-I w/o A-II) and Lp(A-I w A-II) were centrifuged, 14% and 4% of A-I were, respectively, recovered in the D greater than 1.21 g/ml fraction. Only 2% A-II was found in this density fraction. These studies show that the Lp(A-I w/o A-II) particles are less stable than Lp(A-I w A-II) particles upon ultracentrifugation. Among the various Lp(A-I w/o A-II) subpopulations, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 are most labile.

Adult↗
PubMed1972

Artifacts in ultracentrifugal estimation of aqueous fatty acid concentration.

Ultracentrifugation for determination of isotropic concentrations of fatty acids is widely used. However, several artifacts, which would affect the isotropic concentration, could occur if care is not taken. These include sedimentation of micelles, incomplete flotation of unsolubilized oil, and uptake of labeled fatty acid by the walls of centrifuge tubes. The first artifact can be overcome by sampling large volumes from the ultracentrifuged sample, and the second by ultracentrifugation for long periods; a force-duration of 7.2 X 10(7) g-min is suitable. The third artifact cannot be eliminated but may be made constant if the duration of exposure of lipid mixtures to centrifuge tubes is kept constant.

Carbon Isotopes↗
PubMed1983

Quantification of lipoprotein cholesterol in serum from children with different lipoprotein profiles: heparin-calcium precipitation and ultracentrifugation compared.

We compared the serum lipoprotein cholesterol concentrations in subgroups of children (n = 360), ages 5-17 years, as measured by the heparin-Ca2+ and preparative ultracentrifugation methods. Children were grouped from the total population on the basis of their previous results for serum beta- and pre-beta-lipoprotein cholesterol (Group I: low beta- and low pre-beta-; Group II: high beta- and low pre-beta; Group III: high beta- and high pre-beta-; Group IV: low beta- and high pre-beta-). The values for very-low-density (VLDL) cholesterol by ultracentrifugation method were 44, 53, 15, and 10 mg/L greater than the values for pre-beta-lipoprotein cholesterol by the heparin-Ca2+ method in Groups I, II, III, and IV, respectively; the differences were not significant in Group IV. The values of low-density (LDL) cholesterol were 64, 137, 144, and 73 mg/L less than the values for beta-lipoprotein cholesterol in Groups I, II, III, and IV, respectively (p less than 0.005). On the other hand, high-density (HDL) cholesterol concentrations in the respective four groups were 10, 37, 93, and 52 mg/L greater than alpha-lipoprotein cholesterol concentrations; the differences were significant for Groups II, III, and IV (p less than 0.005). Overall, the values for LDL-cholesterol correlated highly with beta-lipoprotein cholesterol (r = 0.94), whereas correlations for VLDL- and HDL-cholesterol values with pre-beta-lipoprotein cholesterol (r = 0.76) and alpha-lipoprotein cholesterol (r = 0.77) were somewhat lower. The differences between these two methods may result from their different operational definitions for measuring serum lipoproteins and the possibility that without appropriate corrections the values obtained by preparative ultracentrifugation do not serve as reference values.

Adolescent↗