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At least 19 recordsLinked to original sources
PubMed1996

Ultracentrifugation systematically overestimates vesicular cholesterol levels in bile.

To accurately determine the cholesterol (Ch) distribution between mixed micelles and vesicles in lithogenic bile, both ultracentrifugation and gel chromatography with the correct intermixed micellar/vesicular bile salt concentration (IMC) have been proposed. We have systematically compared both separation techniques with physiological model biles to ascertain their quantitative separation ability. After determination of optimal ultra-centrifugation conditions in systems containing only micelles or vesicles, Ch-supersaturated model biles [3-10 g/dL, 10 mol percent Ch, taurocholate (TC)]/([TC + egg yolk phosphatidylcholine (EYPC)] = 0.6 and 0.7) were adjusted to a density of 1.03 g/mL, and ultracentrifuged at 42,000 rpm and 37 degrees C for 13 hours. Identical model biles were subjected to gel chromatography with the correct IMC, either directly or after remixing and incubation at 37 degrees C after ultracentrifugation. By ultracentrifugation, 31 percent +/- 2 percent (TC/(TC + EYPC) = 0.6) and 40 percent +/- 5 percent (TC/(TC + EYPC) = 0.7) of total Ch were found in vesicles (Ch/EYPC molar ratios = 1.0 and 1.3, respectively). However, by gel chromatography, only 19 percent +/- 2 percent (Ch/EYPC = 1.0) and 22 percent +/- 2 percent (Ch/EYPC = 1.5) of total Ch were found in the corresponding biles. Gel chromatography of biles (TC/(TC + EYPC) = 0.7) ultracentrifuged for various durations showed a progressive increase in vesicular Ch to 41 percent after 13 hours. On incubation for 11.5 hours after ultracentrifugation, vesicular Ch decreased to 31 percent, thus approaching the initial (gel chromatography) value. Quasielastic light scattering also demonstrated formation of vesicles in ultracentrifuged Ch-unsaturated model bile (cholesterol saturation index (CSI) approximately 0.97). As compared with gel chromatography, ultracentrifugation systematically elevates vesicular Ch, possibly because of induced shifts in lipids between lipid aggregates caused by variation in local bile salt concentration. Because ultracentrifugation can alter the phases present in bile, gel chromatography with the correct IMC more accurately represents the distribution of Ch in biliary lipid aggregates.

Bile↗
PubMed1984

Estimation of lipoprotein and apoprotein distribution in rat plasma by cumulative density ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Lipoprotein distribution in rat plasma determined after sequential ultracentrifugation (requiring 8 days of centrifugation to separate lipoproteins in five density classes), was compared to estimates based upon cumulative density ultracentrifugation (46 hr of ultracentrifugation). In general comparable values were obtained by the two methods with regard to protein, total cholesterol, cholesteryl ester, free cholesterol, and triacylglycerol distribution. However, the HDL3 protein concentration found by sequential ultracentrifugation was only about 50% of that found after the cumulative procedure. Apolipoproteins in lipoproteins isolated by the two methods were well separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Color of the stained bands was extracted and read photometrically. A linear standard curve was obtained with albumin. Absorbance corresponding to 1 microgram/ml was 0.057. Below d = 1.100 g/ml (HDL2b) the two ultracentrifugation methods gave comparable results for all apoproteins. In contrast to this the level of apo A-I, apo E, and apo A-IV in the more dense types of HDL was higher when estimated by cumulative than by sequential ultracentrifugation. In HDL3 isolated by sequential ultracentrifugation the apo A-IV, apo E, and apo A-I concentrations were 51, 31, and 45% respectively, of values found after cumulative ultracentrifugation. The results indicate that cumulative density ultracentrifugation, followed by colorimetric determination of apoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is a useful approach when studying lipoprotein distribution in rat plasma.

Animals↗
PubMed2001

Comparison of direct methods and HPLC for the measurement of HDL- and LDL-cholesterol with ultracentrifugation.

Although ultracentrifugation is the gold standard for lipoprotein analysis, inexpensive and easy direct methods for HDL- and LDL-cholesterol (C) have recently been developed. In this study, we compared representative methods of lipoprotein analysis, namely, ultracentrifugation, direct assay methods, and HPLC, to measure LDL- and HDL-C. A good correlation was observed between HDL-C by ultracentrifugation and HDL-C by direct methods or HPLC. A good correlation was also observed between LDL-C (d1.006-1.063) by ultracentrifugation and LDL-C by direct methods or HPLC. Although the correlation between LDL-C (d1.019-1.063) by ultracentrifugation and LDL-C by direct methods was also good, the correlation coefficient was significantly decreased, suggesting that 'LDL-C' by direct methods correlates better with LDL-C (d1.006-1.063) than LDL-C (d1.019-1.063) by ultracentrifugation. Although the correlation between IDL-C (d1.006-1.019) by ultracentrifugation and the difference in LDL-C by direct methods and LDL-C (d1.019-1.063) by ultracentrifugation was investigated, no significant correlation was observed. The IDL-C contained in LDL-C (d1.006-1.063) varied from 2-28%. In homozygous CETP-deficient and LCAT-deficient subjects, the dissociation was marked. It is crucial to understand that 'LDL-C' in the Guidelines for the Diagnosis and Treatment of Hyperlipidemias in Adults by the Japanese Atherosclerosis Society should be considered to be LDL-C (d1.006-1.063) and that 'LDL-C' by direct assay methods means LDL-C (d1.006-1.063) by ultracentrifugation.

Aged↗
PubMed1971

The isolation of lipoproteins from human plasma by ultracentrifugation in zonal rotors.

The major classes of lipoproteins were isolated from human plasma by ultracentrifugation in continuous density gradients using the Ti-14 and Ti-15 zonal rotors. Chylomicrons + VLDL, LDL, and HDL were separated from each other and from the more dense residual proteins (albumin fraction) of plasma by rate-zonal flotation in NaBr gradients in the density range 1.0-1.4. The chylomicron-VLDL fraction was subfractionated into constituent chylomicrons and VLDL by zonal ultracentrifugation in NaBr gradients in the density range 1.0-1.1. Plasma lipoproteins were analyzed for composition of lipids and content of protein, for electrophoretic mobility on paper, and for antigenic determinants by immunoelectrophoresis and immunodiffusion. Flotation constants (S(f)) of the LDL and HDL were calculated from measurements made in the analytical ultracentrifuge. Lipoproteins isolated from plasma by zonal ultracentrifugation were identical by these criteria to lipoproteins isolated by the usual procedure of sequential ultracentrifugation in solvents of increasing density. The procedure of zonal ultracentrifugation is rapid, quantitative, and less laborious than sequential techniques. Lipoproteins isolated by zonal ultracentrifugation are relatively uncontaminated by other proteins and extensive washing is therefore unnecessary. Zonal ultracentrifugation is more than a preparative method for the plasma lipoproteins; it is also an analytical procedure in that a record is obtained of the distribution and quantity of the lipoprotein within the continuous density gradient.

Blood Proteins↗
PubMed1989

Comparison of ultracentrifugation and a precipitation method for high-density lipoprotein cholesterol quantitation in insulin-dependent diabetic patients.

We compared sodium phosphotungstic acid and magnesium chloride precipitation method for high-density lipoprotein (HDL) cholesterol quantitation with the ultracentrifugation method in 64 insulin-dependent diabetic patients with plasma triglyceride less than 3 mmol/l. The cholesterol content of HDL after precipitation of very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) was 86% +/- 3% of the cholesterol content of HDL (q greater than 1.063) determined after ultracentrifugation at q = 1.063 (1.33 +/- 0.05 mmol/l vs 1.55 +/- 0.06 mmol/l; p less than 0.001). HDL cholesterol determined after precipitation closely correlated to HDL cholesterol determined after ultracentrifugation (r = 0.97; p less than 0.001). The absolute difference between the HDL cholesterol values obtained by the two methods was correlated to HDL cholesterol (ultracentrifugation) (r = 0.75; p less than 0.001), but it was not correlated to VLDL cholesterol, LDL cholesterol, triglyceride, HbA1c, blood glucose or serum albumin. LDL cholesterol calculated by use of Friedewald's formula was 108% +/- 4% of the cholesterol content of LDL (q = 1.019 to 1.063), determined after ultracentrifugation, but the calculated and the ultracentrifugally determined LDL cholesterol values were closely correlated (r = 0.98; p less than 0.001). These results suggest that during sodium phosphotungstic acid and magnesium chloride precipitation of plasma from diabetic patients, a constant fraction of HDL cholesterol is co-precipitated, resulting in a systematic difference in HDL cholesterol quantitation when compared with the ultracentrifugation method.

Adolescent↗
PubMed1977

Evaluation of the dual-precipitation method by comparison with the ultracentrifugation method for measurement of lipoproteins in serum.

We evaluated the dual-precipitation method for quantitative measurement of lipoproteins as described by Wilson and Spiger [J. Lab. Clin. Med. 82, 473 (1973)] for normo- and hyperlipemic sera, by comparison with the results obtained with ultracentrifugation. If serum with an above-normal triglyceride concentration is analyzed, the very-low-density lipoprotein cholesterol value obtained with the precipitation method is usually too low. For measurement of high-density lipoprotein cholesterol the ultracentrifugation and precipitation procedures give comparable results, but the latter method is preferred because sinking pre-beta-lipoproteins present in the high-density lipoprotein fraction isolated by means of the ultracentrifuge may result in falsely high values for cholesterol in that fraction. Therefore, at least for the determination of very-low-density lipoprotein cholesterol in hyperlipemic serum, the use of an ultracentrifuge remains necessary. Because few laboratories have an ultracentrifuge at their disposal, it seemed important to look at the stability of sera in view of the forwarding of samples. Also, a way of increasing the efficiency of the ultracentrifuge was studied. Sera can be stored for a week at 4 degrees C or for 54 h at room temperature without noticeable effect on lipoprotein values. Moreover, reliable values can be obtained with an ultracentrifugation time of 8 h (0.8 X 10(8) g-min).

Chemical Precipitation↗
PubMed1978

Ultracentrifugal inoculation of herpes simplex virus.

By ultracentrifugation of 30 ml of highly dilute suspensions of herpes simplex virus (HSV) directly onto monolayer cultures grown in centrifuge tubes, infectivity was significantly greater than without centrifugation. Ultracentrifugation at 20,000 to 25,000 rpm (28,000 to 45,000 X g) for 1.5 to 2.3 h was utilized with good preservation of cultures. With low-speed centrifugation at 3,000 rpm (1,100 X g), infectivity was almost 10-fold greater than without centrifugation. With ultracentrifugal inoculation, infectivity was about 100-fold greater than without centrifugation. Ultracentrifugal inoculation permitted the detection of HSV at concentrations as low as 0.05 plaque-forming units per ml. Similarly, ultracentrifugal inoculation of cultures was almost 100-fold more sensitive a method of detecting infectious HSV than was pelleting HSV from dilute suspensions followed by resuspension and inoculation of cultures. Ultracentrifugal inoculation of cultures may permit the isolation of HSV in situations where virus cannot be detected by ordinary means and may prove applicable to the study of other viruses.

Cells, Cultured↗
PubMed1980

Mechanisms of herpes simplex virus infectivity enhanced by ultracentrifugal inoculation.

Ultracentrifugation of very dilute suspensions of herpes simplex virus directly onto monolayer cells grown in centrifuge tubes was studied. Enhanced infectivity by ultracentrifugation was similar at 4 degrees C and at 35 to 37 degrees C. The high infectivity levels of cultures centrifuged at 4 degrees C were further examined by infectious center assays. At 4 degrees C, the numbers of infectious centers in control (noncentrifuged) cultures were almost 100-fold fewer than in control cultures at 37 degrees C. However, the numbers of infectious centers in cultures ultracentrifuged at 4 degrees C were similar to those ultracentrifuged at 37 degrees C. The great difference in the numbers of infectious centers between 4 and 37 degrees C control cultures, in contrast to the similarity between 4 and 37 degrees C ultracentrifuged cultures, indicated that ultracentrifugation at 4 degrees C enhanced infectivity possibly by facilitation of herpes simplex virus penetration into monolayer cells.

Animals↗
PubMed1995

Rapid isolation of large amount of plasma VLDL and LDL by a two step ultracentrifugation.

A rapid method to obtain large amount of VLDL and LDL by ultracentrifugation is described. The mixture of VLDL and LDL was isolated and concentrated from plasma by an ultracentrifugation at 265 000 g for 2 h. VLDL and LDL were separated and purified by a further ultracentrifugation at 265 000 g for 3 h. This method combines the advantages of both sequential flotation ultracentrifugation and density gradient ultracentrifugation. It can process a large volume of plasma in a short time. The purity of isolated VLDL and LDL was confirmed by the lipoprotein electrophoresis on agarose gel and PAGE and by the apolipoprotein electrophoresis on SDS-PAGE. This rapid economical method is of great value in practical application.

Humans↗
PubMed1986

Analysis of plasma lipoproteins by ultracentrifugation in a new fixed angle rotor: evaluation of a phosphotungstic acid/MgCl2 precipitation and a quantitative lipoprotein electrophoresis assay.

A routine procedure for the ultracentrifugal analysis of human plasma lipoproteins by means of a fixed angle rotor was developed. It was applied as reference method for the evaluation of two widely used procedures of lipoprotein analysis, i.e. measurement of HDL-cholesterol after precipitation of the apolipoprotein B containing lipoproteins with a new phosphotungstic acid/MgCl2 reagent, and a quantitative lipoprotein electrophoresis system. A new statistical approach to the multivariate comparison of analytical methods, the linear structural relationship model, has been applied. The HDL-cholesterol levels measured after phosphotungstic acid/MgCl2 precipitation agreed well to those determined by ultracentrifugation, whereas electrophoretically quantified alpha-lipoprotein-cholesterol exceeded the ultracentrifugal HDL-cholesterol. The Friedewald formula obviously underestimated LDL-cholesterol due to an overestimation of VLDL-cholesterol, whereas the electrophoretically quantified pre-beta- and beta-lipoprotein-cholesterol levels fairly coincided with the respective ultracentrifugal measurements. The inter-assay reproducibility of the HDL-cholesterol determination after phosphotungstic acid/MgCl2 precipitation and subsequent LDL-cholesterol quantification according to Friedewald was statistically equivalent to that of ultracentrifugation, whereas the quantitative lipoprotein electrophoresis proved less precise.

Animals↗