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PubMed1976

Routine titration of foot and mouth disease virus suspensions by analytical ultracentrifugation. 1: Sedimentation method.

Infectivity and Complement Fixation (CF) tests are commonly used for the routine titration of Foot and Mouth Disease (FMD) virus suspensions. Only recently were techniques published for the routine determination of the virus concentration by the physical properties of the virions (Fayet et al., 1971; Barteling et al., 1974). These techniques are based on the separation of the virions from the culture fluid by sedimentation through a sucrose gradient, in a preparative ultracentrifuge. The ultraviolet absorption pattern of the tube content is recorded by a flow colorimeter. The virus concentration is estimated using either standard curves or direct caculation by the specific extinction coefficient (Bachrach et al., 1964). In our own attempts to develop a preparative ultracentrifugation technique for the routine titration of FMD virus suspensions, we had to deal with some problems such as remixing of the virus band at the end of the run. We therefore turned over to analytical ultracentrifugation methods. The manipulations are less complicated and the virus band is traced and measured while the rotor is spinning. Four samples are analyzed simultaneously and the scans are repeated to follow the move of the virus band. The sedimentation rate of the virus band, calculated from the repeated scans, helps to detect artifacts. The present paper describes the technique we developed for the routine titration of FMD virus suspensions, by the band sedimentation method, using an ultraviolet scanning analytical ultracentrifuge.

Aphthovirus↗
PubMed1991

Precipitation with polyethylene glycol and density-gradient ultracentrifugation compared for determining high-density lipoprotein subclasses HDL2 and HDL3.

The purpose of this study was to compare quantification of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 by precipitation with polyethylene glycol (PEG) with that by density-gradient ultracentrifugation. Fresh serum samples from 32 fasting, obese children were analyzed with precipitation reagent "Quantolip" (Immuno AG), and then fractionated with a Beckman TL 100 ultracentrifuge with a swinging-bucket rotor. After centrifugation we carefully removed the supernate with a syringe and measured the cholesterol from each fraction enzymatically with CHOD-PAP reagent (Boehringer Mannheim). The low-density lipoprotein (LDL-), HDL2-, and HDL3-cholesterol values measured by ultracentrifugation did not differ significantly from those obtained by precipitation; the correlation coefficients (r) between the two methods were 0.96 for LDL, 0.75 for HDL2, and 0.96 for HDL3. The relatively simple PEG precipitation method used in this study measures total HDL and its major subclasses HDL2 and HDL3 with accuracy and precision comparable with those of the well-established ultracentrifugation method.

Adolescent↗
PubMed1990

Separation and quantitation of cholesterol carriers in native bile by ultracentrifugation.

The vesicular and micellar carriers of biliary cholesterol were isolated and quantitated from native bile by a simple and short isopyknic ultracentrifugal method. The method was designed to decrease the potential pitfalls of classic ultracentrifugation: osmotic effects of the centrifugation media and hydrostatic pressure effects generated in the centrifuge tube. This was accomplished by using metrizamide as an inert centrifugation medium for isopyknic separation and a vertical rotor. The buoyant density of vesicles isolated from human native bile varied between 1.010 and 1.030 gm/ml, as determined in preformed bile-metrizamide density gradients after 285 min of centrifugation. When 16% metrizamide was directly dissolved in bile, its density increased to 1.060 gm/ml. After 120 min of centrifugation, it was found that more than 95% of total vesicular cholesterol floated at the top of the centrifuge tube. This fraction appeared as one or two white opalescent bands. The present ultracentrifugal method was validated by gel filtration chromatography. It was found that more than 95% of vesicular cholesterol migrated to the top 0.4 ml of the centrifuge tube after the short-run centrifugation. Approximately 5% of total biliary cholesterol present in the vesicular fractions was in fact solubilized in mixed micelles as assessed by gel filtration chromatography. Although the proportion of vesicles and micelles estimated with the present ultracentrifugal method is in the range reported by other authors using the more common chromatographic method, we believe that our method has two major advantages. First, it eliminates the dilutional effect of buffers necessary for gel filtration chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile↗
PubMed1985

Plasma protein binding of salicylic acid, phenytoin, chlorpromazine, propranolol and pethidine using equilibrium dialysis and ultracentrifugation.

The in vitro plasma protein binding of phenytoin (diphenylhydantoin), salicylic acid, propranolol, pethidine (meperidine) and chlorpromazine was measured using an air-driven, bench-top ultracentrifuge and the results were compared with those obtained by equilibrium dialysis. For all drugs studied, except chlorpromazine, a significant correlation was found between the plasma binding results obtained by the two methods. However, only in the case of propranolol the actual binding values obtained by the two techniques were very similar. In the case of phenytoin, salicylic acid and pethidine, ultracentrifugation gave higher plasma binding values than equilibrium dialysis. Binding values obtained by the two methods for chlorpromazine were not correlated at all. This may be explained by binding of chlorpromazine to the very low density lipoprotein fraction which floats after ultracentrifugation. The described ultracentrifugation binding technique may be useful to rapidly measure the plasma binding of certain drugs in microsamples.

Adolescent↗
PubMed1982

Separation of lipoproteins in newborn plasma using an Airfuge ultracentrifuge.

The isolation of lipoprotein fractions in newborn plasma was performed by ultracentrifugation in an Air-driven ultracentrifuge (Airfuge). The purity and recovery of the fractions was checked by quantitation of the apo A1 and B proteins in the supernatant and infranatant fractions, and by gradient gel electrophoresis in 4-30% polyacrylamide gels. The influence of the duration of the ultracentrifugal run, the temperature and method for recovery of the fractions were tested. Under optimal conditions pure VLDL fractions could be isolated at d = 1.006 milligrams, whereas VLDL + LDL isolated at d = 1.063 milligrams were contaminated with HDL and other plasma proteins. Isolation of total lipoproteins at d = 1.21 milligrams enabled recovery of 75% of total plasma lipoproteins, however strongly contaminated by other plasma proteins. These results indicate that the recovery and purity of the lipoprotein fractions isolated in the Airfuge are not comparable to the results obtained in a conventional preparative ultracentrifuge.

Apolipoprotein A-I↗
PubMed1995

Separation of VLDL subfractions by density gradient ultracentrifugation.

To assess the presence and composition of very-low-density lipoprotein (VLDL) in various types of hyperlipoproteinemia, a method of density gradient ultracentrifugation has been developed. After 2 hours of density gradient ultracentrifugation, human serum VLDL is separated into two distinct VLDL cholesterol peaks (VLDL1 and VLDL2). The two VLDL subfractions were detected in the serum samples from all subjects in the study, including subjects with normolipidemia (n = 10), familial dysbetalipoproteinemia (n = 12), and type IIa (n = 8), type IIb (n = 12), and type IV/V (n = 10) hyperlipoproteinemia. The cholesterol profiles obtained by the density gradient ultracentrifugation technique resembled the band patterns after electrophoresis of identical serum samples on 2% to 16% nondenaturing polyacrylamide gradient gel: VLDL1 represents relatively large VLDL particles (diameter of about 67 nm) and VLDL2 represents relatively small VLDL particles (diameter of about 38 nm). Recentrifugation of isolated VLDL1 and isolated VLDL2 did not result in any change in their density distribution. In all groups studied, the fluidity of VLDL1 was significantly higher than that of VLDL2, in accordance with the finding that VLDL1 particles were relatively rich in triglycerides and VLDL2 particles were relatively rich in cholesteryl esters. These results indicate that the two VLDL subfractions isolated represent distinct VLDL subclasses. The density gradient ultracentrifugation technique presented in this study allows the rapid isolation and characterization of VLDL subfractions from the serum samples of normolipidemic individuals and patients with hyperlipoproteinemia.

Adult↗
PubMed2006

Remnant lipoprotein density profiling by CsBiEDTA density gradient ultracentrifugation.

Remnant lipoproteins (RLPs) are now considered a strong marker of the triglyceride-rich lipoprotein (TRL) class for cardiovascular heart disease. The purpose of this research is to demonstrate the efficacy of a novel method that combines an established immunoseparation assay used to measure the RLP class in human serum with ultracentrifugal density gradient separation. These two methods are combined to obtain an RLP density profile. The immunoseparation effectively removes the non-RLP lipoproteins from serum. The RLPs obtained from the immunoseparation are separated into two density-distinct fractions by ultracentrifugal density gradient separation in CsBiEDTA. It is now clear that IDL is distinct in density and immunoreactivity from the two RLP classes isolated by the immunoseparation and ultracentrifugation. This methodology defines the RLP by density and measures their relative prevalence in the TRL class. When applied to clinical samples, variations in the RLP subclasses in different patients are examined. The differences in the RLP density profile are also examined in fasting and postprandial samples. The RLP density profile significantly increases in the postprandial state versus the fasting state. However, the overall quantity of TRL does not appreciably increase in the postprandial state. This work demonstrates the feasibility of measuring the postprandial state in clinical samples to provide insight into the clearance of RLP by the liver as well as the general atherogenicity of these particles. The major outcome of this research is a novel analytical method that couples immunoseparation and density gradient ultracentrifugation to separate and differentially profile the RLP subclass against its nascent counterparts in the TRL class.

Centrifugation, Density Gradient↗
PubMed1990

Rapid isolation of vesicular and micellar carriers of biliary lipids by ultracentrifugation.

A simple, rapid, and new method has been developed to isolate and to quantitate the vesicular carrier of biliary lipids by isopycnic ultracentrifugation. The method combines the use of Metrizamide, as an inert centrifugation media to change the density of bile for isopycnic separation of vesicles, and a vertical rotor, to decrease both the time of centrifugation and the pressure of the hydrostatic column in the ultracentrifuge tube. Vesicles harvested from bile-Metrizamide density gradients were identified by negative staining electron microscopy. The buoyant densitites of biliary vesicles varied between 1.010 and 1.030 g/ml. The diameter of vesicles in fractions with d less than 1.020 g/ml was 82 +/- 10 nm and in fraction with d approximately 1.030 g/ml was 57 +/- 8 nm. Gel filtration chromatography with Ultrogel AcA 34 was used to validate the quantitive isolation of vesicles by the ultracentrifugal method. In experiments with bile-Metrizamide continuous preformed density gradients, greater than 93% of vesicular cholesterol was found in fractions with d less than 1.030 g/ml after 285 min of centrifugation at 50,000 rpm in a VTi vertical rotor (Beckman Instruments, Inc.). When 16% Metrizamide was dissolved in bile and centrifuged for 120 min, greater than 96% of total vesicular cholesterol was found in the top 0.4 ml of the 5-ml centrifuge tube, as assessed by gel filtration chromatography. This fraction contained less than 8% of cholesterol carried in micelles, as assessed by gel filtration chromatography. The variation coefficient of this short ultracentrifugal method to isolate biliary vesicles was 4.6%.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile↗
PubMed1970

Isolation of plasma lipoproteins by zonal ultracentrifugation in the B14 and B15 titanium rotors.

Lipoproteins were isolated from plasma of man, dog, rabbit, rat, and chicken by ultracentrifugation in continuous density gradients using the B14 titanium and B15 titanium zonal rotors. Both the VLDL and the LDL of human plasma were separated easily from the HDL and from the other more plentiful plasma proteins by centrifugation for only 1 or 2 hr in the B14 or B15 rotor, respectively. Satisfactory separation of the HDL from the more dense plasma proteins was not achieved with these rotors. The human LDL achieved isopycnic equilibrium (d 1.04) on prolonged periods (> 24 hr) of centrifugation in a sucrose-KBr density gradient. The pattern of distribution of cholesterol and phospholipid throughout the density gradient coincided with the pattern of distribution of the lipoprotein-protein measured spectrophotometrically or chemically. The concentration of cholesterol and phospholipid in the lipoproteins isolated by zonal ultracentrifugation agreed with analyses reported for lipoproteins isolated by sequential centrifugation in solutions of increasing density. The lipoproteins isolated by zonal ultracentrifugation were characterized further by their electrophoretic behavior. The fractions which were identified as the LDL (d 1.04-1.05) from all species migrated on paper as a beta-globulin; the LDL from plasma of dogs contained an additional component which has been designated as an alpha(2)-globulin. The fractions which were identified as the HDL from all species migrated as an alpha(1)-globulin. Reaction of human LDL with either rabbit antihuman beta-lipoprotein or rabbit antihuman serum resulted in a single immunodiffusion band. The S(f, 1.063) of the human LDL was calculated to be 6.0. When plasma from humans or rabbits was centrifuged in the B15 rotor, the HDL was not visible as a distinct peak and was not separable from the bulk of the more dense plasma proteins; when plasma from dogs or chickens was centrifuged under identical conditions, the HDL was clearly detectable. Even though the mean density of the HDL from dogs or chickens was not different from that of man or rabbits, the visibility of this lipoprotein in dogs and chickens was probably due to its high concentration in the plasma of these species. When plasma from the rat was centrifuged under similar conditions, the HDL was also clearly in evidence. Although rat plasma contained a relatively small concentration of HDL, the lipoprotein had a lower mean density than did the HDL of the other species and was therefore more easily separable from the dense plasma proteins. The procedure of zonal ultracentrifugation for the isolation of lipoproteins by flotation is simultaneously preparative and analytical and should find useful application in the investigation of the soluble lipoproteins from plasma and tissues.

Adult↗
PubMed1983

Ultracentrifugation in swinging-bucket and fixed-angle rotors evaluated for isolation and determination of high-density lipoprotein subfractions HDL2 and HDL3.

We evaluated the density-gradient ultracentrifugation method in a swinging-bucket rotor (Anal Biochem 111, 149-157, 1981) in a slightly modified version for isolation and determination of high-density lipoprotein (HDL) subfractions. We prestained the serum with Coomassie Brilliant Blue R, which did not change the hydrated densities of the lipoproteins, and after only 2.2 X 10(8) gav . min obtained an equilibrium distribution of the lipoproteins along the gradient. The density distribution of the HDL of 120 sera obtained from apparently healthy persons and from patients with different types of hyperlipoproteinemia was bimodal. The HDL2 could be isolated in the density range 1.072-1.098 kg/L and the HDL3 at 1.100-1.176 kg/L, the latter fraction being more heterogeneous. At a solvent density of 1.100 we obtained similar results for HDL2-and HDL3-cholesterol by ultracentrifugation in two different fixed-angle rotors with tube angles of 15 degrees or 35 degrees. Independent of the rotor and the ultracentrifugation technique, subfractionation at d = 1.100 resulted in more distinct stained entities than ultracentrifugation at d = 1.125. In the swinging-bucket rotor procedure, interference by sinking pre-beta-lipoproteins was minimized because, having hydrated densities between 1.058 and 1.075, they could be removed without aspirating the HDL2. The method is both accurate and precise. For HDL2- and HDL3-cholesterol determined in a thawed frozen serum pool, CVs were 8.8 and 6.3%, respectively (n = 18).

Centrifugation, Density Gradient↗
PubMed1984

The plasma lipoproteins in familial chylomicronemia. Analysis by zonal ultracentrifugation.

Familial chylomicronemia is a rare genetic disorder attributable to the absence of lipoprotein lipase activity or the absence of apo-CII, i.e., the cofactor for the same enzyme. Plasma lipoproteins were analyzed by zonal ultracentrifugation under rate flotation conditions in four patients with lipoprotein lipase deficiency and two patients with apo-CII deficiency. Lipoproteins of density less than 1.006 gm/ml, and particularly lipoproteins with Sf greater than 100, were present in very high concentrations. Low levels of density greater than 1.006 gm/ml lipoproteins were observed. This fraction was composed of some different and discrete lipoprotein populations: intermediate-density lipoproteins (in three of six patients, density = 1.006 to 1.019 gm/ml); low-density lipoprotein LDL2 (in all patients, density = 1.019 to 1.045 gm/ml); low-density lipoprotein LDL3 (in all patients, density = 1.045 to 1.063 gm/ml); high-density lipoprotein HDL2 (in four of six patients); and high-density lipoproteins HDL3 (in all patients). LDL3 was never observed in normal participants by means of zonal ultracentrifugation; this subclass of low-density lipoproteins seems to correspond to LDL particles of very low Sf (2 to 5) previously identified by analytical ultracentrifugation in patients with severe hypertriglyceridemia. LDL3 was isolated by means of zonal ultracentrifugation as a single and discrete peak in all patients. Lipoproteins of density greater than 1.006 gm/ml were rich in triglycerides and poor in cholesterol in comparison with normal lipoproteins. The heterogeneity of low-density lipoproteins (particularly the appearance of LDL3), low levels of total high-density lipoproteins, and lower HDL3 flotation rate than normal are typical aspects of serum lipoproteins in these patients. No significant differences in the lipoprotein profiles of the patients with lipoprotein lipase deficiency in comparison with patients with apo-CII deficiency were found. In both groups of patients, the plasma lipoproteins profile and the altered lipoprotein composition could be related to the impaired catabolism of triglyceride-rich lipoproteins caused by the absence of lipoprotein lipase activity.

Apolipoprotein C-II↗
PubMed1965

ULTRACENTRIFUGATION IN THE CONCENTRATION AND DETECTION OF ENTEROVIRUSES.

Ultracentrifugation has been evaluated as a method of concentrating enteroviruses from suspensions whose initial titers ranged from 1.7 x 10(8) to 1.6 x 10(-2) plaque-forming units (PFU) per ml. A technique employing a "trap" of 0.1 ml of 2% gelatin solution at the point at which the pellet forms in tubes for the number 30 and number 50 rotors of the Spinco model L preparative ultracentrifuge has been tested and found to have a number of advantages. Qualitative studies have been performed to determine the sensitivity of the ultracentrifuge technique in detecting the presence of enteroviruses in very dilute suspensions. There was found to be at least a 50% probability of detecting virus present initially at levels as low as 0.12 PFU per ml by means of the number 50 rotor. The input level for similar results with the number 30 rotor was found to be 0.025 PFU per ml.

Enterovirus↗
PubMed2002

Ultracentrifugation micromethod for preparation of small experimental animal lipoproteins.

Sequential flotation ultracentrifugation is commonly used in the preparation of plasma lipoproteins. However, protocols often require prolonged centrifugation time (48-72 h) and large plasma volumes (2-20 ml), which makes them unsuitable for studies on small laboratory animals. Although analytical techniques such as FPLC have often small sample requirements, further fraction analysis is often limited to the small fraction volume obtained. A sequential ultracentrifugation micromethod is described to obtain rat lipoprotein fractions from 400 microl of plasma in a cumulative centrifugation time of 7.5 h. Fraction volumes were determined and densities were adjusted to those of rat plasma lipoproteins. Polyacrylamide gel electrophoresis and enzymatic measurements of triglycerides, total cholesterol, and phospholipids were used to assess the purity of the lipoprotein fractions. The results were compared with those obtained from a classical sequential ultracentrifugation protocol. The micromethod presented here can be further adapted to other experimental animal species with little modifications.

Animals↗
PubMed1997

Analytical ultracentrifugation and agarose gel electrophoresis as tools for studying chromatin folding in solution.

Analytical ultracentrifugation and agarose gel electrophoresis each can be used to accurately quantify changes in structure that accompany chromatin folding in solution. Analytical ultracentrifugation directly measures the extent of compaction of each species present in a chromatin sample under a wide range of solution conditions. Agarose gel electrophoresis yields information about changes in the average surface charge density, size and/or shape, and conformational flexibility during chromatin folding. When used together, these methodologies are particularly powerful. Protocols for the characterization of chromatin folding by analytical ultracentrifugation and agarose gel electrophoresis are described. Discussion focuses on analysis and interpretation of experimental chromatin folding data.

Chromatin↗
PubMed1983

Comparison of ultracentrifugation and gel filtration for the isolation of bovine lipoproteins.

Lipoproteins from the plasma of three nonlactating Holstein cows were isolated using either preparative ultracentrifugation or gel filtration chromatography. Lipoprotein classes obtained by ultracentrifugation were very low density plus chylomicra, less than 1.006 g/ml; low density, 1.007-1.039 g/ml; high density1, 1.040-1.063 g/ml; and high density, 1.064-1.22 g/ml. These lipoprotein classes were individually applied to an agarose gel column to determine at what volume they eluted in comparison to lipoproteins that were separated after applying total bovine lipoproteins to the column. Three major peaks corresponding to very low density lipoproteins plus chylomicra, low density, and high density lipoproteins resulted after gel filtration of total lipoproteins. Very low density lipoproteins plus chylomicra, obtained by ultracentrifugation, eluted as a single peak, as did low density and high density lipoproteins. However, high density1 lipoproteins eluted as two peaks. The first peak eluted at the same volume as low density lipoproteins, and the second peak eluted at a volume similar to that of the ascending slope of the high density lipoprotein peak. Results from disc polyacrylamide gel electrophoresis, immunoelectrophoresis and double immunodiffusion of lipoprotein fractions, and SDS polyacrylamide gel electrophoresis of their apoproteins, similarly indicated that the lipoproteins present in the 1.040-1.063 g/ml density interval are a mixture of low and high density lipoproteins rather than a unique class of lipoproteins.

Animals↗
PubMed1983

Rapid precision interferometry for the analytical ultracentrifuge. I. A laser controller based on a phase-lock-loop circuit.

This is the first of a series of manuscripts presenting methods to enable rapid reduction of data from the Rayleigh interference optical system of the Beckman Model E analytical ultracentrifuge. Here we present a pulsed laser controller for the ultracentrifuge. This laser controller uses a phase-lock-loop to provide properly timed light pulses over the speed range of 3000 to 60,000 rpm; it effectively resolves one rotor revolution into 4096 discrete angular positions. The circuit has been designed so that the laser light bursts occur at selectable angular positions of the rotor that are independent of rotor speed even under conditions of maximum acceleration or deceleration. We have used this controller in our laboratory over a 7-year period for both photographic and real-time collection at interferometric data from the ultracentrifuge.

Computers↗
PubMed1988

Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation.

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.

Arteriosclerosis↗
PubMed1986

Distribution of lipids and apolipoproteins in human plasma by vertical spin ultracentrifugation.

A study on the fractionation of human plasma into various lipoprotein classes was performed by single spin vertical ultracentrifugation. Detailed apolipoprotein (A-I, A-II, B, C-III, and E) and lipid (cholesterol, cholesterol ester, and triacylglycerol) analyses were performed on the fractions obtained by single spin vertical ultracentrifugation. ApoB was located primarily in the low-density lipoprotein (LDL) fraction whereas Lp(a) was detected in the fractions extending into the high-density lipoprotein region. The distributions of ApoA-I and ApoA-II varied among individuals, suggesting heterogeneity in the composition of HDL, and this was confirmed by the distributions of ApoC-III, ApoD, and ApoE. ApoC-III and ApoD were associated primarily with HDL3 while ApoE showed the most variation among subjects, spreading across the entire density spectrum. Distributions of cholesterol and cholesterol ester coincided with the elution of major apolipoproteins A-I and B while the distribution of triacylglycerol was variable. An immunoassay for lecithin:cholesterol acyltransferase (LCAT) demonstrated that the majority of LCAT was present in the high- to very high-density lipoprotein region, but LCAT was also detected in LDL and the immunoreactivity extended into the very low-density lipoprotein region. The presence of apolipoproteins A-I and A-II, as well as LCAT in the intermediate lipoprotein density region (LDL1) was consistent with these species representing lipolytic remnants. This study indicated that rapid (3 h) single spin vertical ultracentrifugation offers insights into the heterogeneity of lipoproteins and offers a useful tool for monitoring perturbations of the plasma lipid transport system. In conjunction with immunosorbers, immunoassays, and micro lipid analyses, this procedure offers a method to isolate and characterize various lipoprotein subspecies.

Apolipoproteins↗