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PubMed1981

A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum.

A density gradient ultracentrifugal procedure is described for the rapid and reproducible isolation of the major lipoprotein classes, VLDL, LDL, HDL2, and HDL3, from human serum. A step gradient is constructed from four NaCl/KBr solutions varying in density from 1.006 to 1.24 g/ml and from 3 ml of serum adjusted to d 1.21 g/ml. Separation is achieved after a single ultracentrifugation for some 56 x 10(7) gavg min at 15 degrees C in a swinging bucket rotor, at which time the lipoproteins band isopycnically and albumin and other serum proteins are sedimented. Densitometric scanning of gradients revealed a lipoprotein mass profile distinguished by four absorption maxima which fell within the hydrated density ranges of VLDL (d less than 1.016 g/ml), LDL (1.028-1.050 g/ml), HDL2 (1.066-1.100 g/ml), and HDL3 (1.100-1.153 g/ml). Fractionation of gradients on the basis of band distribution, followed by chemical, physical, and immunological analyses of the four principal fractions (i.e., bands) provided data on their electrophoretic mobility, chemical composition, morphology and size distribution, immunological reactivity and apolipoprotein content, thereby confirming their identities as VLDL, LDL, HDL2, and HDL3. The validity of this separation was supported by the quantitative distribution of apo B and apo A-I as assessed by radial immunodiffusion. Lipoprotein quantitation based on chemical analysis of gradient fractions was compared with that by analytical ultracentrifugation for a group of normolipidemic males; results concorded well, giving a similar HDL2:HDL3 ratio (0.35-0.36). Our procedure thus provides a simple and precise manner in which to assess the lipoprotein and apolipoprotein profile of human serum quantitatively and qualitatively.

Adult↗

PubMed1982

Factors affecting the integrity of high density lipoproteins in the ultracentrifuge.

Because of reported losses of apolipoproteins from high density lipoproteins during ultracentrifugation, we studied several factors that could affect the integrity of these lipoprotein complexes. Alteration of temperature, rotor configuration, and composition of the tubes had little effect on loss of apolipoprotein A-I. Interestingly, the high ionic strengths commonly used in ultracentrifugal isolation of these lipoproteins were associated with the smallest loss of apolipoprotein A-I. Losses increased substantially as the ionic strength of the medium was decreased. After repeated ultracentrifugation, apolipoprotein A-I content of high density lipoproteins approached a limiting value of approximately 65% of the original serum value, but no apolipoprotein A-II was lost. Our results imply that the binding environments of these two apolipoproteins in high density lipoproteins differ. Further, they imply that apolipoprotein A-I may exist in more than one type of environment or in more than one form in high density lipoproteins.

Apolipoprotein A-I↗

PubMed1978

[Ultracentrifugation in virus diagnosis].

Ultracentrifugation has assumed growing importance in virus diagnosis as a technique by which to concentrate and purify viruses for immediacy diagnosis on the basis of electron microscopy as well as for purely virological and serological tests. Ultracentrifugation has proved to be helpful for sizeable improvement of sensitivity for detection, which, in turn, has been conducive to time saving. The preparational ultracentrifuge enables also direct diagnosis by determination of isodensities and sedimentation coefficients of viruses and their components. Examples are mentioned, in that context, such as FMD virus, virus of vesicular swine disease, virus of larynx papillomatosis of man, and goose influenza virus. An account is given of isodensities and sedimentation coefficients of animal viruses.

Animals↗

PubMed1982

Correlative design of electrophoretic and ultracentrifugal investigation of metabolic effects of probucol.

Probucol demonstrated a profound metabolic effect on the metabolism of plasma lipoproteins by lowering the levels of circulating low-density lipoproteins (LDL). This main metabolic phenomenon was usually accompanied with other substantial events in lipoprotein patterns. Their significance ought to be confirmed by more than one analytical method. Our primary analytic system used for the basic analysis of plasma lipoproteins consisted of analytical ultracentrifugation, highly standardized agarose-gel electrophoresis and molecular separation of lipoproteins according to their molecular sizes. This paper refers to an attempt to endorse electrophoresis as a supporting technique for ultracentrifugal studies. Correlation between corresponding ultracentrifugal classes and electrophoretic fractions, declared by their coefficients, were therefore studied in order to further examine the usefulness and applicability of agarose-gel electrophoresis in the research, as well as in determining and monitoring patient therapy. Close correlations were found between the group of chylomicrons (CHY), very-very-low-density lipoproteins (VVLDL), very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), their remnants and the entire pre-beta-electrophoretic complex of plasma lipoproteins (r=0.91). CHY, VVLDL and VLDL were also well related to the alpha 2- and pre-beta 2-electrophoretic fraction (r=0.95). However, if we consider concentration units for comparison of the relation between pre-beta 1- and beta-electrophoretic fractions versus all sub-classes of LDL, such correlation was found weaker (r=0.77); results were similar when such correlation was made with HDL2,3 classes versus alpha electrophoretic fractions (r=0.78). On the contrary, when the relative percentage in the spectra of the same classes and fractions were correlated, the correlation coefficients were different: CHY and all subclasses and remnants of VLDL, versus the entire pre-beta-electrophoretic complex demonstrated r=0.87, versus the alpha 2- and pre-beta 2-electrophoretic fraction r=0.73. The electrophoretic pre-beta 1-beta-complex correlated weakly (r=0.50) with low and medium density lipoproteins (MDL). Unexpectedly tight (r=0.89) was the correlation between relative percentages of the electrophoretic alpha 1-fraction and the high-density lipoproteins (HDL).

Blood Protein Electrophoresis↗

PubMed1982

Isolation of human serum HDL1 by zonal ultracentrifugation.

High density lipoprotein subfraction-1 (HDL(1)) is thought to interact with the high-affinity apoprotein B, E receptors of peripheral cells and may act as a modulator of LDL binding and uptake. In the present study the concentration and composition of HDL(1) in normal and hypercholesterolemic sera were studied using zonal ultracentrifugation. To permit separation of the HDL(1) from VLDL, LDL, and Lp(a), the apoB-containing lipoproteins were first precipitated from serum using the phosphotungstic acid/magnesium chloride (PTA/MgCl(2)) method after which the supernatant fraction was subjected to zonal ultracentrifugation. It could be demonstrated that following PTA/MgCl(2) precipitation HDL(1) floats as a single peak at d 1.08-1.09 g/ml (NaBr) and is sufficiently separated from high density lipoprotein-2 (HDL(2)) and high density lipoprotein-3 (HDL(3)). The HDL(2)/HDL(3) subfraction pattern was not affected by the precipitation method. As previously described, in vitro incubation of serum leads to the LCAT-dependent interconversion of HDL(3) or HDL(2). Using the technique described here, it was discovered that a simultaneous elevation of HDL(1) occurred. This increase in HDL(1) concentration could not be observed when LCAT was inhibited by heat inactivation or addition of Ellman's reagent. In normal fresh serum only a small HDL(1) peak could be detected, but in patients with familial hypercholesterolemia (apoB, E receptor deficiency) HDL(1) was elevated five to tenfold compared to normal values and further increased in concentration upon incubation of serum. On the other hand, in sera of patients with familial HDL deficiency (Tangier disease), HDL(1) was undetectable. Analysis of the HDL fractions in serum of a patient with abetalipoproteinemia revealed that following in vitro incubation there was formation of HDL(1) despite the lack of apoprotein B-containing lipoproteins. These data support the concept that HDL(1) formation occurs during LCAT-mediated HDL(3)/HDL(2) interconversion in vitro.-Schmitz, G., and G. Assmann. Isolation of human serum HDL(1) by zonal ultracentrifugation.

Apolipoproteins↗

PubMed1994

Ultracentrifugal subclasses of low and intermediate density lipoproteins.

Low density lipoprotein (LDL) and intermediate density lipoprotein (IDL) classes have been shown to be composed of discrete metabolic entities or subclasses. Present ultracentrifugal methods are unable to precisely determine these subclasses. A new analytical micro-ultracentrifugal method was developed that facilitates the determination of IDL and LDL subclasses and their F1.21 flotation coefficient from ultracentrifugal scans. The method is based on the modification of a published equation (Fujita, H. 1956. J. Chem. Phys. 24: 1084-1090) adapted to calculate concentration gradient boundary curves for IDL and LDL that are approximately Gaussian in form. Using an extension of this modified equation, theoretical distributions of the gradient curves were calculated. By applying the theoretical distributions, IDL and LDL subclasses were resolved from absorbance scans as Gaussian concentration gradient boundary curves. Both theoretically calculated and experimentally determined boundary curves for IDL and LDL lipoproteins were plotted and found to be in excellent agreement. Three subclasses of LDL and four subclasses of IDL were determined. The mean flotation rates of the LDL subclasses were: LDL1 = 37.2 +/- 0.6, LDL2 = 31.1 +/- 0.9, and LDL3 = 26.7 +/- 0.7. The mean flotation rates of the IDL subclasses were: IDL1 = 61.6 +/- 0.9, IDL2 = 53.9 +/- 1.0, IDL3 = 50.1 +/- 0.6, and IDL4 = 45.6 +/- 1.1.

Chemical Phenomena↗

PubMed2005

Evaluation of rat and rabbit sera lipoproteins in experimentally induced hyperlipidemia by analytical ultracentrifugation.

Animals of various species are widely used as models with which to study atherosclerosis and the lipoprotein metabolism. The objective of this study was to investigate the lipoprotein profiles in Wistar rats and New Zealand white rabbits with experimentally induced hyperlipidemia by means of ultracentrifugation. The Schlieren curves were utilized to compare suckling and adult rat sera to determine whether aging causes alterations in lipoprotein profiles. A striking feature of the data is the high concentration of low-density lipoproteins (LDL), (>5.2 mmol/l cholesterol) in the 2-week old rat serum pool which was greatly decreased in the 3-weeks rat serum pool (<1.3 mmol/l cholesterol). Additional experiments were performed to permit a direct comparison of the amounts of lipoprotein present in rat sera in experimental hyperlipidemia post-Triton WR 1339 administration. Rapid changes in concentrations in very low-density lipoproteins (VLDL), LDL and high-density lipoproteins (HDL) were observed after Triton injection. The administration of Triton WR 1339 to fasted rats resulted in an elevation of serum cholesterol levels. Triton physically alters VLDL, rendering them refractive to the action of lipolytic enzymes in the blood and tissues, preventing or delaying their removal from the blood. Whereas the VLDL concentration was increased markedly, those of LDL and HDL were decreased at 20 h after Triton treatment. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate of LDL aliquots, to prepare radioactive-labeled lipoproteins and to study induced hyperlipidemia in rabbits. Analytical ultracentrifugation was applied to investigate the LDL flotation peaks before and after cholesterol feeding of rabbits. Modified forms of LDL were detected in the plasma of rabbits with experimentally induced atherosclerosis. ApoB-containing particles, migrating as LDL, intermediate density lipoproteins and VLDL were the most abundant lipoproteins. Gamma camera in vivo scintigraphy on rabbits with radiolabeled lipoproteins revealed visible signals corresponding to atherosclerotic plaques of the aorta and carotid arteries.

Age Factors↗

PubMed1983

Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients.

Quantitative plasma lipoprotein separation is usually performed by sequential ultracentrifugation which because of the necessity of repeated ultracentrifugation is extremely time consuming and results in changes in lipoprotein composition. We describe a simple, reliable, and rapid method for quantitative plasma lipoprotein separation which is based on ultracentrifugation of the plasma in a discontinuous density gradient media consisting of a saline solution of different densities placed over a plasma solution of 1.250 g/ml (with potassium bromide). After 48 hr of ultra-centrifugation in a swinging bucket rotor excellent lipoprotein separation was demonstrated. The lipoproteins were pure and there was significantly less loss of high density lipoprotein protein to the lipoprotein deficient plasma (d greater than 1.210 g/ml) when compared to the losses incurred during the sequential flotation method. The results of separating the plasma lipoproteins from subjects with dyslipoproteinemias by this method were demonstrated. We conclude that discontinuous density gradient ultra-centrifugation is the method of choice for quantitative plasma lipoprotein separation for both clinical and research use.

Adult↗

PubMed1975

Copper (II) induced polymerization of human albumin, and its depolymerization by diglycyl-L-histidine: a pH static and ultracentrifugation study.

Copper (II) ions successively induce dimers and tetramers of human serum albumin (L) when the Cu (II) concentration is extended beyond that of 200 muM. This is shown by emf titrations and by ultracentrifugation experiments. The emf titrations, which involve a new pH static method, were performed at 25 degrees, in a 0.5 M NaCIO4 medium at pH 6.59, using glass and copper amalgam electrodes. The total concentration of Cu(II) varied from 0.14 to 2.2 mM and the albumin concentration from 0.05 to 0.7 mM. In order to evaluate the formula of the main complexes, without using any a priori assumptions regarding their compositions, a detailed graphic procedure was used. The results, in the form of equilibrium constants for the main species, were refined by the use of a general least squares computer program. The experimental data are found to be consistent with the formation of the monomeric CuL, Cu5L, and Cu6L species and the dimeric Cu3L2, Cu4L, Cu6L, and Cu8L2 species. In addition, there is some indication for a minor species, most probably the Cu12L4 tetramer. The pH static results qualitatively agree with the findings obtained by ultracentrifugation. As indicated by distinct bands and their S-values, ultracentrifugation experiments show not only monomeric and dimeric species of albumin, but also tetrameric species. The polymerization of the albumin is reversible, since diglycyl-L-histidine, a peptide designed to mimic the Cu (II) transport site of albumin, depolymerizes the Cu (II)-albumin polymers.

Binding Sites↗

PubMed2003

Measurement of plasma small-dense LDL concentration by a simplified ultracentrifugation procedure and immunoassay of apolipoprotein B.

BACKGROUND: Existing methods for detecting small-dense low-density lipoprotein (SD-LDL) are either semiquantitative (e.g., gradient gel electrophoresis) or require specialised laboratory methods (e.g., density-gradient ultracentrifugation, DGU). METHODS: We report a method in which plasma was adjusted to a density (D) of 1.044 and 1.060 g/ml, respectively, in two tubes, both of which underwent ultracentrifugation (UC). A measure of SD-LDL apolipoprotein B (apo B) was obtained by subtraction of the apo B concentration in D>1.060 g/ml lipoproteins from that in D>1.044 g/ml lipoproteins to correct for apo B associated with lipoprotein (a) [Lp(a)]. This procedure was evaluated in paired plasma samples in healthy men (n=62) and in age-matched healthy women (n=74) and in age-matched primary dyslipidaemic men (n=72) and women (n=29) and compared with an established density-gradient ultracentrifugation (DGU) method. RESULTS: The dyslipidaemic patients had either decreased high-density lipoprotein cholesterol (HDL-C) and/or increased triglycerides. In dyslipidaemic men, SD-LDL apo B level (23 [5-77] mg/dl) was significantly higher than in healthy men (P<0.001). In dyslipidaemic women, the SD-LDL apo B levels (11 [4-71] mg/dl) were significantly higher than in healthy women (7 [1-45] mg/dl; P<0.005). The concentration of SD-LDL apo B correlated inversely with HDL-C in both women (r=-0.280: P<0.005) and men (r=-0.464; P<0.0001) and positively with triglyceride concentration in both women (r=0.213; P<0.05) and men (r=0.592: P<0.0001). Correction for apo B in Lp(a) increased the analytical variation, which was 12% for apo B at D=1.044-1.060 g/ml and 9% for apo B measured at D>1.044 g/ml. Although the correlation between the new method and DGU results was high (r=0.830; P<0.0001, n=43), the concentration of apo B at D>1.044 g/ml correlated strongly with both corrected results (r=0.978; P<0.0001; n=237) and also with SD-LDL isolated using the DGU method (r=0.832; P<0.0001). Results at D>1.044 g/ml showed the expected correlations both with HDL-C (r=-0.465: P<0.0001) and triglycerides (r=0.526; P<0.0001). CONCLUSIONS: The new method gave results consistent with earlier published findings using other techniques. Further simplification of the method using a single-density spin at D>1.044 g/ml appears feasible and may provide an easier quantitative method for clinical use.

Aged↗

PubMed1994

Recovery of cholesterol and triacylglycerol in very-fast ultracentrifugation of human lipoproteins in a large range of concentrations.

Very-fast ultracentrifugation using a benchtop ultracentrifuge was applied to the analysis of lipoproteins in 0.5 ml of human plasma. VLDL, IDL and LDL were flotated at densities of 1.006, 1.019 and 1.063 kg/l in runs lasting 30, 100 and 100 minutes. Chylomicrons, if present, were flotated in a separate run. HDL were isolated by precipitation of the apolipoprotein B-containing lipoproteins from total plasma using polyethylene glycol. Three series of separations were routinely performed: 1. VLDL run alone (632 samples), 2. VLDL run + LDL run (122 samples), and 3. Chylomicron separation + VLDL run + IDL run (92 samples). The concentrations of cholesterol and triacylglycerol were obtained for plasma, chylomicrons, VLDL, IDL, LDL and HDL. Plasma values ranged from 1.8 to 37.1 mmol/l cholesterol and 0.26 to 50.2 mmol/l triacylglycerol. The plasma triacylglycerol concentrations were corrected for free glycerol by 3% (for triacylglycerols < 2.5 mmol/l) and by 2% (for triacylglycerols > or = 2.5 mmol/l). The recovery rate of lipids after ultracentrifugation was determined by comparing the concentrations in lipoproteins and in plasma. It was near to 100% and decreased for samples with extremely high lipid concentrations.

Cholesterol↗

PubMed1982

Separation of serum lipoproteins of Japanese quail by disc polyacrylamide gel electrophoresis and single discontinuous density gradient ultracentrifugation.

A method based on disc polyacrylamide gel electrophoresis (disc PAGE) for the separation of serum lipoproteins of Japanese quail (Coturnix coturnix japonica) prestained with Sudan Black B is described and evaluated. Good separation was obtained by using 3% separating gel and decreasing the concentration of buffer solution A used for preparation of separating gel solution to one-half that recommended by Narayan (1975). Best resolution of the high density lipoproteins was achieved by using 7 to 10% of separating gel, which separated the high density lipoproteins into three bands. Clear lipoprotein bands were obtained after ultracentrifugation of prestained serum. For corresponding positions, the bands of unstained serum after staining had the same mobility in the disc (PAGE) as prestained lipoprotein bands. In quail, density smaller than 1.006 g/ml contains chylomicrons and very low density lipoproteins (VLDL); density from 1.018 to 1.05 g/ml contains the low density lipoprotein (LDL); and density more than 1.05 g/ml and less than 1.16 g/ml contains the high density lipoproteins (HDL). In the profile of serum lipoproteins after ultracentrifugation, two peaks were observed in the density range of HDL. There was one small peak with density 1.05 to 1.09 g/ml and one large peak with density 1.09 to 1.16 g/ml. Based on the lipoprotein profiles from both disc PAGE and ultracentrifugation, HDL is the predominant form, LDL is intermediate, and VLDL and chylomicrons are smallest in amount.

Animals↗

PubMed1991

A micro-enzymatic method to measure cholesterol and triglyceride in lipoprotein subfractions separated by density gradient ultracentrifugation from 200 microliters of plasma or serum.

A micro-enzymatic method was developed to measure total cholesterol (CHOL) and triglyceride (TG) in lipoproteins and their subfractions separated by density gradient ultracentrifugation. This method had a detection limit and sensitivity below 2 mg/dl and accuracy (bias to reference sera) and imprecision (coefficient of variation) of less than 3% between 2 and 30 mg/dl for both CHOL and TG. In addition, the method was in good agreement with standardized Abell-Kendall CHOL (r = 0.98) and enzymatic TG (r = 0.99) methods. Lipoproteins from 200 microliters of plasma or serum were separated by either equilibrium (EQ)- or rate zonal (RZ)-density gradient ultracentrifugation and the resulting fractions were analyzed for CHOL and TG by the micro-enzymatic method. Lipoprotein measurements by these micro-enzymatic/density gradient methods were highly correlated with standardized Lipid Research Clinic (LRC) procedures and preparative ultracentrifugation. The EQ-density gradient procedure also allowed determination of CHOL and TG in LDL and HDL subfractions within any desired density interval. These methods will facilitate the measurements and study of lipoproteins and their subfractions especially in infants, children, the elderly, and small animals. In addition, the micro-enzymatic method may be adapted to other modes of lipoprotein separation such as liquid chromatography, electrophoresis, and precipitation. CHOL or TG determinations could be made on approximately 500 density gradient fractions per hour.

Adult↗

PubMed1990

Conversion of plasma VLDL and IDL precursors into various LDL subpopulations using density gradient ultracentrifugation.

The contribution of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) to various low density lipoprotein (LDL) subfractions was examined in three normal subjects and two with familial combined hyperlipidemia. Autologous VLDL + IDL (d less than 1.019 g/ml) or VLDL only (d less than 1.006 g/ml; one subject only) were isolated by sequential ultracentrifugation, iodinated, and injected into each subject. The appearance, distribution, and subsequent disappearance of radioactivity into LDL density subpopulations was characterized using density gradient ultracentrifugation. These techniques help determine the contribution of precursors to various LDL subpopulations defined uniquely for each subject. The results from these studies have suggested: 1) it took up to several days of intravascular processing of precursor-derived LDL before it resembled the distribution of the 'steady-state' plasma LDL protein; 2) plasma VLDL and IDL precursors contributed rapidly to a broad density range of LDL; 3) the radiolabeled plasma precursors did not always contribute to all LDL density subfractions within an individual in proportion to their relative LDL protein mass as determined by density gradient ultracentrifugation; 4) with time, the distribution of the precursor-derived LDL became more buoyant or more dense than distribution of the LDL protein mass; and 5) the kinetic characteristics of precursor-derived particles within LDL changed within a relatively narrow density range and were not always related to the LDL density heterogeneity of each subject. These studies emphasize the complexities of apoB metabolism and the need to design studies to carefully examine the production of various LDL subpopulations, the kinetic fate and interconversions among the subpopulations, and ultimately, their relationship to the development of atherosclerosis.

Apolipoproteins B↗

PubMed1984

Advantages and limitations of density gradient ultracentrifugation in the fractionation of human serum lipoproteins: role of salts and sucrose.

Two density gradient ultracentrifugation methods, Redgrave et al. (1975. Anal. Biochem. 65: 42-49) and Nilsson et al. (1981. Anal. Biochem. 110: 342-348), currently used for the separation and analysis of plasma lipoproteins were compared with respect to their resolving power and capacity to obtain pure products as a function of time of ultracentrifugation using the same rotor (Beckman SW-40), speed (150,000 g), and temperature (14 degrees C). The effects of sucrose and salts were also investigated. The Redgrave gradient insured the separation of the major classes of plasma lipoproteins after 24 hr of centrifugation; however, equilibrium conditions were only reached after 48 hr, at which time the lipoproteins were contaminated by albumin. When the effluents from each rotor tube were continuously monitored at 280 nm, each lipoprotein band gave values that were higher than those from mass analyses. This was due to a light scattering effect, the extent of which was dependent on the concentration of lipoproteins and salts. Sucrose prevented the scattering effect and was found to bind irreversibly to the apolipoproteins. In contrast, after 66 hr centrifugation, the lipoproteins obtained from the Nilsson gradient exhibited a close correspondence between protein mass and absorbance values at 280 nm, had no scattering effect, and were uncontaminated by albumin. The difference in spectroscopic behavior between the Redgrave and the Nilsson procedures was attributed to three factors: 1) the presence of sucrose in the latter gradient and incorporation of this sugar into lipoproteins as assessed by mass and radioactivity measurements; 2), the salt density to which the serum samples were exposed to at the beginning of the ultracentrifugation; and 3) the final lipoprotein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Bromides↗

PubMed1977

Fractionation of human serum lipoproteins by single-spin gradient ultracentrifugation: quantification of apolipoproteins B and A-1 and lipid components.

A sensitive and reproducible method has been developed for separation of the major serum lipoproteins from 1 ml or less of human serum by isopycnic density gradient ultracentrifugation. The serum, applied to a step gradient (total volume 12.8 ml), was spun for 48 hr at 38,000 rpm at 10 degrees C and, in each of the fractions, apolipoproteins B and A-I were quantified by the respective radioimmunoassays. The markers for lipid distribution used were [4-(14)C]cholesterol and [U-(14)C]lecithin, each incubated with an aliquot of serum at 20 degrees C for 75 min prior to ultracentrifugation. In control sera, three main fractions, very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins were clearly separated from a bottom fraction. Their flotational, electrophoretic, and chemical properties were in good agreement with those reported for the corresponding lipoproteins separated by conventional ultracentrifugation. Both apo B and apo A-I were fully recovered. Essentially all of the apo B was found in VLDL (9.3 +/- 3.5%) and LDL (87 +/- 4.6%); of the apo A-I, 81.0 +/- 5.7% was in HDL and the remainder (17.0 +/- 5.8%) was in the bottom fraction. The peak activities of [(14)C]cholesterol coincided with the peak of apo B in both LDL and VLDL, and with the peak of apo A-I in HDL. The results with the radiolabeled cholesterol were in good agreement with those obtained by chemical analyses. Carbon 14-labeled lecithin, although fully recovered, was not an accurate marker of phospholipid distribution because, under our experimental conditions, a significant amount of the lecithin was converted into its lyso derivative. The mechanism of the conversion was not established; it appeared to be unrelated to the activities of either lecithin-cholesterol acyl transferase or a Ca(2+)-dependent phospholipase. Besides its validity in the study of control sera, our method also proved successful in the separation of the serum lipoproteins of the few patients with dyslipoproteinemia (abetalipoproteinemia and familial hypercholesterolemia) who were examined. However, the applicability of the method to all dyslipoproteinemias was not assessed. Taken together, the results indicate that the single-spin method could be useful in clinical studies as a complement to other established techniques.

Abetalipoproteinemia↗

PubMed1964

STUDIES ON ISOLATED CELL COMPONENTS. XVII. THE DISTRIBUTION OF CYTOCHROME OXIDASE ACTIVITY IN RAT LIVER BREI FRACTIONATED IN THE ZONAL ULTRACENTRIFUGE.

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.

Cell Nucleus↗

PubMed1994

Development of a density gradient ultracentrifugation technique for the resolution of plasma lipoproteins which avoids apo E dissociation.

A density gradient ultracentrifugation technique for analyzing and isolating plasma lipoproteins was developed that was simple to set up, allowed for the isolation of the plasma lipoproteins in one centrifugal spin, and avoided the dissociation of apolipoprotein E from high-density lipoprotein (HDL) which can occur when plasma is subjected to ultracentrifugation at high concentrations of salt. The density gradient emphasized the resolution of the HDL density region while still enabling the separation of low-density and very low-density lipoprotein.

Apolipoproteins E↗