Separation of human red cells by ultracentrifugation in a discontinuous density gradient and relationship between cell age and lipid peroxidation.
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Cell Separation↗
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Genetic variants of group A apolipoproteins. Rapid methods for screening and characterization without ultracentrifugation.
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Apolipoproteins↗
[Cell culture separation by Ficoll density gradient ultracentrifugation].
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Cell Line↗
Characterization of human high density lipoproteins by zonal ultracentrifugation.
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Apolipoproteins↗
[Nomographs for determination of sedimentation constants of nucleic acids by means of centrifugation in sucrose gradients using the "Sinco" ultracentrifuge].
A set of nomographs for determination of standard sedimentation constant values for DNA and RNA directly from the results of centrifugation without any marker is calculated and plotted for 9 Spinco bucket-rotors, 5 temperatures, and two commonly used sucrose gradients (5--20 and 15--30%).
Centrifugation, Density Gradient↗
[Rubella virus hemagglutination inhibition IgM antibodies: the method of absorption of IgG by staphylococcal protein A as compared with density gradient ultracentrifugation (author's transl)].
The elimination of IgG by adsorption to Protein A of S. aureus was used for the demonstration of rubella virus specific IgM by hemagglutination inhibition. The validity of the method was proved by comparison with the results of density gradient centrifugation. The adsorption procedure led to false positive results in 9 of 56 cases and to false negative results in 12 of 46 cases, i.e. 20.7% of the results were discordant, when the sera were tested with short incubation time (30 min. room temperature) and the residual IgG (and IgA) were not excluded by controls with 2-ME (table 1 and table 2). By prolonged incubation time (18 hrs + 4 degrees C) and controls with 2-ME, false positive results were avoided, and the number of false negative results was reduced to 9 (table 2 and table 3). The latter occured mostly in cases where the disease had started 3 or more weeks ago and the titer in the IgM fraction of the sera was low (fig. 3). Under these conditions the absorption of IgG by staphylococcal Protein A proved to be a simple, specific and reliable procedure for the demonstration of rubella virus IgM within 2-3 weeks after the onset of the disease.
Bacterial Proteins↗
[Formation of iodotyrosines and iodothyronines in low iodinated human thyroglobulin fractionated by isopycnic ultracentrifugation].
Iodotyrosines and iodothyronines formation have been studied in thyroglobulins (Tg) extracted from porrly iodinated human thyroid tissues. After centrifugation in RbCl density gradient, various protein fractions containing different amounts of iodine have been isolated from pure Tg. The results indicate that, even after fractionation of Tg, the synthesis of thyroid hormones takes place at a very low level of iodine content. This suggests that the Tg fractions are still heterogenous and contain a large proportion of non-iodinated molecules.
Centrifugation, Isopycnic↗
Lipid composition of human serum lipoproteins.
1. The lipid compositions of the low-density lipoproteins, the high-density lipoproteins and the ultracentrifugal residue of human serum are presented, with emphasis on certain lipoprotein classes and lipid components not previously described. 2. Except for the lipoproteins with the lowest and highest densities, there is a trend for stepwise successive increase or, respectively, decrease in the relative amounts of the main constituents of lipoproteins. 3. High-density lipoprotein-2 and high-density lipoprotein-3 have different amounts of certain lipids; high-density lipoprotein-2 has relatively more free cholesterol and sphingomyelin; high-density lipoprotein-3 has more free fatty acids, diglycerides and ceramide monohexosides. 4. All the lipoproteins contain hydrocarbons of the alkane series. The greatest amount, which averages 4.4% of total lipid extracted, is in the ultracentrifugal residue; n-alkanes comprise 18-50% of the hydrocarbons. 5. All the lipoproteins contain ceramide monohexosides. The highest relative contents of these glycolipids are in high-density lipoprotein-3 and in the ultracentrifugal residue. 6. The ultracentrifugal residue contains 55% of the total quantity of free fatty acids present in serum. The remaining free fatty acids are distributed among the other lipoprotein classes. 7. The choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) comprise about 90% of the phospholipids in all the lipoprotein classes except the low-density lipoprotein-2, which contains about 80% of these phospholipids. 8. The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultracentrifugal residue was confirmed. 9. The low-density lipoprotein-2 and the ultracentrifugal residue are characterized by relatively high contents of the lower glycerides.
Adult↗
Advances in sedimentation velocity analysis.
On February 20, 1996, a workshop titled "Advances in Sedimentation Velocity Analysis" was held at the Biophysical Society meeting in Baltimore, Maryland, in honor of Professor David Yphantis's 65th birthday. Although he is known more for his work with sedimentation equilibrium, David's work on instrumentation and data analysis is the foundation for many of the recent advances in both equilibrium and velocity sedimentation. Over the years he has trained numerous graduate students, most of whom have gone on to emphasize the use of analytical ultracentrifugation to answer biochemical questions involving macromolecular assembly. His laboratory was one of very few that continued to use and develop analytical ultracentrifugation during its nadir in the 1970s and early 1980s. The rebirth and resurgence of analytical ultracentrifugation owe a great deal to his persistence and enthusiasm. These efforts have borne fruit. In the last five years, through his work at the National Analytical Ultracentrifugation Facility, he has helped train nearly 100 individuals in the delicate art of nonlinear least-squares analysis of equilibrium sedimentation data. Furthermore, the number of researchers using the ultracentrifuge and the number of papers published has skyrocketed in the last few years. This workshop, then, was a way to thank David for his years of devotion to analytical ultracentrifugation.
Biochemistry↗
Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility.
Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (approximately 85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained approximately 18% of the activity) and the d>1.21 g/ml fraction (which contained approximately 32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039-1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Characteristics of human lipoproteins isolated by selected-affinity immunosorption of apolipoprotein A-I.
We have isolated high density lipoproteins (HDL) from human serum by a new strategy, selected-affinity immunosorption, avoiding perturbation from ultracentrifugation or polyanion precipitation. The principle of this strategy is to utilize a subpopulation of monospecific antibodies directed against apolipoprotein A-I (apo A-I), which was preselected for dissociation under mild conditions of elution. Particles containing more than 90% of the apo A-I contained in serum were isolated uncontaminated by any other serum proteins. The immunoaffinity columns have been cycled over 300 times without apparent diminution in capacity. The apo A-I-containing particles sequestered from serum by immunosorption were more polydisperse in diameter and contained more protein than in ultracentrifugally isolated HDL. They also contained minor apolipoproteins that were not observed in ultracentrifuged HDL. Furthermore, the apo A-I-containing particles had different electrophoretic mobilities from those of ultracentrifugally isolated HDL when run under nondenaturing conditions in polyacrylamide gels. The apo A-I-containing particles isolated by our procedure separate into discrete bands similar to the subspecies visualized when whole serum is subjected to electrophoresis, whereas ultracentrifuged HDL migrate as two broad featureless zones. This suggests that selected affinity immunosorption does not subject the lipoproteins to structural disruption like that which occurs during ultracentrifugation. The principle of selected-affinity immunosorption should be of widespread utility in the isolation of fragile biological complexes.
Antigen-Antibody Complex↗
Role of influenza B virus in hepatic steatosis and mitochondrial abnormalities in a mouse model of Reye syndrome.
The hepatic steatosis observed in the influenza B virus mouse model of Reye syndrome has been attributed to infectious virus or, alternately, to decreased food intake in the virus-treated mice or impurities in the virus preparation. To resolve this issue, 4- to 6-wk-old male Balb C mice were given, by intravenous injection, 12,800 hemagglutination units of influenza B Lee/40 virus in phosphate buffered saline/1% bovine serum albumin using virus prepared by ultra-centrifugation from infected allantoic fluid, by sucrose density-gradient purification of virus prepared by ultracentrifugation from infected allantoic fluid or by irradiation of virus prepared by ultracentrifugation from infected allantoic fluid to inactivate virus. The infectivity titer of virus prepared by ultracentrifugation from infected allantoic fluid was much higher than that of sucrose density-gradient purified virus prepared from infected allantoic fluid: 50% egg infectious dose for virus prepared by ultracentrifugation from infected allantoic fluid was 3.9 x 10(4)/hemagglutination unit vs. 8.7 50% egg infectious dose/hemagglutination unit for sucrose density-gradient purified virus prepared from infected allantoic fluid. Control mice received phosphate-buffered saline/1% bovine serum albumin or uninfected allantoic fluid diluted in phosphate-buffered saline/1% bovine serum albumin. Mice were fasted to eliminate dietary variation, and livers were obtained 36 hr after virus administration. Of the above treatments, only virus prepared by ultracentrifugation from infected allantoic fluid caused clinical illness and increased hepatic triglycerides (p less than 0.02) compared with controls. Hepatic triglycerides in virus prepared by ultracentrifugation from infected allantoic fluid correlated with histopathological vacuolization scores (r = 0.5773; p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
T gamma subset specificity of lymphocyte reactive factors in juvenile rheumatoid arthritis and systemic lupus erythematosus sera.
Sera from 34 patients with juvenile rheumatoid arthritis (JRA), 31 patients with systemic lupus erythematosus (SLE), and 22 normal controls were studied for microcytotoxicity before and after clearing in the ultracentrifuge. Normal T cells as well as T gamma and non-T gamma subpopulations were used. Before ultracentrifugation all test sera showed apparent T gamma cell specificity in the microcytotoxicity assay where rabbit complement was added. JRA and SLE sera produced much higher proportions of cell killing than normal controls. Ultracentrifugal clearing resulted in marked diminution in microcytotoxicity of JRA and some SLE sera. However, a considerable proportion of lupus sera continued to show T cell subset cytotoxicity after ultracentrifugal clearing. No evidence for significant alteration of T gamma rosetting capacity was recorded when ultracentrifuge-cleared test sera were preincubated with T cells prior to T gamma EA rosette formation. Apparent T gamma cytotoxic specificity in some uncleared JRA and SLE sera may relate to high molecular weight materials (IgM and immune complexes) present in such samples, whereas in others it relates to lymphocyte reactive antibody with subset reactivity.
Adult↗
Cyclosporin A prevents the anti-murine antibody response to a monoclonal anti-tumour antibody in rabbits.
Repeated therapy of cancer with mouse monoclonal antibodies frequently produces antibodies directed against the administered antibody. We have investigated the ability of cyclosporin A (CsA) and the use of ultracentrifuged antibody to prevent the formation of anti-antibodies in rabbits. Courses of CsA, 20 mgkg-1 day-1, were given intramuscularly for 6 days to rabbits and a mouse monoclonal anti-human chorionic gonadotrophin antibody was given intravenously on day 2 with or without ultracentrifugation to remove microaggregates. The whole course was repeated after an interval of two weeks. Rabbit anti-mouse antibody production was prevented in all 8 animals that were given CsA and ultracentrifuged preparation (non UC-W14). Anti-mouse antibody was produced by all the animals that were not given CsA. A further dose of mouse antibody eight weeks after CsA demonstrated that immunological recovery had occurred in all four animals re-challenged with non UC-W14 but only 3/7 animals given an UC-W14 preparation. This suggests that CsA and ultracentrifugation facilitate the induction of immunological tolerance. The complete suppression of antibody production which could be achieved justifies a clinical trial of CsA and ultracentrifugation of antibody.
Animals↗
Loss of A-apoprotein immunoreactivity during high-density lipoprotein separation.
By use of an electroimmunoassay, concentrations of A-apoproteins were estimated in serum and in corresponding apoprotein fractions isolated by ultracentrifugation. These values were compared with high-density lipoprotein concentrations determined by analytical ultracentrifugation. Concentrations of A-apoproteins estimated in serum were considerably higher than in isolated high-density lipoprotein fractions. These discrepancies could not be accounted for entirely by material losses into other fractions during ultracentrifugal fractionation. No comparable differences in apoprotein-B concentrations were observed during the ultracentrifugal separation of low-density lipoprotein. Concentrations of A-apoproteins estimated in the residual serum after precipitation of low-density lipoproteins by heparin and manganous ions were also lower than in the corresponding whole sera. The discrepancies persisted after treatment of serum and isolated fractions with tetramethylurea, urea (9 mol/l), and by heating at 52 degrees C for 3 hours. It is considered that separation by ultracentrifugation induces subtle alterations in the surface structure of the lipoprotein species which give rise to changes in immunoreactivity.
Antigens↗
Optimization of beta-quantification methods for high-throughput applications.
BACKGROUND: Risk of cardiovascular disease is assessed, in part, by laboratory measurement of the concentrations of several lipoproteins. beta-Quantification is a method of lipoprotein measurement that uses ultracentrifugation to partially separate lipoprotein classes. Although beta-quantification is used largely in clinical and basic research, methods have not been described to allow the analysis of a large number of small-volume specimens with a short turnaround time. We report two variations of the traditional 5-mL method used by the Lipid Research Clinics Program that overcome these shortcomings. METHODS: Two lower-volume modifications of the traditional 5-mL beta-quantification method were developed. The methods used either 1 or 0.23 mL of specimen and required substantially less time for analysis (20 and 6 h, respectively) than the 5-mL method (2.5 days). The goal was to develop ultracentrifugation methods such that the concentration of cholesterol in the bottom fraction, from which LDL-cholesterol concentration is calculated, agreed with the 5-mL method. Fresh serum specimens (n = 45) were analyzed by the three methods to determine comparability of the methods based on the recovery of cholesterol in the bottom fraction after ultracentrifugation. To evaluate intrarun precision, replicate specimens (n = 17) were analyzed in a single run for each method. This experiment also evaluated how quickly the fractions would remix after separation by ultracentrifugation. For the 1-mL method, accuracy of the measurement of LDL- and HDL-cholesterol concentrations and the interrun precision were established by analysis of frozen serum specimens provided by the CDC, which established target values for the pools using reference methods. RESULTS: No clinically significant differences in cholesterol concentrations in the bottom fraction were observed for the 1- and 0.23-mL methods, which had mean biases of 0.8% and 1.5% relative to the 5-mL method, respectively. Intra- and interrun variability was acceptable for each method, e.g., <1.8% for cholesterol in the bottom fraction. Ultracentrifuged specimens were stable for at least 4 h with no evidence of contamination of cholesterol in the bottom fraction. For comparison specimens provided by the CDC, the 1-mL method met the accuracy and precision goals of the National Cholesterol Education Program for the measurement of HDL- and LDL-cholesterol concentrations (goals: total error <13% and <12%, respectively), with total errors of 6.45% and 5.43%, respectively. CONCLUSIONS: Both the 1- and 0.23-mL beta-quantification methods are suitable substitutes for the traditional 5-mL method for use in clinical and basic research for the determination of LDL-cholesterol concentration. Both methods provide much higher throughput and require substantially less specimen volume. The 0.23-mL method can be performed in 1 day, but it is slightly less precise than the 1-mL method. In our laboratory setting, as many as 80 specimens are routinely processed per day using the 1-mL method.
Blood Chemical Analysis↗
Simple method for quantifying alpha-tocopherol in low-density+very-low-density lipoproteins and in high-density lipoproteins.
We assessed the distribution of alpha-tocopherol in serum lipoprotein samples after separating the lipoprotein fractions by either sequential ultracentrifugation or selective precipitation with sodium phosphotungstate-magnesium chloride reagent. alpha-Tocopherol concentrations were determined by reversed-phase high-performance liquid chromatography. After ultracentrifugation, we found that in men, low- and very-low-density serum lipoproteins (LDL-VLDL) contained 53.6% of alpha-tocopherol vs 46.4% in high-density lipoproteins (HDL). In women, serum LDL-VLDL contained 45.6% alpha-tocopherol after ultracentrifugation vs 54.4% in HDL. After selective precipitation, the proportions of alpha-tocopherol in men were 56.1% in LDL-VLDL vs 43.9% in HDL, and in women, 45.4% in LDL-VLDL vs 54.6% in HDL. After selective precipitation, alpha-tocopherol recovery from whole lipoprotein fractions was 97% to 100% vs 80% after ultracentrifugation, thus allowing more accurate alpha-tocopherol quantification than after separation by ultracentrifugation.
Adult↗
Mathematical evaluation of methods for estimation of the concentration of the major lipid components of human serum lipoproteins.
The concentration of total cholesterol and triglycerides in the three major lipoprotein classes of human serum was measured in 136 men, randomly selected from an industrial population, by a quantitative method of lipoprotein electrophoresis on agarose gel and of fractions separated by preparative ultracentrifugation. Correlation coefficients for the two estimates were 0.98 for triglycerides in very low-density lipoproteins, 0.93 for total cholesterol in low-density lipoproteins, and 0.75 for total cholesterol in high-density lipoproteins. Data obtained form the analyses of the ultracentrifugal fractions were used to develop regression equations that predict the concentrations of total cholesterol and triglycerides in the lipoprotein classes from their concentrations in whole serum. These equations take into account the inverse curvilinear relationship between total cholesterol in high-density lipoproteins and serum tiriglyceride concentration. When applied to a separate sample of 530 men, the predicted values for triglycerides in very low-density lipoproteins and total cholesterol in low-density lipoproteins correlated as well with ultracentrifugal values as did the electrophoretic estimates. However, for total cholesterol in high-density lipoproteins, the electrophoretic method was superior. Similar regression equations were developed from ultracentrifugal lipoprotein analyses in 158 women from the same industrial population. Although the concentration of total cholesterol in the low-density lipoproteins estimated by both electrophoresis and the regression equations agreed closely in most cases with the ultracentrifugal values, errors exceeded 10% with sufficient frequency to limit the value of the estimates for this purpose. In both men and women, the ratio of total cholesterol to triglycerides in high-density lipoproteins was a hyperbolic function of serum triglyceride concentration, suggesting that cholesteryl esters in the core of this lipoprotein are progressively replaced by triglycerides as the concentration of triglycerides in very low-density lipoproteins increases. This altered composition of nonpolar lipids accounts, at least in part, for the reduction of cholesterol in high-density lipoproteins in hyperlipemic individuals.
Adolescent↗