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PubMed1986

Simplified methods for measuring cholesterol concentrations of high-density lipoprotein subclasses in serum compared.

We used a combination of heparin micro-affinity column chromatography and heparin-Mn2+/dextran sulfate (DS) precipitation procedures to measure directly the total high-density lipoprotein (HDL)-cholesterol and HDL3-cholesterol in serum. The value for HDL2-cholesterol was obtained by subtracting the value for HDL3-cholesterol from that for total HDL-cholesterol. Results of this methodology and of the original heparin-Mn2+/DS double-precipitation method were compared with those of preparative ultracentrifugation. Concentrations of HDL- and HDL2-cholesterol by the original double-precipitation method were respectively 26 and 33 mg/L lower than by the ultracentrifugation method (p less than 0.001). Corresponding values by the micro-affinity/precipitation method were 36 and 29 mg/L higher than by the ultracentrifugation method (p less than 0.001). The values for HDL3-cholesterol by ultracentrifugation and by precipitation differed only by 7 mg/L (p greater than 0.05). Micro-affinity/precipitation and double-precipitation results both correlated well with those by ultracentrifugation (r = 0.84 to 0.94); the y-intercept of the comparison with the micro-affinity/precipitation method was close to zero. Use of the micro-affinity/precipitation method with samples from black or white adolescents showed that the blacks had higher concentrations of HDL2-(p less than 0.01) and HDL3-cholesterol (p less than 0.02) than did the whites.

Adolescent↗

PubMed1989

Distribution of thiobarbituric acid-reactive substances in lipoproteins and proteins in serum.

We assessed the distribution of malondialdehyde (MDA) in lipoproteins and proteins in serum after using two procedures to separate the lipoproteins: sequential ultracentrifugation or selective precipitation with a sodium phosphotungstate and magnesium chloride reagent followed by ultracentrifugation of the supernate. MDA concentrations were determined by the thiobarbituric acid reaction and quantified by fluorometry. We found that 43% of the thiobarbituric acid-reactive substances (TBARS) was bound to the lipoproteins--27% to very-low- and low-density lipoproteins (VLDL-LDL) and 16% to high-density lipoproteins (HDL)--and from 11.5% to 15.8% to proteins, depending on the separation procedure. Residual unbound TBARS were located in the ultracentrifugation layers that contained no lipoproteins or proteins. The TBARS concentration in serum lipoproteins containing apolipoprotein B (i.e., VLDL-LDL) was the same after ultracentrifugation or selective precipitation. We therefore consider the precipitation method more suitable for routine TBARS determination in these lipoproteins, because it is easier to handle and faster. However, for determination of TBARS in HDL, selective precipitation requires subsequent ultracentrifugation at a density of 1.21 kg/L.

Adult↗

PubMed1970

Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions.

Procedures are described for the isolation of lipoproteins from human serum by precipitation with polyanions and divalent cations. A mixture of low and very low density lipoproteins can be prepared without ultracentrifugation by precipitation with heparin and either MnCl(2) alone or MgCl(2) plus sucrose. In both cases the precipitation is reversible, selective, and complete. The highly concentrated isolated lipoproteins are free of other plasma proteins as judged by immunological and electrophoretic methods. The low density and very low density lipoproteins can then be separated from each other by ultracentrifugation. The advantage of the method is that large amounts of lipoproteins can be prepared with only a single preparative ultracentrifugation. Polyanions other than heparin may also be used; when the precipitation of the low and very low density lipoproteins is achieved with dextran sulfate and MnCl(2), or sodium phosphotungstate and MgCl(2), the high density lipoproteins can subsequently be precipitated by increasing the concentrations of the reagents. These lipoproteins, containing small amounts of protein contaminants, are further purified by ultracentrifugation at d 1.22. With a single preparative ultracentrifugation, immunologically pure high density lipoproteins can be isolated from large volumes of serum.

Acetates↗

PubMed1983

[Apoprotein B in high density lipoproteins: variations according to the type of hyperlipoproteinemia].

Two techniques for the separation of high density lipoproteins are compared: ultracentrifugation and concanavalin A precipitation. There is a significant correlation for the high density lipoproteins cholesterol levels found by the two techniques (r = 0.66, p less than 0.001). Nonetheless ultracentrifugation gives higher values than precipitation. When the ultracentrifuged high density lipoproteins fraction was precipitated with concanavalin A, a highly significant correlation was found between the amounts of apoprotein B and cholesterol present in the precipitate (r = 0.90, p less than 0.001). These results indicate the presence of a subfraction of the ultracentrifuged high density lipoproteins fraction which is precipitated by concanavalin A and which contains apoprotein B. This fraction is present in varying proportions according to the type of hyperlipoproteinemia, types iib and IV having the greatest amounts. In conclusion, only the concanavalin A precipitation technique results in a separation of apoprotein B-containing and non-apoprotein B-containing lipoproteins. This renders it preferable to ultracentrifugation for determining atherogenicity risks.

Adult↗

PubMed1981

Characterization of human high-density lipoproteins by gradient gel electrophoresis.

Gradient gel electrophoresis in conjunction with automated densitometry was applied to the identification and estimation of subpopulations of high-density lipoproteins (HDL) in the ultracentrifugal d less than or equal to 1.200 fraction from human plasma. The frequency distribution of relative migration distances (RF values) of subpopulation peaks in HDL patterns of a group (n = 194) of human subjects showed five apparent maxima: two in the RF range associated with the HDL2 subclass, and three in the RF range of the HDL3 subclass. HDL within RF intervals bounding these maxima were designated (HDL2b)gge, (HDL2a)gge, (HDL3a)gge, (HDL3b)gge and (HDL3c)gge and were shown to correspond approximately to material determined by analytic ultracentrifugation within the HDL2b, HDL2a and HDL3 components. Material represented by the HDL2a component, as resolved by three-component analysis of the ultracentrifugal Schlieren pattern, was found by gradient gel electrophoresis to be polydisperse in particle size. Mean hydrated densities and particle sizes of HDL corresponding to those with RF values of the frequency maxima were: 1.085 g/ml and 10.57 nm in the (HDL2b)gge; 1.115 g/ml and 9.16 nm in the (HDL2a)gge; 1.136 g/ml and 8.44 nm in the (HDL3a)gge; 1.154 g/ml and 7.97 nm in the (HDL3b)gge; and 1.171 g/ml and 7.62 nm in the (HDL3c)gge. The mean hydrated density values of the subpopulations within the (HDL3a)gge and (HDL3b)gge were comparable to those of the HDL3L and HDL3D components recently characterized by zonal ultracentrifugation. High order and statistically significant correlations between densitometric scans of the (HDL2b)gge, (HDL2a)gge and (HDL3)gge material, as obtained from gradient gels, and plasma concentrations of the HDL2b, HDL2a and HDL3 components, as obtained from analytic ultracentrifugation, were demonstrated.

Adult↗

PubMed1975

Determinants of the formation and activity of factor V-phospholipid complexes. II. Molecular properties of the complexes.

The stability, buoyancy, and intrinsic activity for factor V-phospholipid complexes were investigated. The decay of factor V activity in the absence of phospholipid followed first order kinetics; however, in the presence of several phospholipids a biphasic decay curve was observed. The addition of phosphatidylethanolamine to factor V produced only a small loss of activity in the first 2 minutes but decreased the subsequent rate of inactivation fourfold. A PE-factor V complex with a low bouyant density was separated from uncomplexed factor V by sucrose density ultracentrifugation. The association constant for this complex was 5 X 10(6) M-1 with approximately 2 moles of factor V bound per mole of lipid micelle. The isolated complex was capable of increasing prothrombin conversion 10-fold without additional phospholipid. A still lighter complex increased the rate of prothrombin conversion 18-fold. Phosphatidyl serine produced a concentration-dependent loss of up to 95% of the factor V activity in the first 2 minutes. After ultracentrifugation on a sucrose density gradient, a PS-factor V complex of increased density was detected. This complex failed to accelerate prothrombin conversion in the intrinsic two-stage assay. Except at very high concentrations, phosphatidylcholine did not alter the kinetics of inactivation of factor V. A factor V-phosphatidylcholine complex could not be detected after ultracentrifugation. When added to factor V, cardiolipin (200 mug/ml), produced a rapid 50% decline in activity with a subsequent three-fold increase in the rate of inactivation. No activity was recovered after ultracentrifugation of factor V in the presence of cardiolipin. Saturated phosphatidylethanolamine produced a concentration dependent initial loss of activity, but only a minimal increase in the subsequent rate of inactivation. At 200 mug/ml almost no light complex was detected after ultracentrifugation, but at 800 mug/ml a light complex was observed. This behavior corresponds to the ability of saturated phosphatidylethanolamine to accelerate prothrombin conversion only at very high concentrations. Thus, phospholipids combine with factor V to form complexes which differ in their ability to accelerate prothrombin conversion. The most active species are stable lipoprotein complexes of lower buoyant density than factor V.

Animals↗

PubMed1977

Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat.

The feeding of cholesterol to rats resulted in marked alterations in the type and distribution of the plasma lipoproteins and their apoproteins. The hyperlipoproteinemia was characterized by an increase in the d < 1.006 lipoproteins (B-VLDL and VLDL), an increase in the intermediate and low density lipoproteins (LDL), and the appearance of HDL(c). Associated with these lipoproteins was a prominence of the arginine-rich apoprotein. The high density lipoproteins (HDL) were decreased. A two-dimensional immunoelectrophoretic procedure was adapted to quantitate the changes in distribution of the arginine-rich apoprotein in the plasma and various ultracentrifugal fractions obtained from control and cholesterol-fed rats. In rats fed the cholesterol diet, the total plasma arginine-rich apoprotein increased from a control value of approximately 29 mg/dl to 47 mg/dl. The method of ultracentrifugation, however, was found to markedly alter the quantitative results. When the 60 Ti rotor was used at maximum speed to isolate the ultracentrifugal fractions, less than 50% of the total plasma arginine-rich apoprotein was associated with the lipoproteins in the d < 1.006 or the d 1.006-1.02, 1.02-1.063, or 1.063-1.21 ultracentrifugal fractions. By contrast, after limited ultracentrifugation with the 40 rotor, much less arginine-rich apoprotein was lost, with approximately 20% of the arginine-rich apoprotein in control rats and 10% in cholesterol-fed rats found in the d > 1.21 fraction. Significant alterations in the arginine-rich apoprotein quantitation notwithstanding, the observations of increased arginine-rich apoprotein in the B-VLDL, intermediate fraction, and HDL(c) following cholesterol feeding remained valid. However, precise quantitation awaits refinements in lipoprotein isolation techniques.

Animals↗

PubMed1986

Isolation and partial characterization of endothelial cell extracellular complexes.

Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.

Antigens, Surface↗

PubMed1976

A study of the heparin-manganese chloride method for determination of plasma alpha-lipoprotein cholesterol concentration.

To assess the limits of the heparin MnCl2 precipitation method for quantitation of alpha-lipoprotein cholesterol (C-HDL), effects of varying final McCl2 and heparin concentrations were studied, and the precipitation method was compared to preparative ultracentrifugation. In 65 parallel plasma aliquots, C-HDL (X+/-SE) determined by ultracentrifugation (54.3+/-1.8 mg/dl) correlated significantly (r=0.98, P less than 0.001) with the precipitation method (56.0+/-1.9 mg/dl). C-HDL by ultracentrifugal and precipitation methods were also similar in 16 subjects with triglycerides ranging from 150 to 312 mg/dl (41.4+/-2.6, 43.4+/-2.8, r=0.97, P less than .001). A constant amount of cholesterol in the supernatant was measured over a final McCl2 range of 0.046-0.23 M, and cholesterol values in the supernatant at final McCl2 concentrations of 0.046, 0.05, and 0.055 M did not differ from each other, P greater than 0.1. However, cholesterol levels in the supernatant at final MnCl2 concentration of 0.042 M differed from those at concentrations of 0.046, 0.05, and 0.055 M, P less than 0.05 and the amount of supernatant cholesterol increased as the final McCl2 concentration was reduced from 0.042 to 0.02 M. A constant amount of cholesterol in the supernatant was measured over a heparin concentration range of 92-734 USP units/ml. The final MnCl2 and heparin concentrations of 0.046 M and 184 USP units/ml, which are incorporated in widely used procedures, gave C-HDL values for the precipitation method which were in close agreement with the ultracentrifugal method. There is no evidence for a heparin-Mn++ precipitation of HDL and systematic underestimation of HDL by the precipitation method. However, the final MnCl2 concentration is very near the minimum required for accurate measurement of C-HDL. To preclude incomplete precipitation of low and very low density lipoproteins by insufficient manganese concentration, an increase of the manganese concentration should be considered.

Chemical Precipitation↗

PubMed2003

Adair was right in his time.

The subunit molar mass of hemoglobin was established in the 19th century by chemical analysis, the tetramer structure by osmotic pressure determination in 1924 and by the newly developed analytical ultracentrifuge in 1926, which became a powerful tool for biological macromolecule molar mass determinations. The Svedberg equation was derived by eliminating the translational friction coefficient relating to sedimentation and diffusion in the ultracentrifuge in a strictly solute/solvent vanishing concentration two-component system analysis. A differential equation describing the radial equilibrium concentration distribution in the ultracentrifuge was also derived, both yielding the buoyant molar mass (1-nu2rho)M2 term. Many years later it was realized that solutions of biological macromolecules are multicomponent systems and the two-component analysis leads to minor or major erroneous results. Thermodynamic derivation of an equation for multicomponent systems redefines the buoyant molar mass terms by (deltarho/deltac2)muM2, leading to correct molar mass (g/mol) values following determination of the density increment at constant chemical potentials of diffusible solutes, and powerfully connects the analytical sedimentation equation to the osmotic pressure concentration derivative and, in a broad complementary sense, to light, X-ray and neutron scattering experiments. Macromolecular interactions can be studied with high precision and solute-solvent interactions yield powerful information relating to "thermodynamic" hydration, closely related to hydration derived from X-ray diffraction, as well as solute-cosolute interactions. A series of examples is given to demonstrate the correctness and usefulness of the thermodynamic multicomponent system approach. It is a strange fact that in current analytical ultracentrifugation analysis the elegant and powerful multicomponent solution technology is almost totally disregarded and the classical limited validity Svedberg approach is used uniquely.

Biopolymers↗

PubMed1980

Determination of the K2 lipid clearance coefficient by centrifugation. A comparison with the Gordis test and spectrophotometric measurement.

The K2 lipid clearance coefficient was calculated for the intravenous fat tolerance test (iv FTT), after estimating triglycerides by spectrophotometry, the Gordis technique or by preparative ultracentrifugation. The best correlation between the K2 values (r = 0.95, p < 0.001) is for the results of the Gordis test and ultracentrifugation. Spectrophotometry shows poorer correlations: r = 0.66 and p < 0.05 with the Gordis test, and r = 0.78 and p < 0.01 with ultracentrifugation. Spectrophotometry is only suitable for detection but the two other techniques can be applied to more detailed studies. Ultracentrifugation is faster, less laborious and less liable to error.

Centrifugation, Density Gradient↗

PubMed1975

Anomalous sedimentation behaviour of the E.coli ribosomal system: 50S-30S forms 50S PLUS 30S.

An experimenal and theoretical study has been made into the effect of association-dissociation reactions on the sedimentation of the E.coli ribosomal 50S-30S forms 50S plus 30S. It has been found that (a) the sedimentation pattern is strongly dependent on the rotor speed: (b) the ratio of components as measured using high-speed ultracentrifugation (30000-40000 r.p.m.) is independent of rotor speed; and (c) the speed of ultracentrifugation has a strong effect on the sedimentation coefficeint of the ribosomal system as determined by the mean square second moment. The results of this paper demonstrate that ribosome sedimentation at low-speed ultracentrifugation is affected by some artefactual processes. A theoretical analysis of the experimental findings has shown that the observed effects cannot be attributed to the effect of the association-dissociation reaction nor to the pressure dependence of the equilibrium constant of that reaction. On the other hand, at high-speed ultracentrifugation the ribosomal system sediments as a heterogeneous mixture of non-interacting components. Consequently, the shape of the boundary in this case will reflect the equilibrium composition of the ribosomal system.

Binding Sites↗

PubMed2004

The Escherichia coli multidrug transporter EmrE is a dimer in the detergent-solubilised state.

EmrE is a multidrug transporter that utilises the proton gradient across bacterial cell membranes to pump hydrophobic cationic toxins out of the cell. The structure of EmrE is very unusual, because it is an asymmetric homodimer containing eight alpha-helices, six of which form the substrate-binding chamber and translocation pathway. Despite this structural information, the precise oligomeric order of EmrE in both the detergent-solubilised state and in vivo is unclear, although it must contain an even number of subunits to satisfy substrate-binding data. We have studied the oligomeric state of EmrE, purified in a functional form in dodecylmaltoside, by high-resolution size-exclusion chromatography (hrSEC) and by analytical ultracentrifugation. The data from equilibrium analytical ultracentrifugation were analysed using a measured density increment for the EmrE-lipid-detergent complex, which showed that the purified EmrE was predominantly a dimer. This value was consistent with the apparent mass for the EmrE-lipid-detergent complex (137 kDa) determined by hrSEC. EmrE was purified under different conditions using minimal concentrations of dodecylmaltoside, which would have maintained the structure of any putative higher oligomeric states: this EmrE preparation had an apparent mass of 206 kDa by hrSEC and equilibrium analytical ultracentrifugation showed unequivocally that EmrE was a dimer, although it was associated with a much larger mass of phospholipid. In addition, the effect of the substrate tetraphenylphosphonium on the oligomeric state was also analysed for both preparations of EmrE; velocity analytical ultracentrifugation showed that the substrate had no effect on the oligomeric state. Therefore, in the detergent dodecylmaltoside and under conditions where the protein is fully competent for substrate binding, EmrE is dimeric and there is no evidence from our data to suggest higher oligomeric states. These observations are discussed in relation to the recently published structures of EmrE from two- and three-dimensional crystals.

Amino Acids↗

PubMed2000

Chemical precipitation of apolipoprotein B-containing lipoproteins facilitates determination of LDL particle size.

OBJECTIVES: To simplify the determination of low-density lipoprotein (LDL) particle size by eliminating the need for ultracentrifugation. DESIGN AND METHODS: We compared LDL particle size determination by gradient gel electrophoresis using two different methods for separation of LDL: (a) by ultracentrifugation with a density between 1.019 and 1.063 g/mL, and (b) by precipitation of the apolipoprotein B-containing lipoproteins from plasma. LDL particle size was determined for 41 individuals using both methods. RESULTS: The correlation between these two methods was r = 0.98; peak particle diameter (nm) was reproducible with a coefficient of variation of 1. 3% for LDL separated by ultracentrifugation and 1.4% for LDL prepared by precipitation. The intra-assay variation within a single gel was 0.2%. CONCLUSION: Elimination of the need for ultracentrifugation or lipid staining significantly reduces the cost and simplifies the procedure of LDL particle sizing. As a result, larger patient populations can be more readily screened for the determination of LDL particle size.

Adult↗

PubMed1977

Serum immune complexes in membranoproliferative and other glomerulonephritides.

Immune complexes were detected in the serum of patients with membranoproliferative glomerulonephritis (MPGN) Types I and II, systemic lupus erythematosus (SLE), and acute poststreptococcal glomerulonephritis (AGN) by sucrose gradient ultracentrifugation and by measurement of Clq binding activity (Clq-BA). Clq-BA was more sensitive in detecting complexes than was gradient ultracentrifugation. By gradient ultracentrifugation, the sedimentation velocities of the complexes in the three diseases were similar, ranging from 13S to 19S. This range corresponds to that observed by others in experimental chronic serum sickness nephritis. The complexes isolated by gradient ultracentrifugation always contained C3, usually IgG and C4, and in some patients with SLE and AGN, IgM. In MPGN, IgM and IgA could be present in glomerular deposits when not present in circulating complexes. In this disease also, serum complement levels were poor predictors of the presence of complexes. With increased Clq-BA, the levels of Clq, C4, and C2 could be normal or reduced and there was no correlation with C3 levels. With few exceptions, the clinical status of the patients with MPGN correlated well with Clq-BA.

Acute Disease↗

PubMed1988

Prostasome membrane associated enzyme activities and semen parameters in men attending an infertility clinic.

Seminal plasma from 22 men attending an infertility clinic was subjected to preparative ultracentrifugation for 2 h at 105,000 g. The pelleted material as well as the supernatant thus obtained were investigated with regard to prostasome membrane-linked enzyme activities in relation to other semen parameters. The mean activity of Zn2+-dependent adenosine triphosphatase in the sedimented prostasome fraction was 1.45 +/- 1.02 mumol (range 0.29-4.79) orthophosphate released per milligram protein and 20 min, while the corresponding figures for the supernatant were 0.56 +/- 0.30 (range 0.12-1.29). Hence, 72% of the specific activity was sedimented, and 28% remained in the supernatant. The same pattern was recognized with regard to the other two enzymes investigated, although they displayed individual characteristics with regard to distribution after ultracentrifugation. The pelleted prostasome-linked mean aminopeptidase activity was 0.39 U/mg protein (81.9%), with only 0.087 U (18.1%) remaining in the supernatant. The corresponding figures for gamma-glutamyltransferase were 7.89 (60.4%) and 5.17 (39.6%) mu kat/g protein, respectively. The different enzyme activities in the prostasome fraction and supernatant, respectively, were interrelated to each other and correlated significantly with r values between 0.73 and 0.93 (p less than 0.001). It was concluded that a minor fraction of prostasomes remained in the supernatant after ultracentrifugation. A relationship existed between prostasomes and semen volume revealing a rather consistent pattern in that small volumes favoured the presence of comparatively more prostasomes in the supernatant and less prostasomes in the pelleted fraction than large volumes. In addition, the sperm concentration seemed to be another determinant of the distribution of prostasomes in seminal plasma on subsequent ultracentrifugation.

Adenosine Triphosphatases↗

PubMed1968

Irreversibly sickled erythrocytes: a consequence of the heterogeneous distribution of hemoglobin types in sickle-cell anemia.

THE AMOUNT OF FETAL HEMOGLOBIN (HB F) IN ERYTHROCYTES OF PATIENTS WITH SICKLE CELL ANEMIA (HB SS DISEASE) WAS MEASURED BY TWO METHODS: (a) photometry of individual cells stained for Hb F by the Kleihauer-Betke technique; and (b) chemical assay of alkali-resistant hemoglobin in cells distributed according to specific gravity by ultracentrifugation. Irreversibly sickled cells (ISC), which could be identified directly during photometry and which were found to gather in high concentration at the bottom of ultracentrifuged cell columns, contained significantly less Hb F than non-ISC. Cell content of total Hb was constant regardless of cell size, shape, or ultracentrifugal behavior: thus absolute amounts of Hb F and S varied reciprocally from cell to cell. In experiments designed to estimate age, at formation, and rate of destruction of ISC, Hb SS blood was incubated with selenomethionine-(75)Se (which labels reticulocytes) or (51)Cr (which labels erythrocytes at random) and reinfused. Sequential blood samples were separated by ultracentrifugation into fractions rich in reticulocytes, non-ISC, and ISC; and chronological changes in the specific activity of each fraction were determined. Analogous information was obtained from radioautography of sequential blood samples after reinfusion of whole blood labeled with amino acids-(3)H: this technique permitted direct visual characterization of labeled erythrocytes as ISC or non-ISC, all of which had been reticulocytes at the time of reinfusion. The transformation of non-ISC into ISC, presumably a manifestation of membrane damage, proved to begin soon after cell release from the marrow; and ISC subsequently underwent rapid removal from the circulating blood. IT IS THEREFORE APPARENT FROM THESE STUDIES THAT, IN HB SS DISEASE, RELATIVELY SMALL RECIPROCAL CHANGES IN THE AMOUNTS OF THE TWO MAJOR HEMOGLOBINS CARRY PREDICTIVE IMPORTANCE: (a) net synthesis of Hb F is least in erythroid cells destined to become ISC; and (b) these irreversibly deformed erythrocytes suffer preferential destruction.

Adult↗

PubMed1996

Sedimentation of biliary sludge: effect on composition of gallbladder bile from patients with cholesterol, mixed, or pigment stones.

BACKGROUND: Ultracentrifugation of bile has been used extensively to remove insoluble material such as sludge from bile before further studies of cholesterol nucleation. Although it has been recognized that this procedure may affect the composition of gallbladder bile, it has not been studied systematically in different gallstone populations. Therefore, we investigated the concentration of biliary lipids, protein, mucin, and bilirubin before and after ultracentrifugation. METHODS: Gallbladder bile samples were aspirated during laparoscopic surgery from 66 patients (35 with cholesterol, 16 with mixed, and 15 with pigment stones). RESULTS: Whereas the concentrations of bile acids, phospholipids, protein, and bilirubin in gallbladder bile did not change significantly after ultracentrifugation, cholesterol (20.6 +/- 1.6 to 14.8 +/- 1.2 mmol/l) and mucin concentrations (0.99 +/- 0.2 to 0.67 +/- 0.1 mg/ml) and the cholesterol saturation index (1.68 +/- 0.12 to 1.31 +/- 0.10) decreased significantly in gallbladder bile from patients with cholesterol stones. CONCLUSIONS: Sedimentation of biliary sludge may profoundly affect the composition of gallbladder bile, which has to be considered in studies of cholesterol saturation and nucleation. The cholesterol concentration difference between native and ultracentrifuged bile reflects the insoluble crystalline fraction of cholesterol and may be useful for quantitation of the mass of cholesterol crystals in gallstone-associated bile samples.

Bile↗