Solubilisation of phospholipid membranes by human plasma high density lipoproteins.
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Apolipoproteins↗
Biomedical subjects
D M Small
Publications and source records attributed to D M Small.
Production of cholesteryl ester-rich, anisotropic inclusions by mammalian cells in culture.
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Animals↗
Structure of human plasma low-density lipoproteins: molecular organization of the central core.
Human plasma low density lipoprotein (LDL) exhibits a thermal transition over the temperature range 20-40 degrees. This transition is associated with a structural change within the lipoprotein particle and is reflected in the small-angle x-ray scattering profiles from LDL. The scattering profile of the quasispherical LDL particle at 10 degrees shows a relatively intense maximum at 1/36 A-1 which is absent from the scattering of LDL at 45 degrees. Theoretical calculations, using model electron density distributions, have been carried out to describe the packing of arrangement of the cholesterol esters, based on perturbations of the molecular packing of crystalline cholesteryl myristate, adequately reproduces the high relative intensity of the x-ray scattering maximum at 1/36 A-1. The perturbations of the packing in the crystal structure of cholesteryl myristate involve "melting" of the hydrocarbon chains of the esters together with translations of pairs of molecules parallel to the molecular long axis. The interaction of opposing steroid moieties, with C18 and C19 angular methyl groups interlocked, exhibited in the crystal structure is retained in the perturbed arrangement. At 45 degrees, thermally induced disorder of this arrangement averages the electron density of the central core. The x-ray scattering profiles of particles with a homogeneous electron density in the core region do not show a high relative intensity of the subsidiary maxima in the 1/36 A-1 region, in agreement with experimental observation. The results of these calculations support the concept that the thermal transition observed for LDL is due to a smectic leads to disordered transition of the cholesterol esters in the core of the LDL particle.
Cholesterol↗
Biliary lipid secretion in cholesterol gallstone disease. The effect of cholecystectomy and obesity.
Cholesterol gallstone disease is initiated in a liver which produces abnormal bile with excess cholesterol relative to bile salts and phospholipid. To define the responsible secretory mechanism(s), the rate of biliary lipid secretion was measured by a duodenal marker perfusion technique, while the bile salt pool was simultaneously estimated by isotope dilution. Two groups of control patients expected to have normal biliary lipid composition--14 subjects without hepatobiliary disease and 6 patients with pigment gallstones, were compared to two experimental groups expected to have abnormal bile--10 nonobese patients with cholesterol gallstones and 7 obese subjects without gallstones. Both control groups had nearly identical biliary lipid secretion rates, and a corresponding low relative molar concentration of cholesterol. Two different secretory mechanisms were found to be responsible for the abnormal bile in the experimental groups. In the nonobese patients with cholesterol gallstones, bile salt and phospholipid secretion rates were both significantly reduced. Conversely, the grossly obese subjects had an increased cholesterol secretion. To determine how cholecystectomy improves biliary lipid composition, three groups of gallstone patients --6 with pigment stones, 4 grossly obese with cholesterol stones, and 13 nonobese with cholesterol stones --were all examined after full recovery from surgery. In the nonobese patients with cholesterol gallstones, both bile salt and phospholipid secretion significantly increased, causing a definite improvement in bile composition. Cholecystectomy produced a similar but less marked trend in the obese patients with cholesterol stones, and in the patients with pigment stones. Cholesterol secretion, however, was unaffected by surgery. The bile salt pool was definitely small in the nonobese patients with cholesterol gallstones and became slightly smaller after cholecystectomy. The pool was significantly reduced by cholecystectomyin the patients with cholesterol gallstones. Removal of the gallbladder in all three groups caused a greater fraction of the pool to cycle around the enterohepatic circulation each hour. This more rapid cycling produced the increase in bile salt and phospolipid secretion, and was responsible for the improved composition found after cholecystectomy.
Adult↗
The storage lipids in Tangier disease. A physical chemical study.
The physical states and phase behavior of the lipids of the spleen, liver, and splenic artery from a 38-yr-old man with Tangier disease were studied. Many intracellular lipid droplets in the smectic liquid crystalline state were identified by polarizing microscopy in macrophages in both the spleen and liver, but not in the splenic artery. The droplets within individual cells melted sharply over a narrow temperature range, indicating a uniform lipid composition of the droplets of each cell. However different cells melted over a wide range, 20-53 degrees C indicating heterogeneity of lipid droplet composition between cells. Furthermore, most of the cells (81%) had droplets in the liquid crystalline state at 37 degrees C. X-ray diffraction studies of splenic tissue at 37 degrees C revealed a diffraction pattern typical of cholesterol esters in the smectic liquid crystalline state. Differential scanning calorimetry of spleen showed a broad reversible transition from 29-52 degrees C, with a maximum mean transition temperature at 42 degrees C, correlating closely with the polarizing microscopy observations. The enthalpy of the transition, 0.86+/-0.07 cal/g of cholesterol ester, was quantitatively similar to that of the liquid crystalline to liquid transition of pure cholesterol esters indicating that nearly all of the cholesterol esters in the tissue were free to undergo the smectic-isotropic phase transition. Lipid compositions of spleen and liver were determined, and when plotted on the cholesterol-phospholipid-cholesterol ester phase diagram, fell within the two phase zone. The two phases, cholesterol ester droplets and phospholipid bilayers were isolated by ultracentrifugation of tissue homogenates. Lipid compositions of the separated phases approximated those predicted by the phase diagram. Extracted lipids from the spleen, when dispersed in water and ultracentrifuged, underwent phase separation in a similar way. Thus (a) most of the storage lipids in the liver and spleen of this patient were in the liquid crystalline state at body temperature, (b) the phase behavior of the storage lipids conformed to that predicted by lipid model systems indicating lipid-lipid interactions predominate in affected cells, (c) lipid droplets within individual cells have similar compositions, whereas droplet composition varies from cell to cell, and (d) cholesterol ester does not accumulate in the splenic artery. Since Tangier patients lack high density lipoprotein, we conclude that high density lipoprotein-mediated cholesterol removal from cells is essential only for those cells which have an obligate intake of cholesterol (macrophages).
Adult↗
Interaction of cholesterol ester and triglyceride in human plasma very low density lipoprotein.
The properties of human plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL), and their extracted lipids were compared using calorimetric, X-ray scattering, and polarizing microscopy techniques. Intact LDL, and cholesterol esters isolated from LDL and VLDL each undergo reversible changes in their physical state around body temperature. These transitions are associated with ordered liquid crystalline to liquid phase changes of the cholesterol esters. In contrast to LDL, VLDL has no reversible transitions and shows no evidence of ordered liquid crystalline structures between 10 and 45 degrees C. Therefore, unlike LDL, VLDL does not contain a separate cholesterol ester region capable of undergoing cooperative melting. Solubility studies at 37 degrees C of cholesterol esters and triglyceride isolated from VLDL show that even at a weight ratio of 1:1, which greatly exceeds the relative amount of cholesterol esters in VLDL, cholesterol ester is completely soluble in triglyceride. Thus, the cholesterol ester in VLDL is not sequestered in a separate domain within VLDL, but is dissolved in the liquid core of the particle.
Binding Sites↗
Nature of the Thermal pretransition of synthetic phospholipids: dimyristolyl- and dipalmitoyllecithin.
The hydrated synthetic lecithins, dimyristoyl and dipalmitoyllecithins, undergo two thermal transitions, a broad low enthalpy "pretransition" prior to the sharp first-order "chain-melting" transition. Both phospholipids exhibit the same temperature-dependent structural changes associated with the thermal pretransition. At low temperatures, below the pretransition, a one-dimensional lamellar lattice is observed. The hydrocarbon chains are fully extended and tilted with respect to the plane of the lipid bilayer. The hydrocarbon chain packing displays a temperature dependence and the angle of tilt of the hydrocarbon chains decreases with increasing temperature, reaching a minimum value of 30 degrees at the pretransition temperature of both lecithins. The pretransition is associated with a structural transformation from a one-dimensional lamellar to a two-dimensional monoclinic lattice consisting of lipid lamellae distorted by a periodic ripple. The hydrocarbon chains remain tilted in the temperature range intermediate between the pretransition and chain-melting transition. The cell parameters of this two-dimensional lattice exhibit a compositional dependence. The a parameter (proportional to the lamellar repeat distance) increases with increasing water content, while the b parameter (a measure of the ripple periodicity) decreases with increasing water content. At the chain-melting transition, the hydrocarbon chains of the phospholipid melt and assume a liquid-like conformation and the lattice reverts to one-dimensional lamellar. These structural changes observed for dimyristoyl- and dipalmitoyllecithins may be a common feature of all synthetic lecithins exhibiting a thermal pretransition. The appearance of the pretransition and accompanying two-dimensional may arise from specific interactions between the choline moiety of the polar head group and the structured water matrix surrounding it.
Membranes, Artificial↗
Temperature-dependent 13C nuclear magnetic resonance studies of human serum low density lipoproteins.
The natural abundance 13C nuclear magnetic resonance (NMR) spectrum of human serum low density lipoproteins (LDL) shows significant temperature-dependent changes. These temperature-dependent spectra have been used to monitor changes in the organization of cholesterol esters within the LDL particle. Comparison with 13C NMR spectra of both cholesterol linoleate and an aqueous codispersion of cholesterol linoleate and egg phosphatidylcholine suggests that at low temperatures (10 degrees C), the cholesterol esters in LDL are organized in a smectic-like, liquid-crystalline arrangement. At temperatures above the order-disorder transition exhibited by the cholesterol esters of LDL, the cholesterol esters appear to be partially melted but still are motionally restricted compared with liquid cholesterol esters.
Carbon Isotopes↗
Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins.
Conformational changes of apo A-1, the principal apoprotein of human plasma high density lipoprotein, have been studied by differential scanning calorimetry and ultraviolet difference spectroscopy as a function of temperature, pH, concentration of apoprotein, and urea concentration. Calorimetry shows that apo A-1 (5 to 40 mg/ml, pH 9.2) undergoes a two-state, reversible denaturation (enthalpy = 64 +/- 8.9 kcal/mole), between 43--71 degrees (midpoint temperature, Tm = 54 degrees), associated with a rise in heat capacity (deltaCvd) of 2.4 +/- 0.5 kcal/mole/degrees C. Apo A-1 (0.2 to 0.4 mg/ml, pH 9.2) develops a negative difference spectrum between 42--70 degrees, with Tm = 53 degrees. The enthalpy (deltaH = 59 +/- 5.7 kcal/mole at Tm) and heat capacity change (2.7 +/- 0.9 kcal/mole/degrees C) in the spectroscopic experiments were not significantly different from the calorimetric values. Below pH 9 and above pH 11, the calorimetric Tm and deltaH of denaturation are decreased. In the pH range of reversible denaturation (6.5 to 11.8), delatH and Tm are linearly related, showing that the heat capacity change (ddeltaH/dT) associated with denaturation is independent of Tm. In urea solutions, the calorimetric Tm and deltaH of denaturation are decreased. At 25 degrees, apo A-1 develops a negative difference spectrum between 1.4 and 3 M urea. Fifty per cent of the spectral change occurs in 2.4 M urea, which corresponds to the urea concentration obtained by extrapolation of the calorimetric Tm to 25 degrees. In urea solution of less than 0.75 M there is hyperchromicity at 285 nm (delta epsilon = 264 in 0.75 M urea), indicating strong interaction of aromatic amino acid residues in the native molecule with the solvent. Spectrophotometric titration of apo A-1 shows that 6.6 of the 7 tyrosine groups of apo A-1 titrate at pH less than 11.9, with similar titration curves obtained in aqueous solutions and in 6 M urea. The free energy of stabilization (deltaG) of the native conformation of apo A-1 was estimated, (a) at 37 degrees, using the calorimetric deltaA and deltaCvd, and (b) at 25 degrees, by extrapolation of spectroscopic data to zero urea concentration. The values (deltaG (37 degrees) = 2.4 and deltaG (25 degrees) = 2.7 kcal/mole) are small compared to typical globular proteins, indicating that native apo A-1 has a loosely folded tertiary structure. The low values of deltaG reflect the high degree of exposure of hydrophobic areas in the native protein molecule. The loosely folded conformation of apo A-1 allows extensive binding of lipid, since this can involve both surface hydrophobic sites and hydrophobic areas exposed by a cooperative, low energy unfolding process.
Apoproteins↗
The thermal transitions and structural properties of 5alpha-cholestan-3beta-ol esters of aliphatic acids.
5 alpha-Cholestan-3 beta-ol esters of aliphatic acids undergo both enantiotropic and monotropic changes of state. Ten saturated and three unsaturated esters have been examined by differential scanning calorimetry and polarizing microscopy to determine transition temperatures, enthalpies, and entropies. The results are compared with an analogous series of cholesterol esters. All esters of even-numbered n-alkanoic acids from C2 to C20 melt from a crystalline state to an isotropic liquid. The crystalline state has been studied by X-ray powder diffraction. The C8 to C20 esters have progressively increasing crystalline melting transition temperatures from 76 to 99 degrees C and possess similar X-ray powder diffraction patterns, suggesting that these compounds form an isostructural series. Esters of C2, C4, and C6 acids exhibit polymorphism. Crystalline cholestanol oleate melts to an isotropic liquid, whereas cholestanol linoleate and linolenate fail to crystallize, even after several months at -20 degrees C. Esters of the even-numbered saturated acids from C4 to C14 form monotropic cholesteric liquid crystalline phases. Esters C10, C12, and C14 form smectic liquid crystalline phases. Cholestanol oleate, linoleate, and linolenate form both cholesteric and smectic mesophases. The lower smectic to cholesteric and cholesteric to isotropic transition temperatures of the cholestanol esters compared to the corresponding transition temperatures of the analogous cholesterol esters suggest that the delta 5 double bond in cholesterol increases the thermal stability of the mesophases of cholesterol esters.
Binding Sites↗
A study of the physicochemical interactions between biliary lipids and chlorpromazine hydrochloride. Bile-salt precipitation as a mechanism of phenothiazine-induced bile secretory failure.
Since chlorpromazine hydrochloride [2-chloro-10-(3-dimethylaminopropyl)-phenothiazine hydrochloride] is commonly implicated in causing bile-secretory failure in man and is secreted into bile, we have studied the physicochemical interactions of the drug with the major components of bile in vitro. Chlorpromazine hydrochloride molecules are amphiphilic by virtue of possessing a polar tertiary amine group linked by a short paraffin chain to a tricyclic hydrophobic part. At pH values below the apparent pK (pK'a 7.4) the molecules are water-soluble cationic detergents. We show that bile salts in concentrations above their critical micellar concentrations are precipitated from solution by chlorpromazine hydrochloride as insoluble 1:1 salt complexes. In the case of mixed bile-salt/phosphatidylcholine micellar solutions, however, the degree of precipitation is inhibited by the phospholipid in proportion to its mole fraction. With increases in the concentration of chlorpromazine hydrochloride or bile salt, micellar solubilization of the precipitated complexes results. Sonicated dispersions of the negatively charged phospholipid phosphatidylserine were also precipitated, but dispersions of the zwitterionic phospholipid phosphatidylcholine were not. Chlorpromazine hydrochloride efficiently solubilized these membrane phospholipids as mixed micellar solutions when the drug:phospholipid molar ratio reached 4:1. Polarizing-microscopy and X-ray-diffraction studies revealed that the precipitated complexes were amorphous and potentiometric studies confirmed the presence of a salt bond. Some dissociation of the complex occurred in the case of the most polar bile salt (Ks 0.365). As canalicular bile-salt secretion determines much of bile-water flow, we propose that complexing and precipitation of bile salts by chlorpromazine hydrochloride and its metabolites may be physicochemically related to the reversible bile-secretory failure produced by this drug.
Animals↗
Editorial: Hormone use to change normal physiology - is the risk worth it?
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Adolescent↗
Gallstone disease: pathogenesis and management.
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Adult↗
Physical chemistry of the lipids of human atherosclerotic lesions. Demonstration of a lesion intermediate between fatty streaks and advanced plaques.
95 individual human atherosclerotic lesions from 26 persons were classified into three groups under the dissecting microscope: fatty streaks, fibrous plaques, and gruel (atheromatous) plaques. Each lesion was isolated by microdissection, its lipid composition determined by chromatography, and the physical states of the lipids identified by polarizing microscopy and in some cases by X-ray diffraction. The composition of each lesion was plotted on the in vitro phase diagram of the major lipids of plaques: cholesterol, cholesterol ester, and phospholipid. The observed physical states were compared with those predicted by the location of the lipid composition on the phase diagram. The most severe lesions (gruel plaques) had an average lipid composition of cholesterol 31.5+/-1.9%, cholesterol ester 47.2+/-2.3%, and phospholipid 15.3+/-0.5%. Their compositions fell within the three-phase zone of the phase diagram, predicting the lipids to be separated into a cholesterol crystal phase, a cholesterol ester oily phase and a phospholipid liquid crystalline phase. In addition to the phospholipid liquid crystalline phase of membranes and myelin-like figures demonstrable by electron microscopy, polarizing microscopy revealed the other two predicted phases, isotropic cholesterol ester-rich droplets and cholesterol crystals. X-ray diffraction studies verified the identity of the crystals as cholesterol monohydrate. Fibrous plaques also had an average lipid composition within the three-phase zone of the phase diagram. Polarizing microscopy revealed the presence of cholesterol monohydrate crystals and lipid droplets in all of these lesions; the droplets were predominately isotropic in 28 of the 31 fibrous plaques. Although these lesions had less free cholesterol and more cholesterol ester than gruel plaques, they were otherwise similar. Fatty streaks had compositions within both the two- and three-phase zones of the phase diagram. Compared with gruel plaques, the fatty streaks within the two-phase zone, defined as "ordinary," had more cholesterol ester, less free cholesterol, a higher cholesteryl oleate/cholesteryl linoleate ratio, a lower sphingomyelin/lecithin ratio, more anisotropic lipid droplets, and rare or no cholesterol crystals. Those lesions within the three-phase zone had many chemical and physical features intermediate between ordinary fatty streaks and gruel plaques. Moreover, 68% of these "intermediate" lesions had no cholesterol crystals by polarizing microscopy in spite of their compositions being within the three-phase zone, indicating the cholesterol ester oily phase or the phospholipid phase or both were supersaturated with cholesterol. Identification of this group of intermediate lesions provides evidence that some fatty streaks may be precursors of advanced plaques.
Adult↗
The use of phospholipid vesicles for in vitro studies on cholesteryl ester hydrolysis.
Radiolabeled cholesteryl oleate was incorporated into vesicles prepared from egg yolk lecithin and utilized as a substrate for studies of sterol ester hydrolases present in rat liver homogenates. The cholesteryl oleate was shown to be associated with vesicles (unilamellar liposomes) using Sepharose 4B chromatography. With this substrate, two different cholesteryl ester hydrolytic enzymes were demonstrated in subcellular fractions from the liver homogenates. In the lysosome-rich fraction an acid hydrolase was present, while in the cytosol fraction (150,000 g supernatant), hydrolytic activity was shown to occur with an optimum pH between 8 and 8.5. The substrate was characterized by Sepharose chromatography both before and after incubation with the liver fraction and was not dramatically altered even by rigorous incubation conditions. The lysosomal enzyme preparation was capable of hydrolyzing almost all the cholesteryl oleate in the vesicles. Hydrolysis of the phospholipid was proportionately much less than that of the cholesteryl oleate. Comparisons were performed between the vesicle preparation and an alternate substrate preparation involving the direct addition of cholesteryl oleate in acetone solution. The vesicles appeared to be a better substrate for the lysosomal enzyme whereas the activity in the cytosol fraction did not distinguish between the two substrate preparations. Unsonicated suspensions of cholesteryl oleate and lecithin did not serve as suitable substrates for the enzymes. These studies demonstrate the applicability of cholesteryl ester-containing vesicles as a useful substrate for studying cholesteryl ester hydrolysis in vitro.
Animals↗
Part I. The etiology and pathogenesis of gallstones.
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Bile↗
Simultaneous measurement of the pancreatic and biliary response to CCK and secretin. Primate biliary physiology. XIII.
A primate model is described that permits separate but simultaneous sampling of both bile and pancreatic fluid without altering the normal enterohepatic circulation. Simultaneous biliary and pancreatic dose response curves are presented to contrast the production of bile with both pancreatic flow and the secretion of pancreatic protein and bicarbonate. The data show that in the cholecystectomized animal both cholecystokinin and secretin increase bile flow at doses that produce appropriate submaximal responses from the pancreas. Both these hormones, therefore, are physiologically important in regulating flow. Further, over a wide dose range (0.2 to 2.3 U per kg) secretion has the major effect on bile flow, and pancreatic flow, and pancreatic bicarbonate secretion, whereas cholyecystokinin has the major effect on pancreatic protein secretion.
Animals↗
Surface and solution properties of steroid antibiotics: 3-acetoxylfusidic acid, cephalosporin P1 and helvolic acid.
The colloid/chemical properties of the fusidane antibiotics, 3-acetoxylfusidic acid, cephalosporin P1, and helvolic acid, and their sodium salts, were investigated. The sodium salts of 3-acetoxylfusidic acid and cephalosporin P1 were found to be detergent-like molecules with micellar properties comparable to the parent compound sodium fusidate and the bile salt sodium cholate. Critical micellar temperatures (cmt) were less than 0 degrees C except for sodium helvolate which being sparingly soluble did not form micelles between 0 and 50 degrees C. Potentiometric titrations of dilute solutions gave apparent pK values (5.2-6.5) in the range expected for carboxylated steroid detergents. The apparent pK values increased significantly once the detergent concentration exceeded the critical micellar concentration (cmc). Micellar properties were determined by surface tension, titration with a water-soluble dye (Rhodamine 6G), light scattering, and solubilization of lecithin and cholesterol. Cmc's, in the range of 1.5 to 5.6 mM, were found which varied slightly depending on the method employed and in all cases fell slightly in the presence of added NaCl. The number of monomers per micelle (aggregation number) in concentrations well above the cmc was extrapolated from Debye light scattering plots in 0.15 M NaCl. The values varied from 6 for fusidate to 14 for 3-acetoxylfusidate with sodium cephalosporin P1 having an intermediate value. Each detergent readily solubilized the phospholipid lecithin.
Cephalosporins↗