Residual Cholesterol Research Articles

The effects of bile salt hydrophobicity on model bile vesicle morphology

The addition of bile salts to vesicles supersaturated with cholesterol induces cholesterol precipitation, an important step in the formation of cholesterol gallstones. To investigate the effects of bile salt hydrophobicity on vesicle morphology, vesicles obtained from supersaturated model bile by density gradient ultracentrifugation, were incubated with mixtures of deoxycholate (DC) and ursodeoxycholate (UDC) with a constant total bile salt concentration of 30 mM but with a varying hydrophobicity index ranging from −0.31 (UDC alone) up to + 0.72 (DC alone) depending on the composition of the mixture. Five days after addition of bile salts to vesicles, cholesterol precipitation was determined microscopically and incubation samples were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, mixed micelles, and cholesterol crystals. Structure and size of the isolated residual vesicles were studied by freeze fracture electron microscopy. The control, and samples exposed to hydrophilic bile salt mixtures, consisted of unilamellar vesicles of which more than 75% had a diameter of 50–80 nm. After addition of increasingly hydrophobic bile salt mixtures, multilamellar vesicles with progressively greater diameters (up to 1300 nm) were found, suggesting that vesicle fusion and aggregation took place and might hence be important in the cholesterol precipitation process. Accordingly, crystallization was positively correlated with bile salt hydrophobicity. We conclude that cholesterol crystallization from vesicles depends on the hydrophobicity of the bile salts added, and apparently occurs from fused or aggregated vesicles of extended magnitude and with a multilamellar constitution.

Fish eye syndrome: a molecular defect in the lecithin-cholesterol acyltransferase (LCAT) gene associated with normal alpha-LCAT-specific activity. Implications for classification and prognosis.

We have identified the molecular defect in two siblings presenting with classical clinical and biochemical features of Fish Eye disease (FED), including corneal opacities, HDL cholesterol < 10 mg/dl, normal plasma cholesteryl esters, and elevated triglycerides. In contrast to previously reported patients with FED who are unable to esterify HDL-associated cholesterol, our patients' plasma lecithin-cholesterol acetyltransferase (alpha-LCAT)-specific activities assayed using an HDL-like proteoliposome substrate were 12.7-25.7 nmol/micrograms (19.5 +/- 1.8 in controls). In addition, significant residual cholesterol esterification was present in VLDL/LDL-depleted plasma, confirming the presence of HDL-associated alpha-LCAT activity. DNA sequence analysis of the proband's LCAT gene identified deletion of the triplet coding for leu300, which resulted in the loss of a restriction site for MlnI. Digestion of PCR-amplified DNA using MlnI established that both siblings are homozygous for this defect. Expression of LCAT300-del. in human embryonic kidney-293 cells revealed normal mRNA and intracellular LCAT concentrations. However, reduced amounts of LCAT300-del., which had a normal specific alpha-LCAT activity, were present in the media. In summary, we report the first case of FED associated with a mutant enzyme that has a normal alpha-LCAT-specific activity. The functional significance of this LCAT gene defect has been established in an in vitro expression system, which demonstrates that very small amounts of this functional LCAT mutant enzyme accumulate in the media. Characterization of LCAT300-del. established that selective alpha-LCAT deficiency is not a prerequisite for the development of FED. On the basis of our combined results, we propose that the residual amounts of total plasma LCAT activity and not its distribution on lipoproteins primarily determines the heterogeneity in phenotypic expression observed in familial LCAT deficiency syndromes.

Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from adrenocorticotropic hormone-treated rats: Reconstitution of the isocitrate response with succinate and low concentrations of isocitrate

Cholesterol side-chain cleavage in isolated adrenal mitochondria requires unique energy requirements that may determine not only electron transport to P450 but also cholesterol availability. In mitochondria from ACTH-treated rats, two approximately equal pools of reactive cholesterol are indicated by the partial effectiveness of succinate (SU; Type A), and the metabolism of residual cholesterol by 1 m m isocitrate (IC; Type B). Type A metabolism is associated with relatively few initial cholesterol-P450 scc complexes and is rapidly and selectively lost when mitochondria are preincubated without an energy source. We now show that cholesterol metabolism supported by IC resolves into equal high and low affinity components (EC 50 = 10 and 250 μ m) exhibiting, respectively, Type A and Type B characteristics. SU and 50 μ m IC, in combination, provided nearly the same activity characteristics as 1 m m IC, including resistance to preincubation and increased turnover of cholesterol-P450 scc complexes. Much higher (three to six times) and more sustained pregnenolone formation was seen, with all reductants, following either enhancement of the reactive cholesterol pool or addition of 20-α-hydroxycholesterol, indicating that adrenocorticotropic hormone-mitochondria are limited by substrate availability. ATP generation was most effectively supported by SU, and IC was maximally active at 50 μ m, emphasizing differences between respiratory and steroidogenic energy requirements. ATP production and the maintenance of uniform suppression after in vivo cycloheximide treatment indicate the integrity of the mitochondrial interaction with all reductants. Inhibitors of SU oxidation (KCN, malonate) strongly inhibited SU-supported cholesterol metabolism but had little effect on SU synergism with IC. Fumarate (but not α-ketoglutarate or oxaloacetate) was equally effective as a synergist, but was totally ineffective as a reductant. SU or fumarate, therefore, act by a nonreductive pathway to boost NADPH production from low concentrations of IC. This decrease in apparent K m for IC may be mediated by stimulation of mitochondrial uptake of the reductant through the specific transporters.

Effect of cholesterol and growth factors on the proliferation of cultured human skin fibroblasts

A cholesterol-deficient growth medium for human skin fibroblasts was prepared by adding to Eagle's Minimum Essential Medium a bovine serum treated with ultracentrifugation to remove bulk lipoproteins followed by silicic acid adsorption to remove residual lipoproteins and cholesterol. Cell growth was slow, but the daily cell doublings could be increased by 76% by including 7.5 μg purified cholesterol/ml in the medium. Cell growth in cholesterol-deficient culture medium could be increased to that seen with medium containing 15 % untreated fetal bovine serum by the inclusion of the following growth factors: epidermal growth factor (EGF), cortisol, non-essential amino acids, insulin, transferrin and selenium. Cholesterol increased the proliferation of these rapidly-growing cultures by 19%. No effect of cholesterol was observed in transformed L-cell mouse fibroblasts.

Importance of Viscosity in the Dissolution Rate of Cholesterol in Monooctanoin Solutions

Several factors affecting the dissolution rate of cholesterol in monooctanoin were investigated. This solvent is used clinically for dissolution of residual cholesterol gallstones in the bile duct after cholecystectomy. The effect of added water on dissolution rate, measured using the static-or rotating-disk methods, was not consistent with the previously measured solubility. The discrepancy was found to be due to the decreasing viscosity of the solvent as water was added. Addition of cholesterol, however, increased the viscosity of monooctanoin. The viscosity effect on dissolution rate was investigated further by addition of polymers (povidone and poloxamer 237) which increased solvent viscosity. Dissolution rate was proportional to viscosity to the –0.4 power with these polymers. An equation was derived which predicts that dissolution rate should be proportional to viscosity to the −2/3 power. The predicted exponent was very close to reported experimental values for benzoic acid, but the dissolution rate/viscosity relationship for cholesterol in aqueous monooctanoin was nonlinear with apparent exponents of −0.65 to −2.3. Although the Arrhenius activation energies for viscosity (3.79 kcal/mol) and dissolution rate constant (3.66 kcal/mol) were almost equal for benzoic acid, a nonlinear relationship was again observed for cholesterol in aqueous monooctanoin with approximate Ea values of 5.6–10 kcal/mol. The strong influence of viscosity on dissolution rate in this system is attributed to the viscosity-increasing effect of cholesterol in the diffusion layer. The increased viscosity at higher cholesterol concentrations reduces the diffusion coefficient of cholesterol and causes the dissolution rate to be slower even though solubility may have been higher. The data suggest that it may be possible to increase cholesterol gallstone dissolution by addition of water to monooctanoin.

Effects of cholesterol evulsion on susceptibility to perfringolysin o of human erythrocytes

Human erythrocytes preincubated with a phosphatidylcholine suspension (preincubated cells) showed decreased susceptibility to perfringolysin O, the decrease being strongly affected by preincubation time and temperature, and the phosphatidyl choline concentration. The binding of the toxin to the preincubated cells also decreased with the preincubation time and reached minimum at 37°C for 6 h. Through this preincubation, about 30% of cholesterol was removed from cells without lysis. The susceptibility of preincubated cells to the toxin seemed to be affected by the amount of cholesterol removed from cells, but not by the cholesterol content of cell membranes. This indicates that most of the cholesterol interactive with the toxin is removable from cell membranes by preincubation with phosphatidylcholine suspension, and that the residual cholesterol is firmly constituted in the membrane structure and cannot interact with the toxin. After cholesterol evulsion by the preincubated plasma method (Murphy, J.R. (1962) J. Lab. Clin. Med. 60, 86–109 and 60, 571–578), cells also exhibited lower susceptibility to the toxin and to saponins, but higher susceptibility to lysophosphatidylcholine.

The release of an immobilized lipoprotein fraction from atherosclerotic lesions by incubation with plasmin

A large amount of plasma low density lipoprotein is present in human aortic intima, and this can be removed and measured by electrophoresis directly from the minced tissue into an antibody-containing gel. We now find that, in addition to this electrophoretically mobile lipoprotein, there is an immobilized lipoprotein fraction that can be released from lesions by incubation of the tissue sample with plasmin or other proteolytic enzymes after the mobile lipoprotein has been removed. The concentration of immobilized lipoprotein is highly correlated with the concentration of the residual cholesterol (not mobile on electrophoresis) that has accumulated in the tissue ( r = 0.702; P ⪡ 0.001). Thus, in normal intima and early gelatinous lesions it is about 15% of the concentration of mobile lipoprotein, whereas in the atheroma lipid layers of fibrous or gelatinous plaques it may be 2 or 3 times greater than the concentration of mobile lipoprotein. This suggests that immobilization of plasma lipoprotein is an intermediate step in the irreversible deposition of extracellular cholesterol in atherosclerotic lesions. Incubation with plasmin allowed maximum release of lipoprotein: plasmin = crude collagenase > trypsin > “pure” collagenase > chondroitinase ABC in order of their relative effectiveness. The concentration of immobilized lipoprotein was significantly correlated ( r = 0.793; P ⪡ 0.001) with the concentration in the tissue of fibrin or other insoluble derivatives of fibrinogen (“fibrin”). In aliquots of lesions incubated with varying amounts of plasmin for varying times there was a constant relation between release of lipoprotein and release of fibrin-degradation products. Together, these findings suggest that the lipoprotein is associated with insoluble “fibrin”. This appears to be of considerable clinical interest, suggesting a synergism between lipoprotein and fibrinogen in the accumulation of lipid in lesions.

Lipid depletion in atheromatous coronary arteries in rhesus monkeys after regression diets.

Lipids were measured in the coronary arteries of monkeys on an atherogenic diet and in the arteries of matched monkeys on the atherogenic diet followed by regression diets. Cholesterol content was 51 mg/g dry weight in the arteries of monkeys with atheromatosis; after 40 months on the regression diets it was 18 mg/g. Cholesteryl ester was 69% lower and free cholesterol 53% lower after the regression diets. Decreases in triglycerides and phospholipids were not significant. The cholesterol content of the arteries of two monkeys autopsied after 20 months on the regression diets was close to the mean value after 40 months. The data show that cholesterol in both its free form and its ester form is depleted from experimentally induced coronary atheromatosis by dietary regression regimens. The data also suggest that most of the cholesterol depletion occurs during the first half of regression; the susceptibility of the residual excess arterial cholesterol to mobilization from the vessel wall by dietary means is questionable.

RELATIONSHIP BETWEEN LOW-DENSITY LIPOPROTEIN IN AORTIC INTIMA AND SERUM-LIPID LEVELS

The availability of blood-samples drawn from twenty-one patients during the week preceding death and aortic tissue taken at necropsy has made possible a direct study of the relationship between serum-lipid levels and the concentration of intact lipoprotein in the aortic intima. Lipoprotein in the intima was measured by a microimmunoassay method. In normal intima the concentration of intimal lipoprotein was significantly correlated with serum-cholesterol: for every 100 mg. per 100 ml. increase in serum-cholesterol the lipoprotein cholesterol in the intima increased by 1·8 mg. per 100 mg. dry tissue, which represents an increment of nearly 50% in the total intimal cholesterol. In the patient with the lowest serum-cholesterol (55 mg. per 100 ml.) the intima contained 0·7 mg. per 100 mg. dry tissue of lipoprotein cholesterol and 3·8 mg. residual (non-lipoprotein) cholesterol, whereas in the patient with the highest serum-cholesterol (426 mg. per 100 ml.) the intima contained 8·3 mg. lipoprotein cholesterol and 4·3 mg. residual cholesterol. In terms of the patients' own sera there was no significant correlation with serum-cholesterol when hypertensive cases were excluded. This suggests that a rather constant amount of whole plasma may be entering the intima, carrying a variable amount of lipoprotein, dependent on its cholesterol level; there was, however, a substantial increase in the two known hypertensives. In fatty streaks containing numerous fat-filled cells there was a 77% decrease in lipoprotein concentration. A comparison of lipoprotein and albumin was made, using the patients' own sera as the standards for both assays. In terms of microlitres of serum there was about seven times as much lipoprotein as albumin, which strongly suggests some form of preferential retention of the lipoprotein.

ROLE OF BILE AND PANCREATIC JUICE IN CHOLESTEROL ABSORPTION AND ESTERIFICATION.

Absence of bile and pancreatic juice in the intestinal tract totally abolished absorption of cholesterol-4-C14 into thoracic duct lymph. Similarly, intestinal cholesterol esterase activity approached zero in animals lacking both bile and pancreatic juice. Intestinal cholesterol esterase could still be demonstrated in animals deprived of pancreatic juice, but which received an infusion or intragastric administration of bile salts. Absorption of cholesterol was shown to occur even in the complete absence of pancreatic juice, provided bile salts were present in the intestinal tract. Some synthesis of cholesterol esterase by the intestinal or reactivation of residual cholesterol esterase in the presence of bile salts is postulated. Thus, pancreatic secretion is not absolutely required for cholesterol absorption, although it has a stimulating effect in the presence of bile salts. This effect is attributed to its cholesterol esterase content. In the presence of bile salts, the process of esterification is postulated to be a rate-limiting step during intestinal absorption of dietary cholesterol.