Cytotoxicity of oxidation derivatives of cholesterol on cultured aortic smooth muscle cells and their effect on cholesterol biosynthesis.

Aortic smooth muscle cell death is an important initial lesion of atherosclerosis. A number of autooxidation products of cholesterol which has been recognized recently has the capability of inducing rabbits' aortic smooth cell death in vitro. Twelve oxidation derivatives of cholesterol, available commercially, were dissolved in small amounts of ethanol, then added to the culture medium at levels not exceeding 0.8%. The medium contained 10% fetal calf's serum which served as an in situ vehicle for the sterols. The degrees of cytotoxicity were graded and measured as percentage of dying and dead cells in the cultures within 24 hr. 25-Hydroxycholesterol and cholesthan-3 beta, 5 alpha, 6 beta-triol, were the most toxic compounds among the sterols tested. When these oxidation derivatives of cholesterol were added to these cultured cells, they significantly depressed activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a regulatory enzyme of cholesterol biosynthesis (up to 83% inhibition by 25 hydroxycholesterol at a 3 microgram/ml concentration in culture medium) but the sequence of degree of inhibition was not exactly correlated with that of cytotoxicity. Various mechanisms are speculated. Purified cholesterol showed no cytotoxic effect and minimal inhibition of cholesterol biosynthesis.

Carbohydrate and lipid metabolism in the Alaskan Arctic Eskimo.

The effect of a low total carbohydrate low sucrose diet on various parameters of lipid and carbohydrate metabolism was studied among residents of a North Slope Alaskan Eskimo village. For comparative purposes a group of Eskimo youths consuming a higher carbohydrate institutional diet was also stidied. Those Eskimos consuming their native diet had unusually low serum triglycerides and very low density lipoproteins and normal glucose and tolbutamide tolerance tests. Eskimos on a higher carbohydrate diet exhibited significant elevations of triglycerides and minor alterations in glucose tolerance testing. Differences in cholesterol intake between these groups were accompanied by changes in serum cholesterol and low density lipoprotein levels. Unusually high levels of free fatty acids without ketonemia in the North Slope sample were noted as well.

Distribution of 25-hydroxycholesterol in plasma lipoproteins and its role in atherogenesis. A study in squirrel monkeys.

Oxidation products of cholesterol have been shown to be potent inhibitors of cholesterol biosynthesis and also highly toxic to cultured aortic smooth muscle cells. In rabbit experiments, these compounds produced arterial injury resulting in arteriosclerosis. Purified cholesterol only minimally inhibited cholesterol biosynthesis and had no effect on cultured aortic smooth muscle cells. This raises the possibility that plasma lipoproteins containing beta-apoprotein (i.e. LDL and VLDL), which are considered to be atherogenic, may carry more oxidation products than HDL which is not atherogenic [3H]25-hydroxycholesterol and [14C]cholesterol were given only orally to 10 squirrel monkeys (Saimiri sciureus) and blood samples were collected via femoral puncture 24 h after administration. Lipoproteins were separated by ultracentrifugation and the radioactivity in each fraction was counted. Results show that the distribution of labeled cholesterol in VLDL, LDL, and HDL was almost identical to that of unlabeled cholesterol. Most of the radio-activity of 25-hydroxycholesterol was located in LDL & VLDL (55.1% and 34.7%, respectively), only 10.2% was present in HDL. If the radioactivity of 25-hydroxycholesterol were calculated on the basis of the apoprotein content of the lipoprotein micelle, the relative capacity of VLDL and LDL to carry 25-hydroxycholesterol was even greater and more significant than that of HDL (90 X and 42 X, respectively).

Gallbladder bile composition in different ethnic groups.

A total of 194 gallbladder biles from various ethnic groups were analyzed for their composition. The solubility of cholesterol in bile was determined mainly by its relative amounts of phospholipid and bile salt present. Bile from patients with gallstones was usually supersaturated with cholesterol above the boundary of the metastable state. Biles from normal white subjects of Finland, New Zealand, and the United States were already in the metastable state of supersaturation with cholesterol. The Masai of East Africa and black subjects of the Unites States had a bile level below the limit of maximum cholesterol solubility. This study indicates that the difference in the prevalence of cholesterol cholelithiasis in various ethnic groups was related to the difference in their bile compontified factors.

Effect of auto-oxidation products from cholesterol on aortic smooth muscle cells: an in vitro study.

Purified cholesterol is quite unstable when stored in air at room temperature. Products of cholesterol auto-oxidation were concentrated from several lots of USP-grade cholesterol by recrystallizing cholesterol from the methanol extract, retaining the mother liquor, and evaporating the residuum to dryness under vacuum. By application of thin-layer chromatography and gas-liquid chromatography, major, individual, oxidation compounds were identified and quantitated. Biological activities of these oxidation compounds were studied by using cultured rabbits' aortic smooth muscle cells. The concentrate of the auto-oxidation products of cholesterol showed remarkable in vitro cytotoxic effects, whereas purified cholesterol at the same concentration produced no toxic effects. The concentrate was further separated into six thin-layer chromatographical fractions. The results showed that 25-hydroxycholesterol and cholestane-3beta, 5alpha, 6beta-triol were probably responsible for the biological toxicity of the concentrate

Expression of gap junctional connexin proteins in ovine fetal ovaries: effects of maternal diet.

Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and connexin (Cx) proteins have been detected in adult ovaries in several species. Furthermore, it has been shown that several environmental factors, including maternal diet, may affect fetal organ growth and function. To determine whether maternal diet affects expression of Cx26, Cx32, Cx37, and Cx43 in fetal ovaries, sheep were fed a maintenance (M) diet with adequate (A) selenium (Se) or high (H) Se levels from 21 d before breeding to day 132 of pregnancy. From day 50 to 132 of pregnancy (tissue collection day), a portion of the ewes from the ASe and HSe groups was fed a restricted (R; 60% of M) diet. Sections of fetal ovaries were immunostained for the presence of Cxs followed by image analysis. All four Cxs were detected, but the distribution pattern differed. Cx26 was immunolocalized in the oocytes from primordial, primary, secondary, and antral follicles; in granulosa and theca layers of secondary and antral follicles; stroma; and blood vessels. Cx32 was in oocytes, granulosa, and theca cells in a portion of antral follicles; Cx37 was on the borders between oocyte and granulosa/cumulus cells of primordial to antral follicles and in endothelium; and Cx43 was on cellular borders in granulosa and theca layers and between oocyte and granulosa/cumulus cells of primordial to antral follicles. Maternal diet affected Cx26 and Cx43 expression, Cx26 in granulosa layer of antral follicles was decreased (P < 0.01) by HSe in the M and R diets, and Cx43 in granulosa layer of primary and granulosa and theca of antral follicles was increased (P < 0.05) by the M diet with HSe. Thus, Cxs may be differentially involved in regulation of fetal ovarian function in sheep. These data emphasize the importance of maternal diet in fetal growth and development.

Serum cholesterol levels of Seventh-day Adventists.

Serum cholesterol levels and dietary habits were surveyed in 27 male and 34 female Seventh-day Adventist. All subjects studied were lacto-ovo-vegetarians and a few consumed some meat products. Their serum cholesterol levels, significantly lower than those of the United States general population, showed no sex difference but increased with age and were higher in overweight males. Their levels, however, were much higher than those of true vegetarians which was most likely attributable to their consumption, even though to a limited acount, of dairy foods.

Experimental arteriosclerosis in Rhesus monkeys induced by multiple risk factors: cholesterol, vitamin D, and nictotine.

Forty-four rhesus monkeys were given various regimens involving three vasotoxic factors: dietary hypercholesteremia, hypervitaminosis D2 and nicotinism for period ranging from 14 to 62 weeks. When administered singly, no or only early disease was observed. The combined three "risk factor" group demonstrated the most impressive arteriosclerosis. Major arteriosclerotic changes: fat, calcium and mensenchyme of thirty standardized segments of aorta, coronary and limb arteries of all monkeys were graded according to a standardized system. Statistical analysis revealed that the total arteriosclerotic scores of the three vasotoxic factor group was significantly greater than those of any other group and that this was the only group with significant coronary arteriosclerosis as well as complicated lesions such as thrombosis or occlusion of arterial luminae of the extremities. A lowering of the vitamin D2 dosage, resulting in a lengthening of the survival time of animals which should allow sufficient time for complicated disease to develop, is recommended for future studies.