Faculty Bibliography

The use of 2-hydroxypropyl-beta-cyclodextrin has made it possible to prepare stable aqueous solutions of cholesterol, 26-hydroxycholesterol, 7 alpha-hydroxycholesterol, and monohydroxy bile acids such as lithocholic and 3 beta-hydroxy-5-cholenoic acids. These solutions are suitable for cell culture studies and for parenteral administration to animals

Cholest-5-ene-3 beta,26-diol (26-hydroxycholesterol) is synthesized by a mitochondrial P-450 enzyme that appears to be widely distributed in tissues. Together with other C-27 steroid intermediates it is transported to the liver and metabolized to bile acids. Although 26-hydroxycholesterol is transported in plasma lipoproteins mostly as the fatty acid ester, neither its assembly and orientation within lipoproteins nor its mechanism of transport across the sinusoidal liver membrane is known. Cell culture studies indicate that 26-hydroxycholesterol can inhibit both cholesterol synthesis and low density lipoprotein (LDL) receptor activity. Inhibition of DNA synthesis also occurs and may not be related to the reduction in HMG-CoA reductase activity. The relationship of these in vitro activities to the physiologic role(s) of 26-hydroxycholesterol remains to be clarified. A clue to its biologic role is the knowledge that markedly decreased 26-hydroxylase activity appears to be the molecular basis of cerebrotendinous xanthomatosis, an inborn error of metabolism characterized by a significant decrease in 26-hydroxycholesterol and bile acid synthesis and an increase in cholesterol synthesis

Breast cyst fluid (BCF) aspirated from 12 women with fibrocystic disease of the breast and sera obtained simultaneously were analyzed for bile acids. Analysis was performed by gas-liquid chromatography of the acetoxy methyl esters of the bile acids prepared after alkaline hydrolysis of the bile salts. An internal standard served to correct for methodological losses. Low levels of bile acids were found in serum samples, precluding overt hepatobiliary complications. Deoxycholic acid (17-160 mumol/L), chenodeoxycholic acid (18-305 mumol/L), and cholic acid (3-119 mumol/L) were detected in 11 of 12 samples of BCF. In 2 cases, chosen at random, the identities of the bile acids were verified by mass spectrometry. Lithocholic acid (9-23 mumol/L), a reported cocarcinogen, was detected in 6 of the 12 samples of BCF. This is the first report of the presence of lithocholic acid in BCF with confirmation by Mass spectrometry. There was no correlation between the levels of individual bile acids and those of potassium ion, Na+/K+, estriol-3-sulfate, or 16 alpha-hydroxyandrogen sulfates that had been quantified previously in these samples. There was borderline correlation between concentrations of total bile acids and K+ (P less than 0.06) and Na+/K+ (P less than 0.07). Yet to be elucidated are the mechanism of accumulation of bile acids in BCF and whether levels of particular bile acids in BCF may serve to identify that small subset of women with fibrocystic disease at risk for developing breast cancer

Deuterated 26-hydroxycholesterol prepared from diosgenin by modifications of existing methods permitted the determination of mitochondrial cholesterol 26-hydroxylase using endogenous cholesterol as the substrate. Enzyme activity in a group of Syrian hamsters was found to be 10.3 +/- 3.7 pmol.min-1.mg protein-1

Metabolism of 26-hydroxycholesterol to 3 beta-hydroxychol-5-en-24-oic acid and other C24-bile acids has been expected to occur by way of 3 beta-hydroxycholest-5-en-26-oic acid in studies in vitro. 3 beta-Hydroxycholest-5-en-26-oic acid was infused intravenously into bile fistula hamsters and the following C24-bile acids were identified: 3 beta-hydroxychol-5-en-24-oic acid, lithocholic acid, chenodeoxycholic acid, and a small amount of cholic acid

Hep G2, a liver cell line derived from a human hepatoblastoma that is free of known hepatotropic viral agents, has been found to express a wide variety of liver-specific metabolic functions. Among these functions are those related to cholesterol and triglyceride metabolism. Confluent Hep G2 monolayers express normal low-density lipoprotein (LDL) receptors and continue to internalize and metabolize chylomicrons, very low-density lipoproteins (VLDL), LDL, and high-density lipoproteins. In lipoprotein-free medium, apolipoproteins A-I, A-II, B, C, and E accumulate in the medium together with cholesterol, cholesteryl ester, triglyceride, and all the primary bile acids. The regulation of their synthesis and secretion is not fully known and their interrelationships have not been established. Because Hep G2 cells express these and other components of cholesterol and triglyceride metabolism, they are a microcosm for studying the central role of the liver

1. Using a human hepatoma (Hep G2) cell line that continually synthesizes 3 beta-hydroxy-5-cholenoic acid, lithocholic acid, chenodeoxycholic acid and cholic acid we have determined the metabolism and biological effects of 26-hydroxycholesterol and 7 alpha-hydroxycholesterol. 2. Addition of 26-hydroxycholesterol to the medium (6 microM) downregulated cholesterol and chenodeoxycholic acid synthesis. 3. The predominant metabolite of 26-hydroxycholesterol was 3 beta-hydroxy-5-cholenoic acid. 4. Cholesterol synthesis was not affected by the addition of 7 alpha-hydroxycholesterol (6 and 12 microM). The predominant metabolite of 7 alpha-hydroxycholesterol was chenodeoxycholic acid. 5. In Hep G2 cells 7 alpha-hydroxylation of 26-hydroxycholesterol is not well expressed

Human fibroblasts and hepatoma (Hep G2) cells were grown in media containing 25% D2O. Cholesterol extracted from the cells and bile acids obtained from the media were analyzed by gas chromatography/mass spectrometry (GC/MS). Fibroblasts that were transferred serially in media containing D2O continued to grow and to synthesize cholesterol enriched in deuterium. The observed distribution of deuterium-enriched species of cholesterol corresponded to a distribution that was calculated based on C = 27, 13C = 1.107%, D2O/H2O = 0.25, hydrogen derived from water = 20, and is in agreement with the concept that deuterium incorporation occurs randomly and represents mostly the NADPD/NADPH ratio in the medium. The deuterium enrichment of cholesterol from hepatoma cells indicated a shift of the most abundant species from m/z 373 to m/z 375, which corresponds more closely to the derivation of 25 hydrogens from water and implies the formation of deuterated acetate in the medium. Analysis of chenodeoxycholic acid, the predominant bile acid synthesized by Hep G2 cells in vitro, indicates its derivation from both pre-formed and newly synthesized cholesterol and that A ring transformation from cholesterol utilizes deuterium derived from water. Analysis of the bile acids derived from hamster bile following the administration of D2O confirms that similar events occur in vivo

Confluent cultures of Hep G2 cells were found to synthesize chenodeoxycholic and cholic acids continually. Chenodeoxycholic acid was synthesized at the rate of 58 +/- 8.6 micrograms/96 h, a rate more than 7-fold greater than that for cholic acid. Addition of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol but not the -3 alpha, 7 alpha-diol was followed by an increase in cholic acid synthesis, thus indicating a relatively low 12 alpha-hydroxylase activity. Endogenous synthesis of monohydroxy bile acid ester sulfates was found, with maximum rates of 135 and 74 micrograms/96 h for lithocholic and 3 alpha-hydroxy-5-cholenoic acids, respectively. Incubation of Hep G2 cells in medium containing 25% D2O permitted a comparison of the precursor/product relationship of cholesterol with 3 beta-hydroxy-5-cholenoic acid. The pattern of incorporation of deuterium was in accordance with that expected, thus allowing the conclusion that this monohydroxy bile acid is derived from cholesterol and should be considered together with chenodeoxycholic and cholic acids as a primary bile acid