Faculty Bibliography

Sterol 27-hydroxylase activity in bovine aortic endothelial (BAE) cells in culture has been compared with that in HepG2 cells and in Chinese hamster ovary (CHO) cells using identical culture conditions. The total enzyme activity of BAE cells (3.0 nmol/72 h per mg cell protein) was comparable with that of HepG2 cells (4.0 nmol/72 h per mg protein) and both values were significantly greater than that in CHO cells (0.002 nmol/72 h per mg protein). The enzyme was identified in the mitochondria extracted from BAE cells by Western blotting using an antibody of proven specificity, and its metabolites 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid were identified by mass spectrum analysis. The presence of the enzyme in endothelium provides a mechanism for preventing accumulation of intracellular cholesterol by initiating a pathway of bile acid synthesis different from that initiated by 7 alpha-hydroxylation of cholesterol in the liver

Bile acid synthesis from cholesterol can occur via two pathways, one initiated by sterol 27-hydroxylase activity or one initiated by that of cholesterol 7 alpha-hydroxylase. In contrast to cholesterol 7 alpha-hydroxylase, which is found in the liver, sterol 27-hydroxylase is a widely distributed mitochondrial enzyme with high activity in vascular endothelial cells. Although both pathways lead to the production of chenodeoxycholic and cholic acids, the key step, 7 alpha-hydroxylation, is governed by two different enzymes. Both 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, the metabolites of cholesterol occurring via sterol 27-hydroxylase activity, normally circulate in plasma. After their uptake by the liver they are metabolized mostly to chenodeoxycholic acid, which down-regulates the activity of cholesterol 7 alpha-hydroxylase, the rate-limiting step for the production of bile acids in the liver. Because of this relationship and also in view of the accelerated atherosclerosis and cholesterol deposition in tissues that occur as a consequence of genetically determined sterol 27-hydroxylase deficiency and of the potent biologic effect of 27-hydroxycholesterol in cell culture, it is proposed that this metabolic pathway serves a regulatory function. The pathway beginning with cholesterol 7 alpha-hydroxylation is modulated by genetic, hormonal, and probably dietary factors, and becomes most prominent with the interruption of the enterohepatic circulation of bile acids

The notion that a breast-gut connection might modulate the microenvironment of breast tissue was supported by the finding that breast cyst fluid contains bile acids that are characteristically found in the intestines. To establish that the gut, rather than circulating steroid precursors, is the source of bile acids in breast cyst fluid, we gave two patients deuterium-labelled chenodeoxycholic acid (three 200 mg doses by mouth), starting 9 days before aspiration of breast cysts. The chenodeoxycholic acid concentration of seven samples of aspirated cyst fluid ranged from 42 to 94 mumol/L. The corresponding serum concentrations of chenodeoxycholic acid on the same day were 0.8 and 2.9 mumol/L, of which the labelled compound comprised 13.0% (0.38 mumol/L) and 28.2% (0.23 mumol/L). The deuterated chenodeoxycholic acid concentrations in cyst fluid were 0.79 and 1.26 mumol/L in two samples from patient 1 and 3.22 mumol/L in patient 2; these values are equivalent to 11-17% of the serum concentrations [corrected]. This study shows that intestinal bile acids rapidly gain access to cyst fluid. Further studies should investigate the mechanisms that govern the exchange processes and the maintenance of the high cyst fluid to plasma concentration gradients, and the biological half-lives of individual constituents

The use of 2-hydroxypropyl-beta-cyclodextrin as a vehicle for solubilizing cholesterol and 27-hydroxycholesterol has led to a study of their rates of 7 alpha-hydroxylation in microsomal preparations from hamster liver and HepG2 cells. Addition of the vehicle alone to the cholesterol 7 alpha-hydroxylase assay always caused a several-fold increase in activity. Preloading the vehicle with cholesterol further augmented the rate of 7 alpha-hydroxycholesterol formation. Preloading the vehicle with 27-hydroxycholesterol or 27-hydroxycholestanol (molar ratio 1/1.2) minimally decreased cholesterol 7 alpha-hydroxylase activity (-12%), compared with preloading with cholestanol (-50%), a known competitive inhibitor of the enzyme. Microsomes from hamster liver yielded rates of 7 alpha,27-dihydroxcholesterol formation of 1.5 to 3.0 nmol/min per mg protein, compared with 0.3 nmol/min per mg protein for 7 alpha-hydroxycholesterol. Although cholesterol and cholestanol had minimal effects on the rate of 7 alpha-hydroxylation of 27-hydroxycholesterol, addition of an approximately equimolar amount of 27-hydroxycholestanol inhibited the rate of formation by 65%. Attempts to separate and identify the two C-27 sterol 7 alpha-hydroxylases chromatographically led to the finding that Emulgen 913 selectively inactivates 7 alpha-hydroxylation of 27-hydroxycholesterol. These results indicate that the metabolic pathway for bile acid synthesis from 27-hydroxycholesterol is not governed by cholesterol 7 alpha-hydroxylase

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