Faculty Bibliography

We describe a convenient and stereoselective route to the synthesis of 27-hydroxycholesterol. Also its radiolabeled analog, 22, 23 di [(3)H]-27-hydroxycholesterol with high specific radioactivity (55 Ci/mmol) was synthesized by this method. Julia condensation of steroidal 22-sulfone with aldehyde, led to the addition of the 23-27 carbon side chain building block to the steroid backbone. Formed in this reaction beta-hydroxysulfone moiety was reduced by sodium amalgam generate 22-23 unsaturated bond. Further reduction either by hydrogen or tritium furnished substrates for the synthesis of title compounds

Oxysterol 7alpha-hydroxylase has broad substrate specificity for sterol metabolites and may be involved in many metabolic processes including bile acid synthesis and neurosteroid metabolism. The cloned human oxysterol 7alpha-hydroxylase (CYP7B1) cDNA encodes a polypeptide of 506 amino acid residues that shares 40% sequence identity to human cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. In contrast to the liver-specific expression of CYP7A1, CYP7B1 mRNA transcripts were detected in human tissues involved in steroid genesis (brain, testes, ovary, and prostate) and in bile acid synthesis (liver) and reabsorption (colon, kidney, and small intestine). The human oxysterol 7alpha-hydroxylase transiently expressed in 293/T cells was able to catalyze 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone (DHEA). The human CYP7A1 and CYP7B1 both contain six exons and five introns. However, CYP7B1 spans at least 65 kb of the genome and is about 6-fold longer than CYP7A1. The transcription start site (+1) was localized 204 bp upstream of the initiation codon. No TATA box-like sequence was found near the transcription start site. Transient transfection assays of CYP7B1 promoter/luciferase reporter constructs in HepG2 cells revealed that the promoter was highly active. The 5' upstream region from nt -83 to +189 is the core promoter of the gene

We attempted to quantitate production of bile acid via the 27-hydroxylation pathway in six human subjects. After bolus intravenous injection of known amounts of [24-14C]cholic acid and [24-14C]chenodeoxycholic acid, each subject underwent a constant intravenous infusion of a mixture of [22, 23-3H]-27-hydroxycholesterol and [2H]-27-hydroxycholesterol for 6;-10 h. Production rate of 27-hydroxycholesterol was calculated from the infusion rate of [2H]-27-hydroxycholesterol and the serum ratio of deuterated/protium 27-hydroxycholesterol, which reached a plateau level by 4 h of infusion. Conversion of 27-hydroxycholesterol to cholic and chenodeoxycholic acids was determined from the 3H/14C ratio of these two bile acids in bile samples obtained the day after infusion. In five of the six subjects, independent measurement of bile acid synthesis by fecal acidic sterol output was available from previous studies. Endogenous production of 27-hydroxycholesterol averaged 17.6 mg/day and ranged from 5.0 to 28.2 mg/day, which amounted to 8.7% (range 3.0;-17.9%) of total bile acid synthesis. On average 66% of infused 27-hydroxycholesterol was converted to bile acid, of which 72.6% was chenodeoxycholic acid.These data suggest that relatively little bile acid synthesis takes place via the 27-hydroxylation pathway in healthy humans. Nevertheless, even this amount, occurring predominantly in vascular endothelium and macrophages, could represent an important means for removal of cholesterol deposited in endothelium

Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by approximately 2-fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5beta-3-one and 7alpha,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3beta-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7alpha,27-OHC was unable to increase SF-1-dependent StAR promoter activity, we examined 27-OHC 7alpha-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7alpha-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions

BACKGROUND & AIMS: Cyp 7-/- mice lack a functional cholesterol 7alpha-hydroxylase enzyme and develop cholestasis before up-regulation of 27-hydroxycholesterol 7alpha-hydroxylase activity. Because 7alpha-hydroxylation is not the initial step in this metabolic pathway, we tested the hypothesis that cholesterol 27-hydroxylase is expressed at an earlier step and leads to the production of monohydroxy bile acids. METHODS: Polymerase chain reaction with specific oligonucleotides was used to detect messenger RNA (mRNA) coding for cholesterol 27-hydroxylase in 5-day-old normal and Cyp 7-/- mice. Gas-liquid chromatography-mass spectrometry and reverse isotope dilution were used to identify intermediates in the cholesterol 27-hydroxylase metabolic pathway. Light and electron microscopy were used to evaluate the morphological appearance of the liver. RESULTS: mRNA for cholesterol 27-hydroxylase was identified in the liver and spleen. The monohydroxy bile acids 3beta-hydroxy-5-cholenoate and 3alpha-hydroxy-5beta-cholanoate together with their precursor, 27-hydroxycholesterol, were identified in liver homogenates. Cholestasis, present focally, was manifested as dilated bile canaliculi, partial loss of microvilli, and retention of electron-dense biliary material. CONCLUSIONS: The cholesterol 27-hydroxylase metabolic pathway of bile acid synthesis is expressed in neonatal life. The absence of 7alpha-hydroxylase activities unmasks the cholestatic potential of monohydroxy bile acids. The Cyp 7-/- knockout mouse mimics cholestatic events known to occur in humans and provides a unique opportunity for studying regulatory determinants

In contrast to current methods of bile acid analysis that require the separation of bile acids into different groups prior to their analysis, the HPLC method using a reverse phase column and gradient elution that we developed permits the separation and detection of nonconjugated, glycine-conjugated, and esterified bile acids as their fluorescent dimethoxycoumarin esters. The mild conditions for ester formation make possible the identification of allylic bile acids characteristic of metabolic errors in bile acid synthesis. Quantification is obtained using 7 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid as an internal standard. In addition to identification based on retention time, peak-shift strategy is used by treatment of aliquots with cholyglycine hydrolase and/or solvolysis. Loss of the parent peak and appearance of the derivative provide further assurance of the identity of each bile acid in biologic fluids that contain other organic acids