Recent Advances in Metabolic Pathways of Bile Acid Synthesis (original) (raw)
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Journal of Lipid Research, 2003
We describe an accurate method for monitoring the enzymatic activity of hepatic cholesterol 7 ␣-hydroxylase (C7 ␣ OH; CYP7A1), the rate-limiting and major regulatory enzyme in the synthesis of bile acids. Assay of 7 ␣-hydroxy-4-cholesten-3-one (C4), an intermediate in bile acid synthesis, revealed that the level of C4 in peripheral blood serum or plasma showed a strong correlation to the enzymatic activity of hepatic C7 ␣ OH, both at steady-state conditions (r ؍ 0.929) as well as during the rapid changes that occur during the diurnal phases. This assay should be of value in clarifying the regulation of bile acid synthesis in vivo in laboratory animals and humans since it allows for the monitoring of hepatic C7 ␣ OH activity using peripheral blood samples.-Gälman, C., I. Arvidsson, B. Angelin, and M. Rudling. Monitoring hepatic cholesterol 7 ␣-hydroxylase activity by assay of the stable bile acid intermediate 7 ␣-hydroxy-4-cholesten-3-one in peripheral blood.
Hepatology, 2001
To elucidate the feedback control exerted on bile acid biosynthesis in humans with obstructive cholestasis, 16 patients with bile duct obstruction were studied. In vivo 7␣hydroxylation, reflecting bile acid synthesis, was assayed in 13 of them by tritium release analysis. Serum 27-hydroxycholesterol was determined by gas chromatography-mass spectrometry. In a subgroup, hepatic cholesterol 7␣-hydroxylase mRNA was assayed by real-time polymerase chain reaction (PCR), enzyme activity was determined by isotope incorporation, and microsomal cholesterol content was assayed by gas chromatography-mass spectrometry. Agematched control subjects were studied in parallel. Hydroxylation rates were lower in cholestatic patients (108 ؎ 33 mg of cholesterol per day, mean ؎ SEM; controls: 297 ؎ 40 mg/d; P < .01). The reduction was proportional to the severity of cholestasis, and synthetic rates were normalized in 4 subjects restudied after resolution of biliary obstruction. Consistent findings were obtained by analysis of serum 7␣hydroxycholesterol levels. On the other hand, hepatic cholesterol 7␣-hydroxylase mRNA, microsomal enzyme activity, and cholesterol content tended to be increased in cholestasis. Finally, serum 27-hydroxycholesterol levels were slightly reduced in cholestatic subjects and were not related with the severity of the disease. Suppression of in vivo bile acid synthesis with no corresponding reduction in tissue 7␣-hydroxylase expression and activity is consistent with nontranscriptional, posttranslational levels of regulation; these may play a role in the feedback control of bile acid synthesis in particular conditions. Alteration of the alternate biosynthetic pathway seems unlikely according to the present data. (HEPATOLOGY 2001;34:234-242.)
Metabolism-clinical and Experimental, 1999
The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7oL-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7~-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 51~-cholestane-3~,7~-diol, and 51~-cholestane-3~,7~,12~-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial stero127-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7~-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7~-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7~-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.
Hepatology, 2003
The e&cts of newly synthesized cholesterol availability on bile acid synthesis are largely unknown, particularly in humans. The present study was aimed to study the changes induced on bile acid synthesis by simvastatin, a competitive inhibitor of hydroxymethyl glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis, during pharmacologic interruption of the enterohepatic circulation. S i x patients with primary hypercholesterolemia were studied in basal conditions, after treatment with the bile acid binding resin cholestyramine alone (8-16 g/d for 6-8 weeks) and subsequently in combination with simvastatin (40 mg/d for 6-8 weeks). Cholesterol 7a-hydroxylation rate, a measure of total bile acid synthesis, was assayed in vim by tritium release analysis. Serum lathosterol levels were assayed by gas chromatographymass spectrometry as a measure of cholesterol synthesis. Serum total and low-density lipoprotein-cholesterol were reduced significantly after cholestyramine (by 26% and 30%, respectively) and during combined treatment (by 47% and 55%). 7a-Hydroxylation rates increased nearly 4-fold with cholestyramine alone; addition of simvastatin induced a significant decrease of hydroxylation rates (cholestyramine alone, 1,591 2 183 mg/d; plus simvastatin, 1,098 f 232 mg/dj mean 2 SEM; P < .05). Hydroxylation rates significantly correlated with serum lathosterou cholesterol ratio (T = 0.79, P < .05). In conclusion, in conditions of chronic stimulation bile acid synthesis may be affected by changes in newly synthesized cholesterol availability. The finding might relate to the degree of substrate saturation of microsomal cholesterol 7a-hydroxylase; alternatively, newly synthesized cholesterol might induce a stimulatory effkct on cholesterol 7a-hydroxylase transcription. (HEPATOLOGY 2003;38:939-946.) B ile acid production is a major mechanism whereby cholesterol is eliminated from the organism and, therefore, represents a crucial event in the maintenance of cholesterol homeostasis.1-3 Two metabolic path-Abbreviations: HMG-CoA, hydroxymethyl glutaryl-CoA; LDL, low-density lipoprotein; HDL, high-density lipoprotein.
Journal of Biological Chemistry
We measured hepatic cholesterol 7 alpha-hydroxylase activity, mass, and catalytic efficiency (activity/unit mass) in bile fistula rats infused intraduodenally with taurocholate and its 7 beta-hydroxy epimer, tauroursocholate, with or without mevalonolactone to supply newly synthesized cholesterol. Enzyme activity was measured by an isotope incorporation assay and enzyme mass by densitometric scanning of immunoblots using rabbit anti-rat liver cholesterol 7 alpha-hydroxylase antisera. Cholesterol 7 alpha-hydroxylase activity increased 6-fold, enzyme mass 34%, and catalytic efficiency 5-fold after interruption of the enterohepatic circulation for 48 h. When taurocholate was infused to the bile acid-depleted animals at a rate equivalent to the hepatic bile acid flux (27 mumol/100-g rat/h), cholesterol 7 alpha-hydroxylase activity and enzyme mass declined 60 and 61%, respectively. Tauroursocholate did not significantly decrease cholesterol 7 alpha-hydroxylase activity, mass and catalyti...
Journal of lipid research, 1986
The present work describes an accurate assay of the rate-limiting enzyme in bile acid synthesis, the cholesterol 7 alpha-hydroxylase, in human liver. The assay is based on isotope dilution-mass spectrometry, and endogenous microsomal cholesterol is used as the only substrate for the enzyme. Operative liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In ten gallstone patients, the enzyme activity of the microsomal fraction averaged 9.6 +/- 1.4 (mean +/- SEM) pmol X min-1 X mg protein-1 corresponding to a daily synthesis of about 0.5 mmol of bile acids. Three cholestyramine-treated patients displayed a four-fold higher enzyme activity. No evidence was obtained supporting the concept that the cholesterol 7 alpha-hydroxylase is modulated by phosphorylation-dephosphorylation.
We have recently reported that coordinate down-regulation of cholesterol 7a-hydroxylase and sterol 27-hydroxylase by bile acids results in suppression of bile acid synthesis in cultured rat hepatocytes [Twisk, J., De Wit, E. & Princen, H. M. G. (1995) Biochem. J. 305, 505-5111. In the current study, we have assessed the effects of a large group of different bile acids, both naturally occurring and synthetic, on these two key enzymes, to elucidate structural features which render bile acids potent as regulators of bile acid synthesis. Addition of 50 pM deoxycholate or cholate, two relatively hydrophobic bile acids, to the culture medium of hepatocytes resulted in strong suppression of cholesterol 7a-hydroxylase (suppression of 75 % and 88 %, respectively) and sterol 27-hydroxylase activity (suppression of 76 % and 72%, respectively). These effects were also reflected in the mRNA levels and the transcriptional activities of the two enzymes, showing a parallel suppression of both parameters in response to cholate (suppression of 78% and 43% for cholesterol 7a-hydroxylase mRNA and transcription, respectively, and suppression of 76 % and 42 % for sterol 27-hydroxylase mRNA and transcription, respectively). In contrast, no effects were observed with the two hydrophilic bile acids, P-muricholate and ursocholate. Transient expression analysis in cultured rat hepatocytes, using a promoter-reporter construct containing the proximal part of the cholesterol 7a-hydroxylase promoter, demonstrated a reduction of transcriptional activity by cholate (reduction of 72 %), but not by ursocholate. Assessment of the effects of 27 different bile acids, varying in the number, position and orientation (cxlp, of hydroxyl groups on the steroid nucleus of the molecule, on cholesterol 7a-hydroxylase mRNA showed only a moderate correlation with the hydrophobicity index of the bile acid involved (r = 0.61 ; P < 0.0001). Analysis of the three-dimensional structure of a number of these bile acids suggests that hydroxyl groups situated in close proximity to each other within the molecule, creating a hydrophilic environment, as in the case of cholate, may be a prerequisite for a strong inhibitory potency. Deviation from this situation leads to a markedly lesser effect on suppression of cholesterol 7a-hydroxylase and sterol 27-hydroxylase.
European Journal of Biochemistry, 1995
We have recently reported that coordinate down-regulation of cholesterol 7a-hydroxylase and sterol 27-hydroxylase by bile acids results in suppression of bile acid synthesis in cultured rat hepatocytes [Twisk, J., De Wit, E. & Princen, H. M. G. (1995) Biochem. J. 305, 505-5111. In the current study, we have assessed the effects of a large group of different bile acids, both naturally occurring and synthetic, on these two key enzymes, to elucidate structural features which render bile acids potent as regulators of bile acid synthesis.