The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis (original) (raw)
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The Biochemical journal, 1998
To quantify the contribution of newly synthesized cholesterol to total plasma and biliary cholesterol under physiological conditions, unrestrained rats were infused intravenously with [1-13C]acetate (0. 6mmol/h per kg) from 12:00 to 24:00 h, and fractional and absolute cholesterol-synthesis rates were determined by mass isotopomer distribution analysis (MIDA). As bile diversion leads to changes in cholesterol metabolism, rats were equipped with permanent catheters in the bile duct and duodenum, allowing sampling of small amounts of bile from an intact enterohepatic circulation. For comparison, rats with chronic bile diversion were also studied. Fractional synthesis of plasma cholesterol was 10.8+/-1.7% (mean+/-S.D.) after 12 h in rats with intact circulation. Fractional synthesis of biliary cholesterol was significantly higher than that of plasma cholesterol, i.e. 16.5+/-2.0% (P<0.05) after 12 h. In contrast, no differences between fractional synthesis of cholesterol in plasma an...
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 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.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1986
Wistar rats were killed 4 h after an intravenous infusion of [1,2-13C]-and [1-r4C]acetic acid sodium salt (39 mg, 12.5 pCi/ml, constant rate: 1.2 ml/h). At this time, labeled free cholesterol movements between the organs are still weak and cholesterol labeling in each tissue mainly originates from the in situ incorporation of the exogenous substrate. In male rats, the specific radioactivity of free cholesterol was found to be higher in the intestine (mucosa and wall) than in the liver and plasma. In female and in cholestyramine-fed male rats, cholesterol 14C labeling was close to that of male rats in the intestine, and was markedly higher in the liver. The same variations of 13C excess, calculated by mass fragmentography, indicated that there was no isotopic effect between 13C and r4C precursors. The advantage of this method consisted in obtaining the proportions of labeled molecules according to their molecular weight (M + 1-M + 11) for each sample. Then the distribution of 13C atoms in newly synthesized cholesterol was assessed in each sterogenesis site. In the intestine, about 3/4 of the 13C atoms were found in molecules of weight of at least M + 4 (after incorporation of at least two labeled acetate units). This proportion was only l/3 in hepatic and plasma free cholesterol. These distinct '3C-labeling patterns clearly indicate that local variations occurred in the isotopic enrichment of acetyl-CoA used for cholesterol formation. Whatever the experimental conditions of this study, cholesterol was synthesized from an acetyl-CoA more "C enriched in the intestine than in the liver. Such variations probably result from the different dilutions of exogenous acetyl-CoA by the endogenous pool in the liver and intestine. Consequently, the 14C or 13C incorporations measured in the liver and intestinal sterols do not account for absolute rates of cholesterol production by these organs. This study also indicated that after a few hours of infusion, free cholesterol labeling in the plasma originated mainly from cholesterol newly formed in the liver, even when acetate incorporation into cholesterol was higher in the intestine than in the liver. * This work is dedicated to the memory of Franqois Cheval-bile pool of body cholesterol by 'internal secrelier. tion'. This flow generally prevails over the one * * To whom correspondence should be addressed. with a dietary origin [1,2].
Solvent isotope effect on bile formation in the rat
The Biochemical journal, 1995
2H2O affects many membrane transport processes by solvent and kinetic isotope effects. Since bile formation is a process of osmotic filtration where such effects could be important, we investigated the effects of 2H2O on bile formation in the in situ perfused rat liver. Dose finding experiments showed that at high concentrations, 2H2O increased vascular resistance and induced cholestasis; at 60% 2H2O however, a clear dissociation between the vascular and biliary effects was observed. Therefore, further experiments were carried out at this concentration. The main finding was a reduction in bile salt-independent bile flow from 0.99 +/- 0.04 to 0.66 +/- 0.04 microliters.min-1.g-1 (P < 0.001). This was associated with a 40% reduction in biliary bicarbonate concentration (P < 0.001). Choleretic response to neither taurocholate nor ursodeoxycholate was altered by 2H2O; in particular, there was a similar stimulation of bicarbonate secretion by ursodeoxycholate in the presence of 60% ...
Effects of bile salts on rat hepatic acyl CoA:Cholesterol acyltransferase
Lipids, 1995
Acyl CoA:cholesterol acyltransferase (EC2.3.1.26, ACAT), responsible for intracellular esterification of cholesterol, may play an important role in cholesterol trafficking within the cell, and thus, in maintenance of cellular cholesterol homeostasis. Bile acids are potential regulators of cholesterol trafficking in the liver. Therefore, the effect of bile salts on hepatic ACAT activity was studied in the perfused rat liver. ACAT activity was increased after liver perfusion with either taurocholate or taurochenodeoxycholate. However, addition of these bile salts at physiological concentrations in vitro had little effect on microsomal ACAT activity. The increase in hepatic ACAT activity due to perfusion with bile salts was accompanied by reduced accumulation of very low density lipoprotein cholesterol in the perfusate, but there was no effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Hepatic ACAT activity was decreased after bile diversion for four hours in the intact animal. This treatment had no statistically significant effect on 3-hydroxy-3-methylglutaryI-CoA reductase activity. These data suggest that bile salts induce changes in hepatic compartmentation and traffic of cholesterol within the hepatocyte accompanied by response of ACAT activity to maintain cellular cholesterol homeostasis.
Bile Acid Synthesis in Man: Metabolism of 7α
Journal of Clinical Investigation, 1972
The pathways of bile acid synthesis in man were evaluated by studying the metabolism of 7ahydroxycholesterol-4-14C and 26-hydroxycholesterol-16, 22-3H administered parenterally to individuals requiring external biliary drainage. Techniques for the identification of metabolites were thin-layer chromatography, column chromatography, gas-liquid chromatography with stream splitting, and crystallization to constant specific activity. It was found that both compounds were rapidly metabolized to bile acids and excreted in bile. Of the total radioactivity recovered in bile as bile acids, 87% of the 26-hydroxycholesterol-3H and 90% of the 7a-hydroxycholesterol-14C was found to be metabolized to both chenodeoxycholate and cholate. Compared to 7a-hydroxycholesterol, a greater proportion of 26-hydroxycholesterol was found to be metabolized to chenodeoxycholate. These findings indicate that both 7a-hydroxycholesterol and 26-hydroxycholesterol can be intermediates in the metabolism of cholesterol to bile acids in man. The observation that conversion to cholate takes place less readily after C-26 hydroxylation is consistent with previous findings in other species.