Biomedical subjects
Publications and source records attributed to S Shefer.
HMG CoA reductase activity of rat intestinal mucosa has a diurnal rhythm which coincides with the diurnal variation of the hepatic HMG CoA reductase but has a lower amplitude. The rhythmic variation of the intestinal reductase was present in both jejunal and ileal crypt cell microsomes and was not abolished by cholestyramine administration.
The production of bile salts by rat liver is regulated by a feedback mechanism, but it is not known which enzyme controls endogenous bile acid synthesis. In order to demonstrate the biochemical site of this control mechanism, bile fistula rats were infused intravenously with (14)C-labeled bile acid precursors, and bile acid biosynthesis was inhibited as required by intraduodenal infusion of sodium taurocholate. The infusion of taurocholate (11-14 mg/100 g of rat per hr) inhibited the incorporation of acetate-1-(14)C, mevalonolactone-2-(14)C, and cholesterol-4-(14)C into bile acids by approximately 90%. In contrast, the incorporation of 7alpha-hydroxycholesterol-4-(14)C into bile acids was reduced by less than 10% during taurocholate infusion. These results indicate that the regulation of bile acid biosynthesis is exerted via cholesterol 7alpha-hydroxylase provided that hepatic cholesterol synthesis is adequate.
The hepatic biosynthesis of bile salts in the rat has been shown to be controlled homeostatically by the quantity of bile salt returning to the liver via the portal circulation. The feedback mechanism was demonstrated in two kinds of experiments. In the first, rats with bile fistulas were infused intraduodenally with sodium taurocholate 12 hr after surgery. If the rate of infusion was greater than 10 mg per 100 g rat per hr, the increase in bile acid output normally observed in bile fistula rats was prevented. In the second type of experiment, the rats were infused with taurocholate 48-72 hr after biliary diversion, when bile acid output had reached a maximal value. Provided the rate of infusion exceeded 10 mg per 100 g rat per hr, bile acid secretion returned to the low levels observed in intact rats. Previous attempts to demonstrate the feedback control have been unsuccessful because too little bile salt was infused. The taurocholate pool of the experimental animals was measured as approximately 15 mg per 100 g rat; it was calculated from this and the above results that this pool circulated 10-13 times daily.
17alpha-Methyltestosterone-4-(14)C was fed to two dogs in an experiment to determine tissue localization and metabolic disposition of this hypocholesterolemic steroid. No accumulation of the drug was found in any tissue, although a small amount of radioactivity was detected in the liver and the ileal mucosa of one animal. Most of the administered radio-activity was excreted in urine and feces. The urinary metabolites consisted largely of highly polar compounds which appeared resistant to glucuronidase treatment or solvolysis procedures. Analysis of the fecal metabolites showed the presence of unchanged methyltestosterone, of four isomeric methylandrostanediols, and of labeled unidentified polar compounds. Of the four identified methylandrostane-diols, the predominating fecal diols were 17alpha-methyl-5alpha-androstane-3beta,17beta-diol (45-62%) and 17alpha-methyl-5-androstane-3alpha,17-diol (12-28%); 17alpha-methyl-5alpha-androstane-3alpha,-17-diol and the 5beta:3beta isomer were found in very small amounts only.
The role of 7alpha,12alpha-dihydroxycholest-4-en-3-one as an intermediate in the formation of 5alpha-bile acids from cholesterol was investigated with liver preparations of Iguana iguana in vitro. The microsomal fraction of iguana liver catalyzed the transformation of 7alpha,12alpha-dihydroxycholest-4-en-3-one to 5alpha-cholestane-3alpha,7alpha,12alpha-triol in good yield. 7alpha,12alpha-dihydroxy-5alpha-cholestan-3-one served as an intermediate. Under the conditions employed, formation of the corresponding 5beta-isomers could not be detected. High speed supernatant solution and mitochondrial fraction of iguana liver did not reduce 7alpha,12alpha-dihydroxycholest-4-en-3-one to a measurable extent. The microsomal enzyme system required NADPH as hydrogen donor and was inactive in the presence of NADH. It is suggested that 7alpha,12alpha-dihydroxycholest-4-en-3-one may serve as a common intermediate in the formation of 5alpha- and 5beta-bile acids from cholesterol.
In a study of the mechanism whereby 5alpha-bile acids are formed from cholestanol, the 7alpha-hydroxylation of cholestanol was investigated in rat liver preparations in vitro. It was found that in the presence of NADPH and oxygen, rat liver microsomes catalyzed the 7alpha-hydroxylation of cholestanol to the same extent as that of cholesterol. The rate of the hydroxylation was enhanced by prior treatment of the experimental rats with cholestyramine (a bile acid sequestrant) or by establishment of bile fistulas-i.e., by partial or complete removal of bile acids from the enterohepatic circulation. The 7-hydroxylation reaction was further stimulated by pretreatment of the animals with phenobarbital, a drug known to produce increased biosynthesis of hepatic endoplasmic membranes. The 7alpha-hydroxylase was inhibited by the reaction product, by sterols with 7-keto or 7beta-hydroxyl groups, and also by mono- and dihydroxy bile acids of the 5beta-series, although cholic acid or taurocholate produced no inhibition unless added in high concentrations. The results of these studies are in accord with the concept that the presence of a Delta(5)-double bond is not required for the enzymatic formation of the 7alpha-hydroxy derivative. The rate of this hydroxylation reaction in vitro appears to depend on the concentration of bile salts in the enterohepatic circulation of the experimental animals from whom the microsomes were obtained.
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The 3-beta-hydroxysteroid dehydrogenase of rat liver which catalyzes the conversion of 5alpha-cholestan-3-one to 5alpha-cholestan-3beta-ol is localized mainly in the microsomal fraction. The enzyme required NADPH as hydrogen donor and differed from the known 3-beta-hydroxysteroid dehydrogenases of the C(19) series in being inactive in the presence of NADH. The microsomal preparations did not reduce the 3-keto groups of cholest-4-en-3-one, cholest-5-en-3-one, or 5beta-cholestan-3-one to the corresponding 3beta-hydroxy compounds. The conversion of 5alpha-cholestan-3-one to 5alpha-cholestan-3beta-ol was only slightly inhibited by the reaction product or by other monohydroxy steroids, but a strong inhibitory effect was noted with cholest-5-en-3-one, 5alpha-cholestane-3beta, 7alpha-diol and 5alpha-cholestan-7-on-3beta-ol. The microsomes, but not high speed supernatant solution, catalyzed the reverse of the cholestanone reductase reaction, namely the conversion of 5alpha-cholestan-3beta-ol to 5alpha-cholestan-3-one in the presence of oxygen and an NADP-generating system. The action of the microsomal preparations upon 5alpha-cholestan-3-one produced 5alpha-cholestan-3alpha-ol in addition to the 3beta-epimer. The 3-alpha-hydroxysteroid dehydrogenase involved functioned with either NADH or NADPH as hydrogen donor. The ratio of 5alpha-cholestan-3beta-ol to 5alpha-cholestan-3alpha-ol formed from 5alpha-cholestan-3-one was approximately 10:1 and was independent of the sex of the animal from which the microsomes were prepared.
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The effect of high doses of chenodeoxycholic and ursodeoxycholic acids on hepatic morphology and on cholesterol and bile acid metabolism was examined in the rat. After 2 weeks of either cheno or ursodeoxycholic acid feeding, the livers of the treated rats revealed marked proliferation of the smooth endoplasmic reticulum which appeared as an adaptation phenomenon of the microsomal enzyme system in response to bile acid intake. However, the livers of the chenodeoxycholic acid-treated rats showed early alteration that included mild triaditis, swelling of the bile canalicular microvilli, distended Golgi vesicles, whorling of the mitochondria, and presence of large vacuoles bound by single membranes. During cheno- or ursodeoxycholic acid treatment, the administered bile acid predominated in the bile and amounted to 79 or 67% of the biliary bile acids, respectively. At the same time, the concentration of the muricholic acids was also increased. Biliary cholic acid content dropped significantly, but no change in lithocholic acid concentration was observed. In addition, the activity of HMG-CoA reductase as well as that of cholesterol-7 alpha-hydroxylase was reduced by either of the administered bile acids, while no change in hepatic cholesterol content was detected, and intestinal cholesterol absorption was not significantly different from that of controls. These results show that cheno- and ursodeoxycholic acids inhibited hepatic cholesterol and bile acid synthesis but did not increase either intestinal cholesterol absorption or hepatic microsomal cholesterol content. Since the amounts of biliary lithocholic acid were similar in the bile acid-treated animals, the morphologic abnormalities detected in the chenodeoxycholic acid-fed rats are probably due to an increased pool of chenodeoxycholic acid. However, lithocholic acid-induced liver injury cannot be excluded.
We have examined the mechanism for the bacterial transformation of chenodeoxycholic acid and lithocholic acid into the corresponding 3 beta-hydroxy epimers with the use of 3 alpha- and 3 beta-tritiated bile acids. The 3-oxo bile acids were transformed into the 3 alpha- (85%) and 3 beta- (15%) hydroxy bile acids after 20-hr incubation with Clostridium perfringens. Approximately 75% radioactivity was recovered in the aqueous medium when [3 beta-3H]chenodeoxycholic acid or [3 beta-3H]lithocholic acid was incubated with the bacteria, and approximately 15% of radioactivity in the bile acid fraction was associated with the 3 alpha-position of the iso-bile acids. When [3 beta-3H]chenodeoxycholic acid was incubated with unlabeled 3-oxo-5 beta-cholanoic acid, tritiated litho- and iso-lithocholic acids were recovered. These results can be explained only when a 3-oxo intermediate is postulated, and the 3 beta-hydrogen in the bile acids is transferred by the bacterial coenzyme (NAD+ or NADP+) to the 3 alpha-position in the iso-bile acids during the reduction of the 3-oxo compounds.
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This paper describes convenient syntheses for labeled and unlabeled cholest-5-en-3-one, cholest-4-en-3-one, epicholesterol, cholest-4-en-3 beta-ol, and cholest-4-en-3 alpha-ol. The thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography of these compounds and of cholestanol and epicholestanol are also described. The synthesized compounds are potential precursors in the biosynthesis of cholestanol in mammals.
We examined the relationship between cholesterol synthesis and high affinity low density lipoprotein (LDL) catabolism in freshly isolated mononuclear leukocytes and plasma sterols and apolipoprotein concentrations in three homozygous and one heterozygous subject with sitosterolemia with xanthomatosis and in 12 control subjects. Observations in untreated subjects were compared during therapy with lovastatin or interruption of the enterohepatic circulation of bile acids. Plasma cholesterol, plant sterol, and apolipoprotein B concentrations declined more than 50% in the two homozygous sitosterolemic subjects after ileal bypass surgery. In contrast, plasma cholesterol, plant sterol, and apolipoprotein B concentrations remained constant in a homozygous sitosterolemic subject and declined only 7% in a heterozygous sitosterolemic subject during 20 weeks of lovastatin (40 mg/day) treatment compared to a 28% decrease in similarly treated control subjects. Lovastatin treatment decreased cholesterol synthesis more than 60% but did not increase high affinity catabolism of LDL further in the sitosterolemic cells, compared to a more than 20% rise in control mononuclear leukocytes. Conversely, bile acid malabsorption increased cholesterol synthesis 59%, total hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity 13%, and receptor-mediated LDL degradation 41% in control cells, but did not stimulate cholesterol synthesis or microsomal HMG-CoA reductase activity in sitosterolemic mononuclear leukocytes although receptor-mediated LDL catabolism rose an additional 26%. These results demonstrate a greater than expected decrease in plasma sterols and apolipoprotein B concentrations in sitosterolemic subjects after stimulation of bile acid synthesis because of the inability to up-regulate cholesterol production. We suggest that bile acid-sequestering drugs or ileal exclusion surgery may be more effective treatments to mobilize accumulated sterol deposits and prevent atherosclerosis in this disease.
We investigated the effect of sitosterol on hepatic sterol 27-hydroxylase activities in subjects with sitosterolemia, a recessive inherited disease associated with accelerated atherosclerosis and increased levels of sitosterol and other plant sterols and stanols in tissues. Hepatic activities of mitochondrial sterol 27-hydroxylase, which catalyzes the first step in the conversion of cholesterol to bile acids via the acidic bile acid synthetic pathway, were measured in liver tissues and related to hepatic microsomal cholesterol 7 alpha-hydroxylase, which controls the rate of bile acid synthesis via the neutral synthetic pathway. These measurements of cholesterol catabolism were correlated to sterol concentrations and composition in plasma and liver. Sterol 27-hydroxylase activities in liver mitochondria of three homozygous sitosterolemic subjects were 68% lower than in 10 control subjects (p < .05) and were associated with increased levels of plant sterols in both plasma and liver (13% and 16% of total sterols, respectively, compared to trace amounts in controls). Analysis of Lineweaver-Burk double reciprocal plots of sterol 27-hydroxylase activities in control human liver specimens (where mitochondrial sterol 27-hydroxylase activities were measured with increasing concentrations of the cholesterol substrate, in the absence and presence of 100 microM and 300 microM sitosterol) revealed that sitosterol inhibited mitochondrial sterol 27-hydroxylase activity up to 50% by a competitive mechanism. In sitosterolemic subjects, competitive inhibition of hepatic sterol 27-hydroxylase activity by sitosterol was associated with competitively inhibited microsomal cholesterol 7 alpha-hydroxylase activity (averages from 4 sitosterolemic homozygotes and 14 controls were 12.4 +/- 1.9 and 23.6 +/- 2.5 pmol/mg/min, respectively). Furthermore, decreased cholesterol catabolism in sitosterolemic subjects was associated with significantly elevated plasma cholesterol concentrations (232 +/- 17 mg/dl, as compared to 180 +/- 13 mg/dl in controls) but with no change in hepatic cholesterol concentrations. In an animal model (rats infused intravenously with sitosterol-containing liposomes that increased sitosterol in the liver and plasma to levels similar to those found in sitosterolemic subjects), hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7 alpha-hydroxylase activities also decreased significantly and were coupled to markedly elevated plasma sterol concentrations (120.7 +/- 12.5 mg/dl, as compared to 59.2 +/- 6.3 mg/dl in control animals; p < .05) but to no change in hepatic cholesterol concentrations. Thus, decreased cholesterol catabolism due to competitive inhibition of both microsomal cholesterol 7 alpha-hydroxylase and mitochondrial sterol 27-hydroxylase by elevated hepatic sitosterol concentrations contributes to hypercholesterolemia and increased risk of atherosclerosis in sitosterolemia.