Erwin H. Mosbach

Vaccinia as a model for membrane biogenesis

Abstract The biogenesis of vaccinia envelopes was examined by combined chemical and electron microscopic procedures. These lipoprotein membranes develop within discrete viroplasmic foci, initially 3–3.5 hours after infection. Sequential appearance of viral envelopes and immature and mature particles could be arrested at defined stages by means of actinomycin D or streptovitacin A. Application of these compounds in advance or following morphogenesis indicated that transcription into the requisite RNA precedes by 60 minutes, and translation into protein by less than 30 minutes, the assembly of membranes. Proteins required for maturation are synthesized within 30 minutes following morphogenesis of immature virus. Experiments with isotopically labeled choline, a specific precursor of lecithin in these membranes, indicated that nascent phospholipid is preferentially integrated into the progeny, and analyses using thin-layer gas chromatography revealed that the fatty acid composition of vaccinia can be distinguished from that of host cell membranes. These combined results imply that unique membranes of vaccinia can condense de novo from precursors, two of which are lecithin and viral protein(s), to become the envelope surrounding immature particles. Further differentiation into mature virus occurs inside this envelope. The applicability of the vaccinia model to biogenesis of cellular membranes in general is discussed.

A Biochemical Abnormality in Cerebrotendinous Xanthomatosis IMPAIRMENT OF BILE ACID BIOSYNTHESIS ASSOCIATED WITH INCOMPLETE DEGRADATION OF THE CHOLESTEROL SIDE CHAIN

Bile acid production in cerebrotendinous xanthomatosis (CTX) is subnormal, yet the activity of cholesterol 7alpha-hydroxylase, the rate-determining enzyme of bile acid synthesis, is elevated. To explain this discrepancy, bile acid precursors were sought in bile and feces of three CTX subjects. Over 10% of the total sterols excreted in bile and feces consisted of compounds more polar than cholesterol. Chromatographic analysis of the polar fractions in conjunction with gasliquid chromatography (GLC)-mass spectrometry indicated two major constituents, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and 5beta-cholestane-3alpha,7alpha,12alpha,24xi,25-pentol. After i.v. injection of [4-(14)C]cholesterol both bile alcohols were radioactive proving that they were derived from cholesterol. The accumulation of alcohols hydroxylated at C-25 and C-24,25 suggests that decreased bile acid synthesis in CTX results from impaired oxidation of the cholesterol side chain. This finding and the virtual absence of intermediates hydroxylated at C-26 indicate that current views of the major pathway of bile acid synthesis may require revision.

Increased formation of ursodeoxycholic acid in patients treated with chenodeoxycholic acid.

The formation of ursodeoxycholic acid, the 7 beta-hydroxy epimer of chenodeoxycholic acid, was investigated in three subjects with cerebrotendinous xanthomatosis and in four subjects with gallstones. Total biliary bile acid composition was analyzed by gas-liquid chromatography before and after 4 months of treatment with 0.75 g/day of chenodeoxycholic acid. Individual bile acids were identified by mass spectrometry. Before treatment, bile from cerebrotendinous xanthomatosis (CTX) subjects contained cholic acid, 85%; chenodeoxycholic acid, 7%; deoxycholic acid, 3%; allocholic acid, 3%; and unidentified steroids, 2%; while bile from gallstone subjects contained cholic acid, 45%; chenodeoxycholic acid, 43%; deoxycholic acid, 11%, and lithocholic acid, 1%. In all subjects, 4 months of chenodeoxycholic acid therapy increased the proportion of this bile acid to approximately 80% and decreased cholic acid to 3% of the total biliary bile acids, the remaining 17% of bile acids were identified as ursodeoxycholic acid. After the intravenous injection of [3H]chenodeoxycholic acid, the specific activity of biliary ursodeoxycholic acid exceeded the specific activity of chenodeoxycholic acid, and the resulting specific activity decay curves suggested precursor-product relationships. When [3H]7-ketolithocholic acid was administrated to another patient treated with chenodeoxycholic acid, radioactivity was detected in both the ursodeoxycholic acid and chenodeoxycholic acid fractions. These results indicate that substantial amounts of ursodeoxycholic acid are formed in patients treated with chenodeoxycholic acid. The ursodeoxycholic acid was synthesized from chenodeoxycholic acid presumably via 7-ketolithocholic acid.

A 25-hydroxylation pathway of cholic acid biosynthesis in man and rat.

This paper describes a pathway of cholic acid synthesis, in man and in the rat, which involves 25-hydroxylated intermediates and is catalyzed by microsomal and soluble enzymes. The subcellular localization, stereospecificity, and other properties of the enzymes involved were studied with liver fractions of normolipidemic subjects, cerebrotendinous xanthomatosis patients, and rats. 5beta-Cholestane-3alpha,7alpha,12alpha,25-tetrol was converted to 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol by the microsomal fraction in the presence of NADPH and O2. 5beta-Cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol, 5beta-cholestane-3alpha,7alpha,12alpha,-23xi,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,25,26-pentol were also formed. In the presence of NAD, 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol, but not the other 5beta-cholestanepentols formed, was converted to cholic acid by soluble enzymes in good yield. These experiments demonstrate the existence of a pathway for side-chain degradation in cholic acid synthesis which does not involve hydroxylation at C-26 or the participation of mitochondria.

Effect of dietary chenodeoxycholic acid and lithocholic acid in the rabbit

The feeding of chenodeoxycholic acid or lithocholic acid (0.05 or 0.5% of the diet) to rabbits produced cirrhotic and necrotic changes in the liver, accompanied by an increase of secondary bile acids in bile. Animals fed 0.05% lithocholic acid, 0.05% or 0.5% chenodeoxycholic acid, but not those receiving 0.5% lithocholic acid were able to survive for a period of 21 days. The most severe cirrhotic and necrotic changes were observed in the rabbits fed either 0.5% lithocholic acid or 0.5% chenodeoxycholic acid for 6 days or longer. Liver damage appeared to correlate with bile composition. The pathologic involvement was greatest whenever the percentage of lithocholic acid in the bile exceeded 15%. It is concluded that chenodeoxycholic acid (which is not a major constituent of rabbit bile) exerts its hepatotoxic effects largely because it is converted to lithocholic acid by the intestinal bacterial flora.

Hepatic Toxicity in the Rhesus Monkey Treated with Chenodeoxycholic Acid for 6 Months: Biochemical and Ultrastructural Studies

The long term administration of chenodeoxycholic acid in man has to be regarded with caution because chenodeoxycholic acid has caused liver damage in various species of animals, including primates. To study the effect of three doses of chenodeoxycholic acid (10, 40, and 100 mg per kg per day) on hepatic function and morphology, biliary bile acid composition and the reversibility of changes were investigated in 22 rhesus monkeys. After 6 months of treatment with 40 and 100 mg per kg per day, bile duct proliferation, portal tract inflammation and fibrosis, bile canalicular bleb formation, and hypertrophy of the smooth endoplasmic reticulum were associated with elevated serum levels of oxaloacetic transaminase, glutamic pyruvic transaminase, and leucine aminopeptidase. In the bile, the proportion of chenodeoxycholic acid and its bacterial metabolite, lithocholic acid, rose to approximately 85 and 10% of the total bile acids. After chenodeoxycholic acid was withdrawn for 3 months, the hepatic morphological lesions persisted in some animals although biliary bile acid composition returned to normal. No hepatic abnormalities were seen in the animals treated with 10 mg per kg per day. The findings suggest that long term treatment of rhesus monkeys with high doses of chenodeoxycholic acid results in severe hepatic histological lesions that can persist after discontinuation of the bile acid.

An improved method for the isolation, quantitation, and identification of bile acids in rat feces

Abstract An improved method for the isolation and quantitation of bile acids from rat feces was developed. This method employs an initial Soxhlet extraction of the solid fecal material, esterification of the bile acid fraction with dry methanol/HCl and quantitation using a combination of tlc and glc techniques. In addition, identification of the individual components of the fecal bile acid fraction is accomplished by tlc and glc-ms. This method has proven useful for the quantitation and identification of the fecal bile acids during sterol metabolism measurements.

A new method for the determination of dihydrocholesterol in tissues.

Abstract 1. 1. A new method has been developed for the analysis of dihydrocholesterol in the sterol fraction of mammalian tissues and in gallstones. 2. 2. The method is based on the finding that cholesterol-dihydrocholesterol mixtures can be oxidized with performic acid, which converts the cholesterol quantitatively to cholestane-3β,5α,6β-triol, leaving the dihydrocholesterol unchanged. The oxidation products can then be separated by chromatography on silicic acid, and the amount of dihydrocholesterol can be measured by digitonin precipitation either gravimetrically or by the anthrone method. 3. 3. The feasibility of this method has been demonstrated by analysis of known cholesterol-dihydrocholesterol mixtures, by recovery experiments, and by infrared spectrophotometry of the isolated dihydrocholesterol. 4. 4. It has been shown that previous concepts of the high dihydrocholesterol content of gallstones and atheromata may be in error since these materials frequently contain less than 0.5% dihydrocholesterol. 5. 5. It has further been shown than rabbit tissues, particularly the intestinal wall and adrenals, may contain as much as 5–10% of dihydrocholesterol in the total sterol mixture obtained from these tissues.

Bile alcohol metabolism in man. Conversion of 5beta-cholestane-3alpha, 7alpha,12alpha, 25-tetrol to cholic acid.

To study the role of C25-HYDROXY BILE ALCOHOLS AS PRECURSORS OF CHOlic acid, [G-3-H]5beta-cholestane-3alpha,7alpha12alpha,25-tetrol was administered intravenously to two subjects with cerebrotendinous xanthomatosis (CTX) and two normal individuals. One day after pulse labeling, radioactivity was present in the cholic acid isolated from the bile and feces of the subjects with CTX and the bile of the normal individuals. In the two normal subjects, the sp act decay curves of [G-3-H]-cholic acid were exponential, and no traces of [G-3-H]-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol were detected. In contrast, appreciable quantities of labeled 5beta-cholestane-3alpha,-7aopha,12alpha,25-tetrol were present in the bile and feces of the CTX subjects. The sp act vs. time curves of fecal [G-3-H]5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and [G-3-H]-cholic acid showed a precursor-product relationship. Although these results suggest that 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol may be a precursor of cholic acid in man, the possibility that C26-hydroxy intermediates represent the normal pathway can not be excluded.

Effect of dietary bile acids, cholesterol, and β-sitosterol upon formation of coprostanol and 7-dehydroxylation of bile acids by rat

During studies of sterol metabolism in the rat, the fecal neutral sterol fraction was analyzed by a combination of thin layer chromatography and gas liquid chromatography. On a stock diet of rat chow supplemented with 5% corn oil, the rats excreted 14.5 mg/day of total neutral sterols. Coprostanol comprised 35% (5 mg/day) of this fraction. When the diet was supplemented with 0.5% sodium taurochenodeoxycholate, the amount of coprostanol in the feces remained the same as in the controls (3.2 mg/day, 32%). The addition of 0.5% sodium taurocholate to the diet resulted in a fivefold reduction of coprostanol formation (0.6 mg/day, 8%). When 1.2% cholesterol was added to the stock diet, the amount of coprostanol present in the feces decreased to an average of 11% compared to controls, but the absolute amount formed was greater (35 mg/day). On a diet enriched with 0.8% β-sitosterol, the rats, on the average, converted 23% of the cholesterol to coprostanol. Feeding diets enriched with sodium taurochenodeoxycholate and sodium taurocholate reduced the 7-dehydroxylation of primary bile acids in the feces by 28% and 42%, respectively. The conversion of primary bile acids to secondary bile acids in the feces of control, cholesterol, and β-sitosterol fed rats was the same (64%).