Chenotherapy for gallstone dissolution. II. Induced changes in bile composition and gallstone response.

Changes in bile saturation and biliary bile acid composition in patients with gallstones who received chenodeoxycholic ("chenic") acid, cholic acid, or placebo were measured. Chenodeoxycholic induced bile desaturation; this effect was attributable solely to a decrease in the proportion of cholesterol. By gas chromatography, chenodeoxycholic acid increased substantially in the biliary bile acids of patients receiving it, and by mass spectrometry, no unusual bile acids were detected in appreciable amounts. Changes in bile saturation and biliary bile acid composition were then related to chenodeoxycholic acid dosage, and all of these variables were, in turn, related to gallstone response. In general, patients whose gallstones dissolved ingested a higher dose of chenodeoxycholic acid or had bile that contained a higher proportion of this acid and it was more unsaturated, but there were many exceptions, casting doubt on the value of a single analysis of fasting-state bile for predicting gallstone dissolutions. The major factor influencing response, provided dosage is adequate, appears to be gallstone type. Nonetheless, the proportion of chenodeoxycholic acid in biliary bile acids can probably be used to infer patient compliance.

Evaluation of Poly S-179 as a stationary phase for the gas-liquid chromatography-mass-spectrometry of bile acid methyl ester acetates.

The stationary phase Poly S-179 has been found to offer distinct advantages over the previously reported SP-525 for the gas-liquid chromatographic separation of bile acid methyl ester acetates. Relative retention times of these bile acid derivatives are compared on the two phases.

Stereospecificity of the hydrogen transfer catalyzed by human placental aldose reductase.

Placental aldose reductase (EC 1.1.1.21) was incubated with glucose in the presence of [4A-2H] NADPH prepared in the oxidation of [2-2H] isocitrate by isocitrate dehydrogenase (EC 1.1.1.42) or [4B-2H] NADPH prepared in the oxidation of [1-2H] glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The sorbitol formed from [4A-2H] NADPH contained deuterium and from [4B-2H] NADPH it did not. Therefore, aldose reductase in an A-type enzyme.

Effect of deoxycholic acid ingestion on bile acid metabolism and biliary lipid secretion in normal subjects.

The effect of deoxycholate ingestion, 750 mg per day, on bile acid kinetics, biliary bile acid composition, and biliary lipid secretion was studied in 7 healthy volunteers. Bile acid kinetics were measured by isotope dilution, and hourly outputs of bile acid, cholesterol, and phospholipid were quantitated by a duodenal perfusion technique during a 24-hr period which included three liquid meals and an overnight fast. Biliary bile acid composition was assessed by coupled gas chromatography-mass spectrometry. After deoxycholic acid ingestion, biliary bile acids became composed of predominantly deoxycholyl conjugates, and deoxycholic acid pools increased 4-fold. Both chenodeoxycholic and cholic acid pools decreased, and daily synthesis of each of the primary bile acids was inhibited by 50%. Total bile acid pools did not change in any consistent manner. Daily bile acid secretion increased slightly during deoxycholic acid ingestion, and recycling frequency varied reciprocally with the total bile acid pool both before and during deoxycholic acid treatment. Deoxycholic acid ingestion caused no change in either the daily secretion of cholesterol or lecithin, or the cholesterol saturation of fasting-state bile, which remained unsaturated throughout the study. SGOT levels increased to 4 times the upper limits of normal in 2 of 7 subjects, but these levels promptly returned to normal when deoxycholate feeding was stopped. Serum cholesterol levels decreased in every subject (average 15%) during deoxycholic acid administration. No evidence for a direct role of deoxycholate in the pathogenesis of cholesterol cholelithiasis was obtained in these studies.

Characterization of bile acid methyl ester acetate derivatives using gas-liquid chromatography, electron impact, and chemical ionization mass spectrometry.

The gas-liquid chromatographic retention times on 0.5% SP-525 for 48 bile acids and related compounds as their methyl ester acetate derivatives are given. Ion tables for electron impact spectra have been compiled that permit direct access to ion structures for any given ion mass. Chemical ionization yields highly simplified mass spectra with two or three ions predominating for each compound. When the relative retention times of bile acids as their methyl ester acetates are combined with selective ion monitoring techniques in chemical ionization mass spectrometry, the retention time and ion mass number form a coordinate system which can be a powerful tool in the characterization of bile acid mixtures.

Formation of bile acids in man. Metabolism of 7alpha-hydroxy-4-cholesten-3-one in normal subjects with an intact enterohepatic circulation.

The formation of bile acids in man is thought to involve a series of reactions in which the initial steps are the same for both cholic acid and chenodeoxycholic acid. The point of bifurcation of the pathway is postulated to occur after the formation of 7alpha-hydroxy-4-cholesten-3-one. To test the hypothesis that the entire synthesis of both bile acids proceeds through this intermediate we studied the metabolism of labeled 7alpha-hydroxy-4-cholesten-3-one in eight normal subjects with an intact enterohepatic circulation. If all the production of cholic acid and chenodeoxycholic acid takes place via 7alpha-hydroxy-4-cholesten-3-one, the areas under the specific decay curves of cholic acid and chenodeoxycholic acid should be identical following a single injection of this labeled intermediate. However, in 6 of the 8 subjects studied the area under the cholic acid specific activity decay curve was significantly less than the area under the chenodeoxycholic acid specific activity decay curve. These results that the production of cholic acid in man may not always involve the intermediate 7alpha-hydroxy-4-cholesten-3-one.

Validation of use of 11,12-2H-labeled chenodeoxycholic acid in isotope dilution measurements of bile acid kinetics in man.

Chenodeoxycholic acid labeled with 2H in the 11 and positions was prepared by catalytic reduction of delta 11-12 unsaturated derivatives of cholic acid. To validate the use of this stable isotope for the determination of bile acid kinetics by isotope dilution, it was administered to seven normal male volunteers simultaneously with [24-14C]chenodeoxycholic acid. Bile was collected at regular intervals over the following 5 days, and the chenodeoxycholic acid pool size and fractional turnover rate were determined from the specific activity decay curve for 14C and from the isotopic abundance curve for 2H. Estimates of the pool size by both isotopes showed a correlation of r = 0.95 and similar precision. Synthesis rate, the product of pool size and fractional turnover rate, also showed good agreement (r = 0.97), Because previous investigations have shown that bile acids tagged with hydrogen isotopes at the 11 and 12 positions are stable in man, the present data suggest that 11, 12-2H-labeled bile acids may be used in place of radioactive isotopes for valid isotopic measurement of bile acid kinetics in healthy infants and children.

Rapid preparation of tritium-labeled bile acids by enolic exchange on basic alumina containing tritiated water.

When a 3-keto bile acid methyl ester was chromatographed on basic alumina inactivated with tritiated water, the enolic hydrogen atoms at C-2 and C-4 exchanged with tritium atoms. The (3)H-labeled keto ester was reduced with borohydride, and the resultant mixture of 3alpha- and 3-hydroxy epimers was resolved by preparative thin-layer chromatography to yield a pure 2,4-(3)H-labeled bile acid ester. Lithocholic, chenodeoxycholic, deoxycholic, and cholic acids having a specific activity of 1-10 micro c/ micromole were prepared from their 3-keto derivatives. The tritium label remained intact during alkaline saponification in vitro and enterohepatic cycling in vivo in human subjects.