William T. Beher

Rapid analysis of human fecal bile acids

A rapid, accurate, precise method for determining human fecal bile acids is reported. Feces are homogenized and then briefly extracted with boiling absolute ethanol. A portion of the extract is evaporated to dryness and the residue heated with mild alkali to hydrolyze bile acid 3 alpha-hydroxyl esters. Aliquots of hydrolyzed crude extract are treated with resazurin reagent which effects a series of enzyme catalyzed reactions in which bile acid free 3 alpha-hydroxyls are first oxidized to 3-oxo-groups in a reaction catalyzed by 3 alpha-hydroxysteroid dehydrogenase. Resulting protons are transferred to beta-nicotinamide adenine dinucleotide, yielding reduced beta-nicotinamide adenine dinucleotide (beta-NADH). beta-NADH then reduces nonfluorescent resazurin to fluorescent resorufin in a reaction catalyzed by diaphorase. Developed fluorescence, which is proportional to the extract aliquots bile acid content, is excited at 565 nm and read at 580 nm, wavelengths which lie in a spectral region in which there is minimal fecal pigment absorption. 3-Oxo-bile acids and bile acid 3 alpha-sulfates are extracted in the procedure but reduction and/or solvolysis is necessary before quantification.

The Effect of Pituitary and Thyroid Hormones on Bile Acid Metabolism in the Rat

Abstract Bile acid synthesis and bile acid pool turnover rates and sizes were determined in normal (N), thiouracil-treated (TU), hypophysectomized (H), and thyroidtreated hypophysectomized (TH) female albino rats. Thiouracil treatment lowered the rate somewhat: N, 3.4 mg./day; TU, 2.2 mg./day. Hypophysectomy reduced the rate to less than one-third of normal: H, 0.96 mg./day. Five mg./day desiccated thyroid increased bile acid synthesis of hypophysectomized rats to 1.2 mg./day—still far below normal. Since bile acid pool sizes per 100 Gm. body weight were similar in the groups (N, 9.66 mg.; TU, 6.80; H, 9.39; TH, 9.59), these changes in synthesis were due largely to changes in pool half-life (N, 3.7 day; TU, 4.0; H, 8.8; TH, 6.5). It was concluded that hypophysectomy retards bile acid metabolism by decreasing end-product turnover rates. The effects of hypophysectomy differ from those of thiouracil-hypothyroidism, and moderate thyroid treatment does not restore steroid turnover or synthesis rates to normal. Thus other factors than lack of thyroid seem to cause the changes in steroid metabolism effected by hypophysectomy.

A Comparative Study of Bile Acid Metabolism in the Rat, Mouse, Hamster, and Gerbil

Summary A comparative study of bile acid metabolism was made in the rat, mouse, hamster, and gerbil. Bile acid elimination was mainly via fecal excretion; less than 7% was excreted in the urine. Little or no bile acid side-chain oxidation took place in any of the species. A study of bile acid distribution in the rodent tissue showed that from 80 to 90% was present in the small intestine plus gall bladder. The cecum was another site which contained considerable quantities of bile acids. The chief pool bile acid in each case was cholic acid. Chenodeoxycholic acid was present in significant amounts in rat and hamster, and in trace amounts in mouse and gerbil bile. Cholic acid half-lives (days) were: rat, 3.5; mouse, 5.0; gerbil, 2.3; and hamster, 1.0. Chenodeoxycholic half-lives (days) were: rat, 2.0; mouse, 2.5; gerbil, 1.3; and hamster, 1.8. The rat had the largest bile acid pool (27.2 mg); the mouse (5.6 mg) and gerbil (7.12 mg) intermediate amounts; and the hamster the smallest pool (2.3 mg). The relationship between bile acid synthesis rates and the rate of accumulation of tissue cholesterol after feeding cholesterol-supplemented diets was discussed. Positive correlations were noted but further studies are necessary to define the relative importance of this factor in maintaining cholesterol homeostasis in the various rodents.

Quantitative determination of bile acids by direct densitometry of thin-layer chromatograms.

Abstract A method was developed for quantitative analysis of bile acids by chromatography and densitometry. Within the range of experimental error, the results of cholic and chenodesoxycholic acid determinations on biological samples agreed with those obtained by the spectrophotometric method of Ganshirt 1 . Reproducibility was influenced by the uniformity of the plate-coating and of the spraying, by width of channels, and by spot size. Since several samples may be run simultaneously and it is unnecessary to locate and remove them from the plates for spectrophotometric analysis, this method has advantages over methods currently employed for bile acid analysis.

Feedback Control of Cholesterol Biosynthesis in the Mouse.

Summary Effects of cholic acid on biosynthesis of hepatic cholesterol and bile acids were studied. Mice maintained on a basal diet, or this diet supplemented with 0.5% cholic acid, received single intraperitoneal injections of mevalonic acid-2-C14 or choles-terol-4-C14. Feces were collected at 24-hour intervals, fecal steroid-C14 was isolated and fractionated, α + β-sterol-C14 and bile acid-C14 were determined. In control mice the distribution of C14 activity in fecal steroids was 60% in the sterol fraction and 40% in the bile acid fraction. This ratio was constant with time after the first 24-hour interval, regardless of the substance injected. It was shown that cholic acid brings about equal decreases in the rates of both hepatic cholesterol and bile acid biosyntheses. Since a preceding study had shown that the rate of bile acid synthesis in mouse liver is inhibited by cholic acid independently of its effect on cholesterol synthesis, it was concluded that the rate of hepatic cholesterol biosynthesis in mice is controlled by cholic acid and its conjugates by means of a double feedback reaction.

Effect of dietary cholic acid on in vivo cholesterol metabolism.

Summary 1. Dietary cholic acid reduced the rate of in vivo hepatic cholesterol synthesis within 3 days, as indicated by incorporated acetate-1-C14. 2. Continued feeding of cholic acid produced no further decrease in hepatic cholesterol synthesis rate over the 21-day period. 3. Increased serum bile acid level correlated with decreased liver cholesterol synthesis rates at all time intervals. 4. There was a small but significant increase in liver cholesterol concentration caused by dietary cholic acid. This increase remained constant throughout the experimental period. 5. Dietary cholic acid had no effect on in vivo kidney and intestine cholesterol synthesis rates.

Effect of blood high density lipoprotein cholesterol concentration on fecal steroid excretion in humans.

Daily excretion of fecal total bile acids and neutral steroids were compared in five controls and two patients with extremely low concentrations of plasma high density lipoprotein (3 to 11 mg/dl) and severe atherosclerosis. There was no significant difference in steroid excretion rates in the groups. The predominant bile acid excreted in control feces was deoxycholic acid; lithocholic acid was predominant in the patients. The patients showed no signs of significant liver disease.

Effects of cholesterol on bile acid metabolism in the rat.

Summary The effects of cholesterol feeding and accumulated tissue cholesterol on bile acid metabolism in normal female rats were studied. Feeding diets supplemented with cholesterol and corn oil over the entire experimental period resulted in decreases in the cholic and chenodeoxycholic acid pool half-lives of 1.6 and 0.9 days, respectively. In experiments on rats with accumulated tissue cholesterol fed unsupplemented basal rations during the experimental period, the half-lives of these acids also decreased 1.6 and 0.9 days, respectively, from the values for normal control rats. In both the dietary and accumulated tissue cholesterol experiments, the total bile acid pool concentration was unchanged but there was an important decrease in cholic acid concentration accompanied by a compensating increase in chenodeoxycholic concentration. Studies in the animals with elevated tissue cholesterol showed that the effects on bile acid turnover were not due to increased levels of sterols in the gastrointestinal tract but were due per se to the increased tissue cholesterol levels. Dietary corn oil by itself had no effect on either turnover rates or pool sizes of the bile acids.

Bile Acid and Cholesterol Metabolism in the Mouse as Affected by Gholestyramine.

Summary Dietary cholestyramine (MK 135) increased the rate of mobilization of blood and liver cholesterol in mice. The bile acid turnover rate in normal mice was 5 days, in mice treated with 1% cholestyramine, 1¼ days, or 4 times as fast. Bile acid pool size was 5.62 ± 1.09 mg in normals, 4.58 ± 0.65 in cholestyramine-treated mice. The pool was shown to consist almost exclusively of cholic acid and this spectrum was not altered by cholestyramine. The daily rate of synthesis of bile acids in normal mice was 0.56 mg, 1.84 mg in cholestyramine-treated mice. Digi-tonin precipitable fecal sterol excretion was 5.54 mg day in normal mice, 7.58 mg/day in mice treated with 1% MK 135. No conclusion could be reached as to whether the increased rate of mobilization of accumulated cholesterol affected by MK 135 was due to the increased excretion of either the fecal bile acid or sterol fraction exclusively, or was attributable to the excretion of both these fractions. We wish to acknowledge the excellent technical assistance of Mrs. J. Bertasius. We also wish to thank Merck Sharp and Dohme Research Laboratories for a generous gift of cholestyramine.

Effect of Dihydrocholesterol and β-Sitosterol on Cholesterol Atherosclerosis in Rabbits

While dihydrocholesterol lowered total plasma cholesterol in the rabbit, it did not promote the regression of either the aorta lipid fractions or plaque areas in atherosclerotic animals. A comparative study of the toxicities of β-sitosterol and dihydrocholesterol disclosed that dihydrocholesterol, fed for a 7 month period, resulted in aorta plaque formation and caused the development of liver cirrhosis. On the other hand, β-sitosterol produced no toxic effects.