G.J. Lin

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 hydrolysis of bile acid conjugates.

Studies were made of a) the relationship of bile acid structure and analytical recoveries (measured by 3-hydroxysteroid oxidoreductase) following vigorous alkaline hydrolysis of bile acid conjugates and b) the relationship of structure and hydrolysis time of taurine- and glycine bile acid conjugates in a reaction catalyzed by glycocholic acid hydrolase. Alkaline hydrolysis resulted in good recoveries of hydroxy and 7 and 12- oxo-bile acids but poor recoveries of 3-oxo-bile acids. Borohydride reduction of the 3-oxo-acids prevented these losses. Complete enzymatic hydrolysis of glycine conjugated bile acids was about five times more rapid than that of taurine conjugates. Hydrolysis of conjugates containing oxo groups was slow. Borohydride reduction of oxo-acids corrected this and did not inhibit enzymatic hydrolysis. It was concluded that both vigorous alkaline and enzymatic hydrolysis are satisfactory in bile acid assays if borohydride reduction is instituted before the hydrolytic step. However, due to the presence of possible enzyme inhibitors and solubility difficulties, strong alkaline hydrolysis is preferable to enzymatic hydrolysis in fecal bile acid determinations at this time.

Thin-layer separation and quantification of bile acids

A method has been developed for quantification of total free and conjugated bile acids separated on silica gel HR coated thin-layer chromatography plates. Aliquots of bile acid solutions are applied to channeled plates which are developed with either ethyl acetates: isooctane:glacial acetic acid 10:10:2 v/v for free bile acid separation, or chloroform:methanol:glacial acetic acid:water 130:50:4:8 V/V for conjugated bile acid separation. Bile acids are determined directly in serial areas of silica gel by treating gel areas suspended in tris buffer with resazurin reagent. The method is quantitative and as little as 0.1 microgram of bile acid is readily determined. Application of the method to determinations of bile acids in crude fecal extracts is described.

Effects of Sphincter of Oddi Bypass on Bile Acid Metabolism in Fed and Fasted Intact and Cholecystectomized Dogs

The effects of cholecystectomy and sphincter of Oddi bypass on bile acid (BA) metabolism in dogs have been studied. Cholecystectomy and sphincter bypass decreased the BA pool half-life and increased the percent of taurodeoxycholic acid in the pool. A 48-hour fast had no effect on total BA pool size of intact and intact sphincter-of-Oddi-bypassed dogs but caused a marked decrease in cholecystectomized dogs. It was concluded that while the sphincter of Oddi is unnecessary to maintain bile acid pool size in fasting dogs, the gallbladder is. Alimentation is necessary to maintain pool size in cholecystectomized dogs.

Rapid photometric determination of free and esterified 3α-hydroxy fecal bile acids

A rapid, precise, and accurate photometric method for determining free and esterified fecal 3 alpha-hydroxy bile acids is described. Feces are homogenized and (a) extracted with boiling absolute ethanol, or (b) lyophilized and extracted with chloroform:methanol 2:1 (v/v). Hydrolyzed and nonhydrolyzed crude extracts are prepared and aliquots treated with a reagent containing nitro blue tetrazolium (NBT), 3 alpha-hydroxysteroid oxidoreductase, beta-nicotinamide adenine dinucleotide (beta-NAD) and diaphorase. The reagent first oxidizes bile acid 3 alpha-hydroxyls to 3-oxo groups and 3 beta-hydrogen is transferred to beta-NAD yielding beta-NADH. beta-NADH in turn reduces NBT (yellow) to its diformazan (blue). Absorbance is measured at 540 nm and is proportional to the 3 alpha-hydroxy bile acid titer of fecal extract aliquots. Fecal pigments present in crude extracts do not interfere with the assay since they absorb minimally at 540 nm.

Rapid enzymatic determination of 0-3-oxo-bile acids separated by thin-layer chromatography

Abstract A method for the rapid quantification of 3-oxo-5-β-cholan-24-oic acids has been developed. The acids are separated on silica gel G and located using a water spray or iodine vapor. Each oxo acid is eluted from the gel and reduced with sodium borohydride. The resulting α- and β-hydroxy acids are then oxidized in a reaction catalyzed by 3-hydroxysteroid dehydrgenase during which NAD is reduced to NADH. The absorbance of the reaction mixture is determined at 340 nm and is directly proportional to the amount of 3-oxo acid originally present on the thin-layer plate.

The Effects of Cholecystectomy on the Bile Salt Pool of the Syrian Hamster

The half-life, distribution, size and composition of the bile salt pool were determined in intact and cholecystectomized Syrian hamsters. Cholecystectomy had no effect on the half-life of either the cholate or chenodeoxycholate pool. Fasting in intact hamsters resulted, as expected, in a shift of bile salts from the small intestine, cecum and liver to the gallbladder. In cholecystectomized hamsters there was a moderate shift of salts from the liver and small intestine to the cecum. Cholecystectomy had no significant effect on the size of the total bile salt pool. The total bile salt pool size of fed and fasted intact hamsters was the same; fasting in cholecystectomized hamsters resulted in a large decrease in the pool. There was no significant difference in the bile salt pool composition of intact and cholecystectomized hamsters, and hamsters were shown to efficiently convert deoxycholate to cholate.

The determination and excretion of individual human fecal bile acids.

A fluorometric method, using resazurin, for the analysis of individual fecal bile acids separated by thin layer chromatography of crude fecal extracts is described. The method is precise and accurate. The assay was used to investigate the constancy of excretion of individual fecal bile acids in small random stool samples collected over three weeks in six humans. Relatively small but significant variations were found in each case, most were random but one was progressive.

Canine bile acid enterohepatic circulation

The enterohepatic circulation (EHC) of bile acids has been studied in fasting dogs with portacaval shunt maintained in the steady state. In such animals the rate of EHC is proportional to systemic blood bile acid concentration. Bile acid EHC was irregular (20 to 100% variation) when measured at 15 minute or hourly intervals. Studies showed that the variations persisted in cholecystectomized and sphincterectomized animals. The irregularities were enhanced by bethanechol chloride which increases intestinal peristalsis and suppressed by diphenoxylate HCl which slows peristalsis. The variations appear to arise from irregular patterns of intestinal peristalsis. This phenomenon may explain some variations in blood bile acid concentration observed in patients with liver disease.

Comparative studies of the effects of fasting on bile salt pool sizes of hamsters and rats

Comparative studies of the effects of fasting on the total bile salt pool sizes of intact and cholecystectomized hamsters and rats were made. Rats, a species which has no gallbladder, are able to maintain the size of their total bile salt pool during 24, 48 and 72 hour fasts by an undetermined effective mechanism. Intact hamsters fasted 24, 48 and 72 hrs maintained and even increased the size of their bile salt pool. Bile salt conservation was effected by storage of the salts in the gallbladder, and to some extent, the small intestine. Cholecystectomized hamsters apparently lack any mechanism to effect bile salt conservation during fasting since their bile salt pool size decreased precipitously during 24 and 48 hr fasts.