Methods of measuring bile acids in bile and feces.

Methods currently available for the determination of bile acids in bile and feces are discussed. While the enzymatic method is suitable for the routine, rapid analysis of total bile acids in bile, specific information on the significance of individual bile acid isomers in metabolic studies can only be gained by a combination of thin-layer and gas-liquid chromatography. Fecal bile acid analysis is complicated and methods are still undergoing substantial modifica­ tion. Bile acid excretion is of major interest in studies concerning hyperlipoproteinemia and the effects of hypocholesteremic agents and diet. 2 ’ 9 ’36 Bile acids also are of interest in regard to cholestasis, 8 2 other hepato­ biliary disease, 18 and a variety of physio­ logic functions. 15 Two primary bile acids ( cholic and chenodeoxycholic acids) formed from cholesterol in the liver are secreted into the bile as their glycine and taurine conjugates. 4 These bile acids are subjected to bacterial action in the intes­ tine and some of the secondary bile acid thus produced is reabsorbed. 3 Hence, at a given time, bile might contain both pri­ mary and secondary bile acids. Since the bile contains a high concentration of bile acids, their measurement is relatively easy. The feces, however, contain a variety of ketonic and 7<*-dehydroxylated bile acids and their isomers. 6 ’7 Since the bile acids in feces occur along with other lipid classes, purification of them is difficult. In this re­ view, some of the major methods currently available for identifying and measuring bile acids in bile and feces are discussed, including methods that measure the total bile acids and others that give information on a specific bile acid isomer. The choice of method depends on the kind of informa­ tion needed. Biliary Bile Acid D eterm ination A number of methods has been de­ scribed for the measurement of bile acids. Some of the methods have been specific only for certain component bile acids, while others identify and quantitate all the bile acids. 4 2 In this review the methods available for the biliary bile acids will be classified on the basis of the specific tech­ niques used to isolate and identify the bile acids. D if f e r e n t ia l E x t r a c t io n S p e c t r o p h o t o m e t r y This procedure, described by Levin et al, 25 separates glycineand taurine-con­ M ETH O D S O F M EA SU R IN G B IL E A CID S IN B IL E AND F E C E S 3 6 3 jugated bile acids by simple solvent extrac­ tion. The bile samples are deproteinized with an alcohol-zinc sulfate-barium hy­ droxide mixture, which also removes a con­ siderable amount of biliary pigments. The neutral sterols and other contaminants are extracted with diethyl ether and hexane before acidification. After acidification with dilute hydrochloric acid, the glycine con­ jugates are extracted with ethyl acetate. After further acidification, the taurine con­ jugates are extracted with butanol. The bile acid conjugates are then mea­ sured spectrophotometrically. The cholates are measured, after reaction with sulfuric acid and furfural, by their absorbance at 620 nm. The dihydroxycholanates are mea­ sured after reaction with a mixture of water, phosphoric acid, and sulfuric acid, 1 :4 :6 (vol/vol/vol) in the presence of benzaldehyde. The addition of ethyl ace­ tate gives a blue-green color only with dihydroxycholanates. The authors25 also noticed that, under certain conditions, the error caused by chenodeoxycholates can be kept low. This procedure does not measure chenodeoxycholate, lithocholate, and other secondary bile acids that normally are found in hu­ man bile. 22 Levin et al25 reported a re­ covery of 96.8 percent to 104 percent, for the overall procedure, for conjugates added to the bile samples. If specific information is needed on the individual bile acids, the conjugate isolated by this procedure has to be saponified and analyzed by gasliquid chromatography. 2 2 ’23 E n z y m a t ic -S p e c t b o p h o t o m e t b ic M e t h o d This method is based on the concept that an enzyme, /?-hydroxysteroid dehydrogen­ ase (NAD-linked), isolated from Pseudo­ monas testosteronii41 can oxidize the 3/3 and 3a groups of any bile acid in the pres­ ence of NAD. 16 This method originally was applied to bile acids in blood1 6 and now has been applied to bile by Javitt and Emerman. 17 In this procedure, 10 /¿I of bile are added to a reaction mixture consisting of 1 . 0 ml of enzyme solution,* 0.5 ml of 1 M hydra­ zine hydrate, 0.25 ml of 5 mM NADP, and 2.0 ml of 0.1 M potassium phosphate buffer, pH 9.4. The net increase in absorb­ ance at 340 nm (light path = 10 mm), against bile and reagent blanks, is used to calculate the amount of bile acid present: . . . . A X 3 X mol wt Bile add (p.g) = -------^ -------in which A = absorbance of NADH2 formed; mol wt = molecular weight of the individual bile acid; 6.26 = molar absorb­ ance of NADH2; and 3 = volume of the incubation mixture. Using this procedure, Javitt and Emer­ man17 found a recovery of greater than 8 8 percent. For the determination of total bile acids, especially in routine laboratories, this method is fast and accurate. If specific information on the individual bile acids is needed, one has to use thin-layer chroma­ tography and measure individual bile acids after elution. P a p e b C h r o m a t o g r a p h y -S p e c t b o p h o t o m e t b ic M e t h o d Sjôvall’s modification34 of his own orig­ inal method33 of measuring bile acids by spectrophotometry after paper chromatog­ raphy is very elaborate. Various moving phases were used to separate the conju­ gated and free bile acids on paper chro­ matography; then the bile acids were eluted with ethanol and the spectra of the bile acids were measured at various wave­ lengths after reaction with 65 percent sul­ furic acid. The time of exposure of the bile acids to the acid was found to be critical. The contribution of other substances to the absorbance was corrected by measuring the spectra of the bile acids in 80 percent ethanol at corresponding wavelengths and subtracting these from the data obtained. * Worthington Biochemical Corporation, Free­ hold, NJ.