Bile acid conjugates, quantitation

Cholic acid and chenodeoxycholic acid, known as the primary bile acids, are quantitatively the most important metabolites of cholesterol. After being biosynthesized, they are mostly activated with coenzyme A and then conjugated with glycine or the non-pro-teinogenic amino acid taurine (see p. 62). The acid amides formed in this way are known as conjugated bile acids or bile salts. They are even more amphipathic than the primary products. 

Bile consists of a watery mixture of organic and inorganic compounds. Phosphatidylcholine (lecithin, see p. 201) and bile salts (conjugated bile acids) are quantitatively the most important organic components of bile. Bile can either pass directly from the liver where it is synthesized into the duodenum through the common bile duct, or be stored in Ihe gallbladder when not immediately needed for digestion. 

If urine is acidified (pH 1) and then extracted three times with equal volumes of /i-butanol, bile acid conjugates can be quantitatively recovered (28). However, due to the time-consuming evaporation of n-butanol and the formation of emulsions other ways to extract bile acids have been tried. The method described by Hofmann (21) may be useful. 

After deproteinization of bile, Turnberg and Anthony-Mote (94) were able to quantitate bile acids directly with a NAD-dependent steroid oxido-reductase from a Pseudomonas strain. The suitable concentration range was 10-80 mg of bile acids per 100 ml. The amounts of individual bile acids could be determined after preparative thin-layer chromatography, either before or after hydrolysis of bile acid conjugates. 

Some further examples of the application of TLC may be cited a procedure for two dimensional separation [179] analysis of bile from various species [76] fractionation of bile lipids into groups [128] metabolism [73] and analysis of human faecal bile acids [153 a] quantitative determination of free [203] and bile acid conjugates [202] in serum. 

The effects of a short-time interruption (24 hours) of the enterohepatic circulation on bile composition has also bfjeu studied in man. At the time of operation on patients with cholelithiasis, Thurciborn (1962) cannulated the common bile duct with a ballenterohepatic circulation. Interruption of the EHC resulted in a decrease in the secretion of bile acid conjugates in the same way as in the bile fistula rats. In addition, the secretion of phospholipids decreased considerably, but the cholesterol output from the liver was about the same as with an intact enterohepatic circulation. Tlie decrease in phospholipid secretion is an interesting finding. Several explanations are possible. The occurrence of an enterohepatic... 

Most important organic components of bile Bile salts and phosphatidylcholine are quantitatively the most important organic compo nents of bile. Bile salts are conjugated bile acids. 

For the quantitative estimation of bile acids in body fluids and tissues, consideration must be given as to whether (1) an extraction step is necessary to partially purify the bile acids prior to further analysis (2) hydrolysis is required to remove glycine and taurine or other conjugate moieties and (3) the method of analysis will be of the required sensitivity and provide infer-... 

M36, Musial, B. C., and Williams, C. N., Quantitative assay of conjugated and free bile acids as heptafluorobutyrate derivates by gas-liquid chromatography. /. Lipid Res. 20, 78-85... 

Setchell, K. D. R., and Worthington, J., A rapid method for the quantitative extraction of bile acids and their conjugates from serum using commercially available reverse-phase octadecylsilane bonded silica cartridges. Clin. Chim. Acta 125, 135-144 (1982). 

The epimerizalion of the 7a-hydroxyl group can occur either by intra- or interspecies mechanisms [16]. However, it is difficult to quantitatively assess the degree of 7-hydroxy epimerization in vivo because this transformation competes with the irreversible 7-dehydroxylation of bile acids (Section VI). 7a-HSDH activity has been reported in several genera of intestinal bacteria however, the most complete characterization of this enzyme has been carried out with the enzyme isolated from Escherichia coli [37] and Bacteroides sp. [29,38,39] (Table 2). Both enzymes used both free and conjugated bile acids as substrates, showed alkaline pH optima and lower values for dihydroxy than for trihydroxy bile acids. However, cell extracts prepared from Bacteriodes sp. contained both NAD- and NADP-depen-dent 7 -HSDH activities whereas, extracts from E. coli contained only an NAD-de-pendent enzyme activity. Additional studies showed that the two 7a-HSDH activities detected in Bacteriodes sp. differed in molecular weight, differential heat inactivation and Mn " requirement, suggesting the presence of two distinct enzymes [29]. 

Extraction in 20 vol. of ethanol as described above is likely to give the most complete recovery of all types of bile acids. Alkaline conditions favor extraction. Folch extraction has been used to get a simultaneous purification, and the conjugated bile acids are then found in the upper phase (20). A different type of extraction is that described by Hofmann, who used a liquid anion exchanger, tetraheptylammonium chloride (21). This was added to bile and the salts formed with bile acids could be quantitatively extracted with ethyl acetate. The use of this solvent makes the evaporation of bile extracts simple. The amine can be removed with a cation exchanger, a procedure first described by Anderson (22). 

TABLE III. Solvent Systems for Quantitative Analysis of Conjugated Bile Acids by Paper Chromatography... 

Several methods have been used to obtain quantitative extraction of conjugated bile acids from silica layers (4, 11, 12). Palmer (50) moistened the silica (removed from the plate with a razor blade) with water and extracted the bile acid by refluxing with chloroform-methanol (redistilled from 2,4-dinitrophenylhydrazine), 1 1, for one hr in a 250-ml round-bottom flask. He then determined the amount of bile acid with a 3a-hydroxysteroid dehydrogenase reaction. The necessity of using large volumes of polar solvent is also evident from the work by Shioda et al. (20) who used 50 ml of methanol-acetic acid, 99 1, and achieved at least 96% recovery of all common conjugated bile acids in human bile labeled in vivo by cholesterol. 

Other workers have not been successful in eluting conjugated bile acids and have therefore made quantitative determinations by forming sulfuric acid chromogens with the gel still carrying the bile acid (51-53). In such... 

In quantitative studies, Stiehl et al. (91) extracted bile acids from duodenal bile with methanol-acetone and the conjugates were hydrolyzed according to the method of Nair et al. (118). The free bile acids were then separated with thin-layer chromatography and determined spectrophoto-metrically after reaction with a sulfuric acid reagent. 

Ali et al. (128 and 129) suspended freeze-dried human feces in alkaline-50 % ethanol and extracted with petroleum ether-diethyl ether, 1 1, to remove neutral lipids. The bile acids in the aqueous phase were next subjected to conditions for hydrolysis of conjugates. After acidification, the free bile acids were extracted with diethyl ether, methylated, and purified by preparative thin-layer chromatography. Mono-, di-, and trisubstituted methyl cholanoates are eluted together from the thin-layer plate and then quantitated on QF-1 columns before and after conversion into trifluoroacetates. 

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