Extraction of Bile Acids

Bile acids are present from 1-2 iJgmL"1 in plasma and urine to significant amounts in the intestinal content and as much as 80 mg mL"1 in the gall bladder bile. They are present in unconjugated form as well as conjugated with glycine and taurine or as sulfate/glucuronides, often in association with proteins, sterols and their esters, free or esterified fatty acids, bile pigments and water-soluble small mole cules, which may interfere in their analysis. It is often necessary to isolate and at least partially purify bile acids before preparation for GC. Plasma bile acids mainly exist as glycine and taurine conjugates and unconjugated bile acids are present in only small proportions. Plasma is usually passed through Sep-pak, a reversed-phase C18 cartridge, when proteins and most of the cholesterol are removed and bile acids and their conjugates are eluted with methanol. Other methods of concentrating bile acids include lipophilic anion exchange gel, diethylaminohyd-roxypropyl Sephadex LH-20 and, more recently, size exclusion chromatography using Sephadex G-75 gel. Some of these ion exchange resins are also employed for separation of various conjugated bile acids so that conjugation pattern is determined in the sample. The bile acid fraction obtained by any of these methods is treated with strong alkali (4 mol L_1 sodium hydroxide, 115°C at 15 psi pressure) to hydrolyse the conjugates; neutral sterols are extracted out with solvents and the free bile acids are then extracted with ether or ethyl acetate after acidification. Under these conditions, the glucuronides and sulfate esters are also hydrolysed, and a pre-step of solvolysis may not be necessary. As an alternative to alkaline hydrolysis, bile acid conjugates may be hydrolysed with the enzyme, cholylglycine hydrolase, while ^-glucuronidase plus sulfatase is used to hydrolyse glucuronides and sulfates. In order to correct for losses during extraction, an internal standard is added before the hydrolysis step, while an external standard (usually 5a-cho-lestane) corrects for detector responses of the various compounds. The appropriate internal standard is a bile acid that is not present in the bile acid mixture to be quantitated. Nor-deoxycholic acid and nor-cholic acid are usually used as internal standards, but other compounds like hyodeoxycholic acid and 7a, 12a-dihydroxy-5^-cholanoic acid have also been employed.

Biliary bile acids are present predominantly as the glycine and taurine conjugates. Since they are present in high concentrations in the bile, only a few microlitres of bile are usually required for quantitation by

GC. Also, since bile contains only 1-2% of cholesterol, it is much easier to obtain pure bile acids free from cholesterol. Urinary bile acids are also present mainly in conjugated form, and only a few milligrams per day are excreted. However, in hepatobiliary diseases like primary biliary cirrhosis, urine may become a major pathway for bile acid excretion and sulfate conjugation of bile acids is increased. Again, bile acids need to be deconjugated before GC analysis and methods similar to those for plasma can be used to obtain urinary bile acid composition.

Faecal bile acids are mainly found in unconjugated form and the bile acid pattern is highly complex, due to bacterial deconjugation and extensive metabolism during intestinal transit. Most abundant of these secondary bile acids are deoxycholic acid and lithocholic acid, which are reabsorbed from the colon and further modified by hepatic enzymes and circulate in enterohepatic circulation. Whereas scores of metabolites of bile acids are formed in the colon, bile acids in the jejunum remain predominantly conjugated due to the absence of bacteria in the small intestine, and they therefore mirror biliary bile acids. A major difficulty in quantitative analysis of faecal bile acids is their strong binding with the bacterial debris in the stool, and quantitative extraction is difficult. Furthermore, in addition to bile acids, stool contains neutral sterols, including cholesterol and its bacterial metabolites and plant sterols and their bacterial metabolites, and also fatty acids. Several methods have been reported for bile acid extraction from faeces, and most are quite complex. Thus, one method involves continuous Soxhlet extraction of aliquots of homogenized stool with chloroform-methanol; the extracted bile acids are subjected to methyl ester formation followed by preparative thin-layer chro-matography. Grundy et al. extracted neutral sterols from homogenized stool, deconjugated any bile acid conjugates with alkali and finally extracted bile acids with chloroform-methanol after acidification of the solution. After evaporation of solvents, the residue was chromatographed over Florisil and the purified bile acid fraction was subjected to methyl ester formation and then to preparative TLC to separate fatty acids. Bands due to bile acids were isolated and used for GC. In other methods, stools have been extracted with ammoniacal alcohol, methanol-hydrochloric acid, acetic acid-toluene, and bile acids extracted after removal of neutral sterols.

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Solar Panel Basics

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