Bile acids quantitation

In 1963, Carey and Haslewood isolated trace amounts of (25/ )-3a,7 ,12a-trihy-droxy-5 8-cholestan-26-oic acid from human fistula bile [96]. The stereochemistry at C-25 of this bile acid was recently confirmed by direct comparison with reference compounds of known absolute configuration [97], This trihydroxy-5j8-cholestanoic acid also occurs in baboon bile [98]. Hanson and Williams found the corresponding dihydroxy bile acid, 3 ,7a-dihydroxy-5 S-cholestan-26-oic acid, in human bile [99]. The occurrence of these higher bile acids, quantitatively of minor importance, is of interest because they are biosynthetic precursors of two primary bile acids of mammalian species, cholic acid and chenodeoxycholic acid, respectively (Chapter 9). 

Clinical Analysis. A wide range of clinically important substances can be detected and quantitated using chemiluminescence or bioluminescence methods. Coupled enzyme assay protocols permit the measurement of kinase, dehydrogenase, and oxidases or the substrates of these enzymes as exemplified by reactions of glucose, creatine phosphate, and bile acid in the following ... 

Purification of bile acids extracted from human stool quantitation by GC/FID 75... 

Assay of bile acids was an essential tool for the early investigation of the enterohepatic circulation, and proved a focus of attention with the belief that serum bile-acid concentrations would provide a sensitive diagnostic test for liver disease. There are three fundamental assay types, based on enzymatic oxidation of a hydroxyl with linked NAD reduction, chromatographic separations and quantitation, encompassing both gas-liquid and high-performance liquid chromatography, and radioimmunoassay assays. 

K. D. R. Setchell, J. A. Ives, G. C. Cashmore and A. M. Lawson, On the homogeneity of stools with respect to bile-acid composition and normal day-to-day variations A detailed qualitative and quantitative study using capillary column gas chromatography-mass spectrometry, Clin. Chim. Acta, 1987, 162, 257. 

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. 

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. 

The conversion of cholesterol to bile acids is quantitatively the most important mechanism for degradation of cholesterol. In a normal human adult approximately 0.5 g of cholesterol is converted to bile acids each day. The regulation of this process operates at the initial biosynthetic step catalyzed by an enzyme in the endoplasmic reticulum, la-hydroxylase (fig. 20.18). The 7a-hydroxylase is one of a group of enzymes called mixed-function oxidases, which are involved in the hydroxylation of the sterol molecule at numerous specific sites. A mixed-function oxidase is an enzyme complex that catalyzes hydroxylation of a substrate with a concomitant production of H20 from a single molecule of 02- The 7a-hydroxylase is one of several enzymes referred to as cytochrome P450. 

Bile acids have historically received much less attention than other steroids, for example the corticosteroids however, now that useful thertqreutic effects ate being observed from some bile acids, there is fresh interest in Ais area. A strain of Cunninghamella Uakesleeana has been isolated that will 15p-hy-droxylate lithocholic acid (80 equation 26) in 31% yield.Further reaction is possible to give 3a,lip,15p-trihydroxy-5p-cholanic acid (15%), 3a,15p,18a-trihydioxy-5p-cholanic acid (4%) and 3a,lla,15p-trihydroxycholanic acid (9%). Taurolithocholic acid (81 equation 27) can be 7p-hydroxy-lated in virtually quantitative yield by Mortierella ramanniana. ... 

Endogenous clearance excretes physiological metabolites in a timely manner and in quantitatively appropriate proportions (e.g. bilirubin, enzymes, bile pigments, bile acids). 

Mg -ATPase are also detectable on the luminal surface of the ductular epithelia cells). Even under normal conditions, yet more so in cholestasis, the enzymes are released through the detergent effect of bile acids (in quantitative dependence on their concentration) and can thereby pass into the bile and blood. 

However, the precise localization of the enzymatic defect in bile acid synthesis in CTX (microsomal 24S hydroxylation or mitochondrial 26-hydroxylation) awaits the determination of which side chain oxidative mechanism is quantitatively most important in bile acid synthesis. Until then, both mechanisms must be considered (56,64,65). 

Ichimiya, H., Yanagisawa, J. and Nakayama, F. (1987). Altered metabolism of bile alcohol and bile acid in complete extrahepatic cholestasis qualitative and quantitative aspects. J. Lipid Res. 2S 1028-1037. 

In practice, the majority of TLC separations are qualitative or semiquantitative (visual comparison) in nature. However, modern computer-controlled densitometers are now available that scan sample and calibrator chromatograms in tracks on HPTLC plates and provide quantitative capabilities. Clinically relevant analytes that have been measured by TLC include amino acids, bile acids, carbohydrates, drugs, fipids, glycolipids, phospholipids, porphyrins, prostaglandins, steroid hormones, purines, pyrimidines, derivatives of nucleic acid, and urinary organic acids. The advantages of TLC include simphcity, rapidity, versatility, ability to process a large number of samples... 

Attempts to describe the enterohepatic cycle of bile acids in quantitative terms began in 1957 with the description by Lindstedt of an isotope dilution technique to measure the turnover and pool size of individual bile acids during the steady state (LIO). This technique has become a standard procedure in bile acid research and involves the administration of a bile acid, labeled with C or H, and the subsequent collection of a bile sample each day by duodenal intubation over a period of 5 to 7 days. From the decay curve of the specific activity of the bile acid, the fractional turnover rate and pool size of the bile acid can be calculated (LIO). The product of the pool size and daily fractional turnover rate equals the daily synthesis rate. 

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-... 

Detection and quantitation of bile acids can be carried out directly on a thin-layer chromatogram with a suitable spray reagent followed by densitometry. By using an acidified solution of phosphomolybdate and ceric ammonium sulfate and heating chromatograms at 110°C for 2 to 5 min to develop blue spots, as little as 50 pmol of bile acid from duodenal bile was detected (R3). Other reagents used to produce colored or fluorescent bile acid spots include sulfuric acid (L7, T12, V4), acidified anisaldehyde (05),... 

B35. Bruusgaard, A., Quantitative determination of the major 3-hydroxy bile acids in biological material after thin-layer chromatographic separation. Clin. Chkn. Acta 28, 495-504... 

D6. Dewael, J., Raaymakers, C. E., and Endeman, H. J., SimpliRed quantitative determination of total faecal bile acids. Clin. Chim. Acta 79, 465-470 (1977). 

El. Eneroth, F., Hellstrfim, K., and Sjovall, J., A method for quantitative determination of bile acids in human feces. Acta Chem. Scand. 22, 1729-1744 (1968). 

G8. Goldman, M., Vlahcevic, Z. R., Schwartz, C. C., Custafsson, J., and Swell, L., Bile acid metabolism in cirrhosis. VIII. Quantitative evaluation of bile acid synthesis from [TR- H] 7a-hydroxycholesterol and [G- H]26-hydroxycholesterol. Hepatology 2, 59-66 (1982). 

---