Urinary bile acids

Stiehl, A., Rudolph, G., Raedsch, R., Moller, B., Hopf, U., Loiterer, E., Bircher, J., Folsch, U., Klaus, J., Endele, R., Senn, M. UrsodeoxychoUc acid-induced changes of plasma and urinary bile acids in patients with primary biliary cirrhosis. Hepatology 1990 12 492-497... 

Batta, A. K., Shefer, S., Batta, M., and Salen, G. (1985). Effect of chenodeoxycholic acid on biliary and urinary bile acids and bile alcohols in cerebrotendinous xanthomatosis monitoring by high performance liquid chromatography. J. Lipid Res. 26 690-698. 

Reports of bile acid sulfates in human urine [56,57] have prompted studies of urinary bile acids under various conditions [164,191,192], and have stimulated investigators to develop new analytical systems to ascertain their chemical composition. Although most of the 37 bile acids in Table 2A have assigned structures with hydroxyl groups at positions 1, 2, 3, 6, 7,12 and 23, a rather large number of minor urinary bile acids not included in the table await structural elucidation. Data in Table 2A were compiled from studies of urinary samples from healthy patients [164], patients with hepatobiliary diseases [109,123-125,164,191-197], late pregnancy and recurrent cholestasis of pregnancy [125,198], and healthy newborn children [199]. 

In patients treated with phenobarbital, tetrahydroxy bile acids become major urinary bile acids the ratio of the lj8,3a,7a,12a-tetrol to the 3a,6.a,7a,12a-tetrol was about 4 1. Stimulation of 6a-hydroxylase was evident from the presence of hyocholate in urine and plasma after 1.5-4.5 weeks on phenobarbital. Thus, phenobarbital stimulates 1/8- and 6a-hydroxylation [197]. 

After administration of radioactive bile acids to patients with cholestasis, most of the urinary bile acids were sulfated [206], However, the relatively large percentage of disulfates has not been noted by other investigators. Perfusion of an isolated rat kidney with various bile acids showed a decreasing production of monosulfates in relation to an increased number of hydroxyl groups [207]. Perfusion of chenodeo-xycholate afforded the 7-sulfate (71%) and a trace of the 3-sulfate (2%) no disulfate was found [208]. With the successful synthesis of various isomeric sulfates of the common bile acids, definitive identification of the sulfates found in human urine can be anticipated. 

Fecal bile acid elimination is markedly reduced in patients with liver cirrhosis (11,182,191), even if icterus and signs of biliary obstruction are absent (11,182). Since, in addition, the urinary excretion of bile acids is quantitatively, if not proportionally (81), negligible, it can be concluded that the overall bile acid synthesis is markedly depressed in liver cirrhosis (182). The administration of cholestyramine, which is associated with reduction of both serum bile acids (188,192,193) and urinary bile acids (88,182), augmented the fecal bile acid excretion to a very small extent only (182). This indicates that the ability of the parenchymal cells to increase their bile acid production is clearly decreased compared to that of normal subjects. Though the fecal neutral sterol excretion was also increased by cholestyramine, only a relatively small decrease in serum cholesterol was found, suggesting that the patients still were able to increase their cholesterol production (182). 

Serum cholesterol is usually markedly elevated in biliary obstruction, with especially high values in biliary cirrhosis. A positive correlation is found occasionally (196) but not constantly (193) between the serum bile acid and cholesterol levels. An absence of bile acids in the gut lumen and a reduced cholesterol absorption may stimulate, at least initially, both intestinal and hepatic cholesterol production cf. 92,223), and this in association with a block in elimination both as bile acids and as cholesterol itself rapidly raises the serum cholesterol level. However, in biliary obstruction of long duration, e.g., in biliary cirrhosis, sterol balance studies and urinary bile acid measurement indicate that cholesterol synthesis is markedly reduced (88). In parenchymal cell damage of the liver, the serum cholesterol is normal or decreased, probably because the hepatic cholesterol synthesis, due to cell injury, is reduced in proportion to, or proportionally more than, the decreased cholesterol elimination (11,182) and, furthermore, intestinal and hepatic cholesterogenesis may still be under the partial feedback control of bile acids and absorbed cholesterol, respectively. 

Identification and Quantitative Determination of Urinary Bile Acids Excreted in Cholestasis Clin. Chim. Acta 44(2) 199-207 (1972) CA 78 156267p... 

Amount of urinary bile acids in healthy neonates, including 3-oxo-A bile acids, has been shown to be elevated in the first month of life - 7a,12a-diOH-3-oxo-cholenoic acid <13.0 pmol/mmol creat (<30% total bile acid excretion) and 7a-OH-3-oxo-cholenoic acid <0.4 pmol/mmol creat (<1% total bile acid excretion). See ref. [14]. 

GC-MS analysis is used to confirm the identities of ions in the LSI-MS urine spectrum and show that the excretion of abnormal cholanoids is >20 times normal. In the case of 5 ff-reductase deficiency GC-MS analysis should show that S-oxo-A" bile acids account for >70% of the total urinary bile acid excretion. In the case of sterol 27-hydroxylase deficiency (CTX), GC-MS analysis should indicate that the major cholestane pentols in the urine are 3,7,12,22,25 and 3,7,12,23,25-pentols. (One patient has been described who had familial cholestatic liver disease associated with greatly increased urinary excretion of 5jff-cholestane-3a,7a,12a,24 S,25-pentol [see previous table]). Liquid secondary ion-tandem mass spectrometry (LSI-MS/ MS) is an alternative method to GC-MS and can rapidly confirm the identity of a number of diagnostic ions that are found in the LSI-MS spectrum of urine. These include sulphated and taurine-conjugated abnormal metabolites such as those observed in 3 ff-HSDH deficiency (32.1), 5)9-reductase deficiency (32.2), oxysterol 7a-hydroxylase deficiency (32.4) and peroxisomal disorders [13]. 

Kimura A, Mahara R, Inoue T, et al. Profile of urinary bile acids in infants and children developmental pattern of excretion of unsaturated ketonic bile acids and 7 -hydroxylated bile acids. Ped Res (1999) 45(4) 603-609... 

Radioimmunoassay. Antibodies can be produced against bile acid-protein complexes - usually bile acid-bovine serum albumin complex - and used for radioimmunoassay of that bile acid in usual manner (cf. 1). Each bile acid should have its own antibody so that the method is suitable for the quantitation of individual bile acids provided the antibodies are specific enough. One of the major problems actually is the cross-reactivity of antibodies and difficulties in the determination of total bile acids in biological materials. Thus, for the fec il bile acids with mainly bacterial transformation products (see Fig. 1) and to some extent for the urinary bile acids, especially under diseased conditions, this is quite Impossible. 

Gas-liquid chromatography (GLC). The use of capillary columns has markedly increased sensitivity and specificity of GLC in the determination of the bile acids (see Fig. 1). Owing to high resolution power great many individual bile acids can be separated and quantitated in each run yet the complex mixtures of fecal and urinary bile acids still show some overlapping and require preliminary group separation. Also, free and conjugated bile acids... 

In diagnoses of some rare conditions, such as bile acid synthetic defects, MS can also be utilized. Nowadays it is possible to screen and rapidly diagnose potential or real inborn errors in bile acid synthesis from urinary bile acid analysis by means of MS. Specific mutations in the genes that encode the enzymes responsible for bile acid synthesis can be identified by molecular techniques. Of the seven known genetic defects that cause progressive cholestatic Uver disease, syndromes of fat-soluble vitamin malabsorption, and neurological disease, six have been properly characterized [16]. 

Glycine participates in the biosynthesis of heme, purines, and creatine and is conjugated to bile acids and to the urinary metabolites of many drugs. 

Mefenamic acid - A false-positive reaction for urinary bile, using the diazo tablet test, may result. 

Although bile acid conjugates with amino acids are normally excreted into bile, amino acid conjugates of xenobiotics are usually excreted into urine. Conjugation with endogenous amino acids facilitates urinary excretion because of the organic anion transport systems located in the kidney tubules. 

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... 

In liver disorders, serum levels of bile acids are elevated, and their measurement is a sensitive indicator of liver disease. Bile acids are not normally found in urine owing to efficient uptake by the liver and excretion into the intestines. In hepatocellular disease and obstructive jaundice, however, their urinary excretion increases. 

A variety of biochemical defects have been reported in patients with the Zellweger syndrome. These include a defect in the catabolism of pipecolic acid (D2) and increased plasma, biliary, and urinary levels of intermediates in bile acid synthesis (H4, M15, M30). More recently, the accumulation of very-long-chain fatty acids, such as hexacosanoic acid, has been noted (M32) and elevated plasma phytanic acid with decreased fibroblast phytanic acid oxidase activity reported (P13). 

The low concentrations of bile acids in urine have also been measured by radioimmunoassay. In one study, total cholic and chenodeoxycholic acid conjugates were measured after extraction and solvolysis to remove sulfate groups, giving a mean urinary excretion of 0.6 p.mol/24 hours for cholic acid and 1.2 p.mol/24 hours for chenodeoxycholic acid in normal subjects (S7). These estimates can be compared with values of 2.1 xmol/24 hours for conjugated cholic acid and 8.4 xmol/24 hours for sulfoglycolithocholic acid obtained for the urinary excretion of bile acids using commercially available radioimmunoassays (WIO). 

Samuelson, K., Aly, A., Johansson, C., and Norman, A., Serum and urinary bile adds in patients with primary biliary cirrhosis. Scand. J. Gastroentend. 17, 121-128 (1982). Samuelson, K., and Eklund, A., Determination of urinary cholic and chenodeoxycholic acid conjugates with radioimmunoassay. Scand. J. Clin. Lab. Invest. 40,555-561 (1980). 

Interestingly, the number of examples of toxicity biomarkers discovered using nuclear magnetic resonance (NMR)-based and MS-based metabonomics is quite impressive. In metabonomics studies in which a severe toxicity is observed, almost always the relative urinary concentrations of Krebs cycle intermediates are decreased and a concomitant decrease in urinary levels of hippuric acid is observed. These compounds have been proposed to be nonspecific markers of toxicity as they reflect a combination of complex changes in an organism [11,48-51], Taurine has been known to be a specific marker for liver toxicity as its urinary levels are typically increased with necrosis and fatty liver. Several bile acids in the serum such as cholic, glycolic, and taurocholic acids have also been demonstrated to be sensitive markers of liver dysfunction [50], In the literature, there are a few preclinical and clinical examples of... 

Hydroxylation. The 2)8-hydroxylated derivative of cholic acid, 2, 3a,7a,12a-tetrahydroxy-5i8-cholanate, was characterized as a minor component of gastric contents of neonates with duodenal atresia [202], Identification was facilitated by the availability of the authentic compound, arapaimic acid, which was isolated from bile of Arapaima gigas (Chapter 10) and was synthesized [204], This tetrol is also a minor urinary metabolite of cholate in patients with intrahepatic cholestasis [123]. A polar bile acid formulated as a 2,3,6,7-tetrol obtained from urine of healthy newborn infants [199] apparently differs from an unidentified urinary tetrol from patients with cholestasis [164],... 

Hydroxylation. Urinary acids hydroxylated at position 6, especially 6a, include hyodeoxycholate, hyocholate, 3a,6 /, 12a-trihydroxy- and 3a,6a,7a,12 -tetrahy-droxy-5 -cholanates. Hyocholate, the major bile acid in urine, serum and duodenal fluid of a child with intrahepatic cholestasis [109], is a major urinary metabolite of chenodeoxycholate in patients with intrahepatic cholestasis, while 3a,6a,12a-trihy-droxy-5j3-cholanate is only a minor metabolite of deoxycholate [123]. The 3a,6a,7a,12a-tetrahydroxy acid, obtained from urine of patients with liver diseases [194] and from gastric contents of neonates with duodenal atresia [203] is another metabolite of cholic acid [123] several unidentified tetrols have yet to be char-... 

Sulfates. All urinary monohydroxy and most dihydroxy bile acids were mono-sulfated. Position 3 was sulfated in the major bile acids which were also conjugated... 

The amino acid taurine has been suggested as a marker of hepatotoxicity because it is involved in the conjugation of bile acids to bile acids, the conjugation of xenobiot-ics, and the detoxification of reactive metabolites. Taurine is a product of sulfur acid metabolism and may reflect protein synthesis. The measurement of urinary taurine in models of hepatotoxicity has been described (Sanins et al. 1990 Waterfield et al. 1993a, 1993b, 1993c Timbrell, Seabra, and Waterfield 1995 Waterfield, Asker, and Timbrell 1996), but the measurement is reported to show diurnal variation, wide intraanimal variability, and less success at minor levels of hepatic injury (Maxuitenko, North, and Roebuck 1997). 

Tumor promotion activity has been demonstrated for a variety of agents in various organs butylated hydroxytoluene (BHT) in mouse lung (27), bile acids in colon (28), saccharin and cyclamate in rat urinary bladder (29), TPA in an vivo-in vitro rat trachea model (, 31), and phenobarbital (32-36) and polychlorinated biphenyls (34,, 38) in rat liver. 

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