Measurement of bile acids

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. 

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In vivo, measuring bile acids in plasma and urine should be revived as potential biomarkers in the modern metabolomic era. Then the first-order scientific question will become whether early and time-controlled fasting-level measurement of bile acid concentration in plasma and urine can become a sensitive and specific biomarker for drug-induced cholestasis and ultimately liver injury at later time-points [117] Clinical trials should be conducted to evaluate whether such bile acid measurements can be used as part of a predictive panel to identify patients who are at increased risk of drug-induced cholestasis. 

E3. Engert, R., and Turner, M. D., Problems in the measurement of bile acids with 3a-hydroxysteroid dehydrogenase. Anal. Biochem. 51, 399—407 (1973). 

V6. Vantrappen, G., Rutgeerts, P., and Ghoos, Y., A new method for the measurement of bile acid turnover and pool size by a double lable, single intubation technique. J. Lipid Res. 22, 528-531 (1981). 

Studies with radioactive glycocholate or taurocholate demonstrated a virtual absence of the enterohepatic circulation of bile acids in patients with jejunotransversocolostomy (77). The small amount of absorbed bile acids contained some deconjugated cholate and deoxycholate (which had been reconjugated in the liver), indicating a rapid bacterial action during an apparently fast intestinal passage. Under these conditions, steatorrhea is apparently not solely due to bile salt deficiency induced impairment of micelle formation, but reduced absorptive area may play an important contributory role. No direct measurement of bile acid synthesis by fecal determination has been performed in this condition. 

Gan-Schreier, H. Okun, J.G. Kohlmueller, D. Langhans, C.D. Peters, V. Ten Brink, H.J. Verhoeven, N.M. Jakobs, C. Voelkl, A. Hoffmann, G.F. Measurement of bile acid CoA esters by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). J. Mass Spectrom. 2005, 40 (7), 882-889. 

The most common assay uses 3a-hydroxysteroid dehydrogenase to form the 3-keto bile acid that is trapped by, for example, hydrazine hydrate, causing the reaction to go to completion. The co-factor NAD is reduced stoichiometrically and can be measured by ultraviolet absorption or more commonly by fluorescence at an activation of 345 nm and emission of 450 nm. Use of this enzyme measures all bile acids with a 3a-hydroxyl but not cholesterol, which has a 3p-hydroxyl, and does not measure bile acids with a sulphate or glucuronide group conjugated to the 3a-hydroxyl. 

Assays have also made use of 7a-hydroxysteroid dehydrogenase that can measure the primary bile acids, or for more specialised purposes such as differentiating between pathways of bile-acid synthesis to determine the proportion derived from the acid pathway. 

It is well recognised that the faecal bile acid content of random stool samples is highly variable with marked daily variation.Therefore, studies testing the association between luminal bile acid exposure and the presence of colorectal neoplasia have usually measured serum bile acid levels, which demonstrate less variability and are believed to reflect the total bile acid pool more accurately. Serum DCA levels have been shown to be higher in individuals with a colorectal adenoma compared with individuals without a neoplasm. Only one study has assessed future risk of CRC in a prospective study of serum bile-acid levels. The study was hampered by the small sample size (46 CRC cases). There were no significant differences in the absolute concentrations of primary and secondary bile acids or DCA/CA ratio between cases and controls although there was a trend towards increased CRC risk for those with a DCA/ CA ratio in the top third of values (relative risk 3.9 [95% confidence interval 0.9-17.0 = 0.1]). It will be important to test the possible utility of the DCA/ CA ratio as a CRC risk biomarker in larger, adequately powered studies. A recent study has demonstrated increased levels of allo-DCA and allo-LCA metabolites in the stool of CRC patients compared with healthy controls. ... 

High-throughput assay of bile acids is technically demanding and expensive. Combined with high intra- and inter-individual variability in stool bile acid levels, it is unlikely that bile acid measurement will find a role as a biomarker of future colorectal neoplastic risk. 

Plasma Membranes and Derived Transporters Measurement of transported calcium in synaptosomes, 174, 3 glutamate accumulation into synaptic vesicles, 174, 9 identification of bile acid transport protein in hepatocyte sinusoidal plasma membranes,... 

A7b. Arvan, D. A., and Ritz, A., Measurement of serum albumin by the HABA-dye technique A study of the effect of free and conjugated bilirubin, of bile acids, and of certain drugs. Clin. Chim. Acta 26, 505-516 (1969). 

Steatorrhoea is the formation of non-solid faeces. Floating stools, due to excess fat, are oily in appearance and foul smelling. There is increased fat excretion, which can be measured by determining the faecal fat level. Possible biological causes can be lack of bile acids (due to liver damage or hypolipidaemic drugs), defects or a reduction in pancreatic enzymes (lipase), and defective mucosal cells. The absence of bile acids will cause the faeces to turn grey or pale. 

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. 

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. 

Using this technique, pool sizes of cholic acid and chenodeoxycholic acid have been estimated to be similar and around 1.0 to 1.5 g each in healthy subjects, with the total bile acid pool amounting to 2 to 4 g (H18, LIO, VIO). Cholic acid turnover is more rapid than for chenodeoxycholic acid, and the rate of hepatic synthesis of cholic acid (300 to 400 mg/day) is therefore approximately double that for chenodeoxycholic acid (150 to 200 mg/day) (H18, VIO). In the steady state, total bile acid synthesis by the liver should equal bile acid loss in the feces, which is around 400 mg/day. Some studies have found that estimates of bile acid synthesis by the isotope dilution technique give values that are higher than those obtained by direct chemical measurement of fecal bile salts (S45), but good agreement has recently been claimed between the two methods (DIO). ITie Lindstedt technique for measuring bile acid turnover and pool size has been modified so that only one bile sample need be collected after intravenous administration of the labeled bile acid. These modified methods measure either pool size alone (D9) or pool size and turnover if both and bile acid are administered at an interval of 24 hours apart (V6). 

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). 

In an early work by Myant and Eder, a time lag was observed between the increase in synthesis of cholesterol and synthesis of bile acids [221]. Such a time lag has not been reported in later studies, and it seems likely that the two rate-limiting enzymes respond to the same signal. The possibility that an increase of available cholesterol is responsible for the increase of cholesterol 7a-hydroxylation seems excluded by the work by Mitropoulos et al. in which attempts were made to measure the substrate of the enzyme under different condition [59,222]. In view of the time lag between introduction of a bile fistula and rise of enzyme activities, it is likely that protein synthesis is involved in the increase of both enzyme activities. 

The bile acids returning to the liver in the portal blood inhibit both cholesterol 7a-hydroxylase and HMG-CoA reductase. Size, circulation rate and composition of the bile acid pool are of importance. The inhibitory effect of bile acids on cholesterol 7a-hydroxylase is mediated by an effect on synthesis or breakdown of proteins, most likely the specific species of cytochrome P-450 involved in the hydroxylation. Evidence that the rate of synthesis of the specific species of cytochrome P-450 is of major importance can be obtained only when it is possible to measure accurately the amount of specific cytochrome P-450 by means other than enzyme activity. Although less likely from the data available, it cannot be excluded at the present state of knowledge that the inhibitory effect of bile acids on cholesterol 7a-hydroxylase is mediated by the effect of bile acids on HMG-CoA reductase. Since the substrate pool for cholesterol 7 -hydroxylase does not seem to be affected [59,222], a hitherto unknown mechanism must then be responsible for the coupling between the two rate-limiting enzymes. 

A simple method for estimating the pH-solubility relationship of bile acids and salts is to carry out aqueous acidometric titration of a bile salt in water with a stronger mineral acid [5,35], Once the molarities of bile salt and mineral acid are known, the titration curves provide a direct measurement of equivalence, equilibrium and metastable pH values, the pH at which precipitation of the HA species occurs (pHpp,), an estimate of the solubilities of the HA species in water (if the system is < CMC) or in water plus micelles (if the system is > CMC) and a calculation of the apparent pK (pATg). The methods, results and interpretation of such titration curves for the common bile salts, titrated with HCl, have been described in detail elsewhere [5,6]. 

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