Bile acids, labelled

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. 

The value of bile acids labelled with carbon-13 in gastroenterological research has been critically evaluated and compared with radioactive tagging. Stable iso-... 

Figure 1 and Table III give a schematic summary of the fragmentations of bile acid derivatives. It should be stressed that detailed mechanistic studies have not been made, i.e. with bile acids labeled with isotopes. Thus, the... 

Another example of a nudibranch, which probably modifies dietary metabolites to obtain more effective allomones, is seen in Aldisa cooperi (= A. sanguinea cooperi) [155]. It elaborates two fish antifeedant bile acids (104,105) that are absent in its prey, the sponge Anthoarcuata graceae, where the main steroid is 4-cholesten-3-one (106). Biosynthetic experiments starting from both labelled mevalonic acid and labelled 4-eholesten-3-one would definitely clarify, whether, the two allomones (104-105) are biosynthetized de novo by the mollusc, or if they are derived from a food source. 

Vesicular transport of bile acids has not been demonstrated under normal conditions, shown by using isolated rat hepatocyte couplets and fluorescently labelled bile acids. In these experiments confocal microscopy found no evidence of sequestering into clusters and colchicine disruption of microtubular function did not affect bile-acid transport. This makes it unlikely that vesicle transport plays a role and it is now believed that bile acids traverse the hepatocyte by diffusion through the cytosol while bound to soluble proteins. It is worth considering the caveat that fluorescently labelled bile acids, while very useful tools, do differ structurally from endogenous bile acids with increased hydro-phobicity leading to greater retention by cells. ... 

Glutathione S transferases bind bile acids in vitro but doubt has been cast over whether this happens in vivo as these enzymes were not labelled by fluorescently labelled bile acids in experiments to identify the carrier proteins but may play a role with the raised levels in cholestasis. Liver fatty-acid-binding protein has been shown to bind bile acids by using a displacement assay with fluorescent fatty-acid ligand. This work clearly showed displacement to be directly related to hydrophobicity, such that lithocholate conjugates had the greatest effect. This may indicate a mechanism to minimise toxicity within the hepatocyte. 

Carcinogens often interact and bind directly with DNA. For this reason, investigators sought to establish whether bile acids could directly induce DNA adducts. Utilising the P-post labelling technique, Scates et reported... 

Additionally, two studies have measured colorectal epithelial cell proliferation and apoptosis in human non-neoplastic mucosa in combination with serum bile acid quantification. Ochsenkuhn et al have reported a positive correlation between serum DCA levels and proliferation measured by flow cytometric cell cycle analysis. However, a more recent study of colorectal adenoma patients failed to detect a correlation between serum DCA and immuno-histochemical Ki-67 antigen labelling. Instead, this latter study revealed a positive correlation between serum DCA and the degree of TUNEL-positive epithelial cell apoptosis. ... 

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

Kl. Kobayashi, N., Oiwa, H., Kubota, K., Sakoda, S., and Goto, J., Monoclonal antibodies generated against an affinity-labeled immune complex of an anti-bile acid metabolite antibody An approach to noncompetitive hapten immunoassays based on anti-idiotype or anti-metatype antibodies. J. Immunol. Methods 245, 95-108 (2000). 

Kawasaki T, Maeda M, Tsuji A (1983) Immobilized -hydroxysteroid dehydrogenase and dansyl hydrazine as a pre-labeling reagent for high-performance liquid chromatography with fluorescence detection of bile acids. J Chromatogr 272 261-268... 

Key words Cholesterol absorption, phytosterols, cholesterol excretion, reverse cholesterol transport, ACAT, CEL, inhibitors, bile acids, fecal sterols, dual label, obesity, cardiovascular disease. 

Metabolism of deuterium-labelled ethanol in rats has been investigated (530, 531) low levels of deuterium incorporation into bile acids are observed in difference spectra obtained by subtracting from spectra. The difference spectra contain... 

Bile-acid formation in rats involves hydroxylation to give 7a- and 6j8-hydroxy-derivatives. In many cases, no isotope effect was observed on hydroxylation of the appropriate labelled sterol. These examples involve cytochrome P-450 in the oxidation. However, oxidation of [7a- H,24- C]deoxycholic acid or tauro-deoxycholic acid to the corresponding cholic acid showed an isotope effect of 3.8 on examination of recovered starting material. 

Steroids, which are a class of compounds that occur in nature and in synthetic products, have a cyclopen-tanoperhydrophenanthrene skeleton. The carbon atoms and rings are labeled according to the scheme shown in Fig. 1. The following classes of compounds belongs to steroids sterols, bile acids, cardenolides, androgens, estrogens, corticosteroids, steroid sa-pogenins, steroid alkaloids, ecdysteroids, and vitamin D. 

Differences in incorporation of radioactivity from the C-1 and C-2 carbons of propionate into cholesterol and bile acids by biliary fistula rats have been demonstrated. It appears that incorporation of propionate via HCO3" is not the major pathway. Feeding experiments with sodium [ C]bicarbonate produced no labelled cholesterol or bile acids. 

G. Corsiero, D. Girbig, R Konig, W. Weyland, C. Identification of the Bile Acid-Binding Site of the Beal Lipid-Binding Protein by Photoaffinity Labeling, Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry, and NMR Structure, 7. Biol. Chem. 276,7291-7301 (2001). 

The exact contributions of these alternate pathways to total hepatic bile acid synthesis in normal subjects is not certain, although 26-hydroxylation is usually regarded as the major pathway. In addition, it should be pointed out that current views of hepatic cholic acid and chenodeoxycholic acid synthesis are based primarily oh studies carried out in the rat. More recent studies, which have involved the administration of labeled bile acid intermediates to patients, have suggested that bile acid biosynthesis is more complex than previously thought and that multiple pathways exist to convert cholesterol to bile acids (Vll). 

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). 

G13. Goto, J., Saito, M., Chikai, T., Goto, N., and Nambara, T., Studies on steroids. CLXXXVIl. Determination of serum bile acids by high-performance liquid chromatography with fluorescence labeling. J. Chromatogr. 276, 289-300 (1983). 

A coupling appears to exist between the rate-limiting enzyme in the biosynthesis of cholesterol, HMG-CoA reductase, and cholesterol 7a-hydroxylase. The two enzymes seem to be located close to each other on the endoplasmic reticulum membrane [66], and the two activities covariate under most conditions. Results from both in vivo and in vitro experiments show that newly synthesized cholesterol is the preferred substrate for cholesterol 7a-hydroxylase. In an early study by Staple and Gurin, it was shown that the bile acids in bile had a higher radioactivity than cholesterol after administration of labelled acetate to rats [67]. Bjorkhem and Danielsson found that the specific radioactivity of 7a-hydroxycholesterol was higher than that of cholesterol after incubation of labelled mevalonate with the 10000 x g supernatant fluid of a rat liver homogenate [50]. Balasubramaniam et al. showed that 7a-hydroxycholesterol isolated from the livers of rats after intravenous administration of labelled cholesterol had a lower specific radioactivity than cholesterol [58]. Cronholm and collaborators measured the incorporation of isotope from [1- H2]-,... 

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