Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats.

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3alpha-hydroxy-5beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha-(t-butoxycarbonyl)-l-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.

Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15alpha-hydroxylithocholic acid.

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15alpha-hydroxylithocholic acid (3alpha,15alpha-dihydroxy-5beta-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15alpha-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Delta14 intermediate. The synthesis of its C-15 epimer, 15beta-hydroxylithocholic acid (3alpha,15beta-dihydroxy-5beta-cholan-24-oic acid), is also reported. The taurine conjugate of 15alpha-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15alpha-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15alpha-hydroxylation of lithocholic acid that was formed by bacterial 7alpha-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15alpha-hydroxylation as a novel detoxification mechanism for lithocholic acid.

Isolation and HPLC of N-epsilon-lithocholyl lysine as its fluorescamine and dimethylaminoazobenzene isothiocyanate derivatives.

N-epsilon-lithocholyl lysine (NELL) is a component of tissue-bound lithocholic acid (TBL). The isolation of NELL from native protein sources was simulated by hydrolysis of lithocholyl-bovine serum albumin (BSA) (synthesized by coupling lithocholyl-N-hydroxysuccinimide to fatty acid-free BSA) by digestion with a mixture of 6N HCl-propionic acid at 70 C for 3 h under partial vacuum. NELL was isolated on a reversed phase Sep-Pak C18 column and converted to either a fluorophor with fluorescamine or to a chromophor with dimethylaminoazobenzene isothiocyanate for subsequent HPLC using appropriate fluorescence or UV/visible absorption detectors. The procedure described here is quantitative, highly sensitive, and not dependent upon the use of Clostridial cholanoylamino acid hydrolase, the activity of which is sometimes blocked by steric hindrance on the substrate. Using this procedure we have demonstrated the presence of TBL in native histones.

Isolation and identification of delta 6-lithocholenic acid (3 alpha-hydroxy-5 beta-6-cholen-24-oic acid) as an intestinal bacterial metabolite of chenodeoxycholic acid in man.

Fecal bacterial biotransformation studies of chenodeoxycholic acid were performed. Incubations were carried out for 30-s to 12-h time intervals. delta 6-Lithocholenic acid was isolated by thin-layer chromatography. Its structure was confirmed by gas-liquid chromatography and mass spectrometry. The proportion of chenodeoxycholic acid biotransformed to delta 6-lithocholenic acid consistently ranged from 5.5 to 14.0%.

Improved extraction procedure for determination of bile acids in faeces.

A simplified extraction procedure for bile acids from wet faeces, using methanol/hydrochloric acid is described. Extracts were analyzed by gas-liquid chromatography, thin-layer chromatography and an enzymatic assay, with 3 alpha-hydroxysteroid dehydrogenase. Recoveries of some stable bile acids, added to faeces, were studied; extraction efficiency was also investigated with a procedure using radioactive labelled bile acids given orally to patients. Resin treatment of faecal extracts, because of the sometimes hard colour of the extracts, resulted in a slightly lower recovery as determined by the enzymatic method. Recoveries were higher, using the proposed extraction procedure, than those obtained with extracts prepared by the standard procedure of Grundy et al [6].

Nature of tissue-bound lithocholic acid and its implications in the role of bile acids in carcinogenesis.

Lithocholic acid, a monohydroxy secondary bile acid, is present in tissues in two forms. One form is extractable with 95% ethanol-0.1% ammonia (soluble lithocholate), and the other form is firmly bound to tissue residues and can be released only by the bile salt-deconjugating enzyme, clostridial cholanoylamino acid hydrolase (tissue-bound lithocholate). Studies on bile salt-protein interactions revealed that lithocholic acid had amino group-modifying activity specifically directed against the basic side group of lysine residues. Degradative procedures yielded N-epsilon-lithocholyllysine, confirmed by comparison with the authentic compound synthesized in our laboratories. Studies on the distribution of tissue-bound lithocholate in tissues have revealed high concentrations of this form of lithocholate in livers of rats treated with the carcinogen, methylazoxymethanol. In light of these observations, the role of bile acids, and specifically lithocholic acid, as promoters of tumorigenesis must be further investigated.

Thin-layer chromatographic separation of conjugates of ursodeoxycholic acid from those of litho-, chenodeoxy-, deoxy-, and cholic acids.

Separation of the glycine and taurine conjugates of ursodeoxycholic acid from those of lithocholic acid, chenodeoxycholic acid, deoxycholic acid, and cholic acid by thin-layer chromatography is described. Thus, on running a silica gel G plate first in a solvent system of n-butanol-water 20:3 and then in a second solvent system of chloroform-isopropanol-acetic acid-water 30:20:4:1, all the above-mentioned conjugated bile acids are separated from one another. The application of this method to study the change in the biliary bile acid conjugation pattern in ursodeoxycholic acid-fed gallstone patients is described.

Isolation and purification of lithocholic acid metabolites produced by the intestinal microflora.

Lithocholic acid metabolites produced by the intestinal microflora of rats can be isolated from other endogenous lipids using Sephadex LH-20 column chromatography. Analyses of individual metabolites collected from the column by silica gel coated glass fiber paper chromatography result in the resolution of epimeric 3-hydroxy derivatives. In addition, glass fiber paper chromatography is more sensitive and requires less development time than conventional glass-coated thin-layer plates. Further confirmation of the identity of metabolites is achieved by gas-liquid chromatography, which separates both methyl and ethyl esters of lithocholic and isolithocholic acids.

Thin-layer chromatographic separation of sulfated and nonsulfated lithocholic acids and their glycine and taurine conjugates.

A method for superior thin-layer chromatographic separation of lithocholic acid and its N-glycine and N-taurine conjugates, as well as their respective 3alpha-sulfates, is described. A solvent system of chloroform-methanol-acetic acid-water 65:24:15:9 (v/v) is used with air-dried plates of silicic acid containing calcium sulfate (10% by weight) under conditions of chamber saturation.

Conformational changes in fibrous elastin due to calcium ions.

A column packed with calcium-free bovine aorta elastin provided good separations of mixtures of bile salts when water was the moving phase. Tritium-labelled cholesterol was applied to the column using dilute solutions of taurodeoxycholate in Tris-NaCl buffers as solvent. The cholesterol was quantitatively eluted as a narrow peak in a rising gradient of taurodeoxycholate. When Na+ in the buffer was replaced by Ca2+ elution of the labelled cholesterol was delayed. Control experiments in which the elastin fibres were replaced as the column packing by an inert stationary phase consisting of n-butanol immobilized by silane-treated Celite showed that the effect of the change from Na+ to Ca2+ on the solvent properties of taurodeoxycholate was small and in the opposite direction. The experiments indicated that the replacement of sodium by calcium as the ionic environment of fibrous elastin produced a configurational change towards increasing hydrophobic character.

Removing substances from blood by affinity chromatography. I. Removing bilirubin and other albumin-bound substances from plasma and blood with albumin-conjugated agarose beads.

Substances such as bilirubin that bind tightly to plasma proteins cannot readily be removed from blood. We describe here the use of affinity chromatography as a new approach to the removal of proteinbound metabolites and toxins from blood. Agarose beads were coupled via cyanogen bromide to human serum albumin so as to contain 30-50 mg of albumin/g wet wt. Such beads, when exposed to plasma from a patient with congenital nonhemolytic jaundice labeled with [(14)C]-bilirubin, bound more than 150 mug bilirubin/g of beads. The binding was saturable, concentration-dependent, relatively independent of flow rate, and reversible by elution with plasma, albumin, or 50% (vol/vol) ethanol. The beads could be repeatedly reused without loss of efficiency after ethanol elution and long storage in the cold. Salicylate, cortisol, and taurocholate, which bind weakly to albumin, were retarded by the beads but eluted with neutral buffer. Thyroxine, taurolithocholate, chenodeoxycholate, and digitoxin bound tightly but were eluted with 50% ethanol. Digoxin did not bind at all. When whole blood was passed over agarose-albumin beads, bilirubin was removed, calcium and magnesium fell slightly, but red cells, white cells, platelets, clotting factors, and a variety of electrolytes and proteins were substantially unchanged. Agarose-albumin beads may be useful for removing protein-bound substances from the blood of patients with liver failure, intoxication with protein-bound drugs, or specific metabolic deficits. Furthermore, it may be possible to make useful adsorbents by attaching other proteins to agarose or other polymer beads.