Evaluation of tauroursodeoxycholic acid in liver cells' cultures by MEKC: Initial hints to comprehend its role in diabetes mellitus of obese individuals.

Genetic factors, diet, lifestyle, and other factors lead to various complications in the body, such as obesity and other chronic diseases. The inflammatory state caused by excessive accumulation of body fat affects the pathways related to the control of glycemic homeostasis, leading to a high demand for insulin, to subsequent failure of stressed ß cells, and development of type 2 diabetes mellitus (T2DM). The study of new endocrine signalers, such as bile acids (BAs), becomes necessary as it allows the development of alternatives for T2DM treatment. In this work, a methodology was developed to quantify tauroursodeoxycholic BA (TUDCA) in liver cells of the HepG2 strain treated in hyperlipidic medium. This BA helps to improve insulin clearance by increasing the expression of the insulin-degrading enzyme, restoring sensitivity to this hormone, and making it viable for treating T2DM. Herein, a targeted metabolomic method for TUDCA determination in extracellular medium of hepatocyte matrices by micellar electrokinetic chromatography-UV was optimized, validated, and applied. The optimized background electrolyte was composed of 40 mmol/L sodium cholate and 30 mmol/L sodium tetraborate at pH 9.0. The following figures of merit were evaluated: linearity, limit of quantification, limit of detection, accuracy, and precision. Data obtained with the validated electrophoretic method showed a self-stimulation of TUDCA production in media supplemented only with BA. On the other hand, TUDCA concentration was reduced in the hyperlipidic medium. This suggests that, in these media, the effect of TUDCA is reduced, such as self-stimulated production and consequent regulation of glycemic homeostasis. Therefore, the results reinforce the need for investigating TUDCA as a potential T2DM biomarker as well as its use to treat several comorbidities, such as obesity and diabetes mellitus.

FXR in the dorsal vagal complex is sufficient and necessary for upper small intestinal microbiome-mediated changes of TCDCA to alter insulin action in rats.

OBJECTIVE: Conjugated bile acids are metabolised by upper small intestinal microbiota, and serum levels of taurine-conjugated bile acids are elevated and correlated with insulin resistance in people with type 2 diabetes. However, whether changes in taurine-conjugated bile acids are necessary for small intestinal microbiome to alter insulin action remain unknown. DESIGN: We evaluated circulating and specifically brain insulin action using the pancreatic-euglycaemic clamps in high-fat (HF) versus chow fed rats with or without upper small intestinal healthy microbiome transplant. Chemical and molecular gain/loss-of-function experiments targeting specific taurine-conjugated bile acid-induced changes of farnesoid X receptor (FXR) in the brain were performed in parallel. RESULTS: We found that short-term HF feeding increased the levels of taurochenodeoxycholic acid (TCDCA, an FXR ligand) in the upper small intestine, ileum, plasma and dorsal vagal complex (DVC) of the brain. Transplantation of upper small intestinal healthy microbiome into the upper small intestine of HF rats not only reversed the rise of TCDCA in all reported tissues but also enhanced the ability of either circulating hyperinsulinaemia or DVC insulin action to lower glucose production. Further, DVC infusion of TCDCA or FXR agonist negated the enhancement of insulin action, while genetic knockdown or chemical inhibition of FXR in the DVC of HF rats reversed insulin resistance. CONCLUSION: Our findings indicate that FXR in the DVC is sufficient and necessary for upper small intestinal microbiome-mediated changes of TCDCA to alter insulin action in rats, and highlight a previously unappreciated TCDCA-FXR axis linking gut microbiome and host insulin action.

A combination of three plasma bile acids as a putative biomarker for schizophrenia.

The aim of the present study is to determine whether plasma bile acids (BAs) could be used as an auxiliary diagnostic biomarker to distinguish patients with schizophrenia from healthy controls. Seventeen different BAs were quantitatively measured in plasma of 12 healthy participants and 12 patients with schizophrenia. Then, the data were subjected to correlation and linear discriminant analysis (LDA). The concentrations of cholic acid (CA), taurochenodeoxycholic acid (TCDCA) and taurodeoxycholic acid (TDCA) were significantly decreased in plasma of the schizophrenia patients. Correlation analysis showed the concentrations of CA, TCDCA and TDCA were negatively correlated with schizophrenia. In addition, LDA demonstrated that combination of CA, TCDCA and TDCA with a classification formula could predict correctly classified cases and the accuracy of prediction was up to 95.83%. Combination of the three BAs may be useful to diagnose schizophrenia in plasma samples.

[Determination of tauroursodeoxycholic acid in compound bile capsule by HPLC].

OBJECTIVE: To discriminate and determine of the artificial bear bile of the compound bile capsule. METHOD: Taking the pharmacopoeia as reference, the artificial bear bile was discriminated and determined by HPLC. RESULT: The compound bile capsule and the control sample had chromatographic peak at the same time from HPLC. The content of the artificial bear bile was above 10 mg per tablets. CONCLUSION: The artificial bear bile of compound bile capsules can be discriminated effectively and determined accurately by HPLC method.

Single-step analysis of individual conjugated bile acids in human bile using 1H NMR spectroscopy.

1H and 13C NMR spectra of intact human bile were assigned using one-dimensional (1H and 13C) and two-dimensional (1H-1H and 1H-13C) experiments. Individual conjugated bile acids--glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid--were identified. The bile acids were quantified accurately and individually in a single step by using distinct and characteristic amide signals. Making use of 13C NMR, the study also suggests a way to analyze unconjugated bile acids separately, if present. Chemical shift assignments and rapid single-step analysis of individual conjugated bile acids from intact bile presented herein may have immense utility in the study of bile acid metabolism and deeper understanding of hepatobiliary diseases.

Influence of Verapamil and Cyclosporin A on bile acid metabolism and transport in rat liver slices.

Verapamil (V) is a specific inhibitor of the P-glycoprotein (mdr1) in the hepatocyte canalicular membrane. Cyclosporin A (CsA) as an essential immunosuppressive drug has potentially cholestatic adverse effects on the liver, but increases the expression of mdr1. In precision-cut liver slices from 34- to 40-day-old male Wistar rats 26 individual free and conjugated bile acids (BAs) as markers of hepatic transport and synthesis function were analysed after 4 h incubation with V (100 microM) or CsA (5 microM) in Krebs-Henseleit buffer. Some slices were loaded with cholic acid (CA 5 microM) or tauro-ursodeoxycholic acid (T-UDCA 5 microM) to investigate the V and CsA effects under conditions of BA supplementation. BAs were determined in tissue and medium by HPLC with postcolumn derivatisation and fluorescence detection. V and CsA, influencing different targets in BA transport, enhanced slice concentrations of T- and glyco- (G-) conjugated CA only when exogenous CA was given additionally. This BA accumulation in tissue is more reflected at decreased medium concentrations of these BAs after V and CsA incubations. Both V and CsA also inhibited CA uptake into the slices. The acidic chenodeoxycholic acid (CDCA) synthesis pathway is disturbed: T- and G-CDCA concentrations are diminished in slices and medium after V and CsA incubations. T-UDCA plus V or CsA enhanced not only its own slice concentration but also the concentration of the trihydroxylated tauro-muricholic acid (T-beta-MCA), reflecting the conversion of the accumulated dihydroxylated T-UDCA into the T-beta-MCA. The similar effects of V and CsA on BA transport and metabolism can be explained by mdr1 mediated disturbances of cellular ATP transport rather than by inhibition of individual BA transporters.

A rapid, sensitive method for accurate analysis of individual bile acids in biological fluids by high performance thin-layer chromatography and densitometry.

1. A rapid new micromethod for quantitative analysis of individual bile acids in duodenal juice by high performance thin-layer chromatography (HPTLC) and densitometry is described and evaluated by comparison with standard TLC and spectrophotometry. 2. Advantages of HPTLC over TLC include more rapid separation, better resolution and more sensitive detection (5 - 10 fold), without the need for prior extraction. Densitometry provides simple, direct and rapid quantitation. 3. The method is accurate and reliable over a range of bile acid concentrations. In the 0.5 mM range, recovery was greater than 89%, and coefficients of variation for within-day analysis were 2 - 12% and for between-day analysis were 6 - 18% for the individual bile acids. Twenty analyses can be performed by one worker in a single day. 4. We conclude that the method offers several advantages over most currently described techniques, is suitable for routine use and is deserving of wider application.

Steroid metabolism along the gastrointestinal tract of the cannulated pig.

Steroid metabolism along the gastrointestinal tract of the cannulated pig was studied. Thi was achieved by fitting simple gut cannulas in the terminal ileum, caecum and mid-colon of three Landrace x large white boars, which enabled convenient collection of digesta and faecal samples at defined time points. Biochemical analyses showed that the neutral steroid profile of the pig is similar to that of man, dominated by cholesterol and its bacterial metabolite coprostanol. In contrast, pigs consuming a normal diet excrete appreciably lower quantities of neutral sterols in faeces. The major primary bile acids detected were the glycine and taurine amidates of hyocholic and chenodeoxycholic acids, which were rapidly converted to the free bile acids and subsequently dehydroxylated to hyodeoxycholic and lithocholic acids respectively, in the terminal ileum and caecum. Bacterial deconjugation and 7 alpha-dehyrdoxylation are virtually complete in the caecum with negligible further metabolism in the colon and faeces. On a wet weight basis the concentration of both neutral and acid steroids was shown to increase aborally. Inclusion of dietary fibre in the form of cellulose (Solka floc) and guar gum reduced steroid concentration considerably at all sites of the large intestine, which is consistent with their stool bulking effects. In conclusion, this study shows that intestinal steroid metabolism in the pig is similar to that in man despite slightly different bile acid profiles and, therefore, the multicannulated pig may serve as a useful model of man in chemoprevention studies of colorectal cancer.

Black bear (Ursus americanus) bile composition: seasonal changes.

Gallbladder contents from 8 active and 14 dormant black bears were analyzed for individual bile acids by high-performance liquid chromatography and for cholesterol, phospholipids, sodium, potassium, calcium, magnesium, zinc, iron, and copper. Only three bile acids occurring as taurine conjugates were detected: tauroursodeoxycholate, taurochenodeoxycholate, and taurocholate. The proportion of tauroursodeoxycholate to the sum of the three bile acids decreased. Calcium, cholesterol, phospholipids, magnesium, zinc, and copper concentrations were increased in dormancy. Standardized collection and handling procedures yielded more consistent data than previously available. The decrease in tauroursodeoxycholate and absence of deoxycholate and lithocholate are consistent with our working hypothesis that a marked reduction in metabolic activity of the gut flora is an integral part of the adaptation to metabolic stability of the dormant bear.

Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition.

Cholesterol in human bile is solubilized in micelles by (relatively hydrophobic) bile salts and phosphatidylcholine (unsaturated acyl chains at sn-2 position). Hydrophilic tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all decrease cholesterol crystal-containing zones in the equilibrium ternary phase diagram (van Erpecum, K. J., and M. C. Carey. 1997. Biochim. Biophys. Acta. 1345: 269-282) and thus could be valuable in gallstone prevention. We have now compared crystallization in cholesterol-supersaturated model systems (3.6 g/dl, 37 degrees C) composed of various bile salts as well as egg yolk phosphatidylcholine (unsaturated acyl chains at sn-2 position), dipalmitoyl phosphatidylcholine, or sphingomyelin throughout the phase diagram. At low phospholipid contents [left two-phase (micelle plus crystal-containing) zone], tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all enhanced crystallization. At pathophysiologically relevant intermediate phospholipid contents [central three-phase (micelle plus vesicle plus crystal-containing) zone], tauroursodeoxycholate inhibited, but dipalmitoyl phosphatidylcholine and sphingomyelin enhanced, crystallization. Also, during 10 days of incubation, there was a strong decrease in vesicular cholesterol contents and vesicular cholesterol-to-phospholipid ratios (approximately 1 on day 10), coinciding with a strong increase in crystal mass. At high phospholipid contents [right two-phase (micelle plus vesicle-containing) zone], vesicles were always unsaturated and crystallization did not occur. Strategies aiming to increase amounts of hydrophilic bile salts may be preferable to increasing saturated phospholipids in bile, because the latter may enhance crystallization.

Changes in the concentration and composition of biliary and serum bile acids in the young domestic fowl.

1. Concentrations of biliary and serum bile acids, their molecular compositions and serum cholesterol concentrations were determined in chicks at 2, 3, 4 and 6 weeks of age. 2. The concentration of biliary bile acid was maximal at 3 to 4 weeks, decreasing by 6 weeks of age. 3. The serum concentration of bile acid was maximal at three weeks of age. 4. Serum total cholesterol increased from two weeks and was maximal at 6 weeks of age. 5. Chenodeoxycholic acid was the predominant biliary unconjugated bile acid. 6. Tauro-chenodeoxycholic acid and tauro-cholic acid were the dominant molecular species of biliary and serum conjugated bile acid.

High performance liquid-chromatographic analysis of individual bile acids: free, glycine- and taurine-conjugated bile acids.

High performance liquid-chromatographic analyses of individual bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid), free and conjugated with glycine and taurine, are described. The analyses of free and glycine-conjugated bile acids are based on esterification of carboxyl group of bile acids with O-(p-nitrobenzyl)-N, N'-diisopropylisourea (PNBDI). Moreover, ursodeoxycholic acid, hyocholic acid, hyodeoxycholic acid and 3beta-hydroxy-5-cholenoic acid also are able to analyse by this method. These bile acids in biological sample were extracted by an Amberlite XAD-2 column, and separated by DEAE-Sepharose CL-6B into free, glycine- and taurine-conjugated bile acids. After the separation, free and glycine-conjugated bile acids were esterified with PNBDI directly. Because taurine-conjugated bile acids are unable to be esterified with PNBDI, these bile acids were hydrolyzed by NaOH in order to make free bile acids, and then they were esterified. Because the p-nitrobenzyl ester of bile acids has characteristic ultraviolet absorption, these compounds were separated to individual bile acids by high performance liquid-chromatography, and detected by an UV-detector. An analysis of individual bile acids in human bile was demonstrated.

Tauroursodeoxycholate increases rat liver ursodeoxycholate levels and limits lithocholate formation better than ursodeoxycholate.

BACKGROUND & AIMS: To explain the greater hepatoprotective effect of tauroursodeoxycholic acid vs. ursodeoxycholic acid, the absorption, hepatic enrichment, and biotransformation of these bile acids (250 mg/day) were compared in rats. METHODS: Bile acids were determined in intestinal contents, feces, urine, plasma, and liver by gas chromatography-mass spectrometry. RESULTS: The concentration of ursodeoxycholate in the liver of animals administered tauroursodeoxycholic acid (175 +/- 29 nmol/g) was greater (P < 0.05) than in animals administered ursodeoxycholic acid (79 +/- 19 nmol/g). Hepatic lithocholate was substantially higher after ursodeoxycholic acid administration (21 +/- 10 nmol/g) than after tauroursodeoxycholic acid administration (12 +/- 1 nmol/g). A concomitant reduction in the proportion of hydrophobic bile acids occurred that was greatest during tauroursodeoxycholic acid administration. In the intestinal tract, the mass of ursodeoxycholate and its specific metabolites was greater in rats administered tauroursodeoxycholic acid (27.2 mg) than those administered ursodeoxycholic acid (13.2 mg). In feces, the proportion of lithocholate was 21.9% +/- 4.9% and 5.4% +/- 4.0% after ursodeoxycholic acid and tauroursodeoxycholic acid administration, respectively. CONCLUSIONS: Compared with ursodeoxycholic acid, tauroursodeoxycholic acid induces a greater decrease in the percent composition of more hydrophobic bile acids within the pool, limits lithocholate formation, and increases hepatic ursodeoxycholate concentration. These differences are explained by increased hepatic extraction and reduced intestinal biotransformation and not by enhanced absorption of the amidated species.

Effects of biliary bile acid composition on biliary cholesterol saturation in gallstone patients treated with chenodeoxycholic acid and/or ursodeoxycholic acid.

Chenodeoxycholic acid (cheno) and ursodeoxycholic acid (urso) dissolve cholesterol gallstones in humans. In the present study conjugation of biliary bile acids with glycine and taurine and their effects on biliary cholesterol saturation were investigated during treatment with cheno, urso, and cheno-urso. Ten patients were included in this study, and every patient served as his own control. Each of the treatment periods lasted for 3 mo. During treatment with cheno or urso, daily doses of 11.9-15.6 mg/kg were administered, while during treatment with cheno-urso each bile acid was administered at one-half the dose. In the control period biliary bile acids consisted of 31.8 +/- 2.8% glycocheno, 10.9 +/- 1.2% taurocheno, 1.0 +/- 0.1% glycourso, and 0.3 +/- 0.1% taurourso. During the three treatment periods dihydroxy bile acids in bile and glycine conjugation of these dihydroxy bile acids increased significantly (P < 0.05). During treatment with urso the amounts of glycourso in bile were positively correlated to the dose of urso administered (P < 0.05). No correlation existed between urso dose and the amounts of taurourso in bile. Biliary cholesterol was 9.0 +/- 1.0 mol% in the control period and decreased during treatment with cheno, urso, and chenourso to 5.2 +/- 0.5, 3.7 +/- 0.3, and 3.8 +/- 0.3 mol%, respectively. Cholesterol saturation index corrected for the biliary content of glycourso and taurourso was 1.2 +/- 0.1 in the control period and decreased during treatment with cheno, urso, and cheno-urso to 0.8 +/- 0.1, 1.0 +/- 0.1 and 0.7 +/- 0.1, respectively. Thus urso treatment led to the lowest biliary content of cholesterol, but cheno-urso treatment led to significantly lower cholesterol saturation indices than urso treatment (P < 0.05).

Differences in the metabolism and disposition of ursodeoxycholic acid and of its taurine-conjugated species in patients with primary biliary cirrhosis.

The clinical effectiveness of ursodeoxycholate in the treatment of liver disease may be limited by its poor absorption and extensive biotransformation. Because in vitro and in vivo studies suggest that the more hydrophilic bile acid tauroursodeoxycholate has greater beneficial effects than ursodeoxycholate, we have compared for the first time the absorption, metabolism, and clinical responses to these bile acids in patients with primary biliary cirrhosis (PBC). Twelve female patients with PBC were sequentially administered tauroursodeoxycholate and ursodeoxycholate (750 mg/d for 2 months) in a randomized, cross-over study. Bile acids were measured in serum, duodenal bile, urine, and feces by gas chromatography-mass spectrometry (GC-MS). Biliary ursodeoxycholate enrichment was higher during tauroursodeoxycholate administration (32.6% vs. 29.2% during ursodeoxycholate; P <.05). Lithocholic acid concentration was consistently higher in all biological fluids during ursodeoxycholate administration. Fecal bile acid excretion was the major route of elimination of both bile acids; ursodeoxycholate accounted for 8% and 23% of the total fecal bile acids during tauroursodeoxycholate and ursodeoxycholate administration, respectively (P <.05). Tauroursodeoxycholate was better absorbed than ursodeoxycholate, and, although it was partially deconjugated and reconjugated with glycine, it underwent reduced biotransformation to more hydrophobic metabolites. This comparative study suggests that tauroursodeoxycholate has significant advantages over ursodeoxycholate that may be of benefit for long-term therapy in PBC.

Taurohyodeoxycholic acid protects against taurochenodeoxycholic acid-induced cholestasis in the rat.

The prevention of the hepatotoxic effects produced by intravenous infusion of taurochenodeoxycholic acid (TCDCA) by coinfusion with taurohyodeoxycholic acid (THDCA) was evaluated in bile fistula rats; the hepatoprotective effects of the latter were also compared with those of tauroursodeoxycholic acid (TUDCA). Rats infused with TCDCA at a dose of 8 micromol/min/kg showed reduced bile flow and calcium secretion, as well as increased biliary release of alkaline phosphatase (AP) and lactate dehydrogenase (LDH). This was associated with a very low biliary secretion rate of TCDCA (approximately 1 micromol/min/kg). Simultaneous infusion of THDCA or TUDCA at the same dose preserved bile flow and almost totally abolished the pathological leakage of the two enzymes into bile. The effect was slightly more potent for THDCA. The maximum secretion rate of TCDCA increased to the highest value (8 micromol/min/kg) when coinfused with either of the two hepatoprotective bile acids (BA), which were efficiently and completely secreted in the bile, without metabolism. Calcium output was also restored and phospholipid (PL) secretion increased with respect to the control saline infusion. This increase was higher in the THDCA study. These data show that THDCA is highly effective in the prevention of hepatotoxicity induced by intravenous infusion of TCDCA by facilitating its biliary secretion and reducing its hepatic residence time; this was associated with selective stimulation of PL biliary secretion.