Effect of Rifampicin on the Plasma Concentrations of Bile Acid-O-Sulfates in Monkeys and Human Liver-Transplanted Chimeric Mice With or Without Bile Flow Diversion.

The present study examined the significance of enterohepatic circulation and the effect of rifampicin [an inhibitor of organic anion-transporting polypeptide 1B (OATP1B)] on the plasma concentrations of bile acid-O-sulfates (glycochenodeoxycholate-O-sulfate, lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate) in monkeys and human liver-transplanted chimeric mice (PXB mouse). Rifampicin significantly increased the area under the curve of bile acid-O-sulfates in monkeys (13-69 times) and PXB mice (13-25 times) without bile flow diversion. Bile flow diversion reduced the concentration of plasma bile acid-O-sulfates under control conditions in monkeys and the concentration of plasma glycochenodeoxycholate-O-sulfate in PXB mice. It also diminished diurnal variation of plasma lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate in PXB mice under control conditions. Bile flow diversion did not affect the plasma concentration of bile acid-O-sulfates in monkeys and PXB mice treated with rifampicin. Plasma coproporphyrin I and III levels were constant in monkeys throughout the study, even with bile flow diversion. This study demonstrated that bile acid-O-sulfates are endogenous OATP1B biomarkers in monkeys and PXB mice. Enterohepatic circulation can affect the baseline levels of plasma bile acid-O-sulfates and modify the effect of OATP1B inhibition.

Dynamics of Bile Acid Profiles, GLP-1, and FGF19 After Laparoscopic Gastric Banding.

Context: An increase of bile acids (BAs), fibroblast growth factor 19 (FGF19), and glucagon-like peptide 1 (GLP-1) has been implicated in metabolic improvements after Roux-en-Y gastric bypass and vertical sleeve gastrectomy. However, data are still conflicting regarding their role after laparoscopic adjustable gastric banding (LAGB). Objective: To assess the fasting BA, FGF19, and GLP-1 concentrations in plasma before and after LAGB and to test for correlations with immunometabolic parameters. Furthermore, hepatic farnesoid X receptor (FXR) expression and regulation of FXR-dependent genes were analyzed. Design and Setting: Observational study at the University Hospital Innsbruck. Patients: Twenty obese patients. Interventions: Fasting plasma samples were taken before, 3, 6, and 12 months after LAGB. Liver biopsies were obtained at surgery and after 6 months postoperatively. Main Outcome Measures: BA profiles, GLP-1 and FGF19 levels, hepatic FXR expression and regulation of FXR target genes were determined. Results: Total, conjugated, and secondary BAs transiently increased 3 months after LAGB (P < 0.01). Only one BA, glycolithocholic acid sulfate, remained significantly elevated throughout the whole follow-up period (P < 0.05). GLP-1 had increased transiently 3 months after surgery (P < 0.01), whereas FGF19 levels increased continuously (P < 0.05). Insulin, homeostasis model assessment index, C-reactive protein, FGF19, and GLP-1 correlated positively with different BAs. No differences were seen in hepatic FXR expression and FXR-regulated genes. Conclusions: Our study results, not only identified LAGB-induced changes in BAs and BA-induced hormones, but also revealed associations between changes in BA profile with GLP-1 and FGF19.

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate.

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.

Biliary lipid secretion in the rat. The uncoupling of biliary cholesterol and phospholipid secretion from bile acid secretion by sulfated glycolithocholic acid.

Glycolithocholic acid and its sulfated derivative are major metabolites of the secondary bile acid lithocholic acid in man. Both compounds are known to induce cholestasis in experimental animals. We compared the effects of these endogenous hepatotoxins on bile production and biliary lipid composition in rats with chronic biliary drainage. The compounds were administered enterally at relatively low rates (5-50% of the rats' endogenous bile acid secretion in these experiments) to simulate enterohepatic circulation. Both compounds were substantially secreted into bile (more than 90% of dose); sulfated glycolithocholic acid unchanged and glycolithocholic acid after hepatic hydroxylation predominantly in the form of glyco-beta-muricholic acid (cf. Kuipers et al. (1986) Am. J. Physiol. 251, G189-G194). Neither glycolithocholic acid nor its sulfated derivative affected the biliary excretion of endogenous bile acids or bile flow in these experiments. In spite of this, phospholipid and cholesterol secretion were significantly reduced by sulfated glycolithocholic acid but were not altered by glycolithocholic acid. Phospholipid and cholesterol secretion rapidly decreased to 25 and 50% of their initial values, respectively, at biliary output rates of sulfated glycolithocholic acid up to 2 mumol/h, and did not further decrease when this output was increased to 6 mumol/h. Small unilamellar liposomes consisting of cholesterol, [Me-14C]choline-labeled phosphatidylcholine, phosphatidylserine and [3H]cholesteryl oleate in a 5:4:1:0.1 molar ratio were employed to label intrahepatic lipid pools. Administration of sulfated glycolithocholic acid slightly reduced bile acid synthesis from [3H]cholesteryl oleate, but significantly reduced the biliary secretion of [14C]phospholipid. Glycolithocholic acid did not affect the hepatic processing of liposomal lipids. It is concluded that sulfated glycolithocholic acid at low doses causes the uncoupling of biliary lipid secretion from that of bile acids, which might represent in initiating event in sulfated glycolithocholic acid hepatotoxicity.

Cholephilic characteristics of a new cytostatic complex of cisplatin with glycocholate (Bamet-R2).

The aim of this work was to investigate both the existence of enterohepatic circulation of cisplatin-cholylglycinate complex, Bamet-R2, and the relevance of biliary versus urinary excretion of this compound. Two experimental models were used: (i) intraluminal perfusion of 'in situ' ileum in anaesthetized rats bearing a biliary catheter that permitted bile sample collection and (ii) conscious rats in which a permanent intraarterial catheter had been implanted to carry out sequential blood sampling after intravenous (i.v.) or intragastric (i.g.) drug administration. Total platinum in serum, bile, ileum, liver, urine and feces was measured by flameless atomic absorption spectroscopy. Serum concentration versus time curves obtained after i.v. administration of 1 micromol Bamet-R2 or cisplatin revealed that the area under the curve was significantly higher for Bamet-R2 than for cisplatin (+48%). Non-ultrafiltrable platinum accounted for 54.8 and 48.4% of serum platinum 168 h after cisplatin and Bamet-R2 i.v. administration, respectively. When the animals received i.g. 1 micromol cisplatin or Bamet-R2, serum concentrations of total platinum were markedly higher (three-fold) after Bamet-R2 than after cisplatin administration. The area under the curve was, also in this case, significantly higher for Bamet-R2 than for cisplatin (+28%). This was in part due to the enhanced intestinal absorption of Bamet-R2, as confirmed in experiments on perfused rat ileum, where a markedly higher amount of the drug was found in ileum tissue and bile after perfusion with media containing Bamet-R2 as compared with experiments where cisplatin instead of Bamet-R2 was added to perfusion media. Moreover, after i.v. administration to conscious rats, excretion of Bamet-R2 by the kidney was three-fold lower than that of cisplatin, while elimination of the former compound into feces was four-fold higher than that of the latter. In summary, these results indicate that in addition to the previously reported cytostatic activity of Bamet-R2, this complex has interesting cholephilic characteristics typical of bile acids, such as low urinary excretion together with enhanced intestinal absorption and biliary secretion, probably endowed by the cholylglycyl moiety included in the Bamet-R2 molecule.

Radioimmunoassay of urinary 3 beta-hydroxy-5-cholenoyl glycine in hepatobiliary disease.

A radioimmunoassay for 3 beta-hydroxy-5-cholenoyl glycine in human urine has been developed. The antiserum was elicited with the antigen in which the steroid hapten is linked to a bovine serum albumin through the C-19 position. The [125I]-tyrosine derivative of the hapten was used as radioligand. The standard curves were linear ranging from 10 to 320 ng/mL. The cross-reactivities with other bile acids were not detectable and below 0.3% with cholesterol. Sample preparation includes extraction of 3 beta-hydroxy-5-cholenoyl glycine from urine and solvolysis of the sulfates--main form present in urine. Urinary excretion of 3 beta-hydroxy-5-cholenoyl glycine was 0.373 +/- 0.133 mumol/day in healthy adults. Urinary excretion of 3 beta-hydroxy-5-cholenoyl glycine increased in chronic liver dysfunction, hepatoma and obstructive jaundice in this order.

A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test.

Urinary levels of sulfated metabolites of lithocholic acid (LCA) are expected to be a useful index of liver function. Thus, a sensitive, specific, and feasible enzyme-linked immunosorbent assay (ELISA) of these sulfated LCA metabolites (LCA-Suls) should be established. A newly generated monoclonal antibody specific to glycolithocholic acid sulfate (glycine-amidated LCA-Sul (GLCA-Sul)) was immobilized on microtiter plates via a second antibody. A urine specimen and an alkaline phosphatase-labeled antigen were added to the plate, which was then incubated at room temperature for 3h. After this competitive reaction, bound enzyme activity was measured colorimetrically using p-nitrophenyl phosphate as a substrate. The detection limit for GLCA-Sul was 0.4 pg/assay. Nonamidated LCA-Sul and taurine-conjugated LCA-Sul showed 40 and 11% cross-reactivities, respectively, while 3-sulfates of cholic acid (CA; 0.02%), chenodeoxycholic acid (CDCA; 0.63%), and deoxycholic acid (DCA; 2.2%) exhibited very low cross-reactivities. Applicability of the ELISA system to clinical samples was well validated by parallelism, recovery test, and intra/inter-assay variance. Enzymatic deconjugation with bile acids sulfatase resulted in dramatically decreased urinary levels, supporting the specificity of the ELISA toward GLCA-Sul. The mean GLCA-Sul levels in early morning urine from healthy volunteers were 314 ng/mg Ucre (males: n=16) and 507 ng/mg Ucre (females: n=9). Patients with liver diseases, including chronic hepatitis (CH) and liver cirrhosis (LC) exhibited significantly higher values (mean 5222 ng/mg Ucre: n=21). The present 'monoclonal ELISA' is predicted to be useful as a novel noninvasive diagnostic tool for liver function and hepatobiliary diseases.

Evaluation of enzymic and radioimmunoassay methods for measuring serum bile acids.

Serum bile acids were measured in control subjects and in 23 patients with cirrhosis or cholestasis using an enzymic method which measures total non-sulphated bile acids and 2 commercially available radioimmunoassay methods which measure cholylglycine (CG) and sulpholithocholylglycine (SLCG) respectively. All 3 methods gave abnormal values in patients with cholestasis. In cirrhosis the CG method gave abnormal values more often than the enzymic method. The SLCG method was much less sensitive. Measurement of CG in 2 postprandial samples per patient detected only one patient with normal fasting but abnormal postprandial levels. Analysis of samples by gas liquid chromatography (GLC) showed that the enzymic and CG methods gave a good estimation of total non-sulphated bile acids and cholic acid respectively. Values determined by the SLCG method were much higher than the equivalent GLC values. The CG method is faster than the enzymic method and is less expensive provided sample are analysed in sufficiently large batches.

[Pruritus and bile acids. Determination of sulfalithoglycocholate and glycocholate in suction blister fluid and in serum (author's transl)]. / Pruritus und Gallensäuren. Bestimmung von Sulfalithocholylglycin und Cholylglycin in der Saugblasenflüssigkeit und im Serum.

Until now is not clear, whether bile acids play a role in hepatogenic pruritus in patients with cholestasis. A correlation between serum levels and itching does not exist, and therefore an accumulation of bile acids in the skin was made responsible for the etiology of pruritus. However, this could not be proved in skin homogenisates whereas bile acids were found accumulated in the surface lipids of the skin. For this reason, the investigations presented here deal with the determination of glycocholate and sulfalithoglycocholate in suction blister fluid and in serum. The levels of these bile acids were remarkably lower in the fluid of the subepidermally located blisters than in the serum. No significant difference was found between the group of patients with pruritus and cholestasis and the group without cholestasis. These results show that an accumulation of bile acids in the skin does not exist. A correlation between bile acids in the skin and pruritus cannot be proved.

A simplified procedure for the isolation of bile acids from serum based on a batch extraction with the non-ionic resin--Amberlite XAD-7.

A simple and reproducible method using the non-ionic resin, Amberlite XAD-7, for the isolation of bile acids from serum by a batch procedure is described. Recoveries were greater than 95% for the non-sulphated bile acids and greater than 70% for the sulphate esters of bile acids. By using 1 g of resin, recoveries were independent of the mass (0.1-5 mumol) of the bile acid present. Up to 35 samples a day can be extracted without requiring dedication of the operator. When serum extracts were analysed by the 3alpha-hydroxysteroid dehydrogenase procedure for estimation of bile acids, virtually all the background fluorescence was eliminated. These extracts were also suitable for gas liquid chromatography, thin layer chromatography, and radioimmunoassay.

[Clinico-diagnostic significance of the determination of bile acids in chronic liver diseases]. / Significato clinico-diagnostico del dosaggio di alcuni acidi biliari sierici nelle epatopatie croniche.

Certain conjugated biliary acids (total pool - choliglycine - sulpholytic choliglycine) and the following haematochemical parameters: total bilirubin and its direct quota, alkaline phosphatase, albumin, prothrombin activity, gamma globulin, oxalacetic and pyruvic transaminase were radioimmunologically (RIA) studied in 115 subjects. Subjects were divided into the following subgroups: --20 normal controls; --20 cases of persistent chronic hepatitis; --20 cases of active chronic hepatitis; --15 cases of A.C.H. with cirrhosis; --20 cases of cirrhosis without direct hyperbilirubinaemia; --20 cases of cirrhosis with direct hyperbilirubinaemia. Each case was assigned to its particular group on the basis of the histological report on each patient. The following observations were drawn from the results obtained: --there is a progressive increase in above normal biliary acid rate in proportion to the gravity of the liver pathology; --choliglycine especially and to a lesser extent the total pool increased sufficiently to distinguish between normal and hepatopathic subjects (PCH and ACH) and also between PCH and ACH patients; --the combination of cirrhosis and ACH causes a significant increase in total pool and chliglycine over levels noted in ACH alone; --in contrast no difference is found between the levels of these acids in inactive (or minimally active) cirrhosis and ACH with cirrhosis; --gamma globulin, oxalacetic and pyruvic transaminase levels were found to have substantially the same diagnostic significance as choliglycine in the early stages of liver diseases. Significant correlations were also encountered between total conjugated biliary acid pool and choliglycine (not in the group with cirrhosis without direct hyperbilirubinaemia) and between total pool and choliglycine with haematochemical cholestasis test results (alkaline phosphatase and total and direct bilirubin) the latter only in the two cirrhotics groups. In conclusion, choliglycine was found to be the most sensitive of the biliary acids routinely measured by RIA and is valuable in clinical practice not as a substitute for the main liver tests but as an extremely useful and sensitive addition to them. In clinical practice, its use is recommended in the diagnosis and monitoring of healthy subjects at risk and those with chronic liver conditions (PCH, ACH, ACH + C).

[Determination of serum bile acids in hepatobiliary diseases. Clinical applicability]. / La determinazione sierica degli acidi biliari nelle malattie epatobiliari. Applicabilità clinica.

Determination of serum bile acids has long been regarded as the most sensitive indicator of liver function. An assessment was made of the clinical applicability of RIA evaluation of two of these acids, cholylglycine (CG) and sulpholithochocholyglycine (SLGG), on an empty stomach and 2hr after a cholecystokinetic meal in 109 liver patients ans 20 controls. After the meal test, both acids proved more sensitive than the usual liver function indices. Different mean values were observed for different diseases. They were in good correlation with the extent of histological damage. Values were highest in obstructive icterus, cirrhosis and neoplasia of the liver, fairly highly high in steatofibrosis, ACH and PCH, and normal in viral hepatitis in the course of resolution, aspecific reactive hepatitis, and steatosis. The meal test thus proved a good indicator of liver disease. Its wider use is to be hoped for in order that its limits and applications may be better understood.

[Synthesis and properties of fluorescent labeled detergents analogues of glycocholic acid]. / Sintez i svoistva fluorestsentnomechenykh detergentov, analogov glikokholevoi kisloty.

For studying membrane processes with participation of detergents, fluorescent analogues of glycocholic acid containing p-hydroxybenzyl, 7-nitrobenz-2-oxa-1,3-diazol-4-yl, or fluorescein-5-thiocarbamoyl fluorophore in the glycyl moiety attached to glycocholic acid were synthesized. The fluorophores are in the probes near their carboxyl groups and, in membrane systems, should therefore be situated on the interface and be sensitive to phase transitions. The critical micelle concentrations were determined for the analogues and found to be close to those of cholate and glycocholate in the case of the first two compounds. We presume that the behavior of the probes in membrane systems will mimic the behavior of the bile acid salts.

Enhanced transbuccal salmon calcitonin (sCT) delivery: effect of chemical enhancers and electrical assistance on in vitro sCT buccal permeation.

This study investigates the combined effect of absorption enhancers and electrical assistance on transbuccal salmon calcitonin (sCT) delivery, using fresh swine buccal tissue. We placed 200 IU (40 µg/mL) of each sCT formulation--containing various concentrations of ethanol, N-acetyl-L-cysteine (NAC), and sodium deoxyglycocholate (SDGC)--onto the donor part of a Franz diffusion cell. Then, 0.5 mA/cm(2) of fixed anodal current was applied alone or combined with chemical enhancers. The amount of permeated sCT was analyzed using an ELISA kit, and biophysical changes of the buccal mucosa were investigated using FT-IR spectroscopy, and hematoxylin-eosin staining methods were used to evaluate histological alteration of the buccal tissues. The flux (J(s)) of sCT increased with the addition of absorption enhancer groups, but it was significantly enhanced by the application of anodal iontophoresis (ITP). FT-IR study revealed that all groups caused an increase in lipid fluidity but only the groups containing SDGC showed statistically significant difference. Although the histological data of SDGC groups showed a possibility for tissue damage, the present enhancing methods appear to be safe. In conclusion, the combination of absorption enhancers and electrical assistance is a potential strategy for the enhancement of transbuccal sCT delivery.