Effect of beta-muricholic acid on the prevention and dissolution of cholesterol gallstones in C57L/J mice

This study investigated whether beta-muricholic acid, a natural trihydroxy hydrophilic bile acid of rodents, acts as a biliary cholesterol-desaturating agent to prevent cholesterol gallstones and if it facilitates the dissolution of gallstones compared with ursodeoxycholic acid (UDCA). For gallstone prevention study, gallstone-susceptible male C57L mice were fed 8 weeks with a lithogenic diet (2% cholesterol and 0.5% cholic acid) with or without 0.5% UDCA or beta-muricholic acid. For gallstone dissolution study, additional groups of mice that have formed gallstones were fed chow with or without 0.5% beta-muricholic acid or UDCA for 8 weeks. One hundred percent of mice fed the lithogenic diet formed cholesterol gallstones. Addition of beta-muricholic acid and UDCA decreased gallstone prevalence to 20% and 50% through significantly reducing biliary secretion rate, saturation index, and intestinal absorption of cholesterol, as well as inducing phase boundary shift and an enlarged Region E that prevented the transition of cholesterol from its liquid crystalline phase to solid crystals and stones. Eight weeks of beta-muricholic acid and UDCA administration produced complete gallstone dissolution rates of 100% and 60% compared with the chow (10%). We conclude that beta-muricholic acid is more effective than UDCA in treating or preventing diet-induced or experimental cholesterol gallstones in mice.     

General surgery: biliary surgery.

The management of biliary tract disease has changed completely as a result of minimally invasive treatment. For most patients with gallstones that cause symptoms a laparoscopic cholecystectomy will treat the condition with minimal morbidity and a short recovery period. If complications are encountered, conversion to a mini-cholecystectomy gives results that are nearly as good. Acute cholecystitis can be treated by percutaneous drainage followed either by percutaneous cholecystolithotomy or a laparoscopic cholecystectomy. Gallstones in the bile duct are best treated by endoscopic sphincterotomy with duct clearance. The day of the large cholecystectomy scar with its subsequent incisional hernia has gone.     

Bile acids in treatment of ocular disease

Bear bile has been included in Asian pharmacopeias for thousands of years in treatment of several diseases, ranging from sore throat to hemorrhoids. The hydrophilic bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) are the major bile acids of bear bile. Both of these are available as synthetic formulations and are approved by the health administrations of several countries for treatment of cirrhosis and gallstones. This review briefly covers the use of bear bile in Traditional Chinese Medicine, bile acid physiology, approved use of UDCA and TUDCA in Western medicine, and recent research exploring their neuroprotective properties, including in models of ocular disease.     

Clinical evidence of hepatoprotection induced by ursodeoxycholic acid.

Ursodeoxycholic-acid (UDCA) was introduced to the clinical practice as an effective agent for the dissolution of gallstones. The efficacy of UDCA was proved recently in the treatment of patients with chronic cholestatic liver disease. We demonstrate the hepatoprotective effect of UDCA in a patient with chronic cholestatic liver disease. A sixty-nine years old male patient was admitted to our department with severe jaundice. The laboratory and radiologic examinations revealed significant cholestasis without any morphological alterations. Among the serological tests the anti-HCV antibody was positive. Based on these findings and anamnestic data (no blood transfusion and/or operation), sporadic chronic C virus hepatitis was assumed with dominant cholestasis. The corticosteroid therapy even in high doses was ineffective, the liver function parameters worsened. Later UDCA (Ursofalk, Falk Pharma) was given at a dose of 250 mg three times daily. Clinical improvement was seen after the first week of UDCA treatment. The patient's complaints relieved parallel with decrease of serum bilirubin, gamma-glutamyl transferase and transaminase levels. These parameters showed further decrease during the treatment.     

Ursodeoxycholic acid (UDCA) in the treatment of chronic cholestatic diseases.

Several studies suggest that UDCA treatment has beneficial effects in chronic cholestatic diseases. We designed a controlled trial to assess the efficacy and tolerance of UCDA in primary biliary cirrhosis (PBC): 73 patients received UDCA (13-15 mg/kg per day) and 73 a placebo. One side-effect required interruption of therapy in each group. The relative risk of treatment failure (doubling of the bilirubin level or occurrence of a severe complication of cirrhosis) was 3 times higher in the placebo group. Pruritus resolved in 40% of the patients of UDCA group vs 19% in placebo group. Biological and histological parameters significantly improved in the patients receiving UDCA. Unexpectedly, immune parameters, including IgM levels and anti-mitochondrial antibody titers, also improved. The Mayo risk score was significantly different between the two groups at one and two years, suggesting that UDCA could prolong survival in PBC. Recent studies suggest that UDCA could have immunoregulating properties. Abnormal MHC class I expression by hepatocytes, observed in PBC, was dramatically reduced by UDCA treatment. Cholestasis itself induces hepatic MHC expression: hepatocyte MHC class I expression was present in 6/6 cholestatic patients vs 0/8 control subjects. Experimental cholestasis in the rat induced MHC class I expression. Cyclosporin or corticosteroids had no effect on this overexpression, suggesting that an immune mechanism is not involved in this phenomenon. To assess the effect of bile acids on MHC expression, human hepatocytes were incubated with bile acids. Chenodeoxycholic acid (CDCA) (an endogenous bile acid) but not UDCA induced a dose-dependent MHC class I hyperexpression. UDCA suppressed the CDCA-induced MHC hyperexpression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel FXR (farnesoid X receptor) modulators: Potential therapies for cholesterol gallstone disease

Metabolic disorders such as diabetes are known risk factors for developing cholesterol gallstone disease (CGD). Cholesterol gallstone disease is one of the most prevalent digestive diseases, leading to considerable financial and social burden worldwide. Ursodeoxycholic acid (UDCA) is the only bile acid drug approved by FDA for the non-surgical treatment of gallstones. However, the molecular link between UDCA and CGD is unclear. Previous data suggest that the farnesoid X receptor (FXR), a bile acid nuclear receptor, may protect against the development of CGD. In studies aimed at identifying the role of FXR, we recently identify a novel chemical tool, 6EUDCA (6-αethyl-ursodeoxycholic acid), a synthetic derivative of UDCA, for studying FXR. We found that 6EUDCA binds FXR stronger than UDCA in a TR-FRET binding assay. This result was supported by computational docking models that suggest 6EUDCA forms a more extensive hydrogen bound network with FXR. Interestingly, neither compound could activate FXR target genes in human nor mouse liver cells, suggesting UDCA and 6EUDCA activate non-genomic signals in an FXR-dependent manner. Overall these studies may lead to the identification of a novel mechanism by which bile acids regulate cell function, and 6EUDCA may be an effective targeted CGD therapeutic.     

腹腔镜联合十二指肠镜治疗胆结石合并胆总管结石的临床疗效

目的:探讨腹腔镜联合十二指肠镜治疗胆结石合并胆总管结石的临床疗效。方法:收集我院肝胆科2012年6月到2014年3月住院的胆结石合并胆总管结石60例,按照随机数字表法分为试验组和对照组,各30例,试验组给予腹腔镜联合十二指肠镜治疗,对照组给予传统的开腹胆囊切除以及胆总管探查取石,观察对比两组的临床疗效。结果:试验组手术时间、住院时间、术后恢复时间及术中出血量明显的短于或少于对照组,差异均具有统计学意义(均P0.05),试验组治愈率为96.7%明显的高于对照组的66.7%,差异有统计学意义(P0.05)。两组主要不良反应有:结石残留、感染、十二指肠穿孔、恶性呕吐及伤口疼痛等,试验组不良反应发生率为16.67%明显的低于对照组总的73.33%,差异有统计学意义(P0.05)。结论:腹腔镜联合十二指肠镜治疗胆结石合并胆总管结石较传统的开腹手术治疗的效果好、恢复时间短、手术创伤小,且具有一定的安全性,不良反应发生少,值得临床推广使用。     

Variation in Hepatic Bile Composition Following Cholecystectomy in Patients with Previous Gallstones

The composition of fasting hepatic bile was analyzed in 63 samples from 8 patients following cholecystectomy to determine if bile was lithogenic in patients with previous cholesterol gallstones after removal of the gallbladder. Bile specimens were obtained from t-tubes over a 7-20 day study period following re-establishment of the enterohepatic circulation. Bile composition varied on a day to day basis in each patient. 18 of 63 samples were lithogenic according to criteria of Admirand and Small while 35 of 63 samples were lithogenic according to criteria of Hegardt and Dam. Variations in the composition of hepatic bile appeared related to changes in the excretion rate of bile acids. These studies demonstrate that hepatic bile may be lithogenic after cholecystectomy and indicate that factors other than sequestration of the bile acid pool in the gallbladder influence the enterohepatic circulation of bile acids and the lithogenicity of bile.     

Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.     

Increases in biliary cholesterol-to-bile acid ratio in pregnant hamsters fed low and high levels of cholesterol

Gallstones develop when the secretion of cholesterol is elevated compared with the secretion of bile acids into bile. One of the risk factors for the formation of gallstones is pregnancy. Because the pregnancy-induced increase in hepatic cholesterol synthesis rates could play a critical role in the development of cholesterol stones, the aim of the present study was to determine whether stone formation, as assessed by the ratio of cholesterol to bile acids in bile, could be ablated by blocking the pregnancy-induced increase in hepatic sterol synthesis rates. Golden Syrian hamsters were fed either ground chow or chow supplemented with 0.5% cholesterol for 3 wk and studied in the nonpregnant state or in late gestation. In chow-fed animals, a 1.6-fold increase in the ratio of cholesterol to bile acids occurred simultaneously with a sevenfold increase in hepatic sterol synthesis rate and a ninefold increase in the amount of newly synthesized cholesterol secreted into the bile in late gestation. In the cholesterol-fed dams, an increase in the ratio of cholesterol to bile acids occurred even with the lack of induction of hepatic sterol synthesis rates during pregnancy. Thus it appears that the marked induction of hepatic sterol synthesis rates during gestation is not essential for the pregnancy-induced cholesterol saturation of bile when cholesterol is fed to animals.     

Hepatic cholesterol metabolism in cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 +/- 13 vs. 75 +/- 5%, P less than 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 +/- 11 vs. and 109 +/- 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 alpha-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 +/- 1.1 vs. 8.0 +/- 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A:cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 +/- 0.4 vs. 6.7 +/- 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.     

Calcium binding by bile acids: in vitro studies using a calcium ion electrode

In this study, we compared in vitro calcium binding by the taurine and glycine conjugates of the major bile acids in human bile: cholic (CA), chenodeoxycholic (CDCA) and deoxycholic (DCA) acids, together with the cholelitholytic bile acids ursodeoxycholic (UDCA) and ursocholic (UCA) acids. At physiological total calcium (CaTOT) (1-15 mM) and bile acid (BA) (10-50 mM) concentrations, all the bile acids caused concentration-dependent falls in [Ca2+], suggesting calcium binding. Except for glycine-conjugated CDCA, all the other calcium-bile acid complexes were soluble in 150 mM NaCl. The calcium binding affinities followed the pattern: dihydroxy (CDCA, UDCA and DCA) greater than trihydroxy (CA and UCA) bile acids, and glycine conjugates greater than taurine conjugates. The glycine conjugate of UDCA, which increases during UDCA treatment, had the highest calcium binding affinity. Ten-20 mM phospholipid modestly increased calcium binding by CA conjugates, but not by CDCA, UDCA, and DCA conjugates. Phospholipid also prevented the precipitation of glyco-CDCA in the presence of calcium. Bile acid-calcium biding was pH-independent over the range 6.5-8.5. The different calcium binding affinities of the major biliary bile acids may partly explain their varying effects on biliary calcium secretion. The results also suggest that neither precipitation of calcium-bile acid complexes nor impaired calcium binding by bile acids is important in the pathogenesis of human calcium gallstone formation.     

Inhibitory Effects of Bile Acids on Enzymatic and Pharmacological Activities of a Snake Venom Phospholipase A<Subscript>2</Subscript> from Group IIA

Bile acids, such as cholic acid (CA) and ursodeoxycholic acid (UDCA) have shown to decrease or increase the enzymatic activity of group IB pancreatic PLA₂, depending on the concentration used. Studies suggest that the inhibition of hydrolysis rate of the substrate is due to formation in aqueous phase of a complex between bile acid and PLA₂, which is catalytically inert. For this reason, we tested the inhibition of the enzymatic activity of group IIA snake venom PLA₂ by bile acids, using an aqueous phase model. In addition, we measured the ability of bile acids to inhibit the toxic effects caused by the mentioned toxin. UDCA and CA inhibited the enzymatic activity of the PLA₂ in a competitive mode. Moreover, these compounds inhibited myotoxic, cytotoxic and edema-forming activities induced by the toxin, but UDCA was more efficient than CA. It was demonstrated that bile acids interact directly with this protein by causing slight changes in the intrinsic fluorescence spectra. Preliminary molecular docking studies suggest that bile acids interact with amino acids at the active site of the PLA₂ through different interactions, CA showed hydrogen bonds with His48, whereas, UDCA displayed with Asp49. Results obtained herein may turn UDCA and CA into promising models for the development of new molecules with anti-inflammatory and anti-snake venom PLA₂ properties.     

一步法腹腔镜胆囊切术联合胆总管探查取石术在慢性胆囊炎胆囊结石合并胆总管结石治疗中的有效性及安全性探究

摘要 目的：探究一步法腹腔镜胆囊切术（LC）联合胆总管探查取石术（LCBDE）在慢性胆囊炎胆囊结石合并胆总管结石治疗中的有效性及安全性。方法：纳入2018年6月至2021年9月行一步法LC+LCBDE治疗的慢性胆囊炎胆囊结石合并胆总管结石患者49例（观察组）,并以行开腹胆囊切除术+胆总管切开取石治疗的慢性胆囊炎胆囊结石合并胆总管结石患者43例为对照组,比较两组手术疗效及手术相关指标;观察患者手术前后肝功能指标、胆红素水平及免疫功能变化,并统计患者术后并发症发生情况。结果：观察组及对照组手术成功率均为100%,两组对比无明显差异（P>0.05）;观察组手术时间、肠鸣音恢复时间、肛门恢复排气时间及住院时间短于对照组,术中出血量少于对照组（P<0.05）;观察组术后丙氨酸氨基转移酶（ALT）、γ-谷氨酰转肽酶（GGT）、天冬氨酸氨基转移酶（AST）及总胆红素（TBIL）、直接胆红素（DBIL）、间接胆红素（IBIL）水平均低于对照组（P<0.05）;观察组术后7 d的免疫球蛋白A（IgA）、免疫球蛋白G（IgG）、免疫球蛋白M（IgM）水平均高于对照组（P<0.05）;观察组术后并发症发生率低于对照组（P<0.05）。结论：一步法LC+LCBDE治疗慢性胆囊炎胆囊结石合并胆总管结石的成功率高,可促进术后胃肠功能及肝功能恢复,提高机体免疫力,并能降低术后并发症发生率。     

Bile acids with a cyclopropyl-containing side chain. IV. Physicochemical and biological properties of the four diastereoisomers of 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid

To define the influence of the side chain modification on physicochemical and biological properties of bile acids, 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid, a cyclopropyl analog of ursodeoxycholic acid (UDCA) was synthesized in both unconjugated and taurine-conjugated form. The presence of a cyclopropyl ring at C-22, C-23 position introduces chirality generating four diasteroisomers (A, B, C, and D) which greatly differ for the hydrophilicity and critical micellar concentration: A and B are more hydrophilic (K' = 0.21, 0.80 and CMC = 25,20 mM, respectively) than UDCA (K' = 0.95; CMC = 19 mM) while C and D are more hydrophobic and with lower CMC (K' = 1.30, 2.05; CMC = 14, 10 mM, respectively) than UDCA. Differences in these properties are related to the orientation of the C-25 carboxyl which in isomers A and B is oriented toward the back of the steroid body, reducing the continuity of the hydrophobic area. Using the isolated perfused rat liver we found that the isomers inhibited [3H]UDCA uptake differently. Isomer D (noncompetitive) was the most potent (51%) while isomer A (competitive) was the least potent (15%). When infused intravenously to rats, only D isomer and UDCA were quantitatively recovered in bile. They were secreted predominantly as taurine and glycine conjugates. Isomers A, B, C are not conjugated and only partially recovered in bile as unconjugates (less than 15% of the administered dose). The increase in bile flow per unit increase in bile acid secretion induced by isomers A, B, and C, was much greater than that induced by isomer D which is similar to that of UDCA (0.32 +/- 0.04 and 0.22 +/- 0.01, respectively) while it is reduced during infusion of the other isomers. When infused as taurine conjugates they behaved similarly to tauroursodeoxycholic acid. When incubated in anaerobic conditions with human stools only isomer D is partially 7-dehydroxylated (t/2 = 18 hr) even though slower than UDCA (t/2 = 5 hr). The substrate specificity of the taurine conjugated toward cholyglycine hydrolase is very poor, only isomers C and D are partially deconjugated with a kinetics much slower than that of UDCA (10 hr vs. 0.2 hr). By using molecular models it is possible to explain these differences due to the conformation of the side chain that, in the case of isomer D, is quite similar to UDCA. These data are useful to explain the metabolism of dihydroxy bile acids and to design new analogs with enhanced cholelitholytic activity.     

The Reversed Feto-Maternal Bile Acid Gradient in Intrahepatic Cholestasis of Pregnancy Is Corrected by Ursodeoxycholic Acid

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder associated with an increased risk of adverse fetal outcomes. It is characterised by raised maternal serum bile acids, which are believed to cause the adverse outcomes. ICP is commonly treated with ursodeoxycholic acid (UDCA). This study aimed to determine the fetal and maternal bile acid profiles in normal and ICP pregnancies, and to examine the effect of UDCA treatment. Matched maternal and umbilical cord serum samples were collected from untreated ICP (n = 18), UDCA-treated ICP (n = 46) and uncomplicated pregnancy (n = 15) cases at the time of delivery. Nineteen individual bile acids were measured using HPLC-MS/MS. Maternal and fetal serum bile acids are significantly raised in ICP compared with normal pregnancy (p = <0.0001 and <0.05, respectively), predominantly due to increased levels of conjugated cholic and chenodeoxycholic acid. There are no differences between the umbilical cord artery and cord vein levels of the major bile acid species. The feto-maternal gradient of bile acids is reversed in ICP. Treatment with UDCA significantly reduces serum bile acids in the maternal compartment (p = <0.0001), thereby reducing the feto-maternal transplacental gradient. UDCA-treatment does not cause a clinically important increase in lithocholic acid (LCA) concentrations. ICP is associated with significant quantitative and qualitative changes in the maternal and fetal bile acid pools. Treatment with UDCA reduces the level of bile acids in both compartments and reverses the qualitative changes. We have not found evidence to support the suggestion that UDCA treatment increases fetal LCA concentrations to deleterious levels.     

Beneficial effects of UDCA and norUDCA in a rodent model of steatosis are linked to modulation of GPBAR1/FXR signaling

Non-alcoholic steatosis (NAFLD) and steatohepatitis (NASH) are two highly prevalent human disorders for which therapy remains suboptimal. Bile acids play an essential role in regulating liver metabolism, and several bile acids-based therapy are currently investigated for their potential therapeutic efficacy in NAFLD/NASH. Bile acids exert their functions, at least in part, by modulating two main receptors the Farnesoid-x-receptor (FXR) and the G protein-coupled receptor, GPBAR1. In the present study we have compared the pharmacological effects of two bile acids, the ursodeoxycholic acid (UDCA) and its derivative norUDCA, in a model of NAFLD/NASH induced by feeding mice with a Western diet for 12 weeks. The results of these studies demonstrated that both UDCA and norUDCA protected against development of steatosis and fibrosis, but did not reduce the hepatocytes ballooning nor the development of a pro-atherogenic lipid profile. Both agents reduced liver lipogenesis and ameliorated insulin sensitivity and adipocytes signaling as shown by increased expression of adiponectin. Mechanistically, UDCA acts as weak GPBAR1 agonist, while norUDCA exerted no effect on both GPBAR1 and FXR. In vivo administration of UDCA resets bile acid synthesis and promotes a shift toward bile acids species that are GPBAR1 agonists, UDCA, TUDCA and hyodeoxycholic acid, and increases GLP1 expression in the ileum. In contrast norUDCA is poorly metabolized exerting a minimal impact on GPBAR1 signaling. Together, these data, highlight the potential role of UDCA and norUDCA in treating of NAFLD, though these beneficial effects are supported by different mechanisms.     

Isoursodeoxycholic acid: metabolism and therapeutic effects in primary biliary cirrhosis

Significant amounts of ursodeoxycholic acid (UDCA) used for the treatment of patients with primary biliary cirrhosis (PBC) become epimerized at C-3 to isoUDCA. We investigated the metabolism of isoUDCA and a possible pharmacologic effect in five patients (51.4 +/- 5.8 years old; 3 females, 2 males) with PBC and persistent elevations of gamma-glutamyl transpeptidase (gamma-GT) and alkaline phosphatase despite treatment with UDCA for more than one year. Serum samples were analyzed for bile acid metabolites and surrogate markers of cholestasis in 4-week intervals after 1 g/d UDCA, wash-out, 0.5 g/d isoUDCA, 0.75 g/d isoUDCA, 0.75 g/d UDCA, and two further periods with 1 g/d UDCA. Bile acids in urine were analyzed after wash-out, 0.5 and 0.75 g/d isoUDCA, and 0.75 and 1 g/d UDCA. During wash-out, AST, AP, and gamma-GT rose significantly (P < 0.05) but reversed to previous levels during the first isoUDCA period, with 0.5 g/d only. No further improvements were observed after increasing the dose of isoUDCA or switching back to UDCA. In serum, the relative amounts of isoUDCA and UDCA were 8.1 +/- 7.4% and 16.2 +/- 6.4% during 0.5 g/d isoUDCA, 6.2 +/- 2.5% and 45.0 +/- 4.1% during 0.75 g/d isoUDCA, and 0.5;-3% and 56.4;-60.0%, respectively, during UDCA. In urine, UDCA was the predominant bile acid both during isoUDCA and UDCA medications. The similar serum enrichment and urinary excretion of UDCA during administration of either isoUDCA or UDCA together with low concentrations of the intermediate of isomerization, 3-dehydro-UDCA, indicate a first-pass epimerization of isoUDCA to UDCA in the liver. Approximately 25% of serum isoUDCA and 10% of serum UDCA were conjugated with either glucuronic acid or N-acetylglucosamine, indicating hepatic formation and systemic secretion of glycosidic conjugates.In PBC patients, isoUDCA becomes isomerized to UDCA and has similar effects on surrogate markers of cholestasis. Thus, isoUDCA has pro-drug characteristics.     

Dynamic mechanistic modeling of the multienzymatic one‐pot reduction of dehydrocholic acid to 12‐keto ursodeoxycholic acid with competing substrates and cofactors

Ursodeoxycholic acid (UDCA) is a bile acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two‐step reduction of dehydrocholic acid to 12‐keto‐ursodeoxycholic acid (12‐keto‐UDCA), which can be conducted in a multienzymatic one‐pot process using 3α‐hydroxysteroid dehydrogenase (3α‐HSDH), 7β‐hydroxysteroid dehydrogenase (7β‐HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one‐pot reduction which involves three enzymes, four different bile acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi–bi mechanism with EBQ‐type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch‐process was established by coupling rate equations and mass balances. Validation experiments of the one‐pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:375–386, 2015     

Characteristics of apoptosis in HCT116 colon cancer cells induced by deoxycholic acid

Hydrophobic bile acids induce apoptosis in both colon cancer cells and hepatocytes. The mechanism by which colon cancer cells respond to bile acids is thought to be different from that of hepatocytes. Therefore, we investigated the characteristics of apoptosis in colon cancer cell line HCT116. Hydrophobic bile acids, i.e., deoxycholic acid (DCA), and chenodeoxycholic acid, induced apoptosis in HCT116 cells. Apoptotic indications were detectable at as early as 30 min and the extent increased in time- and concentration-dependent manners. SDS and a hydrophilic bile acid, cholic acid, did not induce apoptosis even at cytotoxic concentrations. Pretreatment with cycloheximide failed to inhibit apoptosis, suggesting that protein synthesis is not involved in the apoptotic response. Release of cytochrome c from mitochondria and activation of caspase-9 were detectable after 5 and 10 min, respectively, whereas remarkable activation of Bid was not detected. Ursodeoxycholic acid (UDCA) protected HCT116 cells from DCA-induced apoptosis but a preincubation period of > or =5 h was required. Nevertheless, UDCA did not inhibit cytochrome c release from mitochondria. Our results indicate that hydrophobic bile acids induce apoptosis in HCT116 cells by releasing cytochrome c from mitochondria via an undefined but specific mechanism, and that UDCA protects HCT116 cells by acting downstream of cytochrome c release.