Analysis of bile acid glucuronides in urine. Identification of 3α,6α,12α-trihydroxy-5β-cholanoic acid

doi:10.1016/0022-4731(80)90164-8

Journal of Steroid Biochemistry

Volume 13, Issue 8, August 1980, Pages 907-916

https://doi.org/10.1016/0022-4731(80)90164-8 Get rights and content

Abstract

A method is described for the analysis of bile acid glucuronides in urine. Following extraction on Amberlite XAD-2 and separation of bile acid conjugates on DEAP-LH-20, the fractions containing bile acid glucuronides are methyl esterified and filtered through DEAP-LH-20, yielding a fraction of purified bile acid glucuronide methyl esters. Individual glucuronides are separated in a reversed phase system on Lipidex 1000 and are analyzed as TMS ethers by GC/MS. Oxidation with periodate, which transforms the glucuronyl moiety into a formate ester, followed by oxidation with chromic acid and conversion of keto groups to O-methyloximes, permits determination of the site of conjugation with glucuronic acid.

The daily excretion of bile acid glucuronides in urine of healthy subjects was 0.8–16.2 μmol (12–36% of the total bile acid excretion in urine). Only ether glucuronides were observed, most of which were otherwise unconjugated. A major part of the bile acids were hydroxylated at C-6, and 3α,6α,12α-trihydroxy-5β-cholanoic acid was identified as a quantitatively important new bile acid. The glucuronyl moiety was attached at C-6 of hyodeoxycholic acid and at C-3 of cholic and chenodeoxycholic acids. The results may indicate a possible coupling of glucuronic acid conjugation with 6α-hydroxylation of secondary bile acids.

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UGT enzymes, located in the endoplasmic reticulum, can glucuronidate BAs and, therefore, facilitate their elimination from the liver by increasing their hydrophilicity (Trottier et al., 2006). In addition, in healthy subjects, 12 to 36% of the total BA urinary excretion is represented by BA-glucuronides (Alme and Sjovall, 1980). Sulfation is another phase II conjugation reaction that contributes to bile acid detoxification.
Bile acid (BA) homeostasis is a complex and precisely regulated process to prevent impaired BA flow and the development of cholestasis. Several reactions, namely hydroxylation, glucuronidation and sulfation are involved in BA detoxification. In the present study, we employed a comprehensive approach to identify the key enzymes involved in BA metabolism using human recombinant enzymes, human liver microsomes (HLM) and human liver cytosol (HLC). We showed that CYP3A4 was a crucial step for the metabolism of several BAs and their taurine and glycine conjugated forms and quantitatively described their metabolites. Glucuronidation and sulfation were also identified as important drivers of the BA detoxification process in humans. Moreover, lithocholic acid (LCA), the most hydrophobic BA with the highest toxicity potential, was a substrate for all investigated processes, demonstrating the importance of hepatic metabolism for its clearance. Collectively, this study identified CYP3A4, UGT1A3, UGT2B7 and SULT2A1 as the major contributing (metabolic) processes in the BA detoxification network. Inhibition of these enzymes by drug candidates is therefore considered as a critical mechanism in the manifestation of drug-induced cholestasis in humans and should be addressed during the pre-clinical development.
Synthesis, physicochemical properties, and biological activity of bile acids 3-glucuronides: Novel insights into bile acid signalling and detoxification
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Besides the increased solubility in biological fluids, glucuronides are recognized by specific transporters such as the multidrug resistance related protein 3 (MRP3), that enable their secretion into blood and the subsequent urinary excretion [8,11,12]. Although in humans it is considered a minor pathway for BA metabolism under normal physiological conditions [13], some evidence suggests that glucuronidation may become more relevant under cholestatic conditions, when the reduction of bile flow leads to BA accumulation and liver damage [8,10,14,15]. Glucuronidation is thus considered a protective route against BA hepatoxicity and BA-conjugating UGT enzymes have been proposed as targets for the treatment of cholestasis [16].
Glucuronidation is considered an important detoxification pathway of bile acids especially in cholestatic conditions. Glucuronides are less toxic than the parent free forms and are more easily excreted in urine. However, the pathophysiological significance of bile acid glucuronidation is still controversial and debated among the scientific community. Progress in this field has been strongly limited by the lack of appropriate methods for the preparation of pure glucuronides in the amount needed for biological and pharmacological studies. In this work, we have developed a new synthesis of bile acid C3-glucuronides enabling the convenient preparation of gram-scale quantities. The synthesized compounds have been characterized in terms of physicochemical properties and abilities to modulate key nuclear receptors including the farnesoid X receptor (FXR). In particular, we found that C3-glucuronides of chenodeoxycholic acid and lithocholic acid, respectively the most abundant and potentially cytotoxic species formed in patients affected by cholestasis, behave as FXR agonists and positively regulate the gene expression of transporter proteins, the function of which is critical in human conditions related to imbalances of bile acid homeostasis.
Glucuronides in the gut: Sugar-driven symbioses between microbe and host
2017, Journal of Biological Chemistry
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Bile acids are also glucuronidated in the liver (44), and the resulting conjugates account for between 12 and 36% of the bile acids excreted in the urine. By contrast, sulfate, glycine, and taurine conjugates make up 50–63, 1.8–28, and 4.1–8.3% of excreted bile acids in the urine, respectively (45). Thus, glucuronidated bile acids likely provide a significant energy source to bacteria capable of processing such compounds.
The intestinal milieu is astonishingly complex and home to a constantly changing mixture of small and large molecules, along with an abundance of bacteria, viral particles, and eukaryotic cells. Such complexity makes it difficult to develop testable molecular hypotheses regarding host-microbe interactions. Fortunately, mammals and their associated gastrointestinal (GI) microbes contain complementary systems that are ideally suited for mechanistic studies. Mammalian systems inactivate endobiotic and xenobiotic compounds by linking them to a glucuronic acid sugar for GI excretion. In the GI tract, the microbiota express β-glucuronidase enzymes that remove the glucuronic acid as a carbon source, effectively reversing the actions of mammalian inactivation. Thus, by probing the actions of microbial β-glucuronidases, and by understanding which substrate glucuronides they process, molecular insights into mammalian-microbial symbioses may be revealed amid the complexity of the intestinal tract. Here, we focus on glucuronides in the gut and the microbial proteins that process them.
Nuclear receptors as drug targets in cholestasis and drug-induced hepatotoxicity
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Nuclear receptors are key regulators of various processes including reproduction, development, and metabolism of xeno- and endobiotics such as bile acids and drugs. Research in the last two decades provided researchers and clinicians with a detailed understanding of the regulation of these processes and, most importantly, also prompted the development of novel drugs specifically targeting nuclear receptors for the treatment of a variety of diseases. Some nuclear receptor agonists are already used in daily clinical practice but many more are currently designed or tested for the treatment of diabetes, dyslipidemia, fatty liver disease, cancer, drug hepatotoxicity and cholestasis. The hydrophilic bile acid ursodeoxycholic acid is currently the only available drug to treat cholestasis but its efficacy is limited. Therefore, development of novel treatments represents a major goal for both pharmaceutical industry and academic researchers. Targeting nuclear receptors in cholestasis is an intriguing approach since these receptors are critically involved in regulation of bile acid homeostasis. This review will discuss the general role of nuclear receptors in regulation of transporters and other enzymes maintaining bile acid homeostasis and will review the role of individual receptors as therapeutic targets. In addition, the central role of nuclear receptors and other transcription factors such as the aryl hydrocarbon receptor (AhR) and the nuclear factor-E2-related factor (Nrf2) in mediating drug disposition and their potential therapeutic role in drug-induced liver disease will be covered.
Mechanisms of Cholestasis
2008, Clinics in Liver Disease
Citation Excerpt :
These export systems are normally expressed at very low levels at the basolateral membrane but are dramatically up-regulated after bile acid feeding and in experimental cholestasis in rodents as well as in human cholestatic liver diseases [86,109,110,121,123,125–132]. Since MRP3, MRP4, and OSTα/OSTβ are able to transport sulfated and glucuronidated bile acids that are eliminated into urine during cholestasis, the induction of these transporters may explain the shift toward renal excretion of bile acids as a major mechanism for bile acid elimination in patients with chronic, long-standing cholestasis (see Fig. 3 [E]) [133–137]. In addition to bile acid elimination via the kidney, induction of basolateral efflux pumps and repression of basolateral uptake systems, together with disruption of tight junctions (see below), may also explain the appearance of conjugated bilirubin and development of jaundice and bilirubinuria in cholestasis.
This article gives an overview of the molecular and cellular mechanisms of cholestasis. Topics reviewed include the pathomechanisms of hereditary cholestasis syndromes, such as progressive familial intrahepatic cholestasis, and hepatocellular transporter defects encountered in various acquired cholestatic disorders, such as intrahepatic cholestasis of pregnancy, drug-induced cholestasis, inflammatory cholestasis, primary sclerosing cholangitis, and primary biliary cirrhosis. In addition, current concepts regarding adaptive hepatocellular mechanisms counteracting cholestatic liver damage are discussed.
Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans
2005, Gastroenterology
Background & Aims: Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. Methods: Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4β-hydroxy cholesterol, 4β-OH-C) and bile acid synthesis (7α-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. Results: RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. Conclusions: RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

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^☆: Preliminary results of this study were reported at the 5th Bile Acid Meeting held at Freiburg i.Br., West Germany, June 12–14, 1978.

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Analysis of bile acid glucuronides in urine. Identification of 3α,6α,12α-trihydroxy-5β-cholanoic acid☆

Abstract

Clin. chim. Acta

J. lipid Res.

J. steroid Biochem.

Prostaglandins

J. steroid Biochem.

Biochem. Med.

Biochim. biophys. Acta

Biochim. biophys. Acta

Biochim. biophys. Acta

Bile-salt glucuronides in urine

Hoppe-Setyler's Z. Physiol. Chem.

Bile salt glucuronides: Identification and quantitative analysis in the urine of patients with cholestasis

Europ. J. clin. Invest

3-Keto-6(β)-hydroxycholanic acid and 3(α)-hydroxy-6-ketocholanic acid

J. am. Chem. Soc.