Impact of Blueberry Consumption on the Human Fecal Bileacidome: A Pilot Study of Bile Acid Modulation by Freeze-Dried Blueberry.

Cholesterol-derived bile acids (BAs) affect numerous physiological functions such as glucose homeostasis, lipid metabolism and absorption, intestinal inflammation and immunity, as well as intestinal microbiota diversity. Diet influences the composition of the BA pool. In the present study, we analyzed the impact of a dietary supplementation with a freeze-dried blueberry powder (BBP) on the fecal BA pool composition. The diet of 11 men and 13 women at risk of metabolic syndrome was supplemented with 50 g/day of BBP for 8 weeks, and feces were harvested before (pre) and after (post) BBP consumption. BAs were profiled using liquid chromatography coupled with tandem mass spectrometry. No significant changes in total BAs were detected when comparing pre- vs. post-BBP consumption samples. However, post-BBP consumption samples exhibited significant accumulations of glycine-conjugated BAs (p = 0.04), glycochenodeoxycholic (p = 0.01), and glycoursodeoxycholic (p = 0.01) acids, as well as a significant reduction (p = 0.03) in the secondary BA levels compared with pre-BBP feces. In conclusion, the fecal bileacidome is significantly altered after the consumption of BBP for 8 weeks. While additional studies are needed to fully understand the underlying mechanisms and physiological implications of these changes, our data suggest that the consumption of blueberries can modulate toxic BA elimination.

Omega-3 Polyunsaturated Fatty Acid: A Pharmaco-Nutraceutical Approach to Improve the Responsiveness to Ursodeoxycholic Acid.

Ursodeoxycholic acid (UDCA) is the first line therapy for the treatment of cholestatic and autoimmune liver diseases. Its clinical use is currently limited by a significant proportion of non-responder patients. Polyunsaturated fatty acids (n-3 PUFAs) possess important anti-inflammatory properties and protect liver cells against bile acid (BA)-induced toxicity. The present study was designed to rapidly evaluate whether combining n-3 PUFAs (i.e., eicosapentaenoic [EPA] and docosahexaenoic [DHA] acids) to UDCA would provide additional benefits when compared to the drug alone. The parameters evaluated were (i) the expression of genes governing BA synthesis, transport, and metabolism; (ii) the prevention of BA-induced apoptosis and endoplasmic reticulum (ER)-stress; and (iii) the control of BA- and LPS-dependent inflammation. In the absence of n-3 PUFAs, most of the parameters investigated were unaffected by UDCA or were only altered by the higher dose (500 µM) of the drug. By contrast, in the presence of EPA/DHA (50/50 µM), all parameters showed a strongly improved response and the lowest UDCA dosage (50 µM) provided equal or better benefits than the highest dose used alone. For example, the combination EPA/DHA + UDCA 50 µM caused comparable down-regulation of the CYP7A1 gene expression and of the BA-induced caspase 3 activity as observed with UDCA 500 µM. In conclusion, these results suggest that the addition of n-3 PUFAs to UDCA may improve the response to the drug, and that such a pharmaco-nutraceutical approach could be used in clinic to open the narrow therapeutic dose of UDCA in cholestatic liver diseases.

Differential Role of Liver X Receptor (LXR) α and LXRß in the Regulation of UDP-Glucuronosyltransferase 1A1 in Humanized UGT1 Mice.

Liver X receptors (LXRs), LXRα and LXRß, are nuclear receptors that regulate the metabolism of cholesterol and bile acids and are activated by oxysterols. Humanized UGT1 (hUGT1) mice express the 9-human UGT1A genes associated with the UGT1 locus in a Ugt1-null background. The expression of UGT1A1 is developmentally delayed in the liver and intestines, resulting in the accumulation of serum bilirubin during the neonatal period. Induction of UGT1A1 in newborn hUGT1 mice leads to rapid reduction in total serum bilirubin (TSB) levels, a phenotype measurement that allows for an accurate prediction on UGT1A1 expression. When neonatal hUGT1 mice were treated by oral gavage with the LXR agonist T0901317, TSB levels were dramatically reduced. To determine the LXR contribution to the induction of UGT1A1 and the lowering of TSB levels, experiments were conducted in neonatal hUGT1/Lxrα -/- , hUGT1/Lxrß -/- , and hUGT1/Lxrαß -/- mice treated with T0901317. Induction of liver UGT1A1 was dependent upon LXRα, with the induction pattern paralleling induction of LXRα-specific stearoyl CoA desaturase 1. However, the actions of T0901317 were also shown to display a lack of specificity for LXR, with the induction of liver UGT1A1 in hUGT1/Lxrαß -/- mice, a result associated with activation of both pregnane X receptor and constitutive androstane receptor. However, the LXR agonist GW3965 was highly selective toward LXRα, showing no impact on lowering TSB values or inducing UGT1A1 in hUGT1/Lxrα -/- mice. An LXR-specific enhancer site on the UGT1A1 gene was identified, along with convincing evidence that LXRα is crucial in maintaining constitutive expression of UGT1A1 in adult hUGT1 mice. SIGNIFICANCE STATEMENT: It has been established that activation of LXRα, and not LXRß, is responsible for the induction of liver UGT1A1 and metabolism of serum bilirubin in neonatal hUGT1 mice. Although induction of the human UGT1A1 gene is initiated at a newly characterized LXR enhancer site, allelic deletion of the Lxrα gene drastically reduces the constitutive expression of liver UGT1A1 in adult hUGT1 mice. Combined, these findings indicate that LXRα is critical for the developmental expression of UGT1A1.

N-3 Polyunsaturated Fatty Acids Stimulate Bile Acid Detoxification in Human Cell Models.

Cholestasis is characterized by the accumulation of toxic bile acids (BAs) in liver cells. The present study aimed to evaluate the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, on BA homeostasis and toxicity in human cell models. The effects of EPA and/or DHA on the expression of genes involved in the maintenance of BA homeostasis were analyzed in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells, as well as in primary culture of human intestinal (InEpC) and renal (RPTEC) cells. Extracellular BA species were quantified in culture media using LC-MS/MS. BA-induced toxicity was evaluated using caspase-3 and flow cytometry assays. Gene expression analyses of HepG2 cells reveal that n-3 PUFAs reduce the expression of genes involved in BA synthesis (CYP7A1, CYP27A1) and uptake (NTCP), while activating genes encoding metabolic enzymes (SULT2A1) and excretion transporters (MRP2, MRP3). N-3 PUFAs also generate a less toxic BA pool and prevent the BA-dependent activation of apoptosis in HepG2 cells. Conclusion. The present study reveals that n-3 PUFAs stimulate BA detoxification.

UGT2B17 Expedites Progression of Castration-Resistant Prostate Cancers by Promoting Ligand-Independent AR Signaling.

Castration-resistant prostate cancer (CRPC) is characterized by a shift in androgen receptor (AR) signaling from androgen-dependent to androgen (ligand)-independent. UDP-glucuronosyltransferase 2B17 (UGT2B17) is a key enzyme that maintains androgen homeostasis by catabolizing AR agonists into inactive forms. Although enhanced UGT2B17 expression by antiandrogens has been reported in androgen-dependent prostate cancer, its roles in regulating AR signaling transformation and CRPC progression remain unknown. In this study, we show that higher UGT2B17 protein expression in prostate tumors is associated with higher Gleason score, metastasis, and CRPC progression. UGT2B17 expression and activity were higher in androgen-independent compared to androgen-dependent cell lines. UGT2B17 stimulated cancer cell proliferation, invasion, and xenograft progression to CRPC after prolonged androgen deprivation. Gene microarray analysis indicated that UGT2B17 suppressed androgen-dependent AR transcriptional activity and enhanced of ligand-independent transcriptional activity at genes associated with cell mitosis. These UGT2B17 actions were mainly mediated by activation of the c-Src kinase. In CRPC tumors, UGT2B17 expression was associated positively with c-Src activation. These results indicate that UGT2B17 expedites CRPC progression by enhancing ligand-independent AR signaling to activate cell mitosis in cancer cells. Cancer Res; 76(22); 6701-11. ©2016 AACR.

Role of glucuronidation for hepatic detoxification and urinary elimination of toxic bile acids during biliary obstruction.

Biliary obstruction, a severe cholestatic condition, results in a huge accumulation of toxic bile acids (BA) in the liver. Glucuronidation, a conjugation reaction, is thought to protect the liver by both reducing hepatic BA toxicity and increasing their urinary elimination. The present study evaluates the contribution of each process in the overall BA detoxification by glucuronidation. Glucuronide (G), glycine, taurine conjugates, and unconjugated BAs were quantified in pre- and post-biliary stenting urine samples from 12 patients with biliary obstruction, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The same LC-MS/MS procedure was used to quantify intra- and extracellular BA-G in Hepatoma HepG2 cells. Bile acid-induced toxicity in HepG2 cells was evaluated using MTS reduction, caspase-3 and flow cytometry assays. When compared to post-treatment samples, pre-stenting urines were enriched in glucuronide-, taurine- and glycine-conjugated BAs. Biliary stenting increased the relative BA-G abundance in the urinary BA pool, and reduced the proportion of taurine- and glycine-conjugates. Lithocholic, deoxycholic and chenodeoxycholic acids were the most cytotoxic and pro-apoptotic/necrotic BAs for HepG2 cells. Other species, such as the cholic, hyocholic and hyodeoxycholic acids were nontoxic. All BA-G assayed were less toxic and displayed lower pro-apoptotic/necrotic effects than their unconjugated precursors, even if they were able to penetrate into HepG2 cells. Under severe cholestatic conditions, urinary excretion favors the elimination of amidated BAs, while glucuronidation allows the conversion of cytotoxic BAs into nontoxic derivatives.

Enantiomer selective glucuronidation of the non-steroidal pure anti-androgen bicalutamide by human liver and kidney: role of the human UDP-glucuronosyltransferase (UGT)1A9 enzyme.

Bicalutamide (Casodex(®) ) is a non-steroidal pure anti-androgen used in the treatment of localized prostate cancer. It is a racemate drug, and its activity resides in the (R)-enantiomer, with little in the (S)-enantiomer. A major metabolic pathway for bicalutamide is glucuronidation catalysed by UDP-glucuronosyltransferase (UGT) enzymes. While (S)bicalutamide is directly glucuronidated, (R)bicalutamide requires hydroxylation prior to glucuronidation. The contribution of human tissues and UGT isoforms in the metabolism of these enantiomers has not been extensively investigated. In this study, both (R) and/or (S)bicalutamide were converted into glucuronide (-G) derivatives after incubation of pure and racemic solutions with microsomal extracts from human liver and kidney. Intestinal microsomes exhibited only low reactivity with these substrates. Km values of liver and kidney samples for (S)bicalutamide glucuronidation were similar, and lower than values obtained with the (R)-enantiomer. Among the 16 human UGTs tested, UGT1A8 and UGT1A9 were able to form both (S) and (R)bicalutamide-G from pure or racemic substrates. UGT2B7 was also able to form (R)bicalutamide-G. Kinetic parameters of the recombinant UGT2B7, UGT1A8 and UGT1A9 enzymes support a predominant role of the UGT1A9 isoform in bicalutamide metabolism. Accordingly, (S)bicalutamide inhibited the ability of human liver and kidney microsomes to glucuronidate the UGT1A9 probe substrate, propofol. In conclusion, the present study provides the first comprehensive analysis of in vitro bicalutamide glucuronidation by human tissues and UGTs and identifies UGT1A9 as a major contributor for (R) and (S) glucuronidation in the human liver and kidney.

Differential expression of the androgen-conjugating UGT2B15 and UGT2B17 enzymes in prostate tumor cells during cancer progression.

CONTEXT: Androgens play major roles in prostate cancer initiation and development. In prostate cells, the human uridine diphosphate-glucuronosyltransferase (UGT)2B15 and UGT2B17 enzymes inactivate androgens. OBJECTIVE: We investigated in vivo how UGT2B15 and UGT2B17 expressions are affected during prostate cancer development. DESIGN: We conducted an observational study of the UGT2B15 and UGT2B17 mRNA and protein levels. SETTING: The study was conducted at Laval University (Québec, Canada) and at the University of British Columbia (Vancouver, Canada). PATIENTS/PARTICIPANTS: Participants were from a cohort of prostate cancer patients from the Hôtel-Dieu de Québec hospital (Québec; mRNA analyses) and from the Vancouver Prostate Centre tissue bank (Vancouver; tissue microarray experiments). MAIN OUTCOME MEASURES: UGT mRNA and protein levels were determined using real-time PCR and immunohistochemical analyses, respectively. RESULTS: Both UGT2B15 and UGT2B17 mRNA and protein levels were not significantly associated with Gleason score stratification. However, when protein levels were compared to benign prostatic hyperplasia, UGT2B17 was significantly more abundant in all Gleason-scored tumors. By contrast, UGT2B15 levels were significantly reduced in naive and castration-resistant tumors and undetectable in lymph node metastases. Finally, UGT2B17 proteins were 5-fold more abundant in metastases than in benign samples. CONCLUSIONS: The current study reveals that UGT2B15 and UGT2B17 are differentially regulated during prostate cancer progression. Furthermore, this study also identifies the UGT2B15 gene as a negatively regulated target gene in castration-resistant prostate cancer and lymph node metastases.

PPARalpha L162V polymorphism alters the potential of n-3 fatty acids to increase lipoprotein lipase activity.

Omega-3 fatty acids (FAs) may accelerate plasma triglyceride (TG) clearance by altering lipoprotein lipase (LPL) activity. Yet, the ability of n-3 FAs to increase LPL activity is dependent on transcription factors such as peroxisome proliferator-activated receptor alpha (PPARalpha). The objective was to examine the effects of n-3 FAs on LPL activity considering the occurrence of PPARalpha L162V polymorphism. First, 14 pairs of men either L162 homozygotes or carriers of the V162 allele were supplemented with n-3 FAs. Second, transient transfections in HepG2 cells, for the L162- and V162-PPARalpha variants with the peroxisome proliferator-response element from the human LPL gene, were transactivated with n-3 FAs. In vivo results demonstrate that the LPL activity increased non-significantly by 14.4% in L162 homozygotes compared with 6.6% in carriers of the PPARalpha-V162 allele, after n-3 FA supplementation. Additionally, the L162 homozygotes tended towards an inverse correlation between LPL activities and plasma TG levels. Conversely, carriers of the V162 allele showed no such relationship. In vitro data demonstrates that transcription rates of LPL tended to be higher for the L162-PPARalpha than V162-PPARalpha after n-3 FAs activation. Overall, these results indicate that n-3 FA supplementation increases the transcription rate of LPL to a greater extent in L162-PPARalpha than V162-PPARalpha.

Regulation of endobiotics glucuronidation by ligand-activated transcription factors: physiological function and therapeutic potential.

Recent progresses in molecular pharmacology approaches have allowed the identification and characterization of a series of nuclear receptors (NR) which efficiently control the level UDP-glucuronosyltransferase (UGT) genes expression. These regulatory processes ensure optimized UGT expression in response to specific endogenous and/or exogenous stimuli. Interestingly, numerous endogenous activators of these NRs are conjugated by the UGT enzymes they regulate. In such a case, the NR-dependent regulation of UGT genes corresponds to a feedforward/feedback mechanism by which a bioactive molecule controls its own concentrations. In the present review, we will discuss i) how bilirubin reduces its circulating levels by activating AhR in the liver; ii) how bile acids modulate their hepatic glucuronidation via PXR- and FXR-dependent processes in enterohepatic tissues; and iii) how androgens inhibit their cellular metabolism in prostate cancer cells through an AR-dependent mechanism. Subsequently, with further discussion of the same examples (bilirubin and bile acids), we will illustrate how NR-dependent regulation of UGT enzymes may contribute to the beneficial effects of pharmacological activators of nuclear receptors, such as CAR and PPARa.

Differences in transcriptional activation by the two allelic (L162V Polymorphic) variants of PPARα after Omega-3 fatty acids treatment.

Omega-3 fatty acids (FAs) have the potential to regulate gene expression via the peroxisome proliferator-activated receptor α (PPARα); therefore, genetic variations in this gene may impact its transcriptional activity on target genes. It is hypothesized that the transcriptional activity by wild-type L162-PPARα is enhanced to a greater extent than the mutated variant (V162-PPARα) in the presence of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or a mixture of EPA:DHA. To examine the functional difference of the two allelic variants on receptor activity, transient co-transfections were performed in human hepatoma HepG2 cells activated with EPA, DHA and EPA:DHA mixtures. Results indicate that the addition of EPA or DHA demonstrate potential to increase the transcriptional activity by PPARα with respect to basal level in both variants. Yet, the EPA:DHA mixtures enhanced the transcriptional activity to a greater extent than individual FAs indicating possible additive effects of EPA and DHA. Additionally, the V162 allelic form of PPARα demonstrated consistently lower transcriptional activation when incubated with EPA, DHA or EPA:DHA mixtures than, the wild-type variant. In conclusion, both allelic variants of the PPARα L162V are activated by omega-3 FAs; however, the V162 allelic form displays a lower transcriptional activity than the wild-type variant.

Lipid-activated transcription factors control bile acid glucuronidation.

Bile acids subserve important physiological functions in the control of cholesterol homeostasis. Indeed, hepatic bile acid synthesis and biliary excretion constitute the main route for cholesterol removal from the human body. On the other hand, bile acids serve as natural detergents for the intestinal absorption of dietary cholesterol. However, due to their detergent properties, bile acids are inherently cytotoxic, and their cellular level may be tightly controlled to avoid pathological situations such as cholestasis. Recent investigations have illustrated the crucial roles that a series of ligand-activated transcription factors has in the control of hepatic bile acids synthesis, transport and metabolism. Thus, the lipid-activated nuclear receptors, farnesoid X-receptor (FXR), liver X-receptor (LXR), pregnane X-receptor (PXR) and peroxisome proliferator-activated receptor alpha (PPAR alpha), modulate the expression and activity of genes controlling bile acid homeostasis in the liver. Several members of the UDP-glucuronosyltransferase (UGT) enzymes family are among the bile acid metabolizing enzymes regulated by these receptors. UGTs catalyze glucuronidation, a major phase II metabolic reaction, which converts hydrophobic bile acids into polar and urinary excretable metabolites. This article summarizes our recent observations on the regulation of bile acid conjugating UGTs upon pharmacological activation of lipid-activated receptors, with a particular interest for the role of PPAR alpha and LXRalpha in controlling human UGT1A3 expression.

Calcitrol (1alpha,25-dihydroxyvitamin D3) inhibits androgen glucuronidation in prostate cancer cells.

Calcitriol (1alpha,25-dihydroxyvitamin D(3)), the active metabolite of vitamin D, has recently emerged as a promising therapeutic agent in the treatment of prostate cancer, the second most common cause of cancer death in American males. In the present study, we have analyzed the effects of calcitriol treatment on the expression and activity of the UDP-glucuronosyltransferase (UGT) 2B15 and 2B17 in prostate cancer LNCaP and 22Rv1 cells. These two enzymes share a crucial role in the inactivation of androgens in the human prostate. We report that calcitriol treatment results in lower glucuronide conjugation of the active androgen dihydrotestosterone and its reduced metabolites androstane-3alpha-diol and androsterone in LNCaP cells. The same treatment also drastically decreased the mRNA and protein levels of UGT2B15 and UGT2B17 in LNCaP and 22Rv1 cells. Using casodex, an androgen receptor (AR) antagonist, and AR-specific small interfering RNA probes, we show that calcitriol requires a functional AR to inhibit the expression of the UGT2B17 gene in LNCaP cells. By contrast, transient transfection and site-directed mutagenesis experiments revealed that calcitriol down-regulates UGT2B15 promoter activity through a responsive region between positions -171 and -113 bp. In conclusion, the present study identifies the vitamin D receptor activator calcitriol as a negative regulator of the UGT2B15- and UGT2B17-dependent inactivation of androgens in prostate cancer LNCaP cells. Androgens promote prostate cancer cell proliferation; thus, the reduction of their inactivation could have a limiting effect of the calcitriol antiproliferative properties in prostate cancer cells.

Activators of the farnesoid X receptor negatively regulate androgen glucuronidation in human prostate cancer LNCAP cells.

Androgens are major regulators of prostate cell growth and physiology. In the human prostate, androgens are inactivated in the form of hydrophilic glucuronide conjugates. These metabolites are formed by the two human UGT2B15 [UGT (UDP-glucuronosyltransferase) 2B15] and UGT2B17 enzymes. The FXR (farnesoid X receptor) is a bile acid sensor controlling hepatic and/or intestinal cholesterol, lipid and glucose metabolism. In the present study, we report the expression of FXR in normal and cancer prostate epithelial cells, and we demonstrate that its activation by chenodeoxycholic acid or GW4064 negatively interferes with the levels of UGT2B15 and UGT2B17 mRNA and protein in prostate cancer LNCaP cells. FXR activation also causes a drastic reduction of androgen glucuronidation in these cells. These results point out activators of FXR as negative regulators of androgen-conjugating UGT expression in the prostate. Finally, the androgen metabolite androsterone, which is also an activator of FXR, dose-dependently reduces the glucuronidation of androgens catalysed by UGT2B15 and UGT2B17 in an FXR-dependent manner in LNCaP cells. In conclusion, the present study identifies for the first time the activators of FXR as important regulators of androgen metabolism in human prostate cancer cells.

Human UDP-glucuronosyltransferase (UGT)1A3 enzyme conjugates chenodeoxycholic acid in the liver.

Chenodeoxycholic acid (CDCA) is a liver-formed detergent and plays an important role in the control of cholesterol homeostasis. During cholestasis, toxic bile acids (BA) accumulate in hepatocytes causing damage and consequent impairment of their function. Glucuronidation, a conjugation reaction catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, is considered an important metabolic pathway for hepatic BA. This study identifies the human UGT1A3 enzyme as the major enzyme responsible for the hepatic formation of the acyl CDCA-24glucuronide (CDCA-24G). Kinetic analyses revealed that human liver and UGT1A3 catalyze the formation of CDCA-24G with similar K(m) values of 10.6 to 18.6 mumol/L, respectively. In addition, electrophoretic mobility shift assays and transient transfection experiments revealed that glucuronidation reduces the ability of CDCA to act as an activator of the nuclear farnesoid X-receptor (FXR). Finally, we observed that treatment of human hepatocytes with fibrates increases the expression and activity of UGT1A3, whereas CDCA has no effect. In conclusion, UGT1A3 is the main UGT enzyme for the hepatic formation of CDCA-24G and glucuronidation inhibits the ability of CDCA to act as an FXR activator. In vitro data also suggest that fibrates may favor the formation of bile acid glucuronides in cholestatic patients.

The liver X-receptor alpha controls hepatic expression of the human bile acid-glucuronidating UGT1A3 enzyme in human cells and transgenic mice.

Glucuronidation, an important bile acid detoxification pathway, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT1A3 is considered the major human enzyme for the hepatic C24-glucuronidation of the primary chenodeoxycholic (CDCA) and secondary lithocholic (LCA) bile acids. We identify UGT1A3 as a positively regulated target gene of the oxysterol-activated nuclear receptor liver X-receptor alpha (LXRalpha). In human hepatic cells and human UGT1A transgenic mice, LXRalpha activators induce UGT1A3 mRNA levels and the formation of CDCA-24glucuronide (24G) and LCA-24G. Furthermore, a functional LXR response element (LXRE) was identified in the UGT1A3 promoter by site-directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation experiment. In addition, LXRalpha is found to interact with the SRC-1alpha and NCoR cofactors to regulate the UGT1A3 gene, but not with PGC-1beta. In conclusion, these observations establish LXRalpha as a crucial regulator of bile acid glucuronidation in humans and suggest that accumulation of oxysterols in hepatocytes during cholestasis favors bile acid detoxification as glucuronide conjugates. LXR agonists may be useful for stimulating both bile acid detoxification and cholesterol removal in cholestatic or hypercholesterolemic patients, respectively.

Enzymatic production of bile Acid glucuronides used as analytical standards for liquid chromatography-mass spectrometry analyses.

The present study reports a novel method for the production and purification of analytical standards of glucuronide conjugates of bile acids, chenodeoxycholic (CDCA), lithocholic, (LCA) and hyodeoxycholic (HDCA) acids. CDCA-3G (CDCA-3-glucuronide) and -24G, LCA-3G and -24G, and HDCA-6G and -24G were enzymatically formed by using microsomes from human liver, purified by liquid chromatography, digested with recombinant beta-glucuronidase, and quantified by liquid chromatography/electrospray ionization coupled to mass spectrometry (LC-ESI/MS). The position of the glucuronosyl moiety on the bile acids was determined by analyzing the susceptibility to hydrolysis under elevated pH and temperature conditions of the standards. By using the purified analytical standards, a LC-ESI/MS/MS method was developed for the determination of these glucuronide conjugates in in vitro assays. The linearity of the assay ranged from 0.5 to 40 ng/mL for the six glucuronides, and the limit of quantification (LOQ) was 0.5 ng/mL. Intra- and interday precisions and accuracy values were all lower than 10.2%. Furthermore, processed sample stability analyses revealed that the six standards were stable at 4 degrees C for more than 24 h. This method was successfully used for the quantification of CDCA, LCA, and HDCA glucuronides formed by human liver or hepatoma HepG2 cells. In conclusion, such a method allows the purification of high-quality analytical standards of glucuronide derivatives and may easily be used for the quantification of other endo- and xenobiotics that are glucuronidated.