Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease.

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3ß-hydroxy-5-cholesten-(25R)26-oic acid (3ßHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3ßHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3ßHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1-/- mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3ßHCA levels were maintained at basal levels in ND-fed Cyp7b1-/- mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1-/- mice developed insulin resistance (IR) with subsequent 26HC/3ßHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1-/- mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3ßHCA accumulation. The results suggest 26HC/3ßHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3ßHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.

Coffee modulates insulin-hepatocyte nuclear factor-4α-Cyp7b1 pathway and reduces oxysterol-driven liver toxicity in a nonalcoholic fatty liver disease mouse model.

Oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic pathway" of cholesterol metabolism. Previously, we demonstrated that an inability to upregulate CYP7B1 in the setting of insulin resistance leads to the accumulation of cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) that initiate and promote hepatocyte injury; followed by an inflammatory response. The current study demonstrates that dietary coffee improves insulin resistance and restores Cyp7b1 levels in a well-characterized Western diet (WD)-induced nonalcoholic fatty liver disease (NAFLD) mouse model. Ingestion of a WD containing caffeinated (regular) coffee or decaffeinated coffee markedly reduced the serum ALT level and improved insulin resistance. Cyp7b1 mRNA and protein levels were preserved at normal levels in mice fed the coffee containing WD. Additionally, coffee led to upregulated steroid sulfotransferase 2b1 (Sult2b1) mRNA expression. In accordance with the response in these oxysterol metabolic genes, hepatocellular 26HC levels were maintained at physiologically low levels. Moreover, the current study provided evidence that hepatic Cyp7b1 and Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, hepatocyte nuclear factor (HNF)-4α. We conclude coffee achieves its beneficial effects through the modulation of insulin resistance. Both decaffeinated and caffeinated coffee had beneficial effects, demonstrating caffeine is not fundamental to this effect. The effects of coffee feeding on the insulin-HNF4α-Cyp7b1 signaling pathway, whose dysregulation initiates and contributes to the onset and progression of NASH as triggered by insulin resistance, offer mechanistic insight into approaches for the treatment of NAFLD.NEW & NOTEWORTHY This study demonstrated dietary coffee prevented the accumulation of hepatic oxysterols by maintaining Cyp7b1/Sult2b1 expression in a diet-induced NAFLD mice model. Lowering liver oxysterols markedly reduced inflammation in the coffee-ingested mice. Caffeine is not fundamental to this effect. In addition, this study showed Cyp7b1/Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, HNF4α. The insulin-HNF4α-Cyp7b1/Sult2b1 signaling pathway, which directly correlates to the onset of NASH triggered by insulin resistance, offers insight into approaches for NAFLD treatment.

Development of simultaneous quantitative analysis of tricarboxylic acid cycle metabolites to identify specific metabolites in cancer cells by targeted metabolomic approach.

The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.

Cyclophilin a knokdown inhibits cell migration and invasion through the suppression of epithelial-mesenchymal transition in colorectal cancer cells.

Enhanced expression of cyclophilin A (CypA) in colorectal cancer (CRC) was reported; however, how CypA influences CRC progression is not clear. Therefore, we examine the effects of CypA on CRC cell progression. Knockdown of CypA in SW480 cells significantly inhibited cell migration and invasion but had no effect on cell proliferation. In addition, upregulation of E-cadherin and downregulation of N-cadherin and Snail expression were observed by CypA knockdown. These results suggested that CypA knockdown inhibited cell migration and invasion by suppressing epithelial-mesenchymal transition. CypA knockdown was also associated with increased p38 phosphorylation, and the p38 inhibitor treatment led to increase in the number of invasive CypA-knockdown SW480 cells. Therefore, CypA may be a potential therapeutic target in preventing CRC metastasis.

Correction: Sato, K., et al. Identification of a Novel Oligosaccharide in Maple Syrup as a Potential Alternative Saccharide for Diabetes Mellitus Patients. Int. J. Mol. Sci. 2019, 20, 5041.

The authors wish to make the following corrections to this paper [...].

Identification of a Novel Oligosaccharide in Maple Syrup as a Potential Alternative Saccharide for Diabetes Mellitus Patients.

The incidence of diabetes mellitus (DM) is increasing rapidly and is associated with changes in dietary habits. Although restrictions in the use of sweeteners may prevent the development of DM, this might reduce the quality of life of patients with DM. Therefore, there has been a great deal of research into alternative sweeteners. In the search for such sweeteners, we analyzed the carbohydrate content of maple syrup and identified a novel oligosaccharide composed of fructose and glucose, linked at the C-4 of glucose and the C-6 of fructose. This oligosaccharide inhibited the release of fructose from sucrose by invertase (IC50: 1.17 mmol/L) and the decomposition of maltose by α-(1-4) glucosidase (IC50: 1.72 mmol/L). In addition, when orally administered together with sucrose to rats with DM, the subsequent plasma glucose concentrations were significantly lower than if the rats had been administered sucrose alone, without having any effect on the insulin concentration. These findings suggest that this novel oligosaccharide might represent a useful alternative sweetener for inclusion in the diet of patients with DM and may also have therapeutic benefits.

Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue.

Colorectal cancer (CRC) is one of the most common cancers worldwide, and many patients are already at an advanced stage when they are diagnosed. Therefore, novel biomarkers for early detection of colorectal cancer are required. In this study, we performed a global shotgun proteomic analysis using formalin-fixed and paraffin-embedded (FFPE) CRC tissue. We identified 84 candidate proteins whose expression levels were differentially expressed in cancer and non-cancer regions. A label-free semiquantitative method based on spectral counting and gene ontology (GO) analysis led to a total of 21 candidate proteins that could potentially be detected in blood. Validation studies revealed cyclophilin A, annexin A2, and aldolase A mRNA and protein expression levels were significantly higher in cancer regions than in non-cancer regions. Moreover, an in vitro study showed that secretion of aldolase A into the culture medium was clearly suppressed in CRC cells compared to normal colon epithelium. These findings suggest that decreased aldolase A in blood may be a novel biomarker for the early detection of CRC.

Improvement of solid material for affinity resins by application of long PEG spacers to capture the whole target complex of FK506.

Solid materials for affinity resins bearing long PEG spacers between a functional group used for immobilization of a bio-active compound and the solid surface were synthesized to capture not only small target proteins but also large and/or complex target proteins. Solid materials with PEG1000 or PEG2000 as spacers, which bear a benzenesulfonamide derivative, exhibited excellent selectivity between the specific binding protein carbonic anhydrase type II (CAII) and non-specific ones. These materials also exhibited efficacy in capturing a particular target at a maximal amount. Affinity resins using solid materials with PEG1000 or PEG2000 spacers, bear a FK506 derivative, successfully captured the whole target complex of specific binding proteins at the silver staining level, while all previously known affinity resins with solid materials failed to achieve this objective. These novel affinity resins captured other specific binding proteins such as dynamin and neurocalcin δ as well.

Neokestose suppresses the increase in plasma glucose caused by oral administration of sucrose in a streptozotocin­induced diabetic rat.

Neokestose is considered to have a prebiotic function. However, the physiological activity of neokestose remains unknown. Neokestose has a blastose, a sucrose analog, in its structure. We previously demonstrated that oral administration of blastose to diabetic rats suppressed the increase in plasma glucose (PG) concentration after sucrose administration. Therefore, neokestose might have a similar effect. In this study, we investigated the effects of neokestose on PG concentrations and the mechanism of its action. We first administered neokestose orally to streptozotocin-induced diabetic rats and observed that the expected consequent increase in PG concentration was significantly suppressed. Next, we examined the inhibitory effect of neokestose on glycosidase activity, but observed only a slight inhibitory effect. Therefore, we hypothesized that neokestose might be hydrolyzed by gastric acid to produce blastose. We performed an acid hydrolysis of neokestose using artificial gastric juice. After acid hydrolysis, peaks corresponding to neokestose and its decomposition products including blastose were observed. Therefore, we suggest that neokestose and blastose, a decomposition product, synergistically inhibit glycosidase activity. These findings support the potential use of neokestose as a useful functional oligosaccharide that can help manage plasma glucose concentrations in patients with diabetes mellitus.

Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens.

Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11ß-hydroxyandrost-4-ene-3,17-dione (11ß-OHA) by high-resolution mass spectrometry, (1)H and (13)C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (≈ 1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative "transketolase" (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic "rheostat" controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11ß-OHAD on the host or other gut microbes is currently unknown.

LC/MS/MS of steroids having vicinal diol as electrospray-active boronates.

A derivatization procedure with (3-dimethylaminophenyl)dihydroxyborane (DAPB) was introduced to enhance the detectability of steroids having a vicinal diol in LC/electrospray ionization (ESI)-MS/MS. DAPB reacted with the vicinal diol on the steroids [4ß-hydroxycholesterol (4-HCh), pregnanetriol (PT) and 20R,22R-dihydroxycholesterol] in pyridine at 50°C within 1 h. The resulting DAPB-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using a selected reaction monitoring mode; the detection responses of the DAPB-derivatives were increased by 20-160-fold over those of the intact steroids and the limits of detection were in the low femtomole or attomole range. The derivatization procedure was successfully applied to biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the specific detection of trace amounts of 4-HCh in human plasma and PT in human urine with a small sample volume, simple pretreatment and short chromatographic run time.

Correction: Sato et al. Separation of Fructosyl Oligosaccharides in Maple Syrup by Using Charged Aerosol Detection. Foods 2021, 10, 3160.

In the original publication [...].

Protein components of maple syrup as a potential resource for the development of novel anti­colorectal cancer drugs.

Maple syrup is a natural sweetener consumed worldwide. Active ingredients of maple syrup possess antitumor effects; however, these ingredients are phenolic compounds. The present study aimed to investigate components other than phenolic compounds that may have antitumor effects against colorectal cancer (CRC). Cell proliferation assays demonstrated that treatment with the more than 10,000 molecular weight fraction significantly inhibited viability in DLD­1 cells. Therefore, we hypothesized that the protein components of maple syrup may be the active ingredients in maple syrup. We obtained protein components from maple syrup by ammonium sulfate precipitation, and treatment with the protein fraction of maple syrup (MSpf) was found to exhibit a potential antitumor effect. MSpf­treated DLD­1 colon adenocarcinoma cells exhibited significantly decreased proliferation, migration and invasion. In addition, upregulation of LC3A and E­cadherin and downregulation of MMP­9 expression levels were observed following MSpf treatment. Investigation of the components of MSpf suggested that it was primarily formed of advanced glycation end products (AGEs). Therefore, whether AGEs in MSpf affected the STAT3 pathway through the binding to its receptor, receptor of AGE (RAGE), was assessed. MSpf treatment was associated with decreased RAGE expression and STAT3 phosphorylation. Finally, to determine whether autophagy contributed to the inhibitory effect of cell proliferation following MSpf treatment, the effect of MSpf treatment on autophagy induction following bafilomycin A1 treatment, a specific autophagy inhibitor, was assessed. The inhibitory effect of MSpf treatment on cell proliferation was enhanced through the inhibition of autophagy by bafilomycin A1 treatment. These results suggested that AGEs in MSpf suppressed cell proliferation and epithelial­mesenchymal transition through inhibition of the STAT3 signaling pathway through decreased RAGE expression. Therefore, AGEs in MSpf may be potential compounds for the development of antitumor drugs for the treatment of CRC with fewer adverse effects compared with existing antitumor drugs.

Characterization of non-enzymatic acylation of amino or thiol groups of bionucleophiles by the acyl-adenylate or acyl-CoA thioester of cholic acid.

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are highly electrophilic acyl-linked metabolites which can undergo transacylation reactions with amino and thiol groups of nucleophilic groups on acceptor molecules such as amino acids, peptides, and proteins. Here, non-enzymatic acylation at pH 7.4 of glycine, taurine, glutathione (GSH), and N-acetylcysteine (NAC) by cholyl-adenylate (CA-AMP) was compared with that mediated by cholyl-CoA thioester (CA-CoA) using a 1:1 mixture of stable isotopically labeled CA-AMP and unlabeled CA-CoA. The transacylation products of these substrates were analyzed by liquid chromatography/electrospray ionization linear ion-trap mass spectrometry in negative-ion detection mode. CA-AMP was more reactive than CA-CoA with the amino group of glycine or taurine than with the thiol group of GSH or NAC. In contrast, CA-CoA was more reactive than CA-AMP with the thiol group of GSH or NAC and was far less reactive with the amino group of glycine or taurine. These differences in the reactivity of CA-AMP as compared with that of CA-CoA towards amino and thiol groups may be attributed to the electrophilicity of the carbonyl carbon of these acyl-linked cholic acid metabolites and the nucleophilicity of the amino and thiol group in the bionucleophiles that were studied.

Synthesis of the 3-sulfates of N-acetylcysteine conjugated bile acids (BA-NACs) and their transient formation from BA-NACs and subsequent hydrolysis by a rat liver cytosolic fraction as shown by liquid chromatography/electrospray ionization-mass spectrometry.

Previous work from this laboratory has reported the chemical synthesis of N-acetylcysteine (NAC) conjugates of natural bile acids (BAs) and shown that such novel conjugates can be formed in vivo in rats to which NAC has been administered. The subsequent fate of such novel conjugates is not known. One possible biotransformation is sulfation, a major pathway for BAs N-acylamidates in patients with cholestatic liver disease. Here, we report the chemical synthesis of the 3-sulfates of the S-acyl NAC conjugates of five natural BAs (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic). We also measured the sulfation of N-acetylcysteine-natural bile acid (BA-NAC) conjugates when they were incubated with a rat liver cytosolic fraction. The chemical structures of the BA-NAC 3-sulfates were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation of singly and doubly charged deprotonated molecules, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. When BA-NACs were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate was added, sulfation occurred, but the dominant reaction was hydrolysis of the S-acyl linkage to form the unconjugated BAs. Subsequent sulfation occurred at C-3 on the unconjugated BAs that had been formed from the BA-NACs. Such sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus, NAC conjugates of BAs as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.

Separation of Fructosyl Oligosaccharides in Maple Syrup by Using Charged Aerosol Detection.

Fructosyl oligosaccharides, including fructo-oligosaccharide (FOS), are gaining popularity as functional oligosaccharides and have been found in various natural products. Our previous study suggested that maple syrup contains an unidentified fructosyl oligosaccharide. Because these saccharides cannot be detected with high sensitivity using derivatization methods, they must be detected directly. As a result, an analytical method based on charged aerosol detection (CAD) that can detect saccharides directly was optimized in order to avoid relying on these structures and physical properties to clarify the profile of fructosyl oligosaccharides in maple syrup. This analytical method is simple and can analyze up to hepta-saccharides in 30 min. This analytical method was also reliable and reproducible with high validation values. It was used to determine the content of saccharides in maple syrup, which revealed that it contained not only fructose, glucose, and sucrose but also FOS such as 1-kestose and nystose. Furthermore, we discovered a fructosyl oligosaccharide called neokestose in maple syrup, which has only been found in a few natural foods. These findings help to shed light on the saccharides profile of maple syrup.

Potential corticoid metabolites: chemical synthesis of 3- and 21-monosulfates and their double-conjugates of tetrahydrocorticosteroids in the 5alpha- and 5beta-series.

Here, we describe the chemical synthesis of the complete sets of 18 novel 3- and 21-monosulfates and their double-conjugated form of tetrahydrocortisol (THF), tetrahydro-11-deoxycortisol (THS), and tetrahydrocortisone (THE) in the 5alpha- and 5beta-series. The principal reactions involved are: (1) selective protection of a specific hydroxy group in substrates; (2) catalytic hydrogenation at C-5 of Delta(4)-3-ketosteroids with 10% Pd(OH)(2)/C to yield 3-oxo-5beta-steroids and reductive allomerization with 10% Pd/C to yield 3-oxo-5alpha-isomers; (3) reduction of the resulting 3-oxo-5beta- and 3-oxo-5alpha-steroids to the corresponding 3alpha-hydroxy-compounds with Zn(BH(4))(2) and K-Selectride((R)), respectively; and (4) sulfation of hydroxy groups at C-3 and/or C-21 in the tetrahydrocorticosteroid derivatives with sulfur trioxide-triethylamine complex.

A method for detecting tumor cells derived from colorectal cancer by targeting cell surface glycosylation with affinity capillary electrophoresis.

Circulating tumor cells (CTCs) are involved in metastasis; thus, one of the most important approaches for identifying metastatic cancer is to detect CTCs in blood. In the present study, we examined whether directly analyzing cells with capillary electrophoresis (CE) could distinguish cancer cells from normal cells, based on differences in cell surface glycosylation. We compared human colorectal cancer (CRC) cell lines to a normal colon epithelium cell line. Our results demonstrated that direct CE analysis could successfully distinguish between CRC and normal cells with high reproducibility, based on migration times. We found that the weighted-average migration time was significantly shorter for CRC cells than for normal cells. Next, we observed changes in the electrophoretic behaviors of CRC cells by adding five different types of lectins. When Aleuria aurantia lectin was added, migration delays were observed in CRC cells, but not in normal colon cells. Therefore, by focusing on shifts in migration time after adding specific lectins, we could distinguish cancer cells from normal cells. These findings suggested that this diagnostic method of directly analyzing cells with CE after adding specific lectin(s) could be useful for detecting the difference in the sugar moieties on a surface of normal and cancer cells.

Simultaneous determination of twelve tetrahydrocorticosteroid glucuronides in human urine by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry.

A liquid chromatography/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of 12 tetrahydrocorticosteroid glucuronides in human urine has been developed. The analytes were 3- and 21-monoglucuronides of tetrahydrocortisol, tetrahydrocortisone, tetrahydro-11-deoxycortisol, and their 5alpha-stereoisomers. The mass spectrometric behaviors of these glucuronides in negative-ion ESI-MS/MS revealed the production of intense, structure-specific product ions within the same group of glucuronides. Regioisomeric glucuronides could be distinguished by collision-induced dissociation and tandem mass spectrometry. Using a linear ion trap instrument operating in the negative-ion mode and by monitoring the transition ions of [M - H](-) --> [M - H - CH(2)O](-) for 3-monoglucuronides and [M - H](-) --> [M - H - CH(2)OG](-) for 21-monoglucuronides, a sensitive and specific assay was developed. Initial steps in the assay were a simple solid-phase extraction and the addition of [9,12,12,21,21-d(5)]-tetrahydrocortisone-3-glucuronide (prepared by enzyme-assisted synthesis) as an internal standard. The method was applied to determine the 12 tetrahydrocoticosteroid glucuronides in urine from healthy subjects and from patients with excessive cortisol production. The method described here appears to be useful for clinical and biochemical studies.

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry.

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-(2)H4-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS2. These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.