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Background Hepatobiliary diseases result in the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues which may contribute to an unfavorable prognosis. Aim To discover and validate diagnostic biomarkers of cholestatic liver diseases based on the urinary BA profile. Methods We analyzed urine samples by liquid chromatography-tandem mass spectrometry and compared the urinary BA profile between 300 patients with hepatobiliary diseases vs 103 healthy controls by statistical analysis. The BA profile was characterized using BA indices, which quantifies the composition, metabolism, hydrophilicity, and toxicity of the BA profile. BA indices have much lower inter- and intra-individual variability compared to absolute concentrations of BA. In addition, BA indices demonstrate high area under the receiver operating characteristic curves, and changes of BA indices are associated with the risk of having a liver disease, which demonstrates their use as diagnostic biomarkers for cholestatic liver diseases. Results Total and individual BA concentrations were higher in all patients. The percentage of secondary BA (lithocholic acid and deoxycholic acid) was significantly lower, while the percentage of primary BA (chenodeoxycholic acid, cholic acid, and hyocholic acid) was markedly higher in patients compared to controls. In addition, the percentage of taurine-amidation was higher in patients than controls. The increase in the non-12α-OH BA was more profound than 12α-OH BA (cholic acid and deoxycholic acid) causing a decrease in the 12α-OH/ non-12α-OH ratio in patients. This trend was stronger in patients with more advanced liver diseases as reflected by the model for end-stage liver disease score and the presence of hepatic decompensation. The percentage of sulfation was also higher in patients with more severe forms of liver diseases. Conclusion BA indices have much lower inter- and intra-individual variability compared to absolute BA concentrations and changes of BA indices are associated with the risk of developing liver diseases.

Alcohol-associated liver disease is accompanied by dysregulation of bile acid metabolism and gut barrier dysfunction. Peroxisome proliferator-activated receptor-delta (PPARδ) agonists are key metabolic regulators and have anti-inflammatory properties. Here, we evaluated the effect of the selective PPAR-delta agonist seladelpar (MBX–8025) on gut barrier function and bile acid metabolism in a mouse model of ethanol-induced liver disease. Wild type C57BL/6 mice were fed LieberDe-Carli diet containing 0%–36% ethanol (caloric) for 8 weeks followed by a single binge of ethanol (5 g/kg). Pair fed mice received an isocaloric liquid diet as control. MBX-8025 (10 mg/kg/d) or vehicle were added to the liquid diet during the entire feeding period (prevention), or during the last 4 weeks of Lieber DeCarli diet feeding (intervention). In both prevention and intervention trials, MBX-8025 protected mice from ethanol-induced liver disease, characterized by lower serum alanine aminotransferase (ALT) levels, hepatic triglycerides, and inflammation. Chronic ethanol intake disrupted bile acid metabolism by increasing the total bile acid pool and serum bile acids. MBX-8025 reduced serum total and secondary bile acids, and the total bile acid pool as compared with vehicle treatment in both prevention and intervention trials. MBX-8025 restored ethanol-induced gut dysbiosis and gut barrier dysfunction. Data from this study demonstrates that seladelpar prevents and treats ethanol-induced liver damage in mice by direct PPARδ agonism in both the liver and the intestine.

One of the mechanisms of drug-induced liver injury (DILI) involves alterations in bile acid (BA) homeostasis and elimination, which encompass several metabolic pathways including hydroxylation, amidation, sulfation, glucuronidation and glutathione conjugation. Species differences in BA metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably the DILI during the early stages of drug discovery and development. We developed an in vitro cofactor-fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs (cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid) between humans and several animal species. High- and low-resolution liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance imaging were used for the qualitative and quantitative analysis of BAs and their metabolites. Major species differences were found in the metabolism of BAs. Sulfation into 3-O-sulfates was a major pathway in human and chimpanzee (4.8%-52%) and it was a minor pathway in all other species (0.02%-14%). Amidation was primarily with glycine (62%-95%) in minipig and rabbit and it was primarily with taurine (43%-81%) in human, chimpanzee, dog, hamster, rat and mice. Hydroxylation was highest (13%-80%) in rat and mice followed by hamster, while it was lowest (1.6%-22%) in human, chimpanzee and minipig. C6-β hydroxylation was predominant (65%-95%) in rat and mice, while it was at C6-α position in minipig (36%-97%). Glucuronidation was highest in dog (10%-56%), while it was a minor pathway in all other species (

Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. The marked differences in BA composition between preclinical safety models and humans may play a major role in the poor prediction of drug-induced liver injury using preclinical models. We compared the composition of plasma and urinary BAs and their metabolites between humans and several animal species. Total BA pools and their composition varied widely among different species. Highest sulfation of BAs was observed in human and chimpanzee. Glycine amidation was predominant in human, minipig, hamster and rabbit, while taurine amidation was predominant in mice, rat and dogs. BA profiles consisted primarily of tri-OH BAs in hamster, rat, dog and mice, di-OH BAs in human, rabbit and minipig, and mono-OH BA in chimpanzee. BA profiles comprised primarily hydrophilic and less toxic BAs in mice, rat, pig and hamster, while it primarily comprised hydrophobic and more toxic BAs in human, rabbit and chimpanzee. Therefore, the hydrophobicity index was lowest in minipig and mice, while it was highest in rabbit, monkey and human. Glucuronidation and glutathione conjugation were low in all species across all BAs. Total concentration of BAs in urine was up to 10× higher and more hydrophilic than plasma in most species. This was due to the presence of more tri-OH, amidated, sulfated and primary BAs, in urine compared to plasma. In general, BA profiles of chimpanzee and monkeys were most similar to human, while minipig, rat and mice were most dissimilar to human.

Bile acids (BAs) are post-prandial hormones that play an important role in glucose and lipid homeostasis as well as energy expenditure. Total and glycine-amidated BAs increase after sleeve gastrectomy (SG) and correlate to improved metabolic disease. No specific bile acid subtype has been shown conclusively to mediate the weight loss effect. Therefore, the objective of this study was to prospectively evaluate the comprehensive changes in meal-stimulated BAs after SG and determine if a specific change in the BA profile correlates to the early weight loss response. Patients were prospectively enrolled at the University of Nebraska Medical Center who were undergoing a SG for treatment of morbid obesity. Primary and secondary plasma bile acids and their amidated (glycine, G-, or taurine, T-) subtypes were measured at fasting, 30 and 60 min after a liquid meal performed pre-op, and at 6 and 12 weeks post-op. Area under the curve (AUC) was calculated for the hour meal test for each bile acid subtype. BAs that were significantly increased post-op were correlated to body mass index (BMI) loss. Total BA AUC was significantly increased at 6 (p

Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs that require intracellular phosphorylation to active triphosphate nucleotide metabolites (NMs) for their pharmacological activity. However, monitoring these pharmacologically active NMs is challenging due to their instability, high hydrophilicity, and their low concentrations in blood and tissues. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) is the gold standard technique for the quantification of NRTIs and their phosphorylated NMs. In this review, an overview of the publications describing the quantitative analysis of intracellular and total tissue concentration of NMs is presented. The focus of this review is the comparison of the different approaches and challenges associated with sample collection, tissue homogenization, cell lysis, cell counting, analyte extraction, sample storage conditions, and LC–MS analysis. Quantification methods of NMs via LC–MS can be categorized into direct and indirect methods. In the direct LC–MS methods, chromatographic retention of the NMs is accomplished by ion-exchange (IEX), ion-pairing (IP), hydrophilic interaction (HILIC), porous graphitic carbon (PGC) chromatography, or capillary electrophoresis (CE). In indirect methods, parent nucleosides are 1st generated from the dephosphorylation of NMs during sample preparation and are then quantified by reverse phase LC–MS as surrogates for their corresponding NMs. Both approaches have advantages and disadvantages associated with them, which are discussed in this review.

Accurate quantitative analysis of endogenous analytes is essential for several clinical and non-clinical applications. LC-MS/MS is the technique of choice for quantitative analyses. Absolute quantification by LC/MS requires preparing standard curves in the same matrix as the study samples so that the matrix effect and the extraction efficiency for analytes are the same in both the standard and study samples. However, by definition, analyte-free biological matrices do not exist for endogenous compounds. To address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS, four approaches are used including the standard addition, the background subtraction, the surrogate matrix, and the surrogate analyte methods. This review article presents an overview these approaches, cite and summarize their applications, and compare their advantages and disadvantages. In addition, we discuss in details, validation requirements and compatibility with FDA guidelines to ensure method reliability in quantifying endogenous compounds. The standard addition, background subtraction, and the surrogate analyte approaches allow the use of the same matrix for the calibration curve as the one to be analyzed in the test samples. However, in the surrogate matrix approach, various matrices such as artificial, stripped, and neat matrices are used as surrogate matrices for the actual matrix of study samples. For the surrogate analyte approach, it is required to demonstrate similarity in matrix effect and recovery between surrogate and authentic endogenous analytes. Similarly, for the surrogate matrix approach, it is required to demonstrate similar matrix effect and extraction recovery in both the surrogate and original matrices. All these methods represent indirect approaches to quantify endogenous compounds and regardless of what approach is followed, it has to be shown that none of the validation criteria have been compromised due to the indirect analyses.