Comparison of 1Beta- and 5Beta-hydroxylation of Deoxycholate and Glycodeoxycholate as In Vitro Index Reactions for Cytochrome P450 3A Activities.

Cytochrome P450 3A (CYP3A) participates in the metabolism of more than 30% of clinical drugs. The vast intra- and inter-individual variations in CYP3A activity pose great challenges to drug development and personalized medicine. It has been disclosed that human CYP3A4 and CYP3A7 are exclusively responsible for the tertiary oxidations of deoxycholic acid (DCA) and glycodeoxycholic acid (GDCA) regioselectivity at C-1ß and C-5ß This work aimed to compare the 1ß- and 5ß-hydroxylation of DCA and GDCA as potential in vitro CYP3A index reactions in both human liver microsomes and recombinant P450 enzymes. The results demonstrated that the metabolic activity of DCA 1ß- and 5ß-hydroxylation was 5-10 times higher than that of GDCA, suggesting that 1ß-hydroxyglycodeoxycholic acid and 5ß-hydroxyglycodeoxycholic acid may originate from DCA oxidation followed by conjugation in humans. Metabolic phenotyping data revealed that DCA 1ß-hydroxylation, DCA 5ß-hydroxylation, and GDCA 5ß-hydroxylation were predominantly catalyzed by CYP3A4 (>80%), while GDCA 1ß-hydroxylation had approximately equal contributions from CYP3A4 (41%) and 3A7 (58%). Robust Pearson correlation was established for the intrinsic clearance of DCA 1ß- and 5ß-hydroxylation with midazolam (MDZ) 1'- and 4-hydroxylation in fourteen single donor microsomes. Although DCA 5ß-hydroxylation exhibited a stronger correlation with MDZ oxidation, DCA 1ß-hydroxylation exhibited higher reactivity than DCA 5ß-hydroxylation. It is therefore suggested that DCA 1ß- and 5ß-hydroxylations may serve as alternatives to T 6ß-hydroxylation as in vitro CYP3A index reactions. SIGNIFICANCE STATEMENT: The oxidation of DCA and GDCA is primarily catalyzed by CYP3A4 and CYP3A7. This work compared the 1ß- and 5ß-hydroxylation of DCA and GDCA as in vitro index reactions to assess CYP3A activities. It was disclosed that the metabolic activity of DCA 1ß- and 5ß-hydroxylation was 5-10 times higher than that of GDCA. Although DCA 1ß-hydroxylation exhibited higher metabolic activity than DCA 5ß-hydroxylation, DCA 5ß-hydroxylation demonstrated stronger correlation with MDZ oxidation than DCA 1ß-hydroxylation in individual liver microsomes.

[Analysis of the serum bile acid profile to facilitate diagnosis and differential diagnosis of NA(+)-taurocholate cotransporting polypeptide deficiency].

Objective: This study focuses on Na(+)-taurocholate cotransporting polypeptide (NTCP) deficiency to analyze and investigate the value of the serum bile acid profile for facilitating the diagnosis and differential diagnosis. Methods: Clinical data of 66 patients with cholestatic liver diseases (CLDs) diagnosed and treated in the Department of Pediatrics of the First Affiliated Hospital of Jinan University from early April 2015 to the end of December 2021 were collected, including 32 cases of NTCP deficiency (16 adults and 16 children), 16 cases of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), 8 cases of Alagille syndrome, and 10 cases of biliary atresia. At the same time, adult and pediatric healthy control groups (15 cases each) were established. The serum bile acid components of the study subjects were qualitatively and quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. The data were plotted and compared using statistical SPSS 19.0 and GraphPad Prism 5.0 software. The clinical and bile acid profiles of children with NTCP deficiency and corresponding healthy controls, as well as differences between NTCP deficiency and other CLDs, were compared using statistical methods such as t-tests, Wilcoxon rank sum tests, and Kruskal-Wallis H tests. Results: Compared with the healthy control, the levels of total conjugated bile acids, total primary bile acids, total secondary bile acids, glycocholic acid, taurocholic acid, and glycochenodeoxycholic acid were increased in NTCP deficiency patients (P < 0.05). Compared with adults with NTCP deficiency, the levels of total conjugated bile acids and total primary bile acids were significantly increased in children with NTCP deficiency (P < 0.05). The serum levels of taurochenodeoxycholic acid, glycolithocholate, taurohyocholate, and tauro-α-muricholic acid were significantly increased in children with NTCP deficiency, but the bile acid levels such as glycodeoxycholic acid, glycolithocholate, and lithocholic acid were decreased (P < 0.05). The serum levels of secondary bile acids such as lithocholic acid, deoxycholic acid, and hyodeoxycholic acid were significantly higher in children with NTCP deficiency than those in other CLD groups such as NICCD, Alagille syndrome, and biliary atresia (P < 0.05). Total primary bile acids/total secondary bile acids, total conjugated bile acids/total unconjugated bile acids, taurocholic acid, serum taurodeoxycholic acid, and glycodeoxycholic acid effectively distinguished children with NTCP deficiency from other non-NTCP deficiency CLDs. Conclusion: This study confirms that serum bile acid profile analysis has an important reference value for facilitating the diagnosis and differential diagnosis of NTCP deficiency. Furthermore, it deepens the scientific understanding of the changing characteristics of serum bile acid profiles in patients with CLDs such as NTCP deficiency, provides a metabolomic basis for in-depth understanding of its pathogenesis, and provides clues and ideas for subsequent in-depth research.

Necroptosis of macrophage is a key pathological feature in biliary atresia via GDCA/S1PR2/ZBP1/p-MLKL axis.

Biliary atresia (BA) is a severe inflammatory and fibrosing neonatal cholangiopathy disease characterized by progressive obstruction of extrahepatic bile ducts, resulting in cholestasis and progressive hepatic failure. Cholestasis may play an important role in the inflammatory and fibrotic pathological processes, but its specific mechanism is still unclear. Necroptosis mediated by Z-DNA-binding protein 1 (ZBP1)/phosphorylated-mixed lineage kinase domain-like pseudokinase (p-MLKL) is a prominent pathogenic factor in inflammatory and fibrotic diseases, but its function in BA remains unclear. Here, we aim to determine the effect of macrophage necroptosis in the BA pathology, and to explore the specific molecular mechanism. We found that necroptosis existed in BA livers, which was occurred in liver macrophages. Furthermore, this process was mediated by ZBP1/p-MLKL, and the upregulated expression of ZBP1 in BA livers was correlated with liver fibrosis and prognosis. Similarly, in the bile duct ligation (BDL) induced mouse cholestatic liver injury model, macrophage necroptosis mediated by ZBP1/p-MLKL was also observed. In vitro, conjugated bile acid-glycodeoxycholate (GDCA) upregulated ZBP1 expression in mouse bone marrow-derived monocyte/macrophages (BMDMs) through sphingosine 1-phosphate receptor 2 (S1PR2), and the induction of ZBP1 was a prerequisite for the enhanced necroptosis. Finally, after selectively knocking down of macrophage S1pr2 in vivo, ZBP1/p-MLKL-mediated necroptosis was decreased, and further collagen deposition was markedly attenuated in BDL mice. Furthermore, macrophage Zbp1 or Mlkl specific knockdown also alleviated BDL-induced liver injury/fibrosis. In conclusion, GDCA/S1PR2/ZBP1/p-MLKL mediated macrophage necroptosis plays vital role in the pathogenesis of BA liver fibrosis, and targeting this process may represent a potential therapeutic strategy for BA.

Glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides, but not hexadecanedioate and tetradecanedioate, detected weak inhibition of OATP1B caused by GDC-0810 in humans.

Endogenous biomarkers of drug transporters are promising tools to evaluate in vivo transporter function and potential alterations in the pharmacokinetics of their substrates. We have previously reported that coproporphyrin I/III captured the weak inhibition of OATP1B transporters by GDC-0810. In this study, we measured plasma concentrations of additional biomarkers, namely fatty acids, bile acids and their sulphate or glucuronide conjugates in the presence and absence of GDC-0810. Concentrations of hexadecanedioate and tetradecanedioate did not increase in the presence of GDC-0810. Among bile acids and their conjugates, glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides (GCDCA-3G and GDCA-3G) showed Cmax increases with geometric mean ratio (95% confidence interval) of 1.58 (1.13-2.22) and 1.49 (1.21-1.83), consistent with previous reports from low-dose rifampin co-administration and pharmacogenetic studies. These results suggest that GCDCA-3G and GDCA-3G are two more promising biomarkers that may capture weak OATP1B inhibition in addition to coproporphyrin I/III.

Suppression of Hepatic PPARα in Primary Biliary Cholangitis Is Modulated by miR-155.

BACKGROUND: PPARα is a ligand-activated transcription factor that shows protective effects against metabolic disorders, inflammation and apoptosis. Primary biliary cholangitis and primary sclerosing cholangitis result in the intrahepatic accumulation of bile acids that leads to liver dysfunction and damage. Small, non-coding RNAs such as miR-155 and miR-21 are associated with silencing PPARα. METHODS: The expression of miR-155, miR-21 and PPARα were evaluated using real-time PCR on liver tissue, as well as on human hepatocytes (HepG2) or cholangiocytes (NHCs) following exposure to lipopolysaccharide (LPS), glycodeoxycholic acid (GCDCA), lithocholic acid (LCA) and/or ursodeoxycholic acid (UDCA). RESULTS: A reduction of PPARα in primary biliary cholangitis (PBC) livers was associated with miR-21 and miR-155 upregulation. Experimental overexpression of either miR-155 or miR-21 inhibited PPARα in hepatocytes, whereas, in cholangiocytes, only miR-21 suppressed PPARα. Both GCDCA and LCA induced the cell type-specific upregulation of miR-155 or miR-21. In HepG2, LPS-induced miR-155 expression was blocked by a cotreatment with UDCA and was associated with PPARα upregulation. In NHC cells, the expression of miR-21 was induced by LPS but did not affect PPARα expression. CONCLUSIONS: Hepatic PPARα expression is reduced in PBC livers as a likely result of miR-155 overexpression. UDCA effectively reduced both baseline and LPS-induced miR-155 expression, thus preventing the suppression of PPARα.

L-Malic Acid Protects Lacticaseibacillus paracasei L9 from Glycodeoxycholic Acid Stress via the Malolactic Enzyme Pathway.

Bile stress tolerance is a crucial characteristic of probiotics for surviving in the human gastrointestinal tract. The mechanism underlying the effect of l-malic acid on enhancing the glycodeoxycholic acid (GDCA) tolerance of Lacticaseibacillus paracasei L9 was investigated herein. Bile tolerance specificity assays revealed that Lc. paracasei L9 was more sensitive to GDCA than to taurocholic acid, glycocholic acid, and taurodeoxycholic acid. Notably, l-malic acid significantly enhanced the GDCA tolerance of Lc. paracasei L9 by increasing the pH of the medium. The role of the malolactic enzyme pathway in enhancing GDCA resistance was investigated using molecular techniques. Confocal laser scanning and scanning electron microscopy revealed that l-malic acid preserved membrane permeability and cellular morphology, thereby protecting bacterial cells from GDCA stress-induced damage. The study also demonstrated that l-malic acid enhanced bile tolerance in different species of lactobacilli. These findings provide a novel protective mechanism for coping with bile stress in lactobacilli.

Targeted metabolomics analysis of bile acids and cell biology studies reveal the critical role of glycodeoxycholic acid in buffalo follicular atresia.

The follicular fluid of mammals has a high abundance of bile acids and these have proven to be closely related to the follicular atresia. However, the origin and content of bile acids in follicular fluid and its mechanisms on follicular atresia remain largely unknown. In this work, we analyzed the origin of bile acids in buffalo follicles by using cell biology studies, and quantified the subspecies of bile acids in follicular fluid from healthy follicles (HF) and atretic follicles (AF) by targeted metabolomics. The function of differential bile acids on follicular granulosa cells was also studied. The results showed that the bile acids transporters were abundantly expressed in ovarian tissues, but the rate-limiting enzymes were not, which was consistent with the inability of cultured follicular cells to convert cholesterol into bile acids. Targeted metabolomics analysis revealed thirteen differential subspecies of bile acids between HF and AF. The free bile acids were significant down-regulated and their conjugated forms were significantly up-regulated in AF as compared to HF. Finally, cell biological validation found a specific differentially conjugated bile acid, glycodeoxycholic acid (GDCA), which could promote follicular granulosa cell apoptosis and reduce steroid hormone secretion. In summary, our studies suggest that bile acids in buffalo follicles are transported from the blood rather than being synthesized within the follicles. The conjugated bile acids such as GDCA, accumulate in buffalo follicles, and may accelerate atresia by promoting apoptosis of granulosa cells and inhibiting steroid hormone production. These results will provide new clues for studying the physiological role and mechanism of bile acids involved in buffalo follicular atresia.

Role of glycodeoxycholic acid to induce acute pancreatitis in Macaca nemestrina.

BACKGROUND: Acute pancreatitis exhibits a rapid clinical progression which makes it difficult to observe in human; hence, an experimental animal model is needed. This preliminary study performed an induction of acute pancreatitis using glycodeoxycholic acid (GDOC) in an experimental macaque model. METHODS: GDOC injections (initial dose of 11.20 mg/kg) were administered in an escalating manner at specific time points. The injection was given along the bilio-pancreatic duct, followed by measurement of vital signs, serum amylase-lipase, TNF-α, procalcitonin, oxidative stress parameters, and microscopic and macroscopic findings. RESULTS: The results indicated that acute pancreatitis occurred following induction with low-dose GDOC. Serum amylase and lipase levels increased with subsequent GDOC injections. Blood pressure and heart rate were elevated, indicating abdominal pain. Changes in TNF-α, procalcitonin, and oxidative stress values showed active inflammation. We observed histologic features of pancreatitis and as the dose increased, vasodilation of the splanchnic vasculatures was observed. CONCLUSIONS: Small dose GDOC injection in the bilio-pancreatic duct may have a role to induce acute pancreatitis in Macaca nemestrina.

Serum bile acids in cystic fibrosis patients - glycodeoxycholic acid as a potential marker of liver disease.

BACKGROUND: Cystic fibrosis (CF) and CF-related liver disease can lead to disturbances in bile acid metabolism. AIM: This study determined serum bile acid concentrations in CF to define their usefulness in liver disease assessment. METHODS: Primary, secondary and conjugated bile acid levels were measured in three CF groups (25 patients each) exhibiting: liver cirrhosis, other liver disease, no liver disease, and in 25 healthy subjects (HS). RESULTS: Bile acid levels were higher in CF patients than in HS, except for glycodeoxycholic acid (GDCA). However, bile acid concentrations did not differ between patients with cirrhosis and other liver involvement. GDCA and deoxycholic acid (DCA) differentiated CF patients with non-cirrhotic liver disease from those without liver disease (GDCA-AUC: 0.924, 95%CI 0.822-1.000, p<0.001; DCA-AUC: 0.867, 95%CI: 0.731-1.000, p<0.001). Principal component analysis revealed that in CF liver disease was related to GDCA, GGTP activity, severe genotype and pancreatic insufficiency. CONCLUSIONS: A CF-specific bile acid profile was defined and shown to relate to liver disease. GDCA differentiates patients with non-cirrhotic liver involvement from those with no detectable liver disease. Hence, GDCA is a candidate for validation as a biomarker of non-cirrhotic progression of liver disease in CF.

Glycine-Conjugated Bile Acids Protect RPE Tight Junctions against Oxidative Stress and Inhibit Choroidal Endothelial Cell Angiogenesis In Vitro.

We previously demonstrated that the bile acid taurocholic acid (TCA) inhibits features of age-related macular degeneration (AMD) in vitro. The purpose of this study was to determine if the glycine-conjugated bile acids glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and glycoursodeoxycholic acid (GUDCA) can protect retinal pigment epithelial (RPE) cells against oxidative damage and inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in choroidal endothelial cells (CECs). Paraquat was used to induce oxidative stress and disrupt tight junctions in HRPEpiC primary human RPE cells. Tight junctions were assessed via transepithelial electrical resistance and ZO-1 immunofluorescence. GCA and GUDCA protected RPE tight junctions against oxidative damage at concentrations of 100-500 µM, and GDCA protected tight junctions at 10-500 µM. Angiogenesis was induced with VEGF in RF/6A macaque CECs and evaluated with cell proliferation, cell migration, and tube formation assays. GCA inhibited VEGF-induced CEC migration at 50-500 µM and tube formation at 10-500 µM. GUDCA inhibited VEGF-induced CEC migration at 100-500 µM and tube formation at 50-500 µM. GDCA had no effect on VEGF-induced angiogenesis. None of the three bile acids significantly inhibited VEGF-induced CEC proliferation. These results suggest glycine-conjugated bile acids may be protective against both atrophic and neovascular AMD.

Reduced glycodeoxycholic acid levels are associated with negative clinical outcomes of gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is characterized by glycemia and insulin disorders. Bile acids (BAs) have emerged as vital signaling molecules in glucose metabolic regulation. BA change in GDM is still unclear, which exerts great significance to illustrate the change of BAs in GDM. GDM patients and normal pregnant women were enrolled during the oral glucose tolerance test (OGTT) screening period. Fasting serums were sampled for the measurement of BAs. BA metabolism profiles were analyzed in both pregnant women with GDM and those with normal glucose tolerance (NGT). Delivery characteristics, delivery gestational age, and infant birthweight were extracted from medical records. GDM patients presented distinctive features compared with NGT patients, including higher body mass index (BMI), elevated serum glucose concentration, raised insulin (both fasting and OGTT), and increased hemoglobin A1c (HbA1c) levels. Higher homeostasis model assessment of insulin resistance (HOMA-IR) and decreased ß-cell compensation (i.e., oral disposition index (DIo)) were also prevalent in this group. Total BAs (TBAs) remained stable, but glycodeoxycholic acid (GDCA) and taurodeoxycholic acid (TDCA) levels declined significantly in GDM. GDCA was inversely correlated with HOMA-IR and positively correlated with DIo. No obvious differences in clinical outcome between the GDM and NGT groups were observed. However, GDM patients with high HOMA-IR and low DIo tended to have a higher cesarean delivery rate and younger delivery gestational age. In conclusion, GDCA provides a valuable biomarker to evaluate HOMA-IR and DIo, and decreased GDCA levels predict poorer clinical outcomes for GDM.

Sodium glycodeoxycholate and sodium deoxycholate as epithelial permeation enhancers: in vitro and ex vivo intestinal and buccal bioassays.

Bile salts were first tested as epithelial permeation enhancers (PEs) for the intestine and buccal routes over 20 years ago. They are not as popular as other PEs due to their non-specific mechanism of action and perceived toxicity potential. We revisited two of them by comparing efficacy and toxicity of sodium glycodeoxycholate (SGC) and sodium deoxycholate (DC) for both routes using in vitro and ex vivo methods. Cytotoxicity assays in Caco-2 cells revealed that both agents altered cellular parameters at concentrations >2 mM over 60 min. Both agents reduced the transepithelial resistance (TEER) and doubled the Papp of [3H]-octreotide across isolated rat colonic mucosae mounted in Ussing chambers at 10 mM concentrations. In some studies, 10 mM GDC also increased the Papp of the paracellular marker, FITC-dextran 4000 (FD4) and the fluorescent peptide, FITC-LKP, across colonic mucosae. Tissue histology was intact despite some mild perturbation at 10 mM. In the buccal epithelial cell line, TR146, changes in cell parameters were also seen at 1.5 mM over 60 min for both agents, with slightly more sensitivity seen for DC. In isolated porcine buccal epithelial mucosae, GDC was slightly more potent and efficacious than DC at increasing the Papp of [14C]-mannitol. It also increased the Papp of [3H]-octreotide and FITC-LKP by ∼3-fold across porcine buccal tissue without causing damage. Overall, GDC and DC were efficacious in intestinal and buccal models. Both cause mild perturbation leading to an increase in paracellular fluxes for hydrophilic molecules including peptides. Their moderate efficacy, low potency, and low toxicity in these models are similar to that of more established PEs in clinical trials.

Short communication: Chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli.

Effects of chemical structure, concentration, and pH on antimicrobial activity of conjugated bile acids were investigated in 4 strains of lactobacilli. Considerable differences were observed in the antimicrobial activity between the 6 human conjugated bile acids, including glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid. Glycodeoxycholic acid and glycochenodeoxycholic acid generally showed significantly higher antimicrobial activity against the lactobacilli, but glycocholic acid and taurocholic acid exhibited the significantly lower antimicrobial activity. Glycochenodeoxycholic acid was selected for further analysis, and the results showed its antimicrobial activity was concentration-dependent, and there was a significantly negative linear correlation (R2 > 0.98) between bile-antimicrobial index and logarithmic concentration of the bile acid for each strain of lactobacilli. Additionally, the antimicrobial activity of glycochenodeoxycholic acid was also observed to be pH-dependent, and it was significantly enhanced with the decreasing pH, with the result that all the strains of lactobacilli were unable to grow at pH 5.0. In conclusion, chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli. This study provides theoretical guidance and technology support for developing a scientific method for evaluating the bile tolerance ability of potentially probiotic strains of lactobacilli.

Roasting carob flour decreases the capacity to bind glycoconjugates of bile acids.

Carob is the fruit obtained from Ceratonia siliqua L. and it is a source of bioactive compounds that have been linked to several health promoting effects, including lowering blood cholesterol concentration. The objective of this study was to connect the physicochemical changes of carob flour occurring during roasting with its capacity to bind glycoconjugates of bile acids. Carob flour samples were roasted for different times at 150 °C and chemically characterized by measuring the concentrations of tannins and polyphenols. Data showed that carob flour binds high amounts of bile acids: 732.6 µmol of bound bile acid per g of carob flour which is comparable to the 836.2 µmol per g bound by cholestyramine, a known cholesterol lowering drug. The carob flour ability to bind cholesterol decreases up to 40% during roasting. Data suggested that tannins and insoluble components play a major role in binding bile salts, as a result of hydrophobic interactions.

Bile acid changes after metabolic surgery are linked to improvement in insulin sensitivity.

BACKGROUND: Metabolic surgery is associated with a prompt improvement in insulin resistance, although the mechanism of action remains unknown. The literature on bile acid changes after metabolic surgery is conflicting, and insulin sensitivity is generally assessed by indirect methods. The aim of this study was to investigate the relationship between improvement in insulin sensitivity and concentration of circulating bile acids after biliopancreatic diversion (BPD) and Roux-en-Y gastric bypass (RYGB). METHODS: This was a prospective observational study of nine patients who underwent BPD and six who had RYGB. Inclusion criteria for participation were a BMI in excess of 40 kg/m2 , no previous diagnosis of type 2 diabetes and willingness to participate. Exclusion criteria were major endocrine diseases, malignancies and liver cirrhosis. Follow-up visits were carried out after a mean(s.d.) of 185·3(72·9) days. Fasting plasma bile acids were assessed by ultra-high-performance liquid chromatography coupled with a triple quadrupole mass spectrometer, and insulin sensitivity was measured by means of a hyperinsulinaemic-euglycaemic clamp. RESULTS: A significant increase in all bile acids, as well as an amelioration of insulin sensitivity, was observed after metabolic surgery. An increase in conjugated secondary bile acids was significantly associated with an increase in insulin sensitivity. Only the increase in glycodeoxycholic acid was significantly associated with an increase in insulin sensitivity in analysis of individual conjugated secondary bile acids. CONCLUSION: Glycodeoxycholic acid might drive the improved insulin sensitivity after metabolic surgery.

A multiplex HRMS assay for quantifying selected human plasma bile acids as candidate OATP biomarkers.

AIM: Selected bile acids (BAs) in plasma have been proposed as endogenous probes for assessing drug-drug interactions involving hepatic drug transporters such as the organic anion-transporting polypeptides (OATP1B1 and OATP1B3). MATERIALS & METHODS: Plasma extracts were analyzed for selected BAs using a triple TOF API6600 high-resolution mass spectrometer. RESULTS: Glycodeoxycholic acid 3-sulfate, glycochenodeoxycholic acid 3-sulfate, glycodeoxycholic acid 3-O-ß-glucuronide and glycochenodeoxycholic acid 3-O-ß-glucuronide are presented as potential OATP1B1/3 biomarkers. CONCLUSION: Six BAs are quantified in human plasma using a multiplexed high-resolution mass spectrometry method. Glycodeoxycholic acid 3-sulfate and glycodeoxycholic acid 3-O-ß-glucuronide are proposed as potential biomarkers based on observed four- to fivefold increase in plasma AUC (vs placebo), following administration of a compound known to present as an OATP1B1/3 inhibitor in vitro.

Deoxycholylglycine, a conjugated secondary bile acid, reduces vascular tone by attenuating Ca2+ sensitivity via rho kinase pathway.

Patients with cirrhosis have reduced systemic vascular resistance and elevated circulating bile acids (BAs). Previously, we showed that secondary conjugated BAs impair vascular tone by reducing vascular smooth muscle cell (VSMC) Ca2+ influx. In this study, we investigated the effect of deoxycholylglycine (DCG), on Ca2+ sensitivity in reducing vascular tone. First, we evaluated the effects of DCG on U46619- and phorbol-myristate-acetate (PMA)-induced vasoconstriction. DCG reduced U46619-induced vascular tone but failed to reduce PMA-induced vasoconstriction. Then, by utilizing varied combinations of diltiazem (voltage-dependent Ca2+ channel [VDCC] inhibitor), Y27632 (RhoA kinase [ROCK] inhibitor) and chelerythrine (PKC inhibitor) for the effect of DCG on U46619-induced vasoconstriction, we ascertained that DCG inhibits VDCC and ROCK pathway with no effect on PKC. We further assessed the effect of DCG on ROCK pathway. In ß-escin-permeabilized arteries, DCG reduced high-dose Ca2+- and GTPγS (a ROCK activator)-induced vasoconstriction. In rat vascular smooth muscle cells (VSMCs), DCG reduced U46619-induced phosphorylation of myosin light chain subunit (MLC20) and myosin phosphatase target subunit-1 (MYPT1). In permeabilized VSMCs, DCG reduced Ca2+- and GTPγS-mediated MLC20 and MYPT1 phosphorylation, and further, reduced GTPγS-mediated membrane translocation of RhoA. In VSMCs, long-term treatment with DCG had no effect on ROCK2 and RhoA expression. In conclusion, DCG attenuates vascular Ca2+ sensitivity and tone via inhibiting ROCK pathway. These results enhance our understanding of BAs-mediated regulation of vascular tone and provide a platform to develop new treatment strategies to reduce arterial dysfunction in cirrhosis.

Co-existing colloidal phases in artificial intestinal fluids assessed by AF4/MALLS and DLS: A systematic study into cholate & (lyso-) phospholipid blends, incorporating celecoxib as a model drug.

Colloidal phases (self-assemblies) in aqueous dispersions of selected binary bile salt/phospholipid blends were studied utilizing the combined analytical approach of asymmetrical flow field-flow fractionation (AF4) and multi-angle laser light scattering (MALLS) in order to resolve the co-existence of different colloidal assemblies. The binary blends were prepared by freeze-drying from tert-butanol/water co-solvent solutions. The blends contained one of two bile salts (sodium taurocholate (TC) or sodium glycodeoxycholate (GDX)) and a mono- or di-acyl phospholipid (lyso-phosphatidylcholine (L-PC) and phosphatidylcholine (PC), respectively). Bile salt and phospholipid (PL) concentrations and their respective ratios were varied systematically within the physiological range found in human intestinal fluids. Furthermore, the BCS class II drug Celecoxib was incorporated in selected blends to assess its potential impact on colloidal phases. To further investigate the smallest self-assemblies observed in AF4/MALLS analysis, dispersions of TC and GDX, respectively, were prepared and analyzed by dynamic light scattering (DLS). AF4/MALLS analysis revealed that binary bile-salt/phospholipid blends form three distinct particle fractions, when the concentration of bile-salt was sufficiently high (≥3.5 mM). Those fractions were assumed to be very small pure bile-salt dimeric/oligomeric self-assemblies (Ø ≈ 2-3 nm), mid-sized mixed micelles (Ø ≈ 10-50 nm) and large liposomes/aggregates (Ø ≈ 150-280 nm). If present, Celecoxib was found solubilized within the structures, but at the lowest TC concentration triggered the formation of an additional (vesicular) phase.