Bile Acids In Bile Research Articles

Hypothyroidism Increases Cholesterol Gallstone Prevalence in Mice by Elevated Hydrophobicity of Primary Bile Acids.

Background: Thyroid hormone (TH) deficiency has been associated with increased cholesterol gallstone prevalence. Hypothyroidism impacts hepatic lipid homeostasis, biliary secretion, gallbladder motility, and gallstone (LITH) gene expression, all potential factors contributing to cholesterol gallstone disease (CGD). However, how TH deficiency may lead to gallstone formation is still poorly understood. Therefore, we performed molecular studies in a CGD mouse model under lithogenic conditions and modulation of TH status. Methods: Male, three-month-old C57BL/6 mice were randomly divided into a control (euthyroid) group, a hypothyroid (hypo) group, a gallstone (litho) group, and a gallstone+hypothyroid (litho+hypo) group and were treated for 2, 4, and 6 weeks (n = 8/treatment period). Gallstone prevalence, biliary composition and cholesterol crystals, hepatic expression of genes participating in cholesterol, bile acid (BA), and phosphatidylcholine synthesis (Hmgcr, Cyp7a1, Pcyt1a), and canalicular transport (Abcg5, Bsep, Abcb4) were investigated. Results: Increased cholesterol gallstone prevalence was observed in hypothyroid mice under lithogenic diet after 4 and 6 weeks of treatment (4 weeks: 25% vs. 0%; 6 weeks: 75% vs. 37.5%). Interestingly, neither the composition of the three main biliary components, cholesterol, BAs, and phosphatidylcholine, nor the hepatic expression of genes involved in synthesis and transport could explain the differences in cholesterol gallstone formation in the mice. However, TH deficiency resulted in significantly increased hydrophobicity of primary BAs in bile. Furthermore, downregulation of hepatic sulfonation enzymes Papss2 and Sult2a8 as well as diminished biliary BA sulfate concentrations in mice were observed under hypothyroid conditions all contributing to a lithogenic biliary milieu as evidenced by microscopic cholesterol crystals and macroscopic gallstone formation. Conclusions: We describe a novel pathogenic link between TH deficiency and CGD and suggest that the increased hydrophobic character of biliary BAs due to the diminished expression of hepatic detoxification enzymes promotes cholesterol crystal precipitation and enhances cholesterol gallstone formation in the bile of hypothyroid mice.

Bile acid profiles in bile and feces of obese mice by a high-performance liquid chromatography-tandem mass spectrometry.

Bile acids (BAs) play a pivotal role in manipulating the development of metabolic diseases. However, due to the compositional complexity and functional variation of BAs, it remains unclear about the changes in BA pool for individuals with obesity or metabolic syndrome. We established a high-performance liquid chromatography-mass spectrometer detection system for the simultaneous analysis of both unconjugated and conjugated BAs in the bile and feces of mice. Ten BAs were completely separated, identified, and quantified with low limit of detection (0.5ng/mL) and inter/intraday precision (relative standard deviation<12%). By using this method, these BAs in bile and feces of mice were quantified. The result showed that taurochenodeoxycholic acid, taurine-conjugated α-muricholic acids, and taurine-conjugated β-muricholic acids were the dominated BAs in bile, whereas deoxycholic acid and chenodeoxycholic acid predominated in feces. Further, most of the BA levels were significantly elevated in either bile or fecal samples of high-fat diet-fed mice as compared with those in normal chow diet-fed mice, indicating that excessive production of BAs was closely associated with the occurrence of lipid metabolism disorders. In summary, the present method is practicable for analysis of BAs in bile and fecal samples of patients with obesity.

Chemical profiling for bile acid derivatives in yak bile

Bile acids( BAs),the major constituents of bile,are also known to be potential biomarkers of various diseases,especially liver disease. The systematic analysis of BAs is believed to be of great importance towards the clarification of the effective material basis for bile-type medicines,and the diagnosis and therapy of related diseases as well. As a part of systematic study on bile-type medicine ongoing in our group,this study lays emphasis on the isomer discrimination,and the improvement of analytical method of BAs. Further,this method was subsequently applied to elucidate in depth the chemical profile of BAs in yak bile. Regarding isomer discrimination for BAs,we constructed relative response-collision energy curves( RRCECs) by high performance liquid chromatographyion trap-time of flight-mass spectrometry( HPLC-IT-TOF-MS) in combination with high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry( HPLC-Qtrap-MS). As a result,both the optimum collision energy( OCE) and CE_(50) exhibited great correlations with structural characteristics,thus enabling the isomer distinguishing,such as unconjugated BAs,glycine-conjugated BAs,and taurine-conjugated BAs. According to information provided by mass spectrometry,the comparison of OCE and CE_(50),retention time matching,combined with reference substances and database retrieval,a total of 30 bile acid derivatives were observed and identified in yak bile. The newly developed method could serve as a feasible tool for the in-depth characterization of BAs in bile and biological samples.

Iron depletion induces hepatic secretion of biliary lipids and glutathione in rats

Iron depletion (ID) has been shown to induce the liver expression of Cyp7a1, the rate-limiting enzyme initiating conversion of cholesterol to bile acids (BA), although the effect on bile acids metabolism and bile production is unknown. Therefore, we investigated changes in bile secretion and BA synthesis during diet-induced iron depletion (ID) in rats. ID increased bile flow along with augmented biliary excretion of bile acids, glutathione, cholesterol and phospholipids. Accordingly, we found transcriptional upregulation of the Cyp7a1, Cyp8b1, and Cyp27a1 BA synthetic enzymes, as well as induction of the Abcg5/8 cholesterol transporters in ID rat livers. In contrast, intravenous infusion of 3H-taurocholate failed to elicit any difference in biliary secretion of this compound in the ID rats. This corresponded with unchanged expression of canalicular rate-limiting transporters for BA as well as glutathione. We also observed that ID substantially changed the spectrum of BA in bile and decreased plasma concentrations of BA and cholesterol. Experiments with differentiated human hepatic HepaRG cells confirmed human CYP7A1 orthologue upregulation resulting from reduced iron concentrations. Results employing a luciferase reporter gene assay suggest that the transcriptional activation of the CYP7A1 promoter under ID conditions works independent of farnesoid X (FXR), pregnane X (PXR) and liver X (LXRα) receptors activation. It can be concluded that this study characterizes the molecular mechanisms of modified bile production as well as cholesterol as along with BA homeostasis during ID. We propose complex upregulation of BA synthesis, and biliary cholesterol secretion as the key factors affected by ID.

Quantitative profiling of bile acids in rat bile using ultrahigh-performance liquid chromatography-orbitrap mass spectrometry: Alteration of the bile acid composition with aging.

Bile acids (BAs) play important roles in physiological functions, including the homeostasis of cholesterol and lipids and as ligands for G protein-coupled receptors (GPCRs). With the increasing importance of BAs, analytical methods for their quantification and screening have been developed. However, due to the diverse range and variety of BAs with different activation potency, a simple, effective, and sensitive method is required to screen BAs for accurate quantification and identification. This paper presents an application of ultrahigh-performance liquid chromatography-orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) for profiling BAs in bile. Using this method, along with the accurate quantification of 19 targeted BAs, 22 unknown BAs were detected and characterized by their fragmentation patterns. The method is beneficial for screening most of the BAs (quantitatively and qualitatively) in rat bile with simple preparation in a single run. The sample dilution ranges of each BA were optimized depending on the concentration of BAs in the bile to obtain good peak separation and accurate data. The method validation was performed successfully using charcoal-treated bile and the intra and inter-day coefficients of variation were less than 20% for all BAs while the recovery were above 88.5% except for the lithocholic acid. The method was applied to profile the age-dependent changes in the contents of rat BAs. Through statistical analysis, we found that as the rats aged, unconjugated BAs and glycine-conjugated BAs decreased or were unaffected, while taurine-conjugated BAs were increased in general. Among the unknown BAs, 5 of the taurine-conjugated BAs increased, while a glycine-conjugated BA decreased, in agreement with the trends of the targeted BAs.

Screening for biomarkers of liver injury induced by Polygonum multiflorum: a targeted metabolomic study.

Heshouwu (HSW), the dry roots of Polygonum multiflorum, a classical traditional Chinese medicine is used as a tonic for a wide range of conditions, particularly those associated with aging. However, it tends to be taken overdose or long term in these years, which has resulted in liver damage reported in many countries. In this study, the indicative roles of nine bile acids (BAs) were evaluated to offer potential biomarkers for HSW induced liver injury. Nine BAs including cholic acid (CA) and chenodeoxycholic acid (CDCA), taurocholic acid (TCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), ursodeoxycholic acid (UDCA), and hyodeoxycholic acid (HDCA) in rat bile and serum were detected by a developed LC-MS method after 42 days treatment. Partial least square-discriminate analysis (PLS-DA) was applied to evaluate the indicative roles of the nine BAs, and metabolism of the nine BAs was summarized. Significant change was observed for the concentrations of nine BAs in treatment groups compared with normal control; In the PLS-DA plots of nine BAs in bile, normal control and raw HSW groups were separately clustered and could be clearly distinguished, GDCA was selected as the distinguished components for raw HSW overdose treatment group. In the PLS-DA plots of nine BAs in serum, the normal control and raw HSW overdose treatment group were separately clustered and could be clearly distinguished, and HDCA was selected as the distinguished components for raw HSW overdose treatment group. The results indicated the perturbation of nine BAs was associated with HSW induced liver injury; GDCA in bile, as well as HDCA in serum could be selected as potential biomarkers for HSW induced liver injury; it also laid the foundation for the further search on the mechanisms of liver injury induced by HSW.

Transient changes in the expression pattern of key enzymes for bile acid synthesis during rat liver regeneration

Changes in the expression patterns of genes involved in bile acid (BA) synthesis were investigated during rat liver regeneration that follows two-thirds partial hepatectomy. BAs in bile were measured by GC-MS and the absolute and relative abundance of specific mRNAs in the liver by RT-real-time quantitative PCR. Cyclin E mRNA, used as an indicator of liver cell proliferation, peaked at day 1. The levels of mRNA of α-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) were first reduced (day 1) and then (days 2–3) increased, when those of farnesoid X receptor (FXR) were also transiently enhanced. The early (day 1) up-regulation of Cyp7a1, and Cyp8b1, together with the down-regulation of Cyp27, was consistent with an increased proportion of cholic acid versus chenodeoxycholic acid and a progressive recovery in total BAs secretion. The transient appearance of flat BAs ( allo-BAs plus Δ 4-unsaturated-BAs) during rat liver regeneration was probably due to the changes in the expression ratio of steroid 5α- versus 5β-reductase. Both were first (day 1) down-regulated and then up-regulated (5α-reductase more than 5β-reductase). In conclusion, changes in the expression patterns of nuclear receptors and enzymes involved in BA synthesis are consistent with the transient modifications that occur in BA pool during rat liver regeneration.

Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration

Background/Aims : To investigate changes in nuclear bile acids (BAs) during rat liver regeneration. Methods : Nuclei were isolated from control rat livers and after two-thirds partial hepatectomy (PH). BAs in bile, liver homogenate and nuclei were measured by gas chromatography–mass spectrometry. Nuclear translocation of radiolabeled BAs was determined using fresh isolated hepatocytes from control donors. Results : Liver BA concentrations were transiently reduced after PH. Relative increases in: β-MCA at 1 day, deoxycholic acid at 7 days and cholic acid (CA) at 3 and 14 days were found. Nuclear BAs accounted for <0.5% of liver BAs. Contamination with cytosolic BAs during nuclei isolation was <4%. Unconjugated- and conjugated-CA were able to reach the nucleus with similar efficiency. The pattern of nuclear BAs – CA (80%) and ursodeoxycholic acid (UDCA) (8.5%) being the most abundant – did not match that found in liver or bile. A transient decrease in CA/UDCA ratio, in absence of significant change in total BA content, was observed in nuclei after PH. ‘Flat’ BA species were only detected in homogenate, but not in nuclei, at 1 day after PH. Conclusions : BA pool in nuclei of rat hepatocytes, whose composition is different to that of total liver BA pool, undergoes important changes during liver regeneration.

Bile acid secretion during synchronized rat liver regeneration

One major difficulty in interpreting the changes occurring during liver regeneration is the co-existence of non-activated cells and proliferating hepatocytes at all stages of differentiation. The aim of this study was to investigate bile acid (BA) secretion into bile during normal (NLR) and synchronized (SLR) liver regeneration in rats. Regeneration was synchronized by reversible inhibition of ribonucleotide reductase by 10 h treatment with hydroxyurea (HU) shortly after two-third partial hepatectomy. Total BA output as measured by GC–MS increased immediately after partial hepatectomy. This was followed by a further transient enhancement during the next day in the NLR. HU treatment did not significantly modify total BA output, but after releasing synchronized regeneration a marked reduction was observed. This was followed by a recovery to reach values close to those of NLR on day 7 of the regenerative process in SLR. Amidated BA output as measured by HPLC analysis revealed an early enhancement in the proportion of non-conjugated BAs in bile in NLR. However, the profile of conjugated BAs, which was not affected by HU treatment, matched that of total BAs during the first stage of SLR. By contrast, the increase in BA output observed on day 3 of the regenerative process in this group was accounted for by an enhancement in non-conjugated BA secretion. On day 7 of the regenerative process, the proportion of conjugated BA in bile was restored to approximately 100% in this group. Most BA molecules were conjugated with taurine rather than with glycine in all experimental groups, during both NLR and SLR. GC–MS determinations indicated that the magnitude of the cholic acid predominance in all bile samples was significantly modified during liver regeneration. This was increased early after partial hepatectomy and declined toward control values after few (2–3) days. Enhancement in the cholic acid predominance was due to a reduction in the proportion of all other major BAs, above all ursocholic acid and ω-muricholic acid. By contrast, minor BAs in normal control rat bile such as allo-cholic acid were increased during both NLR and SLR, and remained at detectable levels up to day 7. Changes in the proportion of secreted BA species were similar in SLR and NLR except that the early reduction in the proportion of BAs other than cholic acid was more pronounced in SLR and the quantitative importance of the diversity in BA species was recovered earlier in SLR than in NLR. In summary, these results indicate that profound changes in BA secretion during rat liver regeneration do occur. Most of them are probably related to the existence of retro-differentiation/re-differentiation processes which are speeded up by hydroxyurea-induced synchronization of the wave of hepatocyte proliferation.

Study of CD 4 and CD 8 level and graft versus host reaction during xenotransplantation of UV-B irradiated hepatocytes : [formula omitted]. Dept. of Gastroenterology, Osmania General Hospital, Afzalgunj, Hyderabad, Pin 500 012, INDIA

A series of unconjugated and taurine conjugated bile acids (BAs) differing in water solubility (SWo), critical micellar concentration (CMC), and hydrophilicity (K′) were infused iv to rats at a tracer dose and a dose of 6 μmol/min/kg over a 1-h period. Bile was collected for 3 h to evaluate the role of BA structure on cholesterol, phospholipids secretions, and bile flow. The BAs studied differ in the number (2–3), position (−3, −6, −7, −12), and orientation of the hydroxyls (α/β); the side chain structure was modified by shortening (C-23, nor-BA) and by lengthening (C-25, homo-BA), while maintaining the same structure of nuclear hydroxyls (3α7β). At a “tracer” dose, all C-24 natural BAs are efficiently recovered in bile when administered in both unconjugated and taurine conjugated forms. At a “high dose”, all taurine conjugated BAs are efficiently recovered in bile (80–100%). However, a variable recovery was observed among unconjugated BAs: trihydroxy BAs are efficiently recovered (85–100%), while dihydroxy BAs are only partially recovered (25–40%). The side chain-modified BAs [i.e., C-23 nor and C-25 homo analogs of ursodeoxycholic acid (UDCA)] are partially recovered at a tracer dose (20–30%), but less at a high dose (10–20%) when administered in the unconjugated form. In contrast, the corresponding taurine conjugates are more efficiently recovered in bile (60–80%). Conjugation with taurine increases total recovery of unconjugated BAs in bile by not more than 30–40%. Highly hydrophilic and water-soluble BAs, such as ursocholic acid (SWo = 1.67 mM) and cholic acid (SWo = 0.27 mM), can also be secreted as unconjugates, and this accounts for their complete recovery. The conjugation step is rate limiting for poorly soluble BAs such as ursodeoxycholic acid (SWo = 0.009 mM) when administered at a high dose, and critical for nor and homo analogs which are poorly soluble and whose side chain modification partially suppresses their conjugation with taurine or glycine and thereby induces alternative pathways such as glucuronidation or sulfation. The induced bile flow is directly related to the hydrophilicity of the natural C-24 bile acid. The C-23 analog of UDCA greatly increased bile flow, which reached a maximum at very low BA secretion, and the effect is drastically reduced when it was administered as taurine conjugates. The phospholipids and, to a lesser extent, the cholesterol secretions are poorly related to both the hydrophilicity and CMC of BAs, and hepatic biotransformations play a major role in modulating the biliary lipid secretion. In addition, specific effects not related to physicochemical properties, such as selective inhibition of cholesterol synthesis, were observed (see chenodeoxycholic acid and hyocholic acid). The quantitative parameters here evaluated by means of a factor analysis could be useful in structure–activity relationship studies and in designing BA analogs; the properties of homo-UDCA as a new synthetic BA are also discussed.

Quantitative Relationship between Bile Acid Structure and Biliary Lipid Secretion in Rats

AbstractA series of unconjugated and taurine conjugated bile acids (BAs) differing in water solubility (SWo), critical micellar concentration (CMC), and hydrophilicity (K′) were infused iv to rats at a tracer dose and a dose of 6 μmol/min/kg over a 1-h period. Bile was collected for 3 h to evaluate the role of BA structure on cholesterol, phospholipids secretions, and bile flow. The BAs studied differ in the number (2–3), position (−3, −6, −7, −12), and orientation of the hydroxyls (α/β); the side chain structure was modified by shortening (C-23, nor-BA) and by lengthening (C-25, homo-BA), while maintaining the same structure of nuclear hydroxyls (3α7β). At a “tracer” dose, all C-24 natural BAs are efficiently recovered in bile when administered in both unconjugated and taurine conjugated forms. At a “high dose”, all taurine conjugated BAs are efficiently recovered in bile (80–100%). However, a variable recovery was observed among unconjugated BAs: trihydroxy BAs are efficiently recovered (85–100%), while dihydroxy BAs are only partially recovered (25–40%). The side chain-modified BAs [i.e., C-23 nor and C-25 homo analogs of ursodeoxycholic acid (UDCA)] are partially recovered at a tracer dose (20–30%), but less at a high dose (10–20%) when administered in the unconjugated form. In contrast, the corresponding taurine conjugates are more efficiently recovered in bile (60–80%). Conjugation with taurine increases total recovery of unconjugated BAs in bile by not more than 30–40%. Highly hydrophilic and water-soluble BAs, such as ursocholic acid (SWo = 1.67 mM) and cholic acid (SWo = 0.27 mM), can also be secreted as unconjugates, and this accounts for their complete recovery. The conjugation step is rate limiting for poorly soluble BAs such as ursodeoxycholic acid (SWo = 0.009 mM) when administered at a high dose, and critical for nor and homo analogs which are poorly soluble and whose side chain modification partially suppresses their conjugation with taurine or glycine and thereby induces alternative pathways such as glucuronidation or sulfation. The induced bile flow is directly related to the hydrophilicity of the natural C-24 bile acid. The C-23 analog of UDCA greatly increased bile flow, which reached a maximum at very low BA secretion, and the effect is drastically reduced when it was administered as taurine conjugates. The phospholipids and, to a lesser extent, the cholesterol secretions are poorly related to both the hydrophilicity and CMC of BAs, and hepatic biotransformations play a major role in modulating the biliary lipid secretion. In addition, specific effects not related to physicochemical properties, such as selective inhibition of cholesterol synthesis, were observed (see chenodeoxycholic acid and hyocholic acid). The quantitative parameters here evaluated by means of a factor analysis could be useful in structure–activity relationship studies and in designing BA analogs; the properties of homo-UDCA as a new synthetic BA are also discussed.