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Quantitative profiling of 19 bile acids in rat plasma, liver, bile and different intestinal section contents to investigate bile acid homeostasis and the application of temporal variation of endogenous bile acids
Journal of Steroid Biochemistry and Molecular Biology xxx (xxxx) xxx–xxx
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Quantitative profiling of 19 bile acids in rat plasma, liver, bile and different intestinal section contents to investigate bile acid homeostasis and the application of temporal variation of endogenous bile acids Tingting Yanga, Ting Shua, Guanlan Liua, Huifang Meia, Xiaoyu Zhua, Xin Huanga,b, ⁎ Luyong Zhanga,d, Zhenzhou Jianga,c, a
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China, China Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, China Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, China d State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China b c
A R T I C L E I N F O
A B S T R A C T
Abbreviations: LC–MS/MS high-performance liquid chromatographytandem mass spectrometry BAs bile acids G-BAs glycine-conjugated bile acids T-BAs taurine-conjugated bile acids beta-MCA beta-muricholic acid CA cholic acid UDCA ursodeoxycholic acid HDCA hyodeoxycholic acid DCA deoxycholic acid CDCA chenodeoxycholic acid LCA lithocholic acid IS internal standard MeOH methanol ACN acetonitrile QC quality control
Bile acid homeostasis is maintained by liver synthesis, bile duct secretion, microbial metabolism and intestinal reabsorption into the blood. When drug insults result in liver damage, the variances of bile acids (BAs) are related to the physiological status of the liver. Here, we established a method to simultaneously quantify 19 BAs in rat plasma, liver, bile and different intestinal section contents (duodenum, jejunum, ileum, cecum and colon) using high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) to reveal the pattern of bile acid homeostasis in the enterohepatic circulation of bile acids in physiological situations. Dynamic changes in bile acid composition appeared throughout the enterohepatic circulation of the BAs; taurine- and glycineconjugated BAs and free BAs had different dynamic homeostasis levels in the circulatory system. cholic acid (CA), beta-muricholic acid (beta-MCA), lithocholic acid (LCA), glycocholic acid (GCA) and taurocholic acid (TCA) greatly fluctuated in the bile acid pool under physiological conditions. Taurine- and glycine-conjugated bile acids constituted more than 90% in the bile and liver, whereas GCA and TCA accounted for more than half of the total bile acids and the secretion of bile mainly via conjugating with taurine. While over 80% of BAs in plasma were unconjugated bile acids, CA and HDCA were the most abundant elements. Unconjugated bile acids constituted more than 90% in the intestine, and CA, beta-MCA and HDCA were the top three bile acids in the duodenum, jejunum and ileum content, but LCA and HDCA were highest in the cecum and colon content. As the main secondary bile acid converted by microflora in the intestine, LCA was enriched in the cecum and DCA mostly in the colon. As endogenous substances, the concentrations of plasma BAs were closely related to time rhythm and diet. In conclusion, analyzing detailed BA profiles in the enterohepatic circulation of bile acids in a single run is possible using LC–MS/MS. Based on the physiological characteristics of the metabolic profiling of 19 BAs in the total bile acid pool and the time rhythm variation of the endogenous bile acids, this study provided a new valuable method and theoretical basis for the clinical research of bile acid homeostasis.
Keywords: Bile acid Homeostasis Profile
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Corresponding author at: Jiangsu Key Laboratoty of Drug Screening, China Pharmaceutical University, China E-mail address: [email protected] (Z. Jiang).
http://dx.doi.org/10.1016/j.jsbmb.2017.05.015 Received 22 February 2017; Received in revised form 23 May 2017; Accepted 31 May 2017 0960-0760/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Yang, T., Journal of Steroid Biochemistry and Molecular Biology (2017), http://dx.doi.org/10.1016/j.jsbmb.2017.05.015
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1. Introduction
As shown in Fig. 1, varying with the number and orientation of hydroxyl groups, BAs are complex compounds because of their numerous polarity structural isomers [9]. Therefore, dehydrocholic acid (dhCA) was used as an internal standard (IS) for quantification of the samples as it has similar functional groups as BAs and is not a natural component of rat samples. Individual bile acid has differential effects on bile acid signaling in mice, and the activities of individual bile acids vary markedly under physiological and pathophysiological conditions [10,11]. For example, secondary bile acid (LCA) is the most toxic BA and a potent ligand for pregnenolone X receptor (PXR) [12], whereas chenodeoxycholic acid (CDCA) is less toxic and is a potent farnesoid x receptor (FXR) ligand [13]. Studies have shown that T-beta-MCA and Talpha-MCA are FXR antagonists [14]. BAs are activators of several mammalian nuclear receptors, and the affinities for FXR are as follows: CDCA > LCA > DCA > CA [15]. TGR5 is principally activated by secondary BAs, including DCA and LCA. Bile acid affinities for TGR5 are as follows: LCA > DCA > CDCA > CA. Therefore, bile acid homeostasis and the composition of the bile acid pool must be strictly controlled to maintain physiological levels of BAs in the liver and extrahepatic tissues. BAs also affect the pharmacokinetics of drugs that are mainly excreted through bile. The regulation of hepatic and ileal relevant enzymes and transporters result from the alternations of individual bile acid after cholecystectomy. Of note, the downregulation of hepatic CYP3A11 suggested that undesirable pharmacokinetic alternations of drugs, especially CYP3A11 substrates such as rifampicin, might occur under cholecystectomy conditions [16]. As endogenous substances, serum levels of BAs vary during the day following a rhythm dictated by the ingestion of meals [3]. Studies have shown that the
Bile acids (BAs) play an important role in physiological and pathological processes. BAs are mostly involved in the enterohepatic system not only to facilitate the intestinal absorption of lipids and fatsoluble vitamins and the elimination of cholesterol and to protect against bacterial overgrowth [1,2] but also to regulate energy expenditure, glucose and lipid metabolism, thyroid hormone signaling, and cellular immunity [1,3]. BAs are synthesized in hepatocytes from cholesterol by pathways involving at least 17 different enzymes, and the immediate products of these synthesis pathways are referred to as primary bile acids [4]. Upon conjugation, primary BAs combine with amino acids (mainly the taurine and glycine) before they are excreted into the small intestine via the bile duct. In the gut, primary BAs are deconjugated and converted by microflora to secondary bile acids, which are mainly deoxycholic acid (DCA) and lithocholic acid (LCA). Most BAs in the intestine are reabsorbed into the portal circulation [5]. Following reuptake by the liver, BAs conjugate with taurine and glycine and are excreted into bile again, a process called enterohepatic circulation. The interruption of bile acid homeostasis can cause changes in the bile acid profile and the accumulation of toxic bile acids. A variety of pathologic changes induced by BAs, including cholestasis, bile duct infarction, liver fibrosis, liver cirrhosis, liver cancer and irritable bowel syndrome, were demonstrated in previous studies [6–8]. Meanwhile, the changes in the bile acid profile or some particular BAs may serve as potential pathogenesis-related biomarkers of liver diseases. Thus, investigating the metabolic profiling of bile acids in the total bile acid pool under physiological conditions may provide a new valuable method for the study of liver diseases.
Fig. 1. Chemical structure of the bile acids analyzed in this study. 19 bile acids are complex compounds for their varied number and orientation of hydroxyl groups and binding groups. (A) Backbone and side chain structures of the BAs and their taurine and glycine conjugates. (B) Molecular structure of taurine. (C) Molecular structure of glycine. (D) Structure of the internal standard (dhCA). (E) Structure of major unconjugated, glycine-and taurine-conjugated bile acids.
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(containing 0.1% formic acid), at a total flow rate of 0.1 ml/min. The gradient profile for the LC pumps under the final chromatography conditions are as follows: 0 min, 10:90; 1 min, 50:50; 2 min, 65:35; 3 min, 70:30; 6 min, 75:25; 11 min, 80:20; 20 min, 92:8; 40 min, 95:5; 42.5 min, 30:70; 43.5–45 min, 10:90 (A:B, v/v). The injection volume of all samples was 10 μl. The column temperature was set at 35 °C, and the sample tray temperature was maintained at 8 °C. For MS detection, the ESI source was operated in the negative ion mode to produce MS/ MS spectra and Xcalibur 2.0 software was used (Thermo Fisher). Highpurity nitrogen was used as the sheath (35 arb) and auxiliary (15 arb) gas and high-purity argon was used as the collision gas (1.5 mTorr). The parameters were as follows: spray voltage, 3.5 kV; capillary temperature, 400 °C; scan width for SRM, 0.5 m/z; scan time, 0.2 s. The peak width settings for both Q1 and Q3 were 0.7 m/z. The SRM ion pair transitions and collision energy levels of each component are listed in online resources Table 1.
pharmacokinetics of pethidine in male BALB/c mice is influenced by administration time [17]. The pharmacokinetics of aminoglycosides in humans follow a circadian rhythm, and seriously infected patients are at risk of developing nephrotoxicity after being treated with aminoglycosides [18]. These discoveries shed light on the relationship of pharmacokinetics and circadian rhythm, but the time-based variation of the BAs under normal physiological conditions is still unknown. Understanding the precise metabolism of BAs in the entire bile acid circulation system is essential. However, the detailed physiological circulation patterns of individual bile acids are still unclear. There is a vast literature on bile acid, and publications have studied the composition of bile acid in biological intestinal contents of rats. However, the studies only quantified the composition of bile acid in the large intestine and another studied the composition of bile acid in the gastrointestinal tract [19,20]. Here, we present a bile acid profile analysis of samples containing various solid materials, organs and tissue fluid using LC–MS/MS techniques in a single run. Meanwhile, the validation of BA analytical methods required the use of a matrix free of BAs. Therefore, we used different sample pretreatment methods for various body fluids and tissue samples to obtain the BA-free samples. The aims of this study were as follows: 1) establish a general protocol for BA extraction from various tissues and different intestinal section contents and develop an analytical method for the determination of BA composition using LC–MS/MS and 2) profile 19 BAs in the enterohepatic circulation system to reveal the pattern of bile acid homeostasis and apply a chronopharmacokinetics study of BAs in rats.
2.3. Preparation of BA-free blank plasma, bile, liver and different intestinal content BA-free samples were prepared with a modified method as described in the literature [21]. Briefly, blank rat plasma (1 ml) was mixed with 0.15 g of activated charcoal. The mixture was centrifuged (1000 × g) for 20 min, and the supernatant was collected (this procedure was repeated twice). Then, the supernatant was used as BA-free blank plasma. Blank rat bile (0.04 ml) was mixed with 4 ml of physiological saline, followed by adding 0.6 g of activated charcoal. The mixture was centrifuged (1000 × g) for 20 min, and the supernatant was collected (this procedure was repeated twice). Then, the supernatant was used as BAfree blank bile. Fresh liver samples (200 mg) were homogenized in 1 ml physiological saline. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and mixed with 0.6g of activated charcoal. The mixture was centrifuged (1000 × g) for 20 min, and the supernatant was collected (This procedure was repeated twice). Then, the supernatant was used as BA-free blank liver. Freeze-dried intestinal content samples (200 mg) were homogenized in 1 ml physiological saline. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and mixed with 0.6g of activated charcoal. The mixture was centrifuged (1000 × g) for 20 min, and the supernatant was collected (this procedure was repeated twice). Then, the supernatant was used as BA-free blank intestinal content.
2. Materials and methods 2.1. Reference chemicals and materials Ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA), and deoxycholic acid (DCA) were purchased from National Institutes for Food and Drug Control (Nanjing, China). Cholic acid (CA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), and glycochenodeoxycholic acid (GCDCA) were purchased from Nanjing Jie Bu Instrument Equipment Co., Ltd. (Nanjing, China). Taurocholic acid (TCA) was purchased from Guizhou Dida biological Technology Co., Ltd. (Guizhou, China). Lithocholic acid (LCA), beta-Muricholic acid (beta-MCA), glycolithocholic acid (GLCA), glycohyodeoxycholic acid (GHDCA), and glycoursodeoxycholic acid (GUDCA) were purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Taurodeoxycholic acid (TDCA), taurohyodeoxycholic acid (THDCA), taurolithocholic acid (TLCA), and dehydrocholic acid (dhCA, internal standard, or IS) were purchased from SigmaAldrich company (St. Louis, MO, USA). Taurochenodeoxycholic acid (TCDCA) was purchased from Shanghai future industrial Limited by Share Ltd. (Shanghai, China). Glycodeoxycholic acid (GDCA) was purchased from Aladdin reagent (Los Angeles, CA,USA). The salts for buffers and methanol were of HPLC grade. Methanol, acetonitrile (spectrum, USA), ammonium acetate and formic acid and other chemicals and solvents used were of analytical grade. Ultrapure water was purified using a Milli-Q system (Millipore, Milford, MA, USA).
2.4. Method validation A detailed description of the method validation in this study is provided in the Electronic Supplementary. 2.5. Preparation of standard stock solutions and internal standard stock Bile acid standard stock solutions were prepared by dissolving the respective 19 BAs in the appropriate amount of methanol to obtain individual stock solutions of 1 mg/mL. The 19 individual stock solutions were then further diluted with methanol to obtain standard stock solutions containing 0.05, 0.5, 5, and 50 mg/mL of each bile acid. A 1 mg/mL dhCA solution was prepared in methanol and further diluted with methanol to obtain a 10 μg/mL dhCA internal standard solution.
2.2. HPLC–MS/MS method development Liquid chromatographic separation and mass spectrometric detection were performed using the Thermo Scientific TSQ Quantum Ultra LC–MS/MS system consisting of a Finnigan Surveyor LC pump and a Finnigan Surveyor autosampler and combined with a triple quadrupole TSQ Quantum mass spectrometer via electrospray ionization (ESI) interface (Thermo Fisher, Palo Alto, CA). The chromatographic separation was performed on a Poroshell SB-C18 analytical column (150 × 3.0 mm, i.d.; 2.7 μm). The mobile phase consisted of methanol (A) and 5 mmol/L ammonium acetate in aqueous solution (B)
2.6. Sample preparation Calibrators and quality control samples (QCs) were prepared by adding the appropriate amount of the different standard stock solutions to 100 μl of charcoal treated serum/bile/liver tissue supernatant/ 3
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Fig. 2. LC/MS/MS chromatogram of 19 fully identified bile acids and dehydrocholic acid (Internal standard, IS). A baseline separation of 19 bile acids was achieved in 45 min. (A) Representative chromatogram at the lowest limit of quantification of a mixture of BAs (5 ng/mL), and the IS (dhCA,10 μg/mL) under the final chromatography and detection conditions. (B) Representative chromatogram of BAs in rat plasma.
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point in each matrix from the ratio of the analyte peak area in samples spiked before extraction compared to the corresponding peak area in untreated samples prepared in neat solution.
intestinal content samples homogenate and were extracted using the corresponding sample preparation procedure described below. For plasma samples, simple protein precipitation using ice-cold ACN was used as follows: 1 ml of ice-cold ACN (5% NH4OH in ACN) was added to 100 μl serum spiked with 10 μl IS, and the sample was vortexed and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was aspirated, evaporated under vacuum, reconstituted in 100 μl of MeOH and deionized water (85:15, v:v), and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was collected and 70 μl was injected into the LC–MS/MS system for analysis. Bile samples were diluted 100-fold with deionized water, 1 ml of ice-cold ACN (5% NH4OH in ACN) was added to 100 μl diluted bile samples spiked with 10 μl IS, and the sample was vortexed, and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was aspirated, evaporated under vacuum, reconstituted in 100 μl of 85% MeOH and deionized water (85:15, v:v), and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was collected and 70 μl was injected into the LC–MS/MS system for analysis. Liver samples were extracted by protein precipitation using alkaline ice-cold ACN. Approximately 200 mg of liver was homogenized in 1 ml physiological saline; 100 μl of liver homogenate was spiked with 10 μl IS, 1 ml of ice-cold alkaline ACN was added, and the sample was vortexed, and centrifuged at 14800 rpm and 4 °C for 10 min, reconstituted in 100 μl of MeOH and deionized water (85:15, v:v), and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was collected and 70 μl was injected into the LC–MS/MS system for analysis. Approximately 200 mg freeze-dried intestinal content samples were homogenized in 200 μl physiological saline. The homogenate was brought up to 400 μl with physiological saline and vortexed again. Then, 100 μl of intestinal content homogenate was spiked with 10 μl IS, 1 ml of ice-cold alkaline ACN was added, and the sample was vortexed, and centrifuged at 14800 rpm and 4 °C for 10 min, reconstituted in 100 μl of MeOH and deionized water (85:15, v:v), and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was collected and 70 μl was injected into the LC–MS/MS system for analysis. Extraction recoveries were determined for each quality control (QC)
2.7. Animal studies Eighteen male Sprague-Dawley rats, 5 weeks old (weighing 190–220 g), were purchased from Experimental Animal Center of Zhejiang Province (Hangzhou, China). The animals were housed in a temperature-, light-, and humidity-controlled environment. They were permitted to move freely and fed a standard diet and water ad libitum. Prior to the experiment, the animals were acclimatized to the facilities for 5 days and fasted with free access to water for 12 h. All the experiments in the present study were approved by the Animal Ethics Committee of China Pharmaceutical University. 2.7.1. Rat BA profiles Blank blood samples (approximately 400 μl, n = 6) were collected from the oculi choroidal vein into heparinized polythene tubes with heparin anticoagulant. Plasma samples were immediately obtained by centrifugation at 4000 rpm and 4 °C for 10 min and then stored at −20 °C. The same set of rats was anesthetized with an i.p. injection of 20% ethylurethanm (6 ml/kg), and the common bile duct was cannulated with micro-polyethylene tubing (SAI-infusion, USA) attached to polythene tubing. Bile samples (n = 6) were collected from the cannula for 2 h. The last set of rats (n = 6) was harvested for livers, and different intestinal section contents were also removed from the same animals. All the samples were stored at −20 °C for further analysis. 2.7.2. Application to chronopharmacokinetics study Blank blood samples (approximately 400 μl, n = 6) were drawn in heparinized polythene tubes with heparin anticoagulant before fasting (−12 h) and at 0, 2, 4, 6, 10, 12 and 24 h after feeding. Serum samples were immediately obtained by centrifugation at 4000 rpm and 4 °C for 10 min and then stored at −20 °C.
Fig. 3. The percent ratio of three types of bile acids are various in the total bile acid pool. Liver and bile are abundant in Taurine- and glycine-conjugated bile acids; Unconjugated bile acids play a dominant role in the intestine and plasma. Plasma (A), Liver (B), Bile (C), Duodenum (D), Jejunum (E), Ileum (F), Cecum (G), Colon (H).
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3. Results
3.2. Analysis of BA using LC–MS/MS
3.1. Method validation
We selected 19 different bile acids containing six types of taurine conjugates and six types of glycine conjugates, in addition to the internal standard, dhCA. The BAs in various biological samples were analyzed successfully within 45 min using LC–MS/MS. Representative chromatograms of BAs in the extracts of standards and plasma of SD rats are shown in Fig. 2. In plasma, the greatest proportions of BAs were unconjugated (free) bile acids. Nearly all the BAs were taurine-conjugated bile acids (T-BAs) or glycine-conjugated bile acids (G-BAs) in the liver and bile. A large amount of CA and HDCA was detected in the small intestine. LCA and DCA were abundant in the large intestine, and
The method was validated for specificity, linearity, accuracy, precision, carryover, matrix effects, extraction recovery, freeze and stability according to FDA Guidance for Industry-Bioanalytical Method Validation. Detailed results of the method validation in this study were provided in the Electronic Supplementary.
Fig. 4. Bile acids profiles are dynamically changed in various tissues and fluids of rats. The pattern of bile acid homeostasis is varied throughout the gut-liver axis. Plasma (A), Liver (B), Bile (C), Duodenum (D), Jejunum (E), Ileum (F), Cecum (G), Colon (H).
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the entire intestine. GCA, as the major component of G-BAs, reached its peak value with 28% in bile and declined to the level of plasma with approximately 5% in the jejunum content. Following the gradual decline, it could not be detected in the cecum or colon contents. With no more than 10%, the highest level of GUDCA, GHDCA, GCDCA and GDCA appeared in the liver, followed by a decline to approximately zero in the entire intestine. Thus, these two types of conjugated bile acids had different dynamic homeostasis throughout the circulatory system.
other BAs, such as beta-MCA, HDCA, CA and CDCA, were also found. TLCA and GLCA were undetectable, as they were below the lower limit of quantitation (LLOQ). 3.3. Rat BA profiles BA profiles in rat plasma, bile, liver and intestine contents were characterized using the LC–MS/MS method for six rats. According to the structure, BAs can be divided into two categories: free bile acids and conjugated bile acids (conjugated with taurine and glycine). As shown in Fig. 3, the percent ratio of these three types of BAs in various tissues and fluids of rats varied tremendously. Unconjugated bile acids accounted for 87% of total bile acid in plasma and were the predominant BAs compared with T-BAs at 4% and G-BAs at 9%. Conjugated bile acids played a leading role in the liver and bile, as conjugated bile acids constituted more than 90% of BAs, and taurine-conjugated bile acids were the highest. A comparison of the bile acid percentage of liver and bile indicated that the secretion of bile was mainly via conjugating with taurine and accounted for 80.58%, which was more than the amount of liver synthesis. For the intestine content, the proportion of unconjugated bile acids was up to 90% in all intestinal sections. Sharp changes occurred in the bile from the duodenum content, as conjugated bile acids decreased from 91.16% to 2.36%. However, there was a slight increase in the jejunum and ileum content, which did not exceed 10%. The concentration of conjugated bile acids was no more than 0.1% in the cecum and colon content. As shown in Fig. 4, dynamic changes of bile acid composition appeared throughout the BAs in the enterohepatic circulation. The detailed changes of individual bile acid are shown in Fig. 5. Comparatively speaking, the percent ratio of CA, beta-MCA, LCA, GCA, and TCA fluctuated greatly in the bile acid pool under physiological conditions. The highest percentage of CA was observed in the small intestine and the lowest was in the liver. The ileum had more than 95% BAs reabsorbed into the blood. CA fell even further, from approximately 59% in the small intestine to 5% in the large intestine. The terminal small intestine had the highest percentage of beta-MCA, which was lowest in bile. Converted by microflora in the intestine, LCA and DCA were the main secondary bile acids. The former was mainly formed in the cecum, and the latter was mostly formed in the colon. LCA, the most important hydrophobic bile acid, first appeared in the duodenum and was found at no more than 2% in the ileum with a drastic increase in the cecum of approximately 25-fold and was excreted in the feces, as its percentage in the colon content was higher than in the cecal content. LCA could not be detected in plasma, liver or bile as it was under the detection limit. HDCA, another an important hydrophobic bile acid, has received increasing attention. Its variation trends were similar to that of DCA. With no more than 2%, UDCA had an extra-low content throughout the circulatory system. As the major component of the T-BAs, TCA reached its peak value with 46% in the liver and rapidly declined to the level of plasma with approximately 2% in the entire intestine. Although the content was no more than 10%, the highest composition proportion of TUDCA, THDCA, TCDCA and TDCA appeared in the bile and was followed by a decline in
3.4. BA composition in the intestine Bile acids are transformed by the intestinal microbiota into a variety of metabolically active metabolites, including secondary BAs, DCA and LCA. In abnormally high concentrations, hydrophobic secondary BAs are cytotoxic, leading to DNA damage and cell death [22]. BAs regulate the composition of the gut microbiome at the highest taxonomic levels, resulting in diseases that affect human metabolism. Thus, the content of bile acid should be strictly controlled in the entire intestine. As shown in Fig. 6, the translation of conjugated bile acids synthesized by the liver into unconjugated bile acids began in the duodenum. The transformation from primary bile acids into secondary bile acids mostly occurred in the cecum, and approximately 95% of BAs was reabsorbed into the blood and then entered the systemic circulation in the ileum. As shown in Fig. 7, the relative fold changes of unconjugated bile acids were higher than T-BAs and G-BAs in the jejunum content. The reabsorption of conjugated bile acids was approximately 3 times higher than unconjugated bile acids in the ileum. Comparing the large intestine to the small intestine, the relative fold change of unconjugated bile acids was significantly higher than taurine- and glycine-conjugated bile acids. The transformation from primary bile acids to secondary bile acids mostly occurred in the cecum. Compared with CDCA and HDCA, LCA and DCA had relatively higher transformation by the intestinal microbiota. Although the concentration of beta-MCA was high, microbial metabolic transformation was relatively low and CA was the lowest. The highest reabsorption of colonocytes was UDCA and the lowest was CDCA. 3.5. Application of chronopharmacokinetics study in rats The developed LC–MS/MS method was successfully applied to quantify bile acids in rat serum in 36 h to reveal the effect of fasting and food on the endogenous components over time. The results were presented in Fig. 8. From these three figures, the concentration of several BAs had a transient increase after fasting within 12 h. After providing food, all the BAs had an obvious increase. Unconjugated bile acids gradually reduced at 4 h, but the decrease of conjugated bile acids occurred at 10 h and the steady state was nearly reached at 12 h. In the experiment, the effects of endogenous bile acids on the pharmacokinetics of drugs can be controlled by effective arrangement of diet and administration time (food was given at 4 h after administration). There were significant differences in the levels of bile acids in different individuals, which may be related to the physiological and
Fig. 5. The percent ratio of bile acids are dynamically changed in various tissues and fluids of SD rats. 19 individual bile acid fluctuated greatly in the bile acid pool under physiological conditions; CA, beta-MCA, GCA and TCA have the larger fluctuation throughout the gut-liver axis. (A) Unconjugated bile acids. (B) Taurine conjugated bile acids. (C) Glycine-conjugated bile acids.
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Fig. 6. The three types of BAs are dynamically changed in different intestinal sections of SD rats. Be reabsorbed by ileal epithelial cells and transformed by the intestinal microbiota into a variety of secondary bile acids, a significant decrease of bile acids are observed in ileum and colun and an obviously increase of the unconjugated bile acids is observed in cecum. (A) Unconjugated bile acids. (B) Glycine-conjugated bile acids. (C) Taurine-conjugated bile acids. The results are shown as the mean ± SD (n = 6).
Fig. 7. The concentration of 19 bile acids are dynamically changed in the intestine. The transformation and reabsorption of 19 individual bile acid are different from each other in different intestinal sections. (A) Unconjugated bile acids, (B) Glycine-conjugated bile acids. (C) Taurine-conjugated bile acids. The results are shown as the mean ± SD (n = 6).
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Fig. 8. The dynamic change of BAs in rats in 36 h. The diet and administration time induce fluctuation of the concentration of the endogenous bile acids. (A) Unconjugated bile acids. (B) Taurine-conjugated bile acids. (C) Glycine-conjugated bile acids. The results are shown as the mean ± SD (n = 6).
compared to healthy subjects [8]. A previous study reported that ethanol consumption altered the bile acids in multiple body compartments (liver, gastrointestinal tract, and serum) in rat through the gutliver axis [20]. Heshouwu (HSW), the dry roots of Polygonum multiflorum, significantly changed the secondary bile acids following the ingestion of HEW, indicating that gut microorganisms played an important role in liver injury induced by HSW, the further mechanism of which could be provided by the information from a BA metabolism study [30]. Rhizoma Coptidis (RC, Coptis chinensis Franch) is a widely used traditional Chinese medicine. It alleviated hyperlipidemia by modulating gut microbiota and bile acid pathways in B6 mice [31]. Thus, our technique could be used as a baseline to establish the effects of consuming different traditional Chinese medicines on the intestinal microbiome and on the bile salts that are used by the microbiome to communicate with the host. The primary bile acids in humans are CA and CDCA. In contrast, MCAs are produced in rats from CDCA, due to the existence of 6β-hydroxylase in the liver [32]. Because the 7 position of 5β-cholanic acid can be hydroxylated in both the α and β directions, rats produce four types of primary BAs in their liver [33,34]. Thus, the bile acid composition in rats is more complicated than in humans, especially in the cecum and colon. Conjugated bile acids have been suggested to repress bacterial growth in the small intestine either through direct antimicrobial effects, upregulation of host mucosal defenses, or synergistic action of both mechanisms [35–37]. Deconjugation is catalyzed by bile salt hydrolases (BSHs), which hydrolyze the amide bond and liberate the glycine/taurine moiety from the steroid core by the gut microbiota [38]. Studies have reported that BSHs preferentially hydrolyze G-BAs [39–41]. In the present study, we have identified that T-BAs are the predominant bile acids in the bile acid pool. Therefore, the preferential rate of hydrolysis of glycine over that of taurine by the microbial environment might explain this predominance. There is an interesting clash between T-beta-MCA (FXR inhibitors) and CDCA and the secondary bile acids DCA and LCA (FXR promoters) [42,43]. A previous study reported that gut microbiota exert their effects in parts of the enterohepatic system, such as regulating bile acid synthesis in the liver [14]. Meanwhile, the affinity of secondary bile acid metabolites, as a result of microbial transformations, act as signaling molecules and regulate intestinal homeostasis through the TGR5 and FXR receptors by inhibiting inflammation, preventing pathogen invasion, and maintaining cell integrity [44]. Perturbations of microbiota shape the bile acid pool and modulate the activity of bile acidactivated receptors (BARs) [45]. Thus, through the microbiome, bile acids communicate with the host to maintain homeostasis. Diet considerably influences the microbial composition, function, and effects. Mice fed a high-fat diet had impaired intestinal mucosal barrier integrity, secondary to modification of the BA profile with an increase in the concentration of DCA and decrease in the proportion of a potentially cytoprotective tertiary BA, ursodeoxycholic acid (UDCA) [46]. Consumption of a diet high in certain saturated fats changes the conditions for microbial assemblage and promotes expansion of a low abundance, sulfite-reducing pathobiont, Bilophila wadsworthia, which was associated with a pro-inflammatory Th1 immune response and increased incidence of colitis in genetically susceptible IL-10-/- mice
dietary status. In the process of determining the content of such components, individual differences should be noted to avoid the interference of endogenous components as much as possible. 4. Discussion Differentiation of individual bile acids is not possible using enzymatic methods because total BAs, rather than individual bile acids, are quantified. Therefore, chromatographic techniques are the method of choice for detailed analysis of bile acid profiles. Comparing gas chromatographic-mass spectrometry (GC–MS) and HPLC-UV assays with HPLC-tandem mass spectrometry (HPLC–MS/MS), the latter has the advantage of high sensitivity, specificity, and sample preparation convenience for bile acid analysis in biological fluids. Several methods have been developed and used to quantify bile acids in biological matrices [23–27]. These methods provided valuable data, which deepened our understanding of the various roles of BAs in biological systems. Most MS/MS transitions found in our experiments were consistent with previously published data and with the expected product ions for BAs [24,25]. To obtain chromatograms with optimal resolution and symmetrical peaks of all bile acids, different protein precipitation methods were compared among methanol, acetonitrile and ethylacetate. The compositions of the mobile phase, flow rate and column temperature were also optimized. Better resolution of peaks was achieved with methanol/water and a gradient elution at a flow rate of 0.1 ml/min. Different concentrations of formic acid and ammonium acetate were tested for the mobile phases, and comparisons were also made between a series of concentrations of formic acid (0.01, 0.05, 0.1 and 0.5%, v:v) and ammonium acetate (4, 5, and 7.5 mmol/L). The mobile phase consisted of 0.1% formic acid, and methanol had a higher peak area in our initial assay. Validation required the use of a matrix free of BAs. Li Yang and Aizhen Xiong used an LC–MS method for the analysis of bile acids in the rat serum [21]. To perform the validation of their method, they stripped the serum using charcoal as we did, but in our case, the process had to undergo further stripping cycles to ensure that the serum was free of BAs. Additionally, we made some improvements based on this method to obtain other BA-free samples. The validation of this method was performed completely in plasma. To use this method more accurately, we also verified the specificity and linearity of calibration curves of this method in liver, bile and different intestinal section contents (date not shown). BAs are secreted as glycine, taurine, or sulfate conjugates and then excreted in bile to reach the intestinal tract, where they can be deconjugated by gut microbiota, aided by populations of microorganisms with their own hydrolytic enzymes, such as B-glucuronidase and sulfatase [28]. The intestinal microbiome plays an important role involving metabolism, synthesis of vitamins and immunomodulation and can establish a “cross-talk” with the host [29]. Normally, the healthy state is a homeostasis between the microbiota and the host. Under pathological conditions, the normal balance between the gut microbiota and host is disrupted. Irritable bowel syndrome (IBS) is a multifactorial disease, characterized by a dysbiosis of the gut microbiota. There is an increase of primary bile acid in the feces of diarrhea-predominant IBS patients 9
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and the prevalence of complex immune-mediated diseases, such as inflammatory bowel diseases, in genetically susceptible hosts [47]. These data suggest that the interference of endogenous bile acids should be noted when considering diet.
[18]
5. Conclusion
[19]
[17]
In summary, our method is designed and validated for the quantitative profiling of BAs in rat plasma, liver, bile and different intestinal section contents in a single run and this is the first report in which 19 bile acids have been quantitatively profiled through the gut-liver axis in rats. Dynamic changes in bile acid composition appeared throughout the BAs in the enterohepatic circulation, and individual bile acids had different dynamic homeostasis in this circulatory system. Diet and time rhythm had an impact on BA profiles in rats. This study could be applied to clarify pathological changes in BA profiles in disease, leading to a better understanding of BA metabolism.
[20]
[21]
[22] [23]
[24]
Acknowledgment
[25]
This study was supported by National Natural Science Foundation of China (81320108029 to LZ, 81573514 to ZJ, 81274146 to LZ, 81273604 to ZJ), the Natural Science Foundation of Jiangsu Province (BK20151439 to ZJ), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the National Major Scientific and Technological Special Project for Significant New Drugs project (2015ZX09501004-002-004 to LZ), and Specific Fund for Public Interest Research of Traditional Chinese Medicine, Ministry of finance (201507004-002 to LZ), the Project Program of Jiangsu Key Laboratory of Drug Screening (JKLDS2015ZZ-02 to ZJ).
[26]
[27]
[28]
[29]
[30]
Appendix A. Supplementary data [31]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsbmb.2017.05.015.
[32]
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Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
Quantitative profiling of 19 bile acids in rat plasma, liver, bile and different intestinal section contents to investigate bile acid homeostasis and the application of temporal variation of endogenous bile acids Tingting Yanga, Ting Shua, Guanlan Liua, Huifang Meia, Xiaoyu Zhua, Xin Huanga,b, ⁎ Luyong Zhanga,d, Zhenzhou Jianga,c, a
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China, China Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, China Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, China d State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China b c
A R T I C L E I N F O
A B S T R A C T
Abbreviations: LC–MS/MS high-performance liquid chromatographytandem mass spectrometry BAs bile acids G-BAs glycine-conjugated bile acids T-BAs taurine-conjugated bile acids beta-MCA beta-muricholic acid CA cholic acid UDCA ursodeoxycholic acid HDCA hyodeoxycholic acid DCA deoxycholic acid CDCA chenodeoxycholic acid LCA lithocholic acid IS internal standard MeOH methanol ACN acetonitrile QC quality control
Bile acid homeostasis is maintained by liver synthesis, bile duct secretion, microbial metabolism and intestinal reabsorption into the blood. When drug insults result in liver damage, the variances of bile acids (BAs) are related to the physiological status of the liver. Here, we established a method to simultaneously quantify 19 BAs in rat plasma, liver, bile and different intestinal section contents (duodenum, jejunum, ileum, cecum and colon) using high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) to reveal the pattern of bile acid homeostasis in the enterohepatic circulation of bile acids in physiological situations. Dynamic changes in bile acid composition appeared throughout the enterohepatic circulation of the BAs; taurine- and glycineconjugated BAs and free BAs had different dynamic homeostasis levels in the circulatory system. cholic acid (CA), beta-muricholic acid (beta-MCA), lithocholic acid (LCA), glycocholic acid (GCA) and taurocholic acid (TCA) greatly fluctuated in the bile acid pool under physiological conditions. Taurine- and glycine-conjugated bile acids constituted more than 90% in the bile and liver, whereas GCA and TCA accounted for more than half of the total bile acids and the secretion of bile mainly via conjugating with taurine. While over 80% of BAs in plasma were unconjugated bile acids, CA and HDCA were the most abundant elements. Unconjugated bile acids constituted more than 90% in the intestine, and CA, beta-MCA and HDCA were the top three bile acids in the duodenum, jejunum and ileum content, but LCA and HDCA were highest in the cecum and colon content. As the main secondary bile acid converted by microflora in the intestine, LCA was enriched in the cecum and DCA mostly in the colon. As endogenous substances, the concentrations of plasma BAs were closely related to time rhythm and diet. In conclusion, analyzing detailed BA profiles in the enterohepatic circulation of bile acids in a single run is possible using LC–MS/MS. Based on the physiological characteristics of the metabolic profiling of 19 BAs in the total bile acid pool and the time rhythm variation of the endogenous bile acids, this study provided a new valuable method and theoretical basis for the clinical research of bile acid homeostasis.
Keywords: Bile acid Homeostasis Profile
⁎
Corresponding author at: Jiangsu Key Laboratoty of Drug Screening, China Pharmaceutical University, China E-mail address: [email protected] (Z. Jiang).
http://dx.doi.org/10.1016/j.jsbmb.2017.05.015 Received 22 February 2017; Received in revised form 23 May 2017; Accepted 31 May 2017 0960-0760/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Yang, T., Journal of Steroid Biochemistry and Molecular Biology (2017), http://dx.doi.org/10.1016/j.jsbmb.2017.05.015
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1. Introduction
As shown in Fig. 1, varying with the number and orientation of hydroxyl groups, BAs are complex compounds because of their numerous polarity structural isomers [9]. Therefore, dehydrocholic acid (dhCA) was used as an internal standard (IS) for quantification of the samples as it has similar functional groups as BAs and is not a natural component of rat samples. Individual bile acid has differential effects on bile acid signaling in mice, and the activities of individual bile acids vary markedly under physiological and pathophysiological conditions [10,11]. For example, secondary bile acid (LCA) is the most toxic BA and a potent ligand for pregnenolone X receptor (PXR) [12], whereas chenodeoxycholic acid (CDCA) is less toxic and is a potent farnesoid x receptor (FXR) ligand [13]. Studies have shown that T-beta-MCA and Talpha-MCA are FXR antagonists [14]. BAs are activators of several mammalian nuclear receptors, and the affinities for FXR are as follows: CDCA > LCA > DCA > CA [15]. TGR5 is principally activated by secondary BAs, including DCA and LCA. Bile acid affinities for TGR5 are as follows: LCA > DCA > CDCA > CA. Therefore, bile acid homeostasis and the composition of the bile acid pool must be strictly controlled to maintain physiological levels of BAs in the liver and extrahepatic tissues. BAs also affect the pharmacokinetics of drugs that are mainly excreted through bile. The regulation of hepatic and ileal relevant enzymes and transporters result from the alternations of individual bile acid after cholecystectomy. Of note, the downregulation of hepatic CYP3A11 suggested that undesirable pharmacokinetic alternations of drugs, especially CYP3A11 substrates such as rifampicin, might occur under cholecystectomy conditions [16]. As endogenous substances, serum levels of BAs vary during the day following a rhythm dictated by the ingestion of meals [3]. Studies have shown that the
Bile acids (BAs) play an important role in physiological and pathological processes. BAs are mostly involved in the enterohepatic system not only to facilitate the intestinal absorption of lipids and fatsoluble vitamins and the elimination of cholesterol and to protect against bacterial overgrowth [1,2] but also to regulate energy expenditure, glucose and lipid metabolism, thyroid hormone signaling, and cellular immunity [1,3]. BAs are synthesized in hepatocytes from cholesterol by pathways involving at least 17 different enzymes, and the immediate products of these synthesis pathways are referred to as primary bile acids [4]. Upon conjugation, primary BAs combine with amino acids (mainly the taurine and glycine) before they are excreted into the small intestine via the bile duct. In the gut, primary BAs are deconjugated and converted by microflora to secondary bile acids, which are mainly deoxycholic acid (DCA) and lithocholic acid (LCA). Most BAs in the intestine are reabsorbed into the portal circulation [5]. Following reuptake by the liver, BAs conjugate with taurine and glycine and are excreted into bile again, a process called enterohepatic circulation. The interruption of bile acid homeostasis can cause changes in the bile acid profile and the accumulation of toxic bile acids. A variety of pathologic changes induced by BAs, including cholestasis, bile duct infarction, liver fibrosis, liver cirrhosis, liver cancer and irritable bowel syndrome, were demonstrated in previous studies [6–8]. Meanwhile, the changes in the bile acid profile or some particular BAs may serve as potential pathogenesis-related biomarkers of liver diseases. Thus, investigating the metabolic profiling of bile acids in the total bile acid pool under physiological conditions may provide a new valuable method for the study of liver diseases.
Fig. 1. Chemical structure of the bile acids analyzed in this study. 19 bile acids are complex compounds for their varied number and orientation of hydroxyl groups and binding groups. (A) Backbone and side chain structures of the BAs and their taurine and glycine conjugates. (B) Molecular structure of taurine. (C) Molecular structure of glycine. (D) Structure of the internal standard (dhCA). (E) Structure of major unconjugated, glycine-and taurine-conjugated bile acids.
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(containing 0.1% formic acid), at a total flow rate of 0.1 ml/min. The gradient profile for the LC pumps under the final chromatography conditions are as follows: 0 min, 10:90; 1 min, 50:50; 2 min, 65:35; 3 min, 70:30; 6 min, 75:25; 11 min, 80:20; 20 min, 92:8; 40 min, 95:5; 42.5 min, 30:70; 43.5–45 min, 10:90 (A:B, v/v). The injection volume of all samples was 10 μl. The column temperature was set at 35 °C, and the sample tray temperature was maintained at 8 °C. For MS detection, the ESI source was operated in the negative ion mode to produce MS/ MS spectra and Xcalibur 2.0 software was used (Thermo Fisher). Highpurity nitrogen was used as the sheath (35 arb) and auxiliary (15 arb) gas and high-purity argon was used as the collision gas (1.5 mTorr). The parameters were as follows: spray voltage, 3.5 kV; capillary temperature, 400 °C; scan width for SRM, 0.5 m/z; scan time, 0.2 s. The peak width settings for both Q1 and Q3 were 0.7 m/z. The SRM ion pair transitions and collision energy levels of each component are listed in online resources Table 1.
pharmacokinetics of pethidine in male BALB/c mice is influenced by administration time [17]. The pharmacokinetics of aminoglycosides in humans follow a circadian rhythm, and seriously infected patients are at risk of developing nephrotoxicity after being treated with aminoglycosides [18]. These discoveries shed light on the relationship of pharmacokinetics and circadian rhythm, but the time-based variation of the BAs under normal physiological conditions is still unknown. Understanding the precise metabolism of BAs in the entire bile acid circulation system is essential. However, the detailed physiological circulation patterns of individual bile acids are still unclear. There is a vast literature on bile acid, and publications have studied the composition of bile acid in biological intestinal contents of rats. However, the studies only quantified the composition of bile acid in the large intestine and another studied the composition of bile acid in the gastrointestinal tract [19,20]. Here, we present a bile acid profile analysis of samples containing various solid materials, organs and tissue fluid using LC–MS/MS techniques in a single run. Meanwhile, the validation of BA analytical methods required the use of a matrix free of BAs. Therefore, we used different sample pretreatment methods for various body fluids and tissue samples to obtain the BA-free samples. The aims of this study were as follows: 1) establish a general protocol for BA extraction from various tissues and different intestinal section contents and develop an analytical method for the determination of BA composition using LC–MS/MS and 2) profile 19 BAs in the enterohepatic circulation system to reveal the pattern of bile acid homeostasis and apply a chronopharmacokinetics study of BAs in rats.
2.3. Preparation of BA-free blank plasma, bile, liver and different intestinal content BA-free samples were prepared with a modified method as described in the literature [21]. Briefly, blank rat plasma (1 ml) was mixed with 0.15 g of activated charcoal. The mixture was centrifuged (1000 × g) for 20 min, and the supernatant was collected (this procedure was repeated twice). Then, the supernatant was used as BA-free blank plasma. Blank rat bile (0.04 ml) was mixed with 4 ml of physiological saline, followed by adding 0.6 g of activated charcoal. The mixture was centrifuged (1000 × g) for 20 min, and the supernatant was collected (this procedure was repeated twice). Then, the supernatant was used as BAfree blank bile. Fresh liver samples (200 mg) were homogenized in 1 ml physiological saline. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and mixed with 0.6g of activated charcoal. The mixture was centrifuged (1000 × g) for 20 min, and the supernatant was collected (This procedure was repeated twice). Then, the supernatant was used as BA-free blank liver. Freeze-dried intestinal content samples (200 mg) were homogenized in 1 ml physiological saline. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and mixed with 0.6g of activated charcoal. The mixture was centrifuged (1000 × g) for 20 min, and the supernatant was collected (this procedure was repeated twice). Then, the supernatant was used as BA-free blank intestinal content.
2. Materials and methods 2.1. Reference chemicals and materials Ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA), and deoxycholic acid (DCA) were purchased from National Institutes for Food and Drug Control (Nanjing, China). Cholic acid (CA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), and glycochenodeoxycholic acid (GCDCA) were purchased from Nanjing Jie Bu Instrument Equipment Co., Ltd. (Nanjing, China). Taurocholic acid (TCA) was purchased from Guizhou Dida biological Technology Co., Ltd. (Guizhou, China). Lithocholic acid (LCA), beta-Muricholic acid (beta-MCA), glycolithocholic acid (GLCA), glycohyodeoxycholic acid (GHDCA), and glycoursodeoxycholic acid (GUDCA) were purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Taurodeoxycholic acid (TDCA), taurohyodeoxycholic acid (THDCA), taurolithocholic acid (TLCA), and dehydrocholic acid (dhCA, internal standard, or IS) were purchased from SigmaAldrich company (St. Louis, MO, USA). Taurochenodeoxycholic acid (TCDCA) was purchased from Shanghai future industrial Limited by Share Ltd. (Shanghai, China). Glycodeoxycholic acid (GDCA) was purchased from Aladdin reagent (Los Angeles, CA,USA). The salts for buffers and methanol were of HPLC grade. Methanol, acetonitrile (spectrum, USA), ammonium acetate and formic acid and other chemicals and solvents used were of analytical grade. Ultrapure water was purified using a Milli-Q system (Millipore, Milford, MA, USA).
2.4. Method validation A detailed description of the method validation in this study is provided in the Electronic Supplementary. 2.5. Preparation of standard stock solutions and internal standard stock Bile acid standard stock solutions were prepared by dissolving the respective 19 BAs in the appropriate amount of methanol to obtain individual stock solutions of 1 mg/mL. The 19 individual stock solutions were then further diluted with methanol to obtain standard stock solutions containing 0.05, 0.5, 5, and 50 mg/mL of each bile acid. A 1 mg/mL dhCA solution was prepared in methanol and further diluted with methanol to obtain a 10 μg/mL dhCA internal standard solution.
2.2. HPLC–MS/MS method development Liquid chromatographic separation and mass spectrometric detection were performed using the Thermo Scientific TSQ Quantum Ultra LC–MS/MS system consisting of a Finnigan Surveyor LC pump and a Finnigan Surveyor autosampler and combined with a triple quadrupole TSQ Quantum mass spectrometer via electrospray ionization (ESI) interface (Thermo Fisher, Palo Alto, CA). The chromatographic separation was performed on a Poroshell SB-C18 analytical column (150 × 3.0 mm, i.d.; 2.7 μm). The mobile phase consisted of methanol (A) and 5 mmol/L ammonium acetate in aqueous solution (B)
2.6. Sample preparation Calibrators and quality control samples (QCs) were prepared by adding the appropriate amount of the different standard stock solutions to 100 μl of charcoal treated serum/bile/liver tissue supernatant/ 3
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Fig. 2. LC/MS/MS chromatogram of 19 fully identified bile acids and dehydrocholic acid (Internal standard, IS). A baseline separation of 19 bile acids was achieved in 45 min. (A) Representative chromatogram at the lowest limit of quantification of a mixture of BAs (5 ng/mL), and the IS (dhCA,10 μg/mL) under the final chromatography and detection conditions. (B) Representative chromatogram of BAs in rat plasma.
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point in each matrix from the ratio of the analyte peak area in samples spiked before extraction compared to the corresponding peak area in untreated samples prepared in neat solution.
intestinal content samples homogenate and were extracted using the corresponding sample preparation procedure described below. For plasma samples, simple protein precipitation using ice-cold ACN was used as follows: 1 ml of ice-cold ACN (5% NH4OH in ACN) was added to 100 μl serum spiked with 10 μl IS, and the sample was vortexed and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was aspirated, evaporated under vacuum, reconstituted in 100 μl of MeOH and deionized water (85:15, v:v), and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was collected and 70 μl was injected into the LC–MS/MS system for analysis. Bile samples were diluted 100-fold with deionized water, 1 ml of ice-cold ACN (5% NH4OH in ACN) was added to 100 μl diluted bile samples spiked with 10 μl IS, and the sample was vortexed, and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was aspirated, evaporated under vacuum, reconstituted in 100 μl of 85% MeOH and deionized water (85:15, v:v), and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was collected and 70 μl was injected into the LC–MS/MS system for analysis. Liver samples were extracted by protein precipitation using alkaline ice-cold ACN. Approximately 200 mg of liver was homogenized in 1 ml physiological saline; 100 μl of liver homogenate was spiked with 10 μl IS, 1 ml of ice-cold alkaline ACN was added, and the sample was vortexed, and centrifuged at 14800 rpm and 4 °C for 10 min, reconstituted in 100 μl of MeOH and deionized water (85:15, v:v), and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was collected and 70 μl was injected into the LC–MS/MS system for analysis. Approximately 200 mg freeze-dried intestinal content samples were homogenized in 200 μl physiological saline. The homogenate was brought up to 400 μl with physiological saline and vortexed again. Then, 100 μl of intestinal content homogenate was spiked with 10 μl IS, 1 ml of ice-cold alkaline ACN was added, and the sample was vortexed, and centrifuged at 14800 rpm and 4 °C for 10 min, reconstituted in 100 μl of MeOH and deionized water (85:15, v:v), and centrifuged at 14800 rpm and 4 °C for 10 min. The supernatant was collected and 70 μl was injected into the LC–MS/MS system for analysis. Extraction recoveries were determined for each quality control (QC)
2.7. Animal studies Eighteen male Sprague-Dawley rats, 5 weeks old (weighing 190–220 g), were purchased from Experimental Animal Center of Zhejiang Province (Hangzhou, China). The animals were housed in a temperature-, light-, and humidity-controlled environment. They were permitted to move freely and fed a standard diet and water ad libitum. Prior to the experiment, the animals were acclimatized to the facilities for 5 days and fasted with free access to water for 12 h. All the experiments in the present study were approved by the Animal Ethics Committee of China Pharmaceutical University. 2.7.1. Rat BA profiles Blank blood samples (approximately 400 μl, n = 6) were collected from the oculi choroidal vein into heparinized polythene tubes with heparin anticoagulant. Plasma samples were immediately obtained by centrifugation at 4000 rpm and 4 °C for 10 min and then stored at −20 °C. The same set of rats was anesthetized with an i.p. injection of 20% ethylurethanm (6 ml/kg), and the common bile duct was cannulated with micro-polyethylene tubing (SAI-infusion, USA) attached to polythene tubing. Bile samples (n = 6) were collected from the cannula for 2 h. The last set of rats (n = 6) was harvested for livers, and different intestinal section contents were also removed from the same animals. All the samples were stored at −20 °C for further analysis. 2.7.2. Application to chronopharmacokinetics study Blank blood samples (approximately 400 μl, n = 6) were drawn in heparinized polythene tubes with heparin anticoagulant before fasting (−12 h) and at 0, 2, 4, 6, 10, 12 and 24 h after feeding. Serum samples were immediately obtained by centrifugation at 4000 rpm and 4 °C for 10 min and then stored at −20 °C.
Fig. 3. The percent ratio of three types of bile acids are various in the total bile acid pool. Liver and bile are abundant in Taurine- and glycine-conjugated bile acids; Unconjugated bile acids play a dominant role in the intestine and plasma. Plasma (A), Liver (B), Bile (C), Duodenum (D), Jejunum (E), Ileum (F), Cecum (G), Colon (H).
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3. Results
3.2. Analysis of BA using LC–MS/MS
3.1. Method validation
We selected 19 different bile acids containing six types of taurine conjugates and six types of glycine conjugates, in addition to the internal standard, dhCA. The BAs in various biological samples were analyzed successfully within 45 min using LC–MS/MS. Representative chromatograms of BAs in the extracts of standards and plasma of SD rats are shown in Fig. 2. In plasma, the greatest proportions of BAs were unconjugated (free) bile acids. Nearly all the BAs were taurine-conjugated bile acids (T-BAs) or glycine-conjugated bile acids (G-BAs) in the liver and bile. A large amount of CA and HDCA was detected in the small intestine. LCA and DCA were abundant in the large intestine, and
The method was validated for specificity, linearity, accuracy, precision, carryover, matrix effects, extraction recovery, freeze and stability according to FDA Guidance for Industry-Bioanalytical Method Validation. Detailed results of the method validation in this study were provided in the Electronic Supplementary.
Fig. 4. Bile acids profiles are dynamically changed in various tissues and fluids of rats. The pattern of bile acid homeostasis is varied throughout the gut-liver axis. Plasma (A), Liver (B), Bile (C), Duodenum (D), Jejunum (E), Ileum (F), Cecum (G), Colon (H).
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the entire intestine. GCA, as the major component of G-BAs, reached its peak value with 28% in bile and declined to the level of plasma with approximately 5% in the jejunum content. Following the gradual decline, it could not be detected in the cecum or colon contents. With no more than 10%, the highest level of GUDCA, GHDCA, GCDCA and GDCA appeared in the liver, followed by a decline to approximately zero in the entire intestine. Thus, these two types of conjugated bile acids had different dynamic homeostasis throughout the circulatory system.
other BAs, such as beta-MCA, HDCA, CA and CDCA, were also found. TLCA and GLCA were undetectable, as they were below the lower limit of quantitation (LLOQ). 3.3. Rat BA profiles BA profiles in rat plasma, bile, liver and intestine contents were characterized using the LC–MS/MS method for six rats. According to the structure, BAs can be divided into two categories: free bile acids and conjugated bile acids (conjugated with taurine and glycine). As shown in Fig. 3, the percent ratio of these three types of BAs in various tissues and fluids of rats varied tremendously. Unconjugated bile acids accounted for 87% of total bile acid in plasma and were the predominant BAs compared with T-BAs at 4% and G-BAs at 9%. Conjugated bile acids played a leading role in the liver and bile, as conjugated bile acids constituted more than 90% of BAs, and taurine-conjugated bile acids were the highest. A comparison of the bile acid percentage of liver and bile indicated that the secretion of bile was mainly via conjugating with taurine and accounted for 80.58%, which was more than the amount of liver synthesis. For the intestine content, the proportion of unconjugated bile acids was up to 90% in all intestinal sections. Sharp changes occurred in the bile from the duodenum content, as conjugated bile acids decreased from 91.16% to 2.36%. However, there was a slight increase in the jejunum and ileum content, which did not exceed 10%. The concentration of conjugated bile acids was no more than 0.1% in the cecum and colon content. As shown in Fig. 4, dynamic changes of bile acid composition appeared throughout the BAs in the enterohepatic circulation. The detailed changes of individual bile acid are shown in Fig. 5. Comparatively speaking, the percent ratio of CA, beta-MCA, LCA, GCA, and TCA fluctuated greatly in the bile acid pool under physiological conditions. The highest percentage of CA was observed in the small intestine and the lowest was in the liver. The ileum had more than 95% BAs reabsorbed into the blood. CA fell even further, from approximately 59% in the small intestine to 5% in the large intestine. The terminal small intestine had the highest percentage of beta-MCA, which was lowest in bile. Converted by microflora in the intestine, LCA and DCA were the main secondary bile acids. The former was mainly formed in the cecum, and the latter was mostly formed in the colon. LCA, the most important hydrophobic bile acid, first appeared in the duodenum and was found at no more than 2% in the ileum with a drastic increase in the cecum of approximately 25-fold and was excreted in the feces, as its percentage in the colon content was higher than in the cecal content. LCA could not be detected in plasma, liver or bile as it was under the detection limit. HDCA, another an important hydrophobic bile acid, has received increasing attention. Its variation trends were similar to that of DCA. With no more than 2%, UDCA had an extra-low content throughout the circulatory system. As the major component of the T-BAs, TCA reached its peak value with 46% in the liver and rapidly declined to the level of plasma with approximately 2% in the entire intestine. Although the content was no more than 10%, the highest composition proportion of TUDCA, THDCA, TCDCA and TDCA appeared in the bile and was followed by a decline in
3.4. BA composition in the intestine Bile acids are transformed by the intestinal microbiota into a variety of metabolically active metabolites, including secondary BAs, DCA and LCA. In abnormally high concentrations, hydrophobic secondary BAs are cytotoxic, leading to DNA damage and cell death [22]. BAs regulate the composition of the gut microbiome at the highest taxonomic levels, resulting in diseases that affect human metabolism. Thus, the content of bile acid should be strictly controlled in the entire intestine. As shown in Fig. 6, the translation of conjugated bile acids synthesized by the liver into unconjugated bile acids began in the duodenum. The transformation from primary bile acids into secondary bile acids mostly occurred in the cecum, and approximately 95% of BAs was reabsorbed into the blood and then entered the systemic circulation in the ileum. As shown in Fig. 7, the relative fold changes of unconjugated bile acids were higher than T-BAs and G-BAs in the jejunum content. The reabsorption of conjugated bile acids was approximately 3 times higher than unconjugated bile acids in the ileum. Comparing the large intestine to the small intestine, the relative fold change of unconjugated bile acids was significantly higher than taurine- and glycine-conjugated bile acids. The transformation from primary bile acids to secondary bile acids mostly occurred in the cecum. Compared with CDCA and HDCA, LCA and DCA had relatively higher transformation by the intestinal microbiota. Although the concentration of beta-MCA was high, microbial metabolic transformation was relatively low and CA was the lowest. The highest reabsorption of colonocytes was UDCA and the lowest was CDCA. 3.5. Application of chronopharmacokinetics study in rats The developed LC–MS/MS method was successfully applied to quantify bile acids in rat serum in 36 h to reveal the effect of fasting and food on the endogenous components over time. The results were presented in Fig. 8. From these three figures, the concentration of several BAs had a transient increase after fasting within 12 h. After providing food, all the BAs had an obvious increase. Unconjugated bile acids gradually reduced at 4 h, but the decrease of conjugated bile acids occurred at 10 h and the steady state was nearly reached at 12 h. In the experiment, the effects of endogenous bile acids on the pharmacokinetics of drugs can be controlled by effective arrangement of diet and administration time (food was given at 4 h after administration). There were significant differences in the levels of bile acids in different individuals, which may be related to the physiological and
Fig. 5. The percent ratio of bile acids are dynamically changed in various tissues and fluids of SD rats. 19 individual bile acid fluctuated greatly in the bile acid pool under physiological conditions; CA, beta-MCA, GCA and TCA have the larger fluctuation throughout the gut-liver axis. (A) Unconjugated bile acids. (B) Taurine conjugated bile acids. (C) Glycine-conjugated bile acids.
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Fig. 6. The three types of BAs are dynamically changed in different intestinal sections of SD rats. Be reabsorbed by ileal epithelial cells and transformed by the intestinal microbiota into a variety of secondary bile acids, a significant decrease of bile acids are observed in ileum and colun and an obviously increase of the unconjugated bile acids is observed in cecum. (A) Unconjugated bile acids. (B) Glycine-conjugated bile acids. (C) Taurine-conjugated bile acids. The results are shown as the mean ± SD (n = 6).
Fig. 7. The concentration of 19 bile acids are dynamically changed in the intestine. The transformation and reabsorption of 19 individual bile acid are different from each other in different intestinal sections. (A) Unconjugated bile acids, (B) Glycine-conjugated bile acids. (C) Taurine-conjugated bile acids. The results are shown as the mean ± SD (n = 6).
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Fig. 8. The dynamic change of BAs in rats in 36 h. The diet and administration time induce fluctuation of the concentration of the endogenous bile acids. (A) Unconjugated bile acids. (B) Taurine-conjugated bile acids. (C) Glycine-conjugated bile acids. The results are shown as the mean ± SD (n = 6).
compared to healthy subjects [8]. A previous study reported that ethanol consumption altered the bile acids in multiple body compartments (liver, gastrointestinal tract, and serum) in rat through the gutliver axis [20]. Heshouwu (HSW), the dry roots of Polygonum multiflorum, significantly changed the secondary bile acids following the ingestion of HEW, indicating that gut microorganisms played an important role in liver injury induced by HSW, the further mechanism of which could be provided by the information from a BA metabolism study [30]. Rhizoma Coptidis (RC, Coptis chinensis Franch) is a widely used traditional Chinese medicine. It alleviated hyperlipidemia by modulating gut microbiota and bile acid pathways in B6 mice [31]. Thus, our technique could be used as a baseline to establish the effects of consuming different traditional Chinese medicines on the intestinal microbiome and on the bile salts that are used by the microbiome to communicate with the host. The primary bile acids in humans are CA and CDCA. In contrast, MCAs are produced in rats from CDCA, due to the existence of 6β-hydroxylase in the liver [32]. Because the 7 position of 5β-cholanic acid can be hydroxylated in both the α and β directions, rats produce four types of primary BAs in their liver [33,34]. Thus, the bile acid composition in rats is more complicated than in humans, especially in the cecum and colon. Conjugated bile acids have been suggested to repress bacterial growth in the small intestine either through direct antimicrobial effects, upregulation of host mucosal defenses, or synergistic action of both mechanisms [35–37]. Deconjugation is catalyzed by bile salt hydrolases (BSHs), which hydrolyze the amide bond and liberate the glycine/taurine moiety from the steroid core by the gut microbiota [38]. Studies have reported that BSHs preferentially hydrolyze G-BAs [39–41]. In the present study, we have identified that T-BAs are the predominant bile acids in the bile acid pool. Therefore, the preferential rate of hydrolysis of glycine over that of taurine by the microbial environment might explain this predominance. There is an interesting clash between T-beta-MCA (FXR inhibitors) and CDCA and the secondary bile acids DCA and LCA (FXR promoters) [42,43]. A previous study reported that gut microbiota exert their effects in parts of the enterohepatic system, such as regulating bile acid synthesis in the liver [14]. Meanwhile, the affinity of secondary bile acid metabolites, as a result of microbial transformations, act as signaling molecules and regulate intestinal homeostasis through the TGR5 and FXR receptors by inhibiting inflammation, preventing pathogen invasion, and maintaining cell integrity [44]. Perturbations of microbiota shape the bile acid pool and modulate the activity of bile acidactivated receptors (BARs) [45]. Thus, through the microbiome, bile acids communicate with the host to maintain homeostasis. Diet considerably influences the microbial composition, function, and effects. Mice fed a high-fat diet had impaired intestinal mucosal barrier integrity, secondary to modification of the BA profile with an increase in the concentration of DCA and decrease in the proportion of a potentially cytoprotective tertiary BA, ursodeoxycholic acid (UDCA) [46]. Consumption of a diet high in certain saturated fats changes the conditions for microbial assemblage and promotes expansion of a low abundance, sulfite-reducing pathobiont, Bilophila wadsworthia, which was associated with a pro-inflammatory Th1 immune response and increased incidence of colitis in genetically susceptible IL-10-/- mice
dietary status. In the process of determining the content of such components, individual differences should be noted to avoid the interference of endogenous components as much as possible. 4. Discussion Differentiation of individual bile acids is not possible using enzymatic methods because total BAs, rather than individual bile acids, are quantified. Therefore, chromatographic techniques are the method of choice for detailed analysis of bile acid profiles. Comparing gas chromatographic-mass spectrometry (GC–MS) and HPLC-UV assays with HPLC-tandem mass spectrometry (HPLC–MS/MS), the latter has the advantage of high sensitivity, specificity, and sample preparation convenience for bile acid analysis in biological fluids. Several methods have been developed and used to quantify bile acids in biological matrices [23–27]. These methods provided valuable data, which deepened our understanding of the various roles of BAs in biological systems. Most MS/MS transitions found in our experiments were consistent with previously published data and with the expected product ions for BAs [24,25]. To obtain chromatograms with optimal resolution and symmetrical peaks of all bile acids, different protein precipitation methods were compared among methanol, acetonitrile and ethylacetate. The compositions of the mobile phase, flow rate and column temperature were also optimized. Better resolution of peaks was achieved with methanol/water and a gradient elution at a flow rate of 0.1 ml/min. Different concentrations of formic acid and ammonium acetate were tested for the mobile phases, and comparisons were also made between a series of concentrations of formic acid (0.01, 0.05, 0.1 and 0.5%, v:v) and ammonium acetate (4, 5, and 7.5 mmol/L). The mobile phase consisted of 0.1% formic acid, and methanol had a higher peak area in our initial assay. Validation required the use of a matrix free of BAs. Li Yang and Aizhen Xiong used an LC–MS method for the analysis of bile acids in the rat serum [21]. To perform the validation of their method, they stripped the serum using charcoal as we did, but in our case, the process had to undergo further stripping cycles to ensure that the serum was free of BAs. Additionally, we made some improvements based on this method to obtain other BA-free samples. The validation of this method was performed completely in plasma. To use this method more accurately, we also verified the specificity and linearity of calibration curves of this method in liver, bile and different intestinal section contents (date not shown). BAs are secreted as glycine, taurine, or sulfate conjugates and then excreted in bile to reach the intestinal tract, where they can be deconjugated by gut microbiota, aided by populations of microorganisms with their own hydrolytic enzymes, such as B-glucuronidase and sulfatase [28]. The intestinal microbiome plays an important role involving metabolism, synthesis of vitamins and immunomodulation and can establish a “cross-talk” with the host [29]. Normally, the healthy state is a homeostasis between the microbiota and the host. Under pathological conditions, the normal balance between the gut microbiota and host is disrupted. Irritable bowel syndrome (IBS) is a multifactorial disease, characterized by a dysbiosis of the gut microbiota. There is an increase of primary bile acid in the feces of diarrhea-predominant IBS patients 9
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and the prevalence of complex immune-mediated diseases, such as inflammatory bowel diseases, in genetically susceptible hosts [47]. These data suggest that the interference of endogenous bile acids should be noted when considering diet.
[18]
5. Conclusion
[19]
[17]
In summary, our method is designed and validated for the quantitative profiling of BAs in rat plasma, liver, bile and different intestinal section contents in a single run and this is the first report in which 19 bile acids have been quantitatively profiled through the gut-liver axis in rats. Dynamic changes in bile acid composition appeared throughout the BAs in the enterohepatic circulation, and individual bile acids had different dynamic homeostasis in this circulatory system. Diet and time rhythm had an impact on BA profiles in rats. This study could be applied to clarify pathological changes in BA profiles in disease, leading to a better understanding of BA metabolism.
[20]
[21]
[22] [23]
[24]
Acknowledgment
[25]
This study was supported by National Natural Science Foundation of China (81320108029 to LZ, 81573514 to ZJ, 81274146 to LZ, 81273604 to ZJ), the Natural Science Foundation of Jiangsu Province (BK20151439 to ZJ), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the National Major Scientific and Technological Special Project for Significant New Drugs project (2015ZX09501004-002-004 to LZ), and Specific Fund for Public Interest Research of Traditional Chinese Medicine, Ministry of finance (201507004-002 to LZ), the Project Program of Jiangsu Key Laboratory of Drug Screening (JKLDS2015ZZ-02 to ZJ).
[26]
[27]
[28]
[29]
[30]
Appendix A. Supplementary data [31]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsbmb.2017.05.015.
[32]
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