Semi-quantitative profiling of bile acids in serum and liver reveals the dosage-related effects of dexamethasone on bile acid metabolism in mice
Introduction
Bile acids (BAs) are synthesized from cholesterol in the liver via two major pathways, namely the classic and alternative pathways [1]. Cholesterol is converted to cholic acid (CA) and chenodeoxycholic acid (CDCA), two primary BAs in human liver. In rodents, CDCA could be further metabolized to form α/β‑muricholic acids (α/β‑MCAs). The majority of BAs in the liver are conjugated with taurine and/or glycine, secreted into the bile, and stored in the gallbladder. After a meal, BAs are excreted into the intestine, where they are converted to secondary BAs by bacterial enzymes. Deoxycholic acid (DCA) and lithocholic acid (LCA) are two major secondary BAs in humans.
Individual BAs have distinct physiological and pathophysiological activities, and thus the BA profile determines their hydrophobicity, fat-solubility, and toxicity [2]. The BA profile is important for BA signaling pathways, because individual BAs have distinct potency to activate BA receptors, such as farnesoid X receptor (FXR) and the plasma membrane-bound G-protein-coupled BA receptor 1(TGR5) [3]. In addition, the BA profile could be a reliable hallmark for certain diseases, such as intrahepatic cholestasis of pregnancy, fatty liver disease, and bacterial infection [4]. Consequently, there is a growing interest in the development of methods to quantify and profile individual BAs.
Nuclear receptors are key regulators of various physiological processes, including BA synthesis, transport and metabolism [5]. Activation of pregnane X receptor (PXR) results in an induction of CYP3A, which is sufficient for the hydroxylation and detoxification of BAs in the liver [6]. Glucocorticoid receptor (GR) has been reported to regulate genes involved in BA synthesis and transport [7, 8]. Dexamethasone (DEX), a synthetic glucocorticoid (GC), has been used as an agonist for both PXR and GR in previous studies [9, 10]. DEX at higher doses (>20 mg/kg) was able to activate pregnane X receptor (PXR) in mice [11], whereas lower doses (<6 mg/kg) mainly activate GR signaling [12]. Furthermore, DEX has been used to treat the intrahepatic cholestasis of pregnancy, suggesting an important role of DEX in decreasing BAs [13]. Nevertheless, to our knowledge, it remains largely unknown about the dosage-related effect of DEX on BA homeostasis.
Numerous LC-MS methods have been developed for BA quantification in humans and rodents. Some methods had relatively complicated sample processing and limited BA species coverage (from 11 to 32 BAs quantified) [14, 15]. For instance, Hagio et al. developed a LC-MS/MS method to quantify 22 BAs in liver and intestine, and the extraction procedure included grinding, sonicating, heating, centrifuging, evaporating, and filtering [16]. Wegner et al. developed a rapid LC-MS/MS method to quantify 32 BAs in fermentation broth, which was limited by a strong matrix effect [17]. Some methods were powerful, but had high requirement for the instrumentation. For instance, Sarafian et al. reported a LC-MS method quantifying 146 BA species [18]. However, this method requires two different mass spectrometers (Q-TOF for profiling and TQ-S for targeted detection), which are unaffordable for most laboratories. Additionally, this method included 88 sulfated BAs, which are not commercially available, and are almost undetectable in rodents [19]. Therefore, there is still a need for a simple and rapid method, which should not be limited to certain instruments and could be used as a routine tool for the laboratory. In our previous studies, we have performed BA quantification by using a Waters ACQUITY ultra performance LC system (Waters, Milford, MA) coupled with a Waters Quattro Premier XE triple quadrupole mass spectrometer [15, 20, 21]. In the present study, we developed a simple and semi-quantitative BA profiling method by using an Agilent 1290 UPLC system (Agilent Technologies, USA) coupled to a quadrupole time-of-flight mass spectrometer (Bruker micrOTOF-QII, Germany), which was the only LC-MS system available in the laboratory. With this BA profiling method, we investigated the BA concentrations in serum and livers of mice treated with two dosages of DEX and revealed dosage-related effects of DEX on BA profile.
Section snippets
Chemicals and reagents
Sulfated BAs were kindly provided by Dr. Curtis D. Klaassen at the University of Washington (Seattle, Washington). Other BA standards were purchased from either Sigma-Aldrich (St. Louis, MO) or Steraloids, Inc. (Newport, Rhode Island). Total 39 BAs were quantified in the present study, with their structures and abbreviations shown in Fig. 1. Deuterated internal standard (IS) cholic acid‑2,2,4,4‑d4 was purchased from C/D/N Isotopes, Inc. (Pointe-Claire, Quebec, Canada). Dexamethasone was
Method development and optimization
BAs are extensively metabolized during their enterohepatic circulation, producing a wide range of molecular variants. In the present study, we used Q-TOF MS to detect and profile both known and unknown BA species in serum and livers of mice. Additionally, we took the advantage of extracted ion chromatogram (EIC) function for BA quantification. The present method showed a wide range of linearity (>10 μg/mL) and could be affordable as a routine analysis tool in the laboratory. However, it should
Conclusions
This study reports the development of a simple method for the semi-quantitative profiling of serum and hepatic BAs in mice. It should be noted that the current method is limited by insufficient separation of several BA isomers that are minor metabolites in the body. An important novelty of the current study is that it provides the first comparative analysis of serum and hepatic BA profiles in mice after either a two-week treatment of 1 mg/kg DEX or a 4-day treatment of 75 mg/kg DEX, and reveals
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (No. 81673523) and by the Project of National Basic Research (973) Program of China (2015CB856500).
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Both authors contributed equally to this work.