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Experimental Overview Bile Acid Analysis Chemicals Cholic Acid (CA)

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Experimental Overview Bile Acid Analysis Chemicals Cholic Acid (CA) Supplementary Methods: Experimental overview bile acid analysis Chemicals Cholic acid (CA), taurocholic acid (TCA), glycocholic acid (GCA), deoxycholic acid (DCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), lithocholic acid (LCA), taurolithocholic acid (TLCA), ursodeoxycholic acid (UDCA) and glycoursodeoxycholic acid (GUDCA) were purchased from Sigma-Aldrich (St. Louis, MO). Glycolithocholic acid (GLCA) was purchased from Makaira Ltd (London, England). Tauroursodeoxycholic acid (TUDCA) was purchased from Merck Millipore (Billerica, MA). Alpha-muricholic acid (α-MCA), beta-muricholic acid (β-MCA), omega muricholic acid (ω-MCA), tauro alpha- muricholic acid (Tα-MCA), tauro beta-muricholic acid (Tβ-MCA), hyodeoxycholic acid (HDCA), taurohyodeoxycholic acid (THDCA) and glycohyodeoxycholic acid (GHDCA) were purchased from Steraloids Inc (Newport, RI). D4-cholic acid (D4-CA), D4 chenodeoxycholic acid (D4-CDCA), D4-glycochenodeoxycholic acid (D4-GCDCA) and D4-glycocholic acid (D4-GCA) were purchased from CDN Isotopes (Pointe- Claire, Quebec, Canada). D4 taurochenodeoxycholic acid (D4-TCDCA) and D4- taurocholic acid (D4-TCA) were purchased from Medical Isotopes (Pelham, NH). Sample preparation For sample preparation, after homogenizing and spinning, 25 µL plasma was aliquoted into a clean tube for bile acid analysis. For every 10 samples prepared, one quality control standard plasma was included. To each sample 250 µL internal standard solution was added and vortexed for 60 s. Samples were centrifuged at 15,800 x g and the supernatant poured into a clean glass tube. The fluid was evaporated under nitrogen at 40°C. If samples were not measured immediately, they were stored in this stage at -20°C. Before measuring samples were reconstituted in 200 µL 50% methanol in water, vortexed for 60 s and centrifuged for 3 min at 1,800 x g. The supernatant was transferred into a 0.2 µm spin-filter and centrifuged at 2,000 x g for 10 min. After filtering, the samples were transferred into LC-MS vials and analyzed (10 µL injection volume). Instrumentation For the quantitative determination of bile acids we used a Nexera X2 Ultra High Performance Liquid Chromatography system (SHIMADZU, Kyoto, Japan), coupled to a SCIEX QTRAP 4500 MD triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA) (UHPLC-MS/MS). The LC-MS/MS system is controlled by Analyst MD 1.6.2 software. Liquid chromatographic and mass spectrometric conditions Bile acids were separated with a ACQUITY UPLC BEH C18 Column (1.7 µm x 2.1 x 100 mm) equipped with a ACQUITY UPLC BEH C18 VanGuard Pre-Column (1.7 µm x 2.1 x 5 mm), (Waters, Milford, MA, USA). Separation was achieved in 28 minutes using 10 mM ammonium acetate in 20% acetonitrile (mobile phase A) and 10 mM ammonium acetate in 80% acetonitrile (mobile phase B). The mass spectrometer (MS) parameters such as temperature, ion spray voltage, gas pressures, etc., were optimized by infusing the bile acids and internal standards (IS) in a 50% MeOH solution via the internal syringe pump of the mass spectrometer. Bile acids with the same molar mass were infused as a mixture. All bile acids and internal standards were detected in negative mode with the mass spectrometer settings shown in Table 1 and Table 2. Supplementary Methods Table 1. Mass spectrometer source settings API4500 Detection mode MRM, negative mode Resolution Q1 Unit (scan speed 10 Da/s) Resolution Q3 Unit (scan speed 10 Da/s) IonSpray Voltage (IS) -4500 Temperature 500oC Curtain gas (CUR) 40 CAD gas (CAD) -3 Gas 1 (GS1) 40 Gas 2 (GS2) 70 The multiple reaction monitoring (MRM) transitions for each bile acid and internal standard, as well as their optimum MS parameters such as collision energy (CE), declustering potential (DP), entrance potential (EP) and collision cell exit potential (CXP) are shown in Table 2. Supplementary Methods Table 2. MRM settings API4500 Q1 mass Q3 mass Dwell Component (Da) (Da) DP CE EP CXP (msec) UDCA, HDCA, CDCA, DCA 391.3 391.3 -140.0 -12.0 -8.0 -10.0 50.0 CA, MCA’s 407.3 407.3 -120.0 -12.0 -8.0 -10.0 50.0 GUDCA, GHDCA, GCDCA, GDCA 448.4 74.0 -120.0 -80.0 -8.0 -10.0 50.0 GCA 464.2 74.0 -120.0 -85.0 -8.0 -10.0 50.0 TUDCA, THDCA, TCDCA, TDCA 498.3 80.0 -120.0 -110.0 -8.0 -10.0 50.0 TCA, TMCA’s 514.3 79.9 -120.0 -115.0 -8.0 -10.0 50.0 LCA 375.2 375.2 -120.0 -25.0 -8.0 -10.0 50.0 GLCA 432.2 74.0 -120.0 -75.0 -8.0 -10.0 50.0 TLCA 482.2 79.9 -120.0 -110.0 -8.0 -10.0 50.0 D4-CA 411.3 411.3 -120.0 -12.0 -8.0 -10.0 50.0 D4-GCA 468.2 74.0 -120.0 -85.0 -8.0 -10.0 50.0 D4-TCA 518.3 79.9 -120.0 -115.0 -8.0 -10.0 50.0 D4-GCDCA 452.4 74.0 -120.0 -80.0 -8.0 -10.0 50.0 D4-TCDCA 502.3 80.0 -120.0 -110.0 -8.0 -10.0 50.0 D4-CDCA 395.3 395.3 -120.0 -12.0 -8.0 -10.0 50.0 .
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