Bile Acid Profiling in Mouse Biofluids and Tissues

Abstract

Bile acids (BAs) serve as important signaling molecules and are endogenous ligands of nuclear and cell membrane receptors to regulate physiological and pathological processes. BA synthesis and metabolism have been impaired in NASH patients because of liver injury, inflammation or obstruction of bile ducts. On the other hand, the changes in BA composition might alter the activation status of various cell signaling pathways and contribute to NASH pathogenesis. Due to the rapidly increasing interests in the roles of individual BAs in disease development, this chapter will focus on the method for analyzing individual BA profile in mouse biofluids and tissues by high performance liquid chromatography coupled with ion trap mass spectrometry (HPLC-MS).

1. Introduction

Bile Acids (BAs) are physiological molecules synthesized from cholesterol in the liver via two major pathways, the classical pathway initiated with the cholesterol 7α-hydroxylase (CYP7A1) to produce cholic acid (CA) and the alternative pathway initiated with the cholesterol 27α-hydroxylase (CYP27A1) to produce chenodeoxycholic acid (CDCA)[2, 3]. In rodents, CDCA will be converted to isoforms of muricholic acid (mCA). These three BAs are termed primary BAs that are conjugated to taurine or glycine by bile acid coenzyme A: amino acid N-acyltransferase (BAAT) and transported out of the hepatocytes mainly by bile salt efflux pump (BSEP; ABCB11). BAs will be secreted to the intestine upon food consumption to facilitate the digestion and absorption of lipids and lipid-soluble vitamins. In the gut, primary BAs will be deconjugated, and transformed into secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA), by bile salt hydrolase (BSH) synthesized by gut microflora [16]. A small fraction of CDCA is also converted into a tertiary BA, ursodeoxycholic acid (UDCA), in the liver. Recent scientific discoveries have identified the key enzymes that convert CDCA to mCA and DCA to CA in mice, namely CYP2C70 and CYP2A12, respectively [9, 13, 14]. These aforementioned BAs are major BAs in the body. However, there are numerous minor BAs present in various species, including humans and rodents, and the roles of these minor BAs are far from being understood. Identifying BAs species and composition provides great values to understand the roles of BAs in physiology and pathogenesis.

In general. BAs serve critical physiological functions, including elimination of cholesterol, maintenance of bile flow, absorption of lipids and lipid-soluble vitamins, regulation of gut microbiome, and serving as important signaling molecules. BAs are endogenous ligands of several nuclear receptors, including farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR)[6]. In addition, BAs activate membrane G-protein coupled receptors, including Takeda-G-protein-receptor-5 (TGR5) and sphingosine-1-phosphate receptor 2 (S1PR2) [1, 8]. By signaling pathways initiated from the liver and intestine, BAs suppress their own synthesis, regulate carbohydrate and lipid metabolism, and suppress inflammation and fibrogenesis. Disruption of BA homeostasis leads to severe liver and intestine diseases, including cholestasis, steatosis, inflammation, fibrosis and tumor [4, 5, 7, 10, 11, 15]. Targeting critical factors in the BA regulatory pathways becomes a novel strategy to treat cholestasis, non-alcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma [17].

There is an increasing awareness of the importance of determining not only the total BA (TBA) levels but also individual BA species and composition during health and disease state. Methods that have been widely used to analyze serum TBA include liquid chromatography coupled with ion trap mass spectrometry (LC-MS) and enzymatic assays. The latter assay is commonly used in the clinical and research settings for a quick assessment of BA levels without the knowledge of individual BA composition. Due to the rapidly increasing interests in the roles of individual BAs in disease development, this chapter focuses on the method using LC-MS to profile individual BA species in mouse tissues [12].

2. Materials

2.1. Chemicals and Reagents

The method requires 23 BAs standards, 2 internal BA standards, and reagents for LC-MS/MS analysis (see Note 1):

1. Cholic acid (CA).

2. Chenodeoxycholic acid (CDCA).

3. Deoxycholic acid (DCA).

4. Lithocholic acid (LCA).

5. Ursodeoxycholic acid (UDCA).

6. Taurocholic acid (TCA).

7. Taurochenodeoxycholic acid (TCDCA).

8. Taurodeoxycholic acid (TDCA).

9. Taurolithocholic acid (TLCA).

10. Tauroursodeoxycholic acid (TUDCA).

11. Glycocholic acid (GCA).

12. Glycochenodeoxycholic acid (GCDCA).

13. Glycodeoxycholic acid (GDCA).

14. Glycolithocholic acid (GLCA).

15. β-muricholic acid (β-MCA).

16. α-muricholic acid (α-MCA).

17. ω-muricholic acid (ω-MCA).

18. Tauro-β-muricholic acid (T-β-MCA).

19. Tauro-α-muricholic acid (T-α-MCA).

20. Tauro-ω-muricholic acid (T-ω-MCA).

21. Hyodeoxycholic acid (HDCA).

22. Taurohyodeoxycholic acid (THDCA).

23. Glycoursodeoxycholic acid (GUDCA).

24. Chenodeoxycholic-2,2,4,4-d4 acid (2H4-CDCA).

25. Glycochenodeoxycholic-2,2,4,4-d4 acid (2H4-GCDCA).

26. 50% methanol in water.

27. Methanol

28. Activated charcoal.

29. 5% Ammonium hydroxide (NH₄OH) solution.

30. Phosphate buffered saline (PBS).

31. H₂O, LC/MS reagent grade.

32. Acetonitrile (ACN), LC/MS reagent grade.

33. Methanol with 0.15% formic acid.

34. 0.10% formic acid in water.

35. Kimwipes.

2.2. Equipment

1. Thermo Ultra Performance Liquid Chromatography (UPLC) system coupled with a LTQ XL Ion Trap Mass Spectrometer (ITMS) (Finnigan, Thermo Fisher Scientific).

2. Reverse phase C18 column from Phenomenex Kinetex (1.3 μm, 50mm × 2.1mm).

3. 0.22μm Costar Spin-X centrifuge tube.

4. Refrigerated centrifuge.

5. Sonicator with small probe.

6. Tissue Homogenizer.

7. Lab tube roller or rotator.

8. Speed Vacuum Chemical Concentrator Systems.

3. Methods

As the master BA regulator, FXR maintains BA homeostasis (see Note 2). The total BA pool size is defined as the total amount of BAs in the serum, liver, gallbladder, and small intestine (gut) including the gut contents (see Notes 2 & 3).

3.1. Mouse Tissues and Biofluids Collection

1. Record Mouse body weight and liver weight during necropsy for the normalization of BA concentrations (see Note 4).

2. Euthanize the mouse according to animal protocol approved by IACUC.

3. Collect blood, and allow blood samples to clot at room temperature for 90 minutes.

4. Collect Liver, small intestine (including luminal content), intact gallbladders, and colon, and snap freeze in liquid nitrogen (see Note 5).

5. Centrifuge the completely clotted blood at 8,000x g at room temperature for 10 mins to precipitate blood cells.

6. Store all the collected tissues at −80 °C until use (see Note 6).

3.2. Preparation of BA Standards for LC/MS

Tissue matrixes are prepared from wild-type (WT) mice and then spiked with BA standard solutions to prepare BA standards for different tissues (see Note 7).

3.2.1. Preparation of BA standard solutions

1. Dissolve each of 23 BA standards and 2 deuterated BAs (2H4-CDCA) and 2H4-GCDCA) in 50% methanol to make each BA stock solution at final concentration of 10 mg/ml.

2. Pool 50 μl each BA stock solution and fill up to 5 ml with 50% methanol to make BA Standard AAAA. The concentration of each BA is 100 ng/ml.

3. Make a series of two-fold dilutions using 50% methanol starting from Standard AAAA and label the standards as Std-AAA, -AA, -A, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N.

4. Choose eight BA standards (Std AA, A, C, E, G, I, K, M) and zero (50% methanol) to make standard curve (see Note 8).

5. Use BA standard Std-F as internal standard, and label it as IS F. The final concentration of all the BAs in standards and tissue homogenate after spiking 20 μl IS F are shown in Table 1.

Table 1.

The BAs concentration in BA standard and tissue extract

Standard Name	BA Standard Solution (μg/ml)	BA Final Conc. in Serum/Bile/Urine Extract (ng/ml)	BA Final Conc. in Liver/Intestine/Colon Extract (ng/ml)
50% MeOH	0.000	0.000	0.000
IS F	0.391	86.806	26.042
Std AA	25	5555.556	1666.667
Std A	12.5	2777.778	833.333
Std C	3.125	694.444	208.333
Std E	0.781	173.611	52.083
Std G	0.195	43.403	13.021
Std I	0.049	10.851	3.255
Std K	0.012	2.713	0.814
Std M	0.003	0.678	0.203

3.2.2. Preparation of serum/plasma/urine tissue matrix

1. Collect plasma/serum/urine from at least 5 mice (see Note 9).

2. Pool total 3.0 ml of plasma/serum/urine from 5 mice into a conical tube (600 μl from each mouse).

3.2.3. Preparation of gallbladder (GB) tissue matrix

1. Collect gallbladder from at least 5 mice (see Note 9).

2. Add 1.5 ml 1x PBS to each tube with intact GB per mouse.

3. Break the GB in the solution with forceps.

4. Dilute the GB samples 50 fold in a new tube (mix 40 μl sample with 1960 μl 1x PBS).

5. Pool total 3.0 ml diluted GB bile samples from 5 mice into a new tube (600 μl from each mouse).

3.2.4. Preparation of liver tissue matrix

1. Collect liver from at least 5 mice (see Note 9).

2. Pool total 1.375 g liver samples from 5 mice (~275 mg per mouse) in a conical tube.

3. Add 7.5 ml 1x PBS (300 μl 1x PBS for every 55 mg liver).

4. Homogenize liver sample on ice with powered homogenizer (see Note 10).

5. Pool total 8.0 ml liquid samples from 5 mice into a new conical tube (1600 μl from each mouse).

3.2.5. Preparation of tissue matrix from small intestine

1. Collect the whole intestine from at least 5 mice (see Note 9).

2. Add 6 ml 1x PBS to the tube with the whole intestine and its contents for each mouse.

3. Homogenize the whole intestine with a powered homogenizer.

4. Centrifuge the homogenate at 4°C at 8,000g for 10 mins, and transfer the supernatant to a new conical tube.

5. Repeat step 2 to 4 above two more times, and pool the extraction solution. Total volume is approximately 18 ml.

6. Pool total 8.0 ml liquid samples from 5 mice into a new conical tube (1600 μl from each mouse).

3.2.6. Preparation of colon tissue matrix

1. Collect the whole colon from at least 5 mice (see Note 9).

2. Add 1.5 ml 1x PBS to the tube with the whole colon and its contents for each mouse.

3. Homogenize the whole colon with a powered homogenizer.

4. Centrifuge the homogenate at 4°C at 8,000g for 10 mins, and transfer the supernatant to a new conical tube.

5. Repeat step 2 to 4 above two more times, and pool the extraction solution. Total volume is approximately 4.5 ml.

6. Pool total 8.0 ml liquid samples from 5 mice into a new conical tube (1600 μl from each mouse).

3. 3. Preparation of BA standards using tissue matrix

1. Incubate and rotate each pooled tissue sample with activated charcoal (100 mg/ml) for 1 hour at room temperature to strip the endogenous BAs.

2. Aliquot the charcoal treated solution to several 1.5 ml micro-centrifuge tubes.

3. Centrifuge the tubes at 4°C at 13,000 g for 10 mins to pellet the charcoal.

4. Transfer the supernatant to fresh 1.5 ml micro-centrifuge tubes and repeat step 3.

5. Transfer the supernatant to the filtered column of Spin-X centrifuge tube.

6. Centrifuge the filtered tubes at 4°C at 13,000g for 10 mins.

7. Remove and discard the filter columns from the centrifugation tubes.

8. Prepare 20 micro-centrifuge tubes for two sets of standards, and label them according to Table 1.

9. Aliquot 90 μl (serum/plasma/urine/GB) or 300 μl (liver/intestine/colon) filtered tissue matrix into new micro-centrifuge tubes.

10. Proceed to step 4 (spiking with 20 μl of appropriate standard solutions) in Section 3.5 (Extraction of BAs from biofluids and tissue homogenates).

3.4. Preparation of tissues from experimental mice for BA extraction

3.4.1. Preparation of serum/plasma/urine samples

Aliquot 90 μl liquid sample each into a micro-centrifuge tube.

3.4.2. Preparation of bile samples

1. Add 1.5 ml 1x PBS to the tube with GB.

2. Break the GB in the solution by forceps with sharp tip.

3. Take 40 μl sample and mix with 1960 μl 1x PBS to dilute 50 times in a new tube.

4. Aliquot 90 μl diluted bile samples into a new micro-centrifuge tube (see Note 3).

3.4.3. Preparation of liver homogenate

1. Weigh 55–60 mg frozen liver, record weight, and add 300 μl 1x PBS buffer.

2. Homogenize liver sample on ice with powered homogenizer (see Note 10).

3. Aliquot 300 μl liver extraction solution into a new micro-centrifuge tube.

3.4.4. Preparation of small intestine homogenate

1. Add 6 ml 1x PBS buffer to the tube with the whole intestine and its contents.

2. Homogenize the whole intestine with a powered homogenizer.

3. Centrifuge the homogenate at 4°C at 8,000g for 10 mins, and transfer the supernatant to a new conical tube.

4. Repeat step 1 to 3 above two more times, and pool the extraction solution. Total volume is approximately 18 ml.

5. Aliquot 300 μl intestine extraction solution into a new micro-centrifuge tube (see Note 3).

3.4.5. Preparation of colon homogenate

1. Add 1.5 ml 1x PBS buffer to the tube with the whole colon and its contents.

2. Homogenize the whole colon with a powered homogenizer.

3. Centrifuge the homogenate at 4°C 8,000g for 10 mins, and transfer the supernatant to a new conical tube.

4. Repeat step 1 to 3 above two more times, and pool the extraction solution. Total volume is approximately 4.5 ml.

5. Aliquot 300 μl colon extraction solution into a new micro-centrifuge tube.

3.5. Extraction of BAs from biofluids and tissue homogenates

1. Make ACN with 5% ammonia hydroxide (NH₄OH) and store at −20°C.

2. Pre-chill ACN on ice before extraction (see Note 11).

3. Spike each sample with 20 μl of Internal Standard F (IS F) shown in Table 1.

4. Spike each tissue matrix with 20 μl of appropriate standard solution as indicated in Table 1 to make Standards for each tissue. Prepare Standards and samples in the exact same manner from this point forward.

5. Vortex and equilibrate on ice for 5 mins.

6. Add 900 μl ice-cold ACN to serum/plasma/urine /GB samples, and 1.5 ml ice-cold ACN to liver/small intestine/colon samples.

7. Vortex immediately to precipitate protein.

8. Incubate the tubes on ice for at least 60 mins (see Note 12).

9. Place all samples on tube rotator, and rotate at room temperature for 1 hour.

10. Centrifuge all the samples at 4°C at 13,000g for 15 mins (see Note 13).

11. Transfer the supernatant to a 5 ml glass tube which fits the SpeedVac system. Keep on ice.

12. Resuspend the protein pellet from liver/intestine/colon samples in 0.75 ml methanol by vigorous vortex. Use the pipette tip to break the pellet if necessary.

13. Sonicate the protein resuspension on ice for 1 min by a sonicator (see Note 14).

14. Centrifuge the protein resuspension at 4°C at 13,000g for 15 mins.

15. Transfer the supernatant and pool them with previous supernatant in the 5 ml glass tube.

16. Dry all the samples under SpeedVac system (see Note 15).

17. Add 200μl 50% methanol to the tube after complete drying to reconstitute the sample.

18. Break the dried pellet by pipette tip and vortex.

19. Transfer the solution to the 0.22 μm filtered Spin-X centrifuge tube.

20. Add 200μl 50% methanol to the tube, and repeat the reconstitution by pipetting and vortex. Transfer the solution to the same filtered Spin-X centrifuge tube.

21. Centrifuge all the filtered tubes at 4°C at 13,000g for 10 mins.

22. Remove and discard the filtered columns, and centrifuge the tubes at 4°C at 16,000g again for 15 mins.

23. Transfer 350μl sample solution to autosampler glass vial with insert and store at −20°C until UPLC-MS/MS analysis is conducted.

3.6. BA Profile analysis by Ultra Performance Liquid Chromatography system (UPLC) / electrospray ionization (ESI) / Ion Trap Mass Spectrometer (ITMS)

The configuration and operation for Liquid Chromatography / Mass Spectrometry (LC-MS) are platform-specific. All the settings described here are based on a system consisting of a Thermo Finnigan Ultra Performance Liquid Chromatography system (UPLC) with a refrigerated auto sampler, and a Thermo Scientific LTQ XL Ion Trap Mass Spectrometer (ITMS).

1. Assess and tune system UPLC/MS performance before injecting samples onto the system.

2. Install reverse phase C18 column for BAs separation.

3. Make mobile phase solution. Mobile phase A: Methanol with 0.15% formic acid. Mobile phase B: Water with 0.10% formic acid. Mobile phase C: 100% ACN.

4. Set pump flow rate 270 μl/min and mobile solution gradient as shown in Table 2.

5. Inject a known solution and comparing its response to previous data. Use a blank sample and a sample just containing internal standard to demonstrate selectivity.

6. Load BA standard and the samples on the UPLC/MS system using auto sampler. Perform sample analysis using ESI/ITMS in Selective Ion Monitoring (SIM) mode for simultaneous determination of 23 BAs. Total running time for each sample is 20 mins (11 mins retention time and 9 mins preparation for next run). Fig. 1 shows the chromatogram of 23 BAs obtained by UPLC/ESI/ITMS analysis (see Note 17).

7. Process chromatogram data and quantify the BAs in the samples. Table 3 shows the parameters summary for UPLC/ESI/ITMS analysis (see Note 18).

Table 2.

Gradient program for the UPLC pump

	Time	A %	B %	C %	D %	μl/min
0	0.0	10.0	70.0	20.0	0.0	270.0
1	4.0	15.0	55.0	30.0	0.0	270.0
2	6.0	20.0	40.0	40.0	0.0	270.0
3	7.5	27.5	22.5	50.0	0.0	270.0
4	9.5	37.5	12.5	50.0	0.0	270.0
5	12.0	30.0	5.0	65.0	0.0	270.0
6	13.0	0.0	5.0	95.0	0.0	270.0
7	16.0	0.0	5.0	95.0	0.0	270.0
8	16.0	10.0	70.0	20.0	0.0	270.0
9	22.5	10.0	70.0	20.0	0.0	270.0
10		100.0	0.0	0.0	0.0	270.0

Fig.1.

A chromatogram of 23 bile acids obtained by UPLC/ESI/ITMS analysis in SIM mode of a mixture of bile acids standards. A reverse phase C18 column is used in separation. BAs are eluted by gradually increasing the amount of methanol in the mobile phase.

Table 3.

Summary of parameters for UPLC/ESI/ITMS analysis

BA	Retention Time (min)	Q ions (m/z)	Reference SIM ions (m/z)
T-ω-MCA	2.88	514	514.4, 515.4, 516.41, 582.31
T-α-MCA	3.06	514	514.43, 515.38, 516.41, 582.31
T-β-MCA	3.19	514	514.43, 515.38, 516.41, 582.31
TUDCA	4.59	498	498.33, 499.40, 566.24
THDCA	4.87	498	498.33, 499.40, 566.24
TCA	5.13	514	514.39, 515.50, 582.29, 583.29
GUDCA	6.02	448	448.53, 449.53, 516.37
ω-MCA	6.12	407	407.46, 408.46, 453.23, 475.43, 476.48
α-MCA	6.31	407	407.46, 408.46, 453.23, 475.43, 476.48
GCA	6.38	464	464.50, 465.50, 532.50
β-MCA	6.56	407	407.46, 408.46, 453.23, 475.43, 476.48
TCDCA	6.67	498	98.47, 499.47, 566.38
TDCA	6.95	498	498.47, 499.47, 566.38
UDCA	7.49	391	391.33, 392.33, 459.39, 460.39
CA	7.65	407	407.43, 408.43, 475.33, 476.43
HDCA	7.72	391	391.33, 392.33, 459.39, 460.39
d4-GCDCA (IS)	7.74	452	452.33, 453.40
GCDCA	7.77	448	448.41, 449.41, 516.40, 517.40
GDCA	7.98	448	448.41, 449.41, 516.40, 517.40
TLCA	8.19	482	482.44, 550.33
CDCA	8.82	391	391.37, 392.37
d4-CDCA (IS)	8.83	395	395.53, 396.50
DCA	8.97	391	391.37, 392.37, 459.37, 460.44
GLCA	9.02	432	432.43, 433.35
LCA	10.31	375	375.40, 376.34, 433.24, 511.26

4. Notes

1. All 23 BA standards can be purchased from Steraloids, and 2 internal standards, chenodeoxycholic-2,2,4,4-d4 acid (2H4-CDCA) and glycochenodeoxycholic-2,2,4,4-d4 acid (2H4-GCDCA), can be purchased from CDN Isotopes.

2. Regarding BA metabolism, there are differences between humans and rodents. The predominant BAs in human are CA and CDCA with glycine-conjugation being favored, mice predominantly have MCA and CA with >95% being taurine-conjugated. The method described here focuses on mouse tissues. However, it can be adapted to human biological specimen.

3. In mice, the majority of BAs reside in intestine (approximately 90%) and gallbladder (approximately 10%), and only less than 1% of total BAs are in the liver and serum. For the same reason, diluted samples from intestine and gallbladder are used for BA extraction.

4. Total BA amount in liver, bile, intestine and colon are calculated according to the dilution factors, and final BA concentration is normalized to liver weight or body weight (mg/g), while BA levels in serum/plasma are presented as mg/ml.

5. 90 μl serum/plasma/urine, 55 – 60 mg liver/intestine/colon, and the whole gallbladder are required for BA profiling in this protocol.

6. BA content in the intestine is heterogeneous, so the whole intestine will be used for homogenization. Minimal tissue (less than 1 cm) from duodenum, jejunum or ileum can be saved for RNA or protein isolation.

7. Except for the quick spin, all other procedures described in this protocol are recommended to be carried out on ice or in the 4°C cold room.

8. BA concentration in each tissue is quite different, therefore, appropriate dilutions have been determined to fit the methodology described here.

9. We recommend to prepare two sets of BA standards during every preparation, as one set is saved as a backup, so there are total 20 standards each time. This protocol uses 90 μl serum/plasma/urine or 300 μl tissue homogenate from liver/intestine/colon for BA extraction, and thus it will need at least 2 ml serum/plasma/urine and 6 ml tissue homogenate to prepare the BA standards. Moreover, due to sample-to-sample variations in tissue matrix, pooled samples from at least 5 mice are suggested to be used for standard preparation.

10. Liver tissue should be homogenized until the homogenate is clear and no tissue chunks are left.

11. ACN with 5% ammonia hydroxide (NH₄OH) can be made and stored in −20°C before use, but do not use it immediately after taking from −20°C, otherwise the tissues will form clots. Keep ACN on ice till the temperature is close to ice-cold, and keep it on ice during the extraction process.

12. Longer incubation on ice after adding ACN might help the protein precipitate.

13. There should be a visible protein pellet at the bottom of each tube in liver/intestine/colon samples, but the protein pellets in serum/plasma/bile could be small. Repeat centrifugation once if needed.

14. The setting for sonicator might need optimization, starting from sonicating the solution for 1 min (55% AMP, 30 sec on, 59 sec off, then a final 30 sec on). To limit cross contamination in homogenization and sonication steps, rinse the homogenizer probe twice in ddH₂O between samples, rinse the sonicator probe twice in ddH₂O and 50% methanol, respectively, between samples and wipe with a Kimwipes after each rinse.

15. If the pooled volume from liver/intestine/colon is greater than the volume of tube used in the SpeedVac, the samples can be continually added to the tube used for drying the sample until all of the pooled sample has been dried in the same tube.

16. Make the standard solution by serial dilution of the stock BA solution AAA (50 μg/ml) and each bile acid in the standard solution has the same concentration. Based on the spiking protocol (20 μl standard F (IS F) BA solution (0.391 μg/ml) is used to spike in 90 μl biofluids extract and 300 μl solid tissue extract), Table 1 shows the final BA concentration in extracts and homogenates.

17. BAs are amphipathic and have both a polar and non-polar face. Conjugations and de-hydroxylations increase BAs polarity and their affinity to columns. More hydrophobic BAs in the solution tend to be adsorbed to the column and are eluted last, such as LCA and non-conjugated BAs, while the hydrophilic BAs, such as taurine-conjugated BAs, have less affinity for the column, so they pass through the column faster and are eluted first.

18. Total volume of tissue homogenate is 75 ml for gallbladder, 18 ml for whole intestine, and 4.5 ml for colon, which are used for calculation and normalization. The detection limit for this method is 4.34 ng/g for the liver; 7.88 ng/g for the intestine; 3.11 ng/ml for the gall bladder and 3.11 ng/ml for the plasma.