Comprehensive Characterization of Bile Acids in Human Biological Samples and Effect of 4-Week Strawberry Intake on Bile Acid Composition in Human Plasma

Abstract

Primary bile acids (BAs) and their gut microbial metabolites have a role in regulating human health. Comprehensive characterization of BAs species in human biological samples will aid in understanding the interaction between diet, gut microbiota, and bile acid metabolism. Therefore, we developed a qualitative method using ultra-high performance liquid chromatography (UHPLC) coupled with a quadrupole time-of-flight (Q-TOF) to identify BAs in human plasma, feces, and urine samples. A quantitative method was developed using UHPLC coupled with triple quadrupole (QQQ) and applied to a previous clinical trial conducted by our group to understand the bile acid metabolism in overweight/obese middle-aged adults (n = 34) after four weeks strawberry vs. control intervention. The qualitative study tentatively identified a total of 81 BAs in human biological samples. Several BA glucuronide-conjugates were characterized for the first time in human plasma and/or urine samples. The four-week strawberry intervention significantly reduced plasma concentrations of individual secondary BAs, deoxycholic acid, lithocholic acid and their glycine conjugates, as well as glycoursodeoxycholic acid compared to control (p < 0.05); total glucuronide-, total oxidized-, total dehydroxyl-, total secondary, and total plasma BAs were also lowered compared to control (p < 0.05). The reduced secondary BAs concentrations suggest that regular strawberry intake modulates the microbial metabolism of BAs.

Keywords: UHPLC-Q-TOF; UHPLC-QQQ; bile acid; feces; human; microbial metabolites; plasma; polyphenols; strawberry; urine.

Figure 1

Distribution of BAs in human feces, plasma, and urine samples: (a) primary and secondary BAs; (b–d) different BAs subgroups, categorized based on their conjugates. Data are presented as number of BA species detected in each subgroup of BAs.

Figure 2

Effect of ammonium acetate concentration and mobile phase A (0.01% formic acid in water) pH on bile acids peak area. (a) Ammonium acetate concentration; (b) mobile phase A pH. Data for ammonium acetate concentration at 1 mM, 10 mM and mobile phase A pH 3.8 not shown due to co-elution of TUDCA and THDCA. Data are presented as mean and standard deviation based on three measurements. * p < 0.05, ** p < 0.01. Abbreviations: murideoxycholic acid (MuriDCA), ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), chenodeoxycholic acid-D4 (CDCA-D4), tauroursodeoxycholic acid (TUDCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), glycoursodeoxycholic acid (GUDCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid-2,2,4,4-D4 (GCDCA-D4), β-muricholic acid (β-MCA), hyocholic acid (HCA), cholic acid (CA), glycocholic acid (GCA), taurocholic acid (TCA), lithocholic acid (LCA), glycolithocholic acid (GLCA), taurolithochoic acid (TLCA).

Figure 2

Effect of ammonium acetate concentration and mobile phase A (0.01% formic acid in water) pH on bile acids peak area. (a) Ammonium acetate concentration; (b) mobile phase A pH. Data for ammonium acetate concentration at 1 mM, 10 mM and mobile phase A pH 3.8 not shown due to co-elution of TUDCA and THDCA. Data are presented as mean and standard deviation based on three measurements. * p < 0.05, ** p < 0.01. Abbreviations: murideoxycholic acid (MuriDCA), ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), chenodeoxycholic acid-D4 (CDCA-D4), tauroursodeoxycholic acid (TUDCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), glycoursodeoxycholic acid (GUDCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid-2,2,4,4-D4 (GCDCA-D4), β-muricholic acid (β-MCA), hyocholic acid (HCA), cholic acid (CA), glycocholic acid (GCA), taurocholic acid (TCA), lithocholic acid (LCA), glycolithocholic acid (GLCA), taurolithochoic acid (TLCA).

Figure 3

MRM chromatograms using UHPLC-QQQ: (a) BA standards in blank plasma; (b) BA compounds in human plasma. Abbreviations: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), glycoursodeoxycholic acid (GUDCA), hyocholic acid (HCA), hyodeoxycholic acid (HDCA), lithocholic acid (LCA), β-muricholic acid (β-MCA), murideoxycholic acid (MuriDCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurolithochoic acid (TLCA), tauroursodeoxycholic acid (TUDCA), taurohyodeoxycholic acid (THDCA), ursodeoxycholic acid (UDCA).

Figure 4

Changes in concentrations of (a) individual bile acids (b) bile acids in different subgroups after 4-week control or strawberry beverage intake compared to baseline (week 0). Data are presented as the mean and standard error of the mean. * p < 0.05, ** p < 0.01. The black dots are the outliers, which are defined as outside 1.5 times the interquartile range (IQR) above the upper quartile (Q3) and below the lower quartile (Q1) (Q1 − 1.5 × IQR or Q3 + 1.5 × IQR).

Figure 4

Changes in concentrations of (a) individual bile acids (b) bile acids in different subgroups after 4-week control or strawberry beverage intake compared to baseline (week 0). Data are presented as the mean and standard error of the mean. * p < 0.05, ** p < 0.01. The black dots are the outliers, which are defined as outside 1.5 times the interquartile range (IQR) above the upper quartile (Q3) and below the lower quartile (Q1) (Q1 − 1.5 × IQR or Q3 + 1.5 × IQR).

Figure 5

Tentatively proposed structure of glucuronide-GLCA (a), glucuronide-GCDCA (b), and glucuronide-GCA (c). MS/MS spectra of glucuronide-GLCA (m/z 608.34) (d), glucuronide-GCDCA (m/z 624.34) (e), and glucuronide-GCA (m/z 640.33) (f). The MS/MS spectra showing product ions 432.31, 448.31, 464.30 are corresponding to GLCA, GCDCA, and GCA moiety after the loss of the glucuronide group. The fragment 74.02 is corresponding to the conjugate base of glycine.

Figure 5

Tentatively proposed structure of glucuronide-GLCA (a), glucuronide-GCDCA (b), and glucuronide-GCA (c). MS/MS spectra of glucuronide-GLCA (m/z 608.34) (d), glucuronide-GCDCA (m/z 624.34) (e), and glucuronide-GCA (m/z 640.33) (f). The MS/MS spectra showing product ions 432.31, 448.31, 464.30 are corresponding to GLCA, GCDCA, and GCA moiety after the loss of the glucuronide group. The fragment 74.02 is corresponding to the conjugate base of glycine.

Figure 6

Microbial transformation of bile acids. CDCA and CA with 3, 7 and 3, 7, 12 α-hydroxyl groups are dehydroxylated by gut bacteria with 7α-dehydroxylase to lose the hydroxyl group at the 7-α carbon position, forming LCA and DCA. Gut bacteria can further oxidize the hydroxyl groups at 3, 7, and 12 α-carbon positions with hydroxysteroid dehydrogenases (HSDH) activity to form oxo-bile acids. The resulting oxo groups may then undergo epimerization and reduction to form 3, 7, or 12 β-hydroxyl groups. Figure modified based on Ikegami and Honda, 2018 [35].

Figure 7

Study schema of the 4-week randomized, double-blind, placebo-controlled, crossover trial.