Abstract
We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. Initially, before enzymatic deconjugation, 19 unconjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 μmol/l. Sixty-seven conjugated BAs were identified by LC-MRM-MS analysis before and after enzymatic deconjugation. Four enzymatic assays were used to deconjugate the BA conjugates. FBAs in urine after cholylglycine hydrolase/sulfatase treatment were 33.40 μmol/l, indicating the urinary BAs were comprised of 29.75% FBAs and 70.25% conjugated BAs in single and multiple conjugated forms. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by β-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. These results support that CDCA is a precursor of γ-muricholic acid in BA biosynthesis in piglets.
Highlights
We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated trihydroxycholanoic acid (BA) either in the absence of their standards, or when present in low concentrations
Twenty-four of the standards were purchased from Steraloids, Inc. (Newport, RI): MCA, MCA, MCA, MCA, murideoxycholic acid (MDCA), 5 -cholanic acid-3,12 -diol, 3-deoxycholic acid (3-DCA), isolithocholic acid, allolithocholic acid, taurocholic acid (T-CA), sodium tauro -muricholic acid (T- MCA), tauro -muricholic acid (T- MCA), sodium tauro -muricholic acid (T- MCA sodium salt), taurohyodeoxycholic acid (T-HDCA), taurochenodeoxycholic acid (T-CDCA), tauroursodeoxycholic acid (T-UDCA), taurodeoxycholic acid (T-DCA), taurolithocholic acid (T-LCA), sodium glycohyocholic acid (G- MCA sodium salt), glycohyodeoxycholic acid (G-HDCA), sodium glycochenodeoxycholate (G-CDCA sodium salt), glycoursodeoxycholic acid (G-UDCA), glycodeoxycholic acid (G-DCA), and glycolithocholic acid (G-LCA)
The results indicated that 80% MeOH/H2O was more efficient for extraction of free and conjugated BAs from the lyophilized urine powder
Summary
We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by -glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. Quantitative comparison of BAs from different synthetic pathways in the urine of piglets can be used to determine the effect of different diets on the BA metabolism and to further evaluate the nutritional value of different diets for infants
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