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Title: Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS

item FANG, NIANBAI - Arkansas Children'S Nutrition Research Center (ACNC)
item YU, SHANGGONG - University Arkansas For Medical Sciences (UAMS)
item Ferruzzi, Mario
item RONIS, MARTIN - Louisiana State University Medical Center
item BADGER, THOMAS - Arkansas Children'S Nutrition Research Center (ACNC)

Submitted to: Journal of Lipid Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2016
Publication Date: 8/18/2016
Citation: Fang, N., Yu, S., Adams, S.H., Ronis, M.J., Badger, T.M. 2016. Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS. Journal of Lipid Research. doi:10.1194/jlr.D069831.

Interpretive Summary: We present a method using a combination of enzymatic deconjugation and targeted LC-MRM-MS analysis for analyzing all possible 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 un-conjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 umol/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 was 33.40 umol/l, indicating the urinary BAs was 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 was 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by beta-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs which constituted 16.67% of urinary BAs. Notably, CDCA was initially detected only in trace amounts in urine, but was found in significant levels after the enzymatic assays above. These results support that CDCA is a precursor of yMCA in BA biosynthesis in piglets.

Technical Abstract: Bile acids (BAs) have an important role in the control of fat, glucose and cholesterol metabolism. Synthesis of bile acids is the major pathway for the metabolism of cholesterol and for the excretion of excess cholesterol in mammals. Bile acid intermediates and/or their metabolites are excreted in part in urine, and the patterns of these molecules provides insight into changes in the formation or breakdown of specific bile acids in the body or by intestinal bacteria (microbiome). Interestingly, insulin-resistant individuals had a blunted increase in blood glycochenodeoxycholic acid (a common bile acid) following oral glucose tolerance test. Hence, the differentiated quantification of bile acid profiles may be an important tool for determining the impact of diet, health status and other factors in bile acid biosynthetic pathways. Bile acid metabolites facilitate the absorption of dietary lipids and fat-soluble vitamins by formation of micelles, and diet has been reported to affect bile acid metabolism in infants. Thus, quantitative comparison of BAs from different synthetic pathways in urine of piglets (as a model of newborn metabolism) can be used to determine the effect of different diets on the BA metabolism and to further evaluate nutritional value of different diets for infants. However, there is currently no well-established method in the literature for the detection of all possible BAs because the standards of BA conjugates, and in particular the standards of the conjugates in double and triple conjugated forms, are not available. The purpose of the present work was to develop a novel method to analyze the BAs in biological samples and obtain a comprehensive profile of urinary BAs in piglets. It was discovered that BAs in triple-conjugated forms (when a different molecule is attached to the BA to modify it) accounted for a major portion of the urinary BAs in piglets. This suggests that multiple conjugated forms constitute one of the major pathways for the excretion of excess cholesterol in the body of infants. This research will provide a new analytical toolset to study the multiple conjugated metabolites of other chemical classes in children, which have not been well studied due to the absence of their standards commercially.