Quantification of multiple bile acids in uninephrectomized rats using ultra-performance liquid chromatography-tandem mass spectrometry

Abstract

In order to study the roles of individual BAs and due to limited blood sample volumes available from experimental animals, improved methods for the simultaneous quantification of multiple BAs are needed. We developed and validated an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of 24 BAs, including 11 unconjugated, 6 glycine-conjugated and 7 taurine-conjugated BAs, in 50 mL of rat serum or plasma. The UPLC-MS/MS method, operated in negative and positive ion mode, allows quantification of BAs using multiplereaction monitoring (MRM), with specific fragmentation of BAs. The method showed acceptable intraand inter-day accuracy, precision, extraction recovery and high sensitivity, with a lower limit of quantification (LLOQ) in the pM range for several taurine-conjugated BAs. We applied the established method to investigate potential time-dependent changes of BAs in plasma from sham-operated and uninephrectomized male Sprague-Dawley rats. The levels of several primary and secondary BAs were transiently elevated one week after uninephrectomy, followed by normalization thereafter. In contrast, several conjugated BAs were slightly increased after the second week post-surgery. The established UPLC-MS/MS method, employing specific fragmentation of free and conjugated BAs by MRM, allows the simultaneous quantification of multiple BAs in 50 mL serum or plasma samples, and can be used to assess BA profiles in patho-physiological situations.

Uninephrectomy in rats on a fixed food intake results in adipose tissue lipolysis implicating spleen cytokines

“…Since we had previously shown increases in bile salts following UniNX, we chose to investigate whether bile salt receptor FXR (Penno et al, 2013 ; Gai et al, 2014a ) and its potential regulator SIRT1 (Garcia-Rodriguez et al, 2014 ) were altered in various tissues. Here we show that both were modified in tissues by UniNX.…”

“…Activation of SIRT1 and FXR can counter the metabolic syndrome by acting on lipid and glucose metabolism. We and others have recently shown that bile salts and their receptor FXR are modified by UniNX (Penno et al, 2013 ; Gai et al, 2014a , b ; Chin et al, 2015 ). It has also been reported that SIRT1 may regulate FXR activity (Liu et al, 2014 ).…”

Effect of chronic renal failure on the hepatic, intestinal, and renal expression of bile acid transporters

“…Plasma total cholesterol and creatinine concentrations were measured using a synchro CX3 autoanalyzer (Beckman Instruments). Plasma total bile acids were measured using a kit from Crystal Chemistry, and individual bile acid species were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (27). Urinary protein concentrations were determined using a kit from Wako Chemical Industries (Tokyo, Japan).…”

Impaired oxidoreduction by 11β-hydroxysteroid dehydrogenase 1 results in the accumulation of 7-oxolithocholic acid

“…Concomitantly, lysates were used in parallel experiments to assess the conversion of cortisone to cortisol. Reaction products were directly measured by mass spectrometry as described previously ( 26,31 ). The rate of all reactions was kept below 25% of substrate conversion, with the exception of the comparison of product formation from 7-oxoLCA shown in Fig.…”

“…Extraction and quantifi cation of bile acids in plasma samples was performed essentially as described previously ( 31 ). The extraction of bile acids from liver tissue was performed by homogenizing 100 mg of tissue in 200 µl 50% methanol.…”