Determination of obeticholic acid and its glycine conjugate in human plasma using liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography/tandem mass spectrometry method was recommended by the authors for the determination orally bioavailable farnesoid X receptor agonist obeticholic acid along with its glycine conjugate glycoobeticholic acid human plasma. Obeticholic acid_d5 and glycoobeticholic acid_d5 were used as internal standards, respectively. After extraction with a mixture of tert-butyl methyl ether and dichloromethane, samples were separated on a phenyl column with a mobile phase of 2mM ammonium acetate with 0.01% formic acid and methanol (15:85, v/v). Analysis was performed on an AB Sciex 4500 triple quadrupole mass spectrometer and data acquisition was performed by multiple reaction monitoring (MRM) in the negative ionization mode. Obeticholic acid and its conjugate were quantified in the linearity range of 0.50-100.00 ng/mL and the correction coefficients were≥0.99 during the validation. Precision and accuracy in different days (inter-day) and single day (intra-day) were meeting the acceptance criteria specified in the recent US FDA bioanalytical method validation guidelines. A variety of stability experiments in neat samples and plasma samples were conducted and the results are meeting the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing productivity. This method could be useful to quantitate obeticholic acid and glycoobeticholic acid in real clinical samples.

Intra- and inter-individual metabolic profiling highlights carnitine and lysophosphatidylcholine pathways as key molecular defects in type-2 diabetes

ABSTRACTType-2 diabetes (T2D) mellitus is a complex metabolic disease commonly caused by insulin resistance in several tissues. We performed a matched two-dimensional metabolic screening in tissue samples from a cohort of 43 multi-organ donors. The intra-individual analysis was assessed across five key-metabolic tissues (serum, adipose tissue, liver, pancreatic islets and muscle), and the inter-individual across three different groups reflecting T2D progression. We identified 92 metabolites differing significantly between non-diabetes and T2D subjects. Carnitines were significantly higher in liver, while lysophosphatidylcholines significantly lower in muscle and serum. An investigation of the progression to overt T2D showed that deoxycholic acid glycine conjugate was significantly higher in liver of pre-diabetes samples while additional increase in T2D was insignificant. A subset of lysophosphatidylcholines were significantly lower in the muscle of pre-diabetes subjects. Overall, the analysis of this unique dataset can increase the understanding of the metabolic interplay between organs in the development of T2D.

On the Conformation of Glycobilirubin

The first optically active glycine conjugate 1 of a bilirubin was prepared in several steps from (S)-β-methylxanthobilirubic acid glycine conjugate 8. The latter was synthesized by reaction of benzyl glycinate tosylate with the mixed anhydride formed in the reaction of (S)-β-methylxanthobilirubic acid 6 with isobutyl chloroformate. Spectroscopic analysis of the circular dichroism spectra of 1 in various solvents, including aqueous buffer, indicate a conformational preference for the M-helical ridge-tile conformation, thus providing the first spectroscopic evidence on the conformation of glycobilirubins.

Excretion of benzoic acid derivatives in urine of sheep given intraruminal infusions of 3-phenylpropionic and cyclohexanecarboxylic acids

The quantitative relationship between the urinary excretion of benzoic acid (BA)and the uptake of 3-phenylpropionic (PPA) and cyclohexanecarboxylic (CHCA) acids was assessed.PPA and CHCA are produced in the rumen by microbial fermentation of lignocellulosic feeds and metabolized, after absorption, to BA which is excreted in the urine mainly as its glycine conjugate hippuric acid (HA). Four sheep nourished by intragastric infusions of all nutrients weregiven continuous ruminal infusions of PPA (8,16 or 24 mmol/d) either alone or with CHCA (8 or 16 mmol/d) in a factorial experiment. The treatments were allocated to ten consecutive 6 d periods, with a control being repeated at periods 1, 5 and 10. PPA and CHCA ruminal absorption rates, estimated using the liquid-phase marker Cr-EDTA, were 0·78 (SD 0·29)/h and 0·88 (SD 0·28)/h respectively. For the control, HA excretion was only 0·22 (SD 0·33) mmol/d and free BA was absent. For the other treatments, both HA and free BA were present and HA accounted for 0·85 (SD 0·05) of total BA. The urinary excretion of total BA showed a significant linear correlation (r = 0·997, P<0·001) with the amounts of PPA and CHCA infused. The urinary recovery of infused PPA and CHCA as total BA was 0·79 (SE 0·01). Faecal excretion of BA and its precursors was negligible. Results of this study show that urinary total BA is a potential estimator of the absorption of PPA + CHCA produced in the rumen