Advances in methods for characterization of hepatic urea cycle enzymatic activity in HepaRG cells using UPLC-MS/MS

•Novel assays using quantitative UPLC-MS/MS allow the assessment of four enzymatic reactions of urea cycle in human liver.•The bioanalytical approach is highly sensitive not requiring as much protein and reagents than classical techniques.•The assays are important for the study of samples with low activity of urea cycle, including the human liver cell line HepaRG.•Decreased activity of CPS I, OTC or ARG may underlie the limited nitrogen fixation of HepaRG cells versus liver tissue.

Current methodologies for the assessment of urea cycle (UC) enzymatic activity are insufficient to accurately evaluate this pathway in biological specimens where lower UC is expected. Liver cell lines, including HepaRG, have been described to have limited nitrogen fixation through the UC, limiting their applicability as biocomponents for Bioartificial Livers (BAL). This work aims to develop novel and sensitive analytical solutions using Mass Spectrometry-based methodology to measure the activity of four UC enzymes in human liver and HepaRG cells. Activity of carbamoyl-phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL) and arginase (ARG I and II) was determined on homogenates from normal human liver and HepaRG cells cultured in monolayer or in the AMC-BAL. Enzyme products were determined by stable-isotope dilution UPLC-MS/MS. Activity of CPS I, OTC and ARG I/II enzymes in HepaRG monolayer cultures was considerably lower than in human control livers albeit an increase was achieved in HepaRG-BAL cultures. Improved analytical assays developed for the study of UC enzyme activity, contributed to gain understanding of UC function in the HepaRG cell line. The decreased activity of CPS I suggests that it may be a potential rate-limiting factor underlying the low UC activity in this cell line.

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