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Anal Biochem. 2017 Oct 15;535:47-55. doi: 10.1016/j.ab.2017.07.025. Epub 2017 Jul 27.

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

Author information

1
Research Institute for Medicines - iMed.ULisboa, Metabolism and Genetics Group, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
2
Tytgat Institute for Liver and Intestinal Research and Surgical Laboratory, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
3
Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
4
Research Institute for Medicines - iMed.ULisboa, Metabolism and Genetics Group, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; Department of Biochemistry and Human Biology, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. Electronic address: mbsilva@ff.ulisboa.pt.

Abstract

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.

KEYWORDS:

Bioartificial liver; HepaRG cell line; Hepatotoxicity; Hyperammonemia; Targeted metabolomics; Urea cycle enzymes

PMID:
28757091
DOI:
10.1016/j.ab.2017.07.025
[Indexed for MEDLINE]

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