title: Comparison of in vitro methods for carboxylesterase activity determination in immortalized cells representative of the intestine, liver and kidney.
abstract: Herein we compare the fluorimetric determination of total and specific carboxylesterase activity in immortalized human derived living cells and in cell lysates. The cell lines chosen are representative of metabolism occurring in the intestine (Caco-2 and HT-29), kidney (HEK-293T) and liver (Hep G2). Caco-2 and HT-29, as cells prone to differentiation, were tested along the differentiation time. For evaluation of both methods when distinguishing activity of different carboxylesterases, HEK-293T transfected with the human carboxylestarase-2 (hCES2) were also tested. Application to Caco-2 or HT-29 cells demonstrated higher activity detected in cell lysates than in cell monolayers. The difference is most striking when comparing the methods at different stages of Caco-2 and HT-29 cell maturation, highlighting substrate accessibility as a limiting step in the in vivo hydrolysis rates (possibly limited by plasma and Endoplasmic Reticulum membrane permeability) with increasing relevance as the cells differentiate. Application to Hep G2 or to hCES2 transfected and non-transfected HEK-293T cells, demonstrated a tendency for higher sensitivity in living cell suspensions than that obtained with the cell lysates which indicates the importance of cell environment in the maintenance of enzyme activity. However, quantification of hCES2 activity relative to total esterase, or to total carboxylesterase activity, was not significantly different in any case. The results herein presented help to clarify which method is best suited for evaluation of carboxylesterase activity in vitro depending on the final goal of the study.
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa, Apartado 12, 2780-901, Oeiras, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Lamego
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa, Apartado 12, 2780-901, Oeiras, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Ferreira
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa, Apartado 12, 2780-901, Oeiras, Portugal.
authors.forename: Márcia
authors.initials: M
authors.lastname: Alves
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa, Apartado 12, 2780-901, Oeiras, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Matias
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa, Apartado 12, 2780-901, Oeiras, Portugal. Electronic address: anas@itqb.unl.pt.
authors.forename: Ana Luisa
authors.initials: AL
authors.lastname: Simplício
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002457:Cell Extracts; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology / Q000201:enzymology*; D007668:Kidney / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D002265:Carboxylic Ester Hydrolases
keywords: Caco-2; Carboxylesterase (CES); HEK-293T; HT-29; Hep G2; Human carboxylesterase 2 (hCES2); Whole living cells