Please use this identifier to cite or link to this item:
https://hdl.handle.net/10316/101007
Title: | Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples | Authors: | Grilo, Luís F Martins, João D Cavallaro, Chiara H Nathanielsz, Peter W Oliveira, Paulo J Pereira, Susana P |
Keywords: | Antioxidant defense; Catalase; Hydrogen peroxide; Oxidative stress; Toxicity screening | Issue Date: | Dec-2020 | Volume: | 69 | Abstract: | Oxidative stress biomarkers are powerful endpoints in toxicological research. Cellular reductive/oxidative balance affects numerous signaling pathways involving H2O2. Detoxification and control of H2O2 levels results mainly from catalase activity. The aim of this work was to develop a precise, simple, cost-effective microassay to measure catalase activity in small tissue samples and cell extracts. We developed a protocol that quantifies H2O2 decomposition by intrinsic catalase in biological samples. Catalase activity was calculated based on rate of decomposition of H2O2, following absorbance at 240 nm. We developed a multi-well spectroscopic approach, reducing sample quantity requirements and allowing simultaneous assessment of large number of samples. The protocol is sensitive across a wide range of catalase activity (11.5-7575 U). The assay presents a 95% confidence interval with an intra-assay coefficient of variation of 3.7%, an inter-assay coefficient of variation of 6.2% and good correlation with a commercial kit. The assay was established and validated for different biological samples, including sheep hepatic tissue and human tumor and non-tumor cell lines. This high-throughput method is robust, sensitive, time-saving and cost-effective, generating highly reproducible results with precision and good correlation with a commercial kit reinforcing the method's validity for research and toxicological applications. | URI: | https://hdl.handle.net/10316/101007 | ISSN: | 08872333 | DOI: | 10.1016/j.tiv.2020.104996 | Rights: | openAccess |
Appears in Collections: | I&D CNC - Artigos em Revistas Internacionais |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
Grilo et al. - 2020 - Toxicology in Vitro-annotated.pdf | 1.55 MB | Adobe PDF | View/Open |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.