Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples

• Published in: Toxicology in vitro 2020-12, Vol.69, p.104996-104996, Article 104996
• Main Authors: Grilo, Luís F., Martins, João D., Cavallaro, Chiara H., Nathanielsz, Peter W., Oliveira, Paulo J., Pereira, Susana P.
• Format: Article
• Language: English
• Subjects: Antioxidant defense; Assaying; Biological activity; Biological properties; Biological samples; Biomarkers; Catalase; Coefficient of variation; Confidence intervals; Decomposition; Detoxification; Hydrogen peroxide; Mathematical analysis; Oxidative stress; Sheep; Toxicity screening; Tumor cell lines; Tumors
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2020.104996

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. [Display omitted] •New high-sensitivity and fast methodology for catalase peroxidase activity assessment was developed;•This assay is reliable for tissue and cell samples and a wide catalase activity range;•The developed protocol shows small intra- and inter-assay variations.