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

Luís F. Grilo; João D. Martins; Chiara H. Cavallaro; Peter W. Nathanielsz; Paulo J. Oliveira; Susana P. Pereira

doi:10.1016/j.tiv.2020.104996

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

Luís F. Grilo, João D. Martins, Chiara H. Cavallaro, Peter W. Nathanielsz, Paulo J. Oliveira, Susana P. Pereira

Obstetrics & Gynecology

Research output: Contribution to journal › Article › peer-review

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.

Original language	English (US)
Article number	104996
Journal	Toxicology in Vitro
Volume	69
DOIs	https://doi.org/10.1016/j.tiv.2020.104996
State	Published - Dec 2020

Keywords

Antioxidant defense
Catalase
Hydrogen peroxide
Oxidative stress
Toxicity screening

ASJC Scopus subject areas

Toxicology

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@article{45f93c85665f4b2b9594f00b930f4dc0,

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

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.",

keywords = "Antioxidant defense, Catalase, Hydrogen peroxide, Oxidative stress, Toxicity screening",

author = "Grilo, {Lu{\'i}s F.} and Martins, {Jo{\~a}o D.} and Cavallaro, {Chiara H.} and Nathanielsz, {Peter W.} and Oliveira, {Paulo J.} and Pereira, {Susana P.}",

note = "Funding Information: Funded by ERDF funds through the Operational Programme Competitiveness Factors – COMPETE2020 and national funds by Foundation for Science and Technology under FCT-Post-doctoral Fellowship ( SPP, SFRH/BPD/116061/2016 ), project grant PTDC/DTP-DES/1082/2014(POCI-01-0145-FEDER-016657) , PTDC/BTM-SAL/29297/2017-POCI-01-0145-FEDER-029297 , PTDC/MED-FAR/29391/2017-POCI-01-0145-FEDER-029391 , PTDC/BIA-MOL/28607/2017 , POCI-01-0145-FEDER-028607 , LFG was supported by project “Summer Course in Interdisciplinary Research, Development and Innovation in Cellular and Molecular Metabolism” (15-20/7/245), funded by the Portuguese Foundation for Science and Technology (FCT) and Directorate General for Higher Education (DGES), strategic project UIDB/04539/2020 , and NIH grant R01HD070096-01A1 . The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of this document. ",

year = "2020",

month = dec,

doi = "10.1016/j.tiv.2020.104996",

language = "English (US)",

volume = "69",

journal = "Toxicology in Vitro",

issn = "0887-2333",

publisher = "Elsevier Limited",

}

TY - JOUR

T1 - Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples

AU - Grilo, Luís F.

AU - Martins, João D.

AU - Cavallaro, Chiara H.

AU - Nathanielsz, Peter W.

AU - Oliveira, Paulo J.

AU - Pereira, Susana P.

N1 - Funding Information: Funded by ERDF funds through the Operational Programme Competitiveness Factors – COMPETE2020 and national funds by Foundation for Science and Technology under FCT-Post-doctoral Fellowship ( SPP, SFRH/BPD/116061/2016 ), project grant PTDC/DTP-DES/1082/2014(POCI-01-0145-FEDER-016657) , PTDC/BTM-SAL/29297/2017-POCI-01-0145-FEDER-029297 , PTDC/MED-FAR/29391/2017-POCI-01-0145-FEDER-029391 , PTDC/BIA-MOL/28607/2017 , POCI-01-0145-FEDER-028607 , LFG was supported by project “Summer Course in Interdisciplinary Research, Development and Innovation in Cellular and Molecular Metabolism” (15-20/7/245), funded by the Portuguese Foundation for Science and Technology (FCT) and Directorate General for Higher Education (DGES), strategic project UIDB/04539/2020 , and NIH grant R01HD070096-01A1 . The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of this document.

PY - 2020/12

Y1 - 2020/12

N2 - 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.

AB - 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.

KW - Antioxidant defense

KW - Catalase

KW - Hydrogen peroxide

KW - Oxidative stress

KW - Toxicity screening

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U2 - 10.1016/j.tiv.2020.104996

DO - 10.1016/j.tiv.2020.104996

M3 - Article

AN - SCOPUS:85090892879

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JO - Toxicology in Vitro

JF - Toxicology in Vitro

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