ArticleFree radical biology & medicine2016
Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H2O2 release assays.
Article in Free radical biology & medicine, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 49 papers.
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Who cites it
49 citing papers in PubMed, 107 citations in OpenAlex.
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- Effect of metformin on intact mitochondria from liver and brain: Concept revisited.European journal of pharmacology · 2022Article
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- Hepatocyte-specific perturbation of NADThe Journal of biological chemistry · 2021Article
- Exercise prevents fatty liver by modifying the compensatory response of mitochondrial metabolism to excess substrate availability.Molecular metabolism · 2021Article
- Increased plasma disequilibrium between pro- and anti-oxidants during the early phase resuscitation after cardiac arrest is associated with increased levels of oxidative stress end-products.Molecular medicine (Cambridge, Mass.) · 2021Article
- Detection of Oxidative Stress Induced by Nanomaterials in Cells-The Roles of Reactive Oxygen Species and Glutathione.Molecules (Basel, Switzerland) · 2021Review
- Lactate dehydrogenases amplify reactive oxygen species in cancer cells in response to oxidative stimuli.Signal transduction and targeted therapy · 2021Article
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Authors and funding
7 authors at 4 institutions in 2 countries.
Funding
Abstract
Amplex Red is a fluorescent probe that is widely used to detect hydrogen peroxide (H2O2) in a reaction where it is oxidised to resorufin by horseradish peroxidase (HRP) as a catalyst. This assay is highly rated amongst other similar probes thanks to its superior sensitivity and stability. However, we report here that Amplex Red is readily converted to resorufin by a carboxylesterase without requiring H2O2, horseradish peroxidase or oxygen: this reaction is seen in various tissue samples such as liver and kidney as well as in cultured cells, causing a serious distortion of H2O2 measurements. The reaction can be inhibited by Phenylmethyl sulfonyl fluoride (PMSF) at concentrations which do not disturb mitochondrial function nor the ability of the Amplex Red-HRP system to detect H2O2.In vitro experiments and in silico docking simulations indicate that carboxylesterases 1 and 2 recognise Amplex Red with the same kinetics as carboxylesterase-containing mitochondria. We propose two different approaches to correct for this problem and re-evaluate the commonly performed experimental procedure for the detection of H2O2 release from isolated liver mitochondria. Our results call for a serious re-examination of previous data.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.