Simultaneous detection of the enzyme activities of GPx1 and GPx4 guide optimization of selenium in cell biological experiments
Year: 2020
Type: article
Abstract: Selenium is a metalloid trace element essential for maintaining the optimal redox environment in cells and tissues. It is structurally incorporated into over 25 selenoproteins and enzymes. The glutathione peroxidase (GPx) family of enzymes has a critical role in human health because of its antioxidant function. The recommended daily allowance (RDA) for selenium intake in humans was established to maximize the activity of GPx in plasma. Suboptimal availability of selenium can limit the expression and activities of GPxs leading to a compromised redox environment. This can cause detrimental oxidative distress that could be prevented by increasing the availability of selenium. In cell culture studies, the medium is typically deficient in selenium; supplementation with selenium can increase selenoenzyme activities. However, the optimal level of supplementation in cell culture media has not been well characterized. We performed dose-response experiments for the activities of GPx1 and GPx4 vs. the level of selenium supplementation in cell culture medium. For this, we advanced an assay to determine the activities of both GPx1 and GPx4 efficiently in a single run. During the optimization process, we found that the observed activities of GPx1 and GPx4 depend greatly on the pH of the assay buffer; the observed activities increase with increasing pH, with pH 8 being optimal. Using the combination assay, we also found that the expression and activities for both GPx1 and GPx4 can be maximized in exponentially growing cells by supplementing cell culture media with ≈ 200 nM seleno-l-methionine, without concerns for toxicity. Optimizing the availability of selenium in cell culture to maximize the expression and activities GPx1 and GPx4 may allow for better translation of information from cell culture work to in vivo settings.
Source: Redox Biology
Institution: University of Iowa
Language: English
Subfield: Nutrition and Dietetics
Field: Nursing
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,850
Funders: Roy J. Carver Charitable Trust, Houston Center for Contemporary Craft, National Institutes of Health, Holden Comprehensive Cancer Center, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, National Eye Institute, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Environmental Health Sciences
Awards: Treatment of a Complex Retinal Degenerative Syndrome, Exploiting Redox Metabolism Using Pharmacological Ascorbate for Cancer Therapy, G3423802299, The chemical biology of pharmacological ascorbate in cancer treatment, Complex Mechanisms in Bardet-Biedl Syndrome Retinopathy, Quantitative Redox Biology, Iowa Superfund Research Program: Airborne PCBs: Sources, Exposures, Toxicities, Remediation, Environmental Health Sciences Research Center
OpenAlex ID: W3013127633