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Detection of hydrogen peroxide production in the isolated rat lung using Amplex red. Free Radic Res 2018 Sep;52(9):1052-1062

Date

09/04/2018

Pubmed ID

30175632

Pubmed Central ID

PMC6298832

DOI

10.1080/10715762.2018.1511051

Scopus ID

2-s2.0-85054191562   4 Citations

Abstract

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H2O2) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H2O2 using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR's highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O2) concentration of ventilation gas on lung rate of H2O2 release into the perfusate. Baseline lung rate of H2O2 release was 8.45 ± 0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O2 in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O2-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial H2O2, and that most of acute hyperoxia-enhanced lung rate of H2O2 release is from nonmitochondrial rather than mitochondrial sources.

Author List

Audi SH, Friedly N, Dash RK, Beyer AM, Clough AV, Jacobs ER

Authors

Said Audi PhD Professor in the Biomedical Engineering department at Marquette University
Andreas M. Beyer PhD Associate Professor in the Medicine department at Medical College of Wisconsin
Ranjan K. Dash PhD Professor in the Biomedical Engineering department at Medical College of Wisconsin
Elizabeth R. Jacobs MD Professor in the Medicine department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Antimycin A
Electron Transport Chain Complex Proteins
Hydrogen Peroxide
Lung
Mitochondria
Organ Culture Techniques
Oxazines
Oxidation-Reduction
Oxidative Stress
Potassium Cyanide
Rats
Reactive Oxygen Species
Rotenone
Thenoyltrifluoroacetone