Detection of hydrogen peroxide production in the isolated rat lung using Amplex red

摘要

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H 2 O 2 ) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H 2 O 2 using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. ARs 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 (O 2 ) concentration of ventilation gas on lung rate of H 2 O 2 release into the perfusate. Baseline lung rate of H 2 O 2 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 %O 2 in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O 2 -dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial H 2 O 2 , and that most of acute hyperoxia-enhanced lung rate of H 2 O 2 release is from nonmitochondrial rather than mitochondrial sources. ?2018, ?2018 Informa UK Limited, trading as Taylor & Francis Group.