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

Abstract

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (HO) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured HO 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 (O) concentration of ventilation gas on lung rate of HO release into the perfusate. Baseline lung rate of HO 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 in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial HO, and that most of acute hyperoxia-enhanced lung rate of HO release is from nonmitochondrial rather than mitochondrial sources.

Curcumin analogs (B2BrBC and C66) supplementation attenuates airway hyperreactivity and promote airway relaxation in neonatal rats exposed to hyperoxia

“…Hyperoxia causes the intrinsic imbalance between proand antioxidant mechanisms leading to oxidative stress and production of specific oxidative molecules on the level of the airways, such as H 2 O 2 (Audi et al, 2018;Gil-Ortega et al, 2014). Hyperoxia decreased CAT activity, an enzyme that scavenges H 2 O 2 .…”

Analisis Molekular dan Karakteristik Hidrogen Sianida (HCN)

“…Hidrogen sianida (7; 10; 26; 27; 32; [46][47][48][49][50][51][52][53][54][55][56][57][58][59][60] atau biasa disebut formic anammonide, asam hidrosianat, atau asam sianida yang merupakan suatu monobasa dari asam Lewisboron lemah yang dapat digunakan sebagai penyepuhan emas,dan pembuatan pewarna prussian blue (blau), (108) . Asam sianida akan larut dalam air (109; 110) , senyawa ini didalam air akanmembentuk ion CNdan ion H + .Senyawa dengan rumus kimia HCN cairan tak bewarna dan sangat beracun.…”

Moderate Hydrogen Peroxide Postconditioning Ameliorates Ischemia/Reperfusion Injury in Cardiomyocytes via STAT3-Induced Calcium, ROS, and ATP Homeostasis

“…H 2 O 2 or superoxide production in isolated cardiomyocytes during simulated I/R was detected by Amplex Red or MitoSOX Red (Thermo Fisher, MA, USA) as previously described [20, 21]. The detailed method is described in online suppl.…”