Abstract
Reactive oxygen/nitrogen species (ROS/RNS) have been increasingly recognized as important mediators and play a number of critical roles in cell injury, metabolism, disease pathology, diagnosis, and clinical treatment. Electron paramagnetic resonance (EPR) spectroscopy enables the spectral information at certain spatial position, and, from the observed line-width and signal intensity, the localized tissue oxygenation, and tissue redox status can be determined. We applied in vivo EPR oximetry and redoximetry technique and implemented its physiological/pathophysiological applications, along with the use of biocompatible lithium pthalocyanine (liPc) and nitroxide redox sensitive probes, on in vivo tissue oxygenation and redox profile of the ischemic and reperfused heart in living animals. We have observed that the hypoxia during myocardial ischemia limited mitochondrial respiration and caused a shift of tissue redox status to a more reduced state. ROS/RNS generated at the beginning of reperfusion not only caused a shift of redox status to a more oxidized state which may contribute to the postischemic myocardial injury, but also a marked suppression of in vivo tissue O2 consumption in the postischemic heart through modulation of mitochondrial respiration based on alterations in enzyme activity and mRNA expression of NADH dehydrogenase (NADH-DH) and cytochrome c oxidase (CcO). In addition, ischemic preconditioning was found to be able to markedly attenuate postischemic myocardial hyperoxygenation with less ROS/RNS generation and preservation of mitochondrial O2 metabolism, due to conserved NADH-DH and CcO activities. These studies have demonstrated that EPR oximetry and redoximetry techniques have advanced to a stage that enables in-depth insight in the process of ischemia reperfusion injury.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Zweier JL, Kuppusamy P, Lutty GA (1988) Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues. Proc Natl Acad Sci U S A 85(11):4046–4050
Zhao X, He G, Chen YR et al (2005) Endothelium-derived nitric oxide regulates postischemic myocardial oxygenation and oxygen consumption by modulation of mitochondrial electron transport. Circulation 111(22):2966–2972
Lizasoain I, Moro MA, Knowles RG et al (1996) Nitric oxide and peroxynitrite exert distinct effects on mitochondrial respiration which are differentially blocked by glutathione or glucose. Biochem J 314(Pt 3):877–880
Wang P, Zweier JL (1996) Measurement of nitric oxide and peroxynitrite generation in the postischemic heart. Evidence for peroxynitrite-mediated reperfusion injury. J Biol Chem 271(46):29223–29230
Wolin MS, Xie YW, Hintze TH (1999) Nitric oxide as a regulator of tissue oxygen consumption. Curr Opin Nephrol Hypertens 8(1):97–103
Ohnishi ST, Ohnishi T, Muranaka S et al (2005) A possible site of superoxide generation in the complex I segment of rat heart mitochondria. J Bioenerg Biomembr 37(1):1–15
Ferdinandy P, Schulz R (2003) Nitric oxide, superoxide, and peroxynitrite in myocardial ischaemia-reperfusion injury and preconditioning. Br J Pharmacol 138(4):532–543
Moncada S, Erusalimsky JD (2002) Does nitric oxide modulate mitochondrial energy generation and apoptosis? Nat Rev Mol Cell Biol 3(3):214–220
Beltran B, Mathur A, Duchen MR et al (2000) The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death. Proc Natl Acad Sci U S A 97(26):14602–14607
Cleeter MW, Cooper JM, Darley-Usmar VM et al (1994) Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett 345(1):50–54
Klawitter PF, Murray HN, Clanton TL et al (2002) Reactive oxygen species generated during myocardial ischemia enable energetic recovery during reperfusion. Am J Physiol Heart Circ Physiol 283(4):H1656–H1661
Zweier JL, Chzhan M, Ewert U et al (1994) Development of a highly sensitive probe for measuring oxygen in biological tissues. J Magn Reson B 105(1):52–57
Swartz HM, Dunn JF (2003) Measurements of oxygen in tissues: overview and perspectives on methods. Adv Exp Med Biol 530:1–12
Halpern HJ, Yu C, Peric M et al (1994) Oxymetry deep in tissues with low-frequency electron paramagnetic resonance. Proc Natl Acad Sci U S A 91(26):13047–13051
Liu KJ, Gast P, Moussavi M et al (1993) Lithium phthalocyanine: a probe for electron paramagnetic resonance oximetry in viable biological systems. Proc Natl Acad Sci U S A 90(12):5438–5442
Stoner JD, Angelos MG, Clanton TL (2004) Myocardial contractile function during postischemic low-flow reperfusion: critical thresholds of NADH and O2 delivery. Am J Physiol Heart Circ Physiol 286(1):H375–H380
Brandes R, Bers DM (1996) Increased work in cardiac trabeculae causes decreased mitochondrial NADH fluorescence followed by slow recovery. Biophys J 71(2):1024–1035
Riess ML, Camara AK, Chen Q et al (2002) Altered NADH and improved function by anesthetic and ischemic preconditioning in guinea pig intact hearts. Am J Physiol Heart Circ Physiol 283(1):H53–H60
Eng J, Lynch RM, Balaban RS (1989) Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes. Biophys J 55(4):621–630
Al-Mehdi AB, Shuman H, Fisher AB (1997) Intracellular generation of reactive oxygen species during nonhypoxic lung ischemia. Am J Physiol 272(2 Pt 1):L294–L300
Budd SL, Castilho RF, Nicholls DG (1997) Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells. FEBS Lett 415(1):21–24
Zhao H, Kalivendi S, Zhang H et al (2003) Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med 34(11):1359–1368
Tanoue Y, Herijgers P, Meuris B et al (2002) Ischemic preconditioning reduces unloaded myocardial oxygen consumption in an in-vivo sheep model. Cardiovasc Res 55(3):633–641
An J, Camara AK, Rhodes SS et al (2005) Warm ischemic preconditioning improves mitochondrial redox balance during and after mild hypothermic ischemia in guinea pig isolated hearts. Am J Physiol Heart Circ Physiol 288(6):H2620–H2627
Zhu X, Zuo L, Cardounel AJ et al (2007) Characterization of in vivo tissue redox status, oxygenation, and formation of reactive oxygen species in postischemic myocardium. Antioxid Redox Signal 9(4):447–455
Swartz HM, Bacic G, Friedman B et al (1994) Measurements of pO2 in vivo, including human subjects, by electron paramagnetic resonance. Adv Exp Med Biol 361:119–128
Bolli R (1996) The early and late phases of preconditioning against myocardial stunning and the essential role of oxyradicals in the late phase: an overview. Basic Res Cardiol 91(1):57–63
Angelos MG, Kutala VK, Torres CA et al (2006) Hypoxic reperfusion of the ischemic heart and oxygen radical generation. Am J Physiol Heart Circ Physiol 290(1):H341–H347
Zuo L, Clanton TL (2005) Reactive oxygen species formation in the transition to hypoxia in skeletal muscle. Am J Physiol Cell Physiol 289(1):C207–C216
Shen W, Xu X, Ochoa M et al (1994) Role of nitric oxide in the regulation of oxygen consumption in conscious dogs. Circ Res 75(6):1086–1095
Al-Obaidi MK, Etherington PJ, Barron DJ et al (2000) Myocardial tissue oxygen supply and utilization during coronary artery bypass surgery: Evidence of microvascular no-reflow. Clin Sci (Lond) 98(3):321–328
Trochu JN, Bouhour JB, Kaley G et al (2000) Role of endothelium-derived nitric oxide in the regulation of cardiac oxygen metabolism: implications in health and disease. Circ Res 87(12):1108–1117
Roy S, Khanna S, Bickerstaff AA et al (2003) Oxygen sensing by primary cardiac fibroblasts: a key role of p21(Waf1/Cip1/Sdi1). Circ Res 92(3):264–271
Zhu X, Liu B, Zhou S et al (2007) Ischemic preconditioning prevents in vivo hyperoxygenation in postischemic myocardium with preservation of mitochondrial oxygen consumption. Am J Physiol Heart Circ Physiol 293(3):H1442–H1450
Swartz HM, Boyer S, Brown D et al (1992) The use of EPR for the measurement of the concentration of oxygen in vivo in tissues under physiologically pertinent conditions and concentrations. Adv Exp Med Biol 317:221–228
Ilangovan G, Zweier JL, Kuppusamy P (2004) Mechanism of oxygen-induced EPR line broadening in lithium phthalocyanine microcrystals. J Magn Reson 170(1):42–48
Hirata H, He G, Deng Y et al (2008) A loop resonator for slice-selective in vivo EPR imaging in rats. J Magn Reson. 190(1):124–134
He G, Evalappan SP, Hirata H et al (2002) Mapping of the B1 field distribution of a surface coil resonator using EPR imaging. Magn Reson Med 48(6):1057–1062
Kuppusamy P, Li H, Ilangovan G et al (2002) Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels. Cancer Res 62(1):307–312
Chen YR, Deterding LJ, Tomer KB et al (2000) Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical. Biochemistry 39(15):4415–4422
Gong X, Xie T, Yu L et al (2003) The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli. J Biol Chem 278(28):25731–25737
Teng RJ, Ye YZ, Parks DA et al (2002) Urate produced during hypoxia protects heart proteins from peroxynitrite-mediated protein nitration. Free Radic Biol Med 33(9):1243–1249
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
He, G. (2010). Electron Paramagnetic Resonance Oximetry and Redoximetry. In: Armstrong, D. (eds) Advanced Protocols in Oxidative Stress II. Methods in Molecular Biology, vol 594. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-411-1_6
Download citation
DOI: https://doi.org/10.1007/978-1-60761-411-1_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-410-4
Online ISBN: 978-1-60761-411-1
eBook Packages: Springer Protocols