Piperidine nitroxides have considerable clinical potential, both as antioxidant therapeutic compounds and contrast agents in magnetic resonance imaging. However, their development has thus far been limited by their rapid bioreduction in vivo. Recently, it was reported that polynitroxyl albumin (PNA) can reverse the bioreduction of the reduced 4‐hydroxy‐2,2,6,6‐tetramethylpiperidine‐N‐oxyl (Tempol) in the rat heart, enabling the performance of high resolution EPR imaging for prolonged time (Kuppusamy et al., Biochemistry 35, 7051–7057 (1996)). In this report, the efficacy of PNA in maintaining Tempol concentrations in vivo in mice was demonstrated, using L‐band (1.25 GHz) EPR spectroscopy and imaging. The EPR signal of intravenous Tempol had a half‐life of 1.0 ± 0.2 min and became undetectable within 6 min. Subcutaneous Tempol, however, decayed at a slower rate (half‐life, 5.0 ± 0.5 min) suggesting that Tempol had been bioreduced to the corresponding hydroxylamine form, Tempol‐H. Subcutane‐ously injected PNA restored 20% of the Tempol signal in the vicinity of the PNA deposit. In vivo topical EPR imaging demonstrated that the Tempol signal was restored at the site of PNA injection, but not at locations remote from the PNA injection site. The ability of PNA to maintain Tempol in its paramagnetic state in vivo should enable a wide range of therapeutic and diagnostic applications of piperidinyl nitroxides.

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper, zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.

The application of electron paramagnetic resonance imaging (EPRI) to obtain information from biological samples has been limited by the lack of ideal single line radical labels, The commonly used nitroxides exhibit multiple lines causing either hyperfine-based limitations in the maximum obtainable image resolution or hyperfine-based artifacts in the reconstructed image. The use of a novel single-line triarylmethyl paramagnetic label that enables marked enhancement in image quality and resolution is reported. This label exhibits a single line EPR spectrum that is sharp (linewidth similar to 60 mG in the absence of oxygen) and relatively stable in tissues. The potential of this label in enabling high resolution EPR imaging of biological samples was demonstrated in a series of phantoms and isolated biological organs such as the rat kidney, The images demonstrate that resolutions better than 100 mu m could be obtained at L-band on samples of up to 20 mm in size.

In vivo or ex vivo EPR imaging, EPRI, has been established as a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues, While instrumentation capable of performing EPR imaging of free radicals in whole tissues and isolated organs has been previously reported, it was not possible to image rapidly moving organs such as the beating heart, Therefore instrumentation was developed to enable the performance of gated-spectroscopy and imaging on isolated beating rat hearts at L-band. A synchronized pulsing and timing system capable of gated acquisitions of up to 256 images per cycle, with rates of up to 16 Hz was developed, The temporal and spatial accuracy of this instrumentation was verified using a specially designed beating heart-shaped isovolumic phantom with electromechanically driven sinusoidal motion at a cycle rate of 5 Hz. Gated EPR imaging was performed on a series of isolated rat hearts perfused with nitroxide spin labels. These hearts were paced at a rate of 6 Hz with either 16 or 32 gated images acquired per cardiac contractile cycle. The images enabled visualization of the time-dependent alterations in the free radical distribution and anatomical structure of the heart that occur during the cardiac cycle.

Electron paramagnetic resonance (EPR) imaging utilizing stable nitroxyl radicals is a promising technique for measuring free radical distribution, metabolism, and tissue oxygenation in organs and tissues [Kuppusamy, P., Chzhan, M., Vij, K., Shteynbuk, M., Lefer, D. J., Giannella, E., & Zweier, J. L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3388-3392]. However, the technique has been limited by the rapid reduction of nitroxide in vivo to its hydroxylamine derivative, a diamagnetic, EPR-inactive species. In this report a novel, polynitroxylated derivative of human serum albumin is shown to be capable of reoxidizing the hydroxylamine back to nitroxide in vivo. Polynitroxyl-albumin (PNA) is shown to be effective in maintaining the signal intensity of the nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL or TPL) in the ischemic isolated rat heart, allowing the acquisition of high-resolution three-dimensional (3D) EPR images of the heart throughout a prolonged 2.5 h period of global cardiac ischemia. In serial transverse sections of the 3D image, TPL intensity maps of the heart showed cardiac structure with submillimeter resolution. TPL intensities in coronary arteries and myocardium showed that nitroxide concentration decreases with increasing distance from large blood vessels. These results demonstrate that EPR imaging in vivo is possible using nitroxides in conjunction with PNA. In addition to its utility in the emerging technology of EPR imaging, the greatly prolonged half-life of TPL observed in the presence of PNA may facilitate the therapeutic application of nitroxides in a variety of disease processes.

A burst of endothelial derived oxidants including hydrogen peroxide (H2O2) and superoxide (. O-2(-)) occurs on reperfusion of ischemic tissues that directly causes injury; however, it is not known if this also triggers further injury due to subsequent leukocyte adhesion and adhesion molecule expression. Therefore, studies were performed in an isolated heart model developed to enable study of the role of isolated cellular and humoral factors in the mechanism of postischemic injury. Isolated rat hearts were, subjected to 20 min of 37 degrees C-global ischemia followed by reperfusion with polymorphonuclear leukocytes (PMNs) and plasma in the presence or absence of superoxide dismutase (SOD), 200 U/ml, or catalase, 500 U/ml. Measurements of contractile function, coronary flow, high-energy phosphates, free radical generation, and PMN accumulation were performed. Adhesion molecule expression was measured on the surface of effluent PMNs by fluorescence flow cytometry and within the tissue using immunohistochemistry. SOD or catalase treatment resulted in 2- to 3-fold higher recoveries of contractile function, coronary flow, and high energy phosphates. EPR spin trapping measurements demonstrated that SOD totally quenched the free radical generation observed upon reperfusion while catalase prevented the formation of hydroxyl and alkyl radicals derived from superoxide. SOD or catalase treatment decreased PMN accumulation in the reperfused heart and prevented the marked upregulation of CD18 expression seen after reperfusion. These experiments demonstrate that in addition to their direct antioxidative actions, SOD and catalase each decrease PMN adhesion and CD18 expression resulting in marked suppression of PMN-mediated injury in the postischemic heart. Thus, endothelial derived H2O2 and . O-2(-) further amplify postischemic injury by triggering CD18 expression on the surface of PMNs leading to increased PMN adhesion within the heart.

Altered nitric oxide (NO.) production is a critical factor in tissue reperfusion injury; however, controversy remains regarding these alterations and how they cause injury. Since superoxide (O-2(.-)) generation is triggered during the early period of reperfusion the cytotoxic oxidant peroxynitrite (ONOO-) could be formed, but it is not known if this occurs. Therefore electron paramagnetic resonance and chemiluminescence studies were performed of the magnitude and time course of NO., O-2(.-), and ONOO- formation in the postischemic heart. Isolated rat hearts were subjected either to normal perfusion or to reperfusion after 30 min of ischemia in the presence of the NO. trap Fe2+-N-methyl-D-glucamine dithiocarbamate with electron paramagnetic resonance measurements performed on the effluent. Although only trace signals were present prior to ischemia, prominent NO. adduct signals were seen during the first 2 min of reflow which were abolished by nitric oxide synthase (NOS) inhibition. Similar studies with the O-2(.-) trap 5,5 -dimethyl-1-pyrroline N-oxide demonstrated a burst of O-2(.-) generation over the first 2 min of reflow. Chemiluminescence measurements using 5-amino-2,2-dihydro-1,4-phthalazinedione (luminol) demonstrated a similar marked increase in ONOO- which was blocked by NOS inhibitors or superoxide dismutase. NOS inhibition or superoxide dismutase greatly enhanced the recovery of contractile function in postischemic hearts. Immunohistology demonstrated that the ONOO--mediated nitration product nitrotyrosine was formed in postischemic hearts but not in normally perfused controls. Thus, NO. formation is increased during the early period of reflow and reacts with O-2(.-) to form ONOO-, which results in amino acid nitration and cellular injury.

The development and application of four-dimensional spectral-spatial electron paramagnetic resonance imaging (EPRI) techniques for unambiguous determination of spectral shape and spin distribution of paramagnetic samples are described. Strategies for optimizing acquisition and computation times, image resolution, and data presentation are described. The feasibility of studying small linewidth differences of approximately 0.1 G in samples of up to 25 mm in size was tested by computer simulations and by measurements on phantoms containing 0.5 mM nitroxide spin label. One thousand projections were used to reconstruct 32 x 32 x 32 x 32 pixel images. Similar 4D imaging experiments were performed on an isolated rat heart infused with a suspension of glucose char. The 4D image of the rat heart clearly showed the three-dimensional spatial structure of the heart and the spectral shape at each spatial point. Thus, we have demonstrated, for the first time, that 4D spectral-spatial EPRI could be performed on lossy biological samples or tissues at L-band frequencies, enabling the mapping of spectral information over the entire three-dimensional spatial structure of the object.

While instrumentation capable of performing three-dimensional EPR imaging of free radicals in whole tissues and isolated organs has been developed at L-band, important questions remain regarding the resolution and image quality that can be obtained in practice using the presently available free radical labels. Therefore, studies were performed applying three-dimensional spatial EPR imaging at L-band to image the distribution of free radical labels in the isolated heart and in phantoms of similar size. With nitroxide labels the obtainable resolution is limited by the presence of hyperfine structure in the EPR absorption function that in turn limits the maximum applicable gradient. The authors observed that with the nitroxide labels, resolutions in the range of 1-2 mm are possible, while with a single line glucose char label, resolutions of 0.2 mm are obtained. With the nitroxides, images were of sufficient resolution to resolve the overall global shape of the heart and the location of the left and right ventricular cavities; however, finer structures could not be resolved. With the glucose char much finer resolution could be obtained enabling visualization of the ventricles, aortic root, and proximal coronary arteries.

Electron paramagnetic resonance spectroscopy at the L-band (1.2 GHz) was used to investigate the redox metabolism and oxygenation within ischemic rat hearts loaded with a variety of six- and five-membered carbocyclic nitroxide spin labels. Both cardiopleged and non-cardiopleged hearts were studied at room temperature and at physiological temperatures. The results indicate that nitroxides that have greater intracellular partitioning are reduced faster than those that are extracellular. In addition, the six-membered rings are metabolized much faster than the five-membered rings. Increased nitroxide radical metabolism was observed in non-cardiopleged compared with cardiopleged hearts. The oxygen utilization in the ischemic hearts was studied by using the oxygen-dependent broadening of the nitroxide signals that occurs due to Heisenberg spin exchange, These structurally different nitroxides were evaluated for their sensitivity and suitability as probes of myocardial redox metabolism and oxygenation in the heart.