Enzyme-independent formation of nitric oxide in biological tissues.

The gaseous free radical nitric oxide (NO.) is an important regulator of a variety of biological functions and also has a role in the pathogenesis of cellular injury. It has been generally accepted that NO. is solely generated in biological tissues by specific nitric oxide synthases, NOSs, which metabolize arginine to citrulline with the formation of NO.. We report that NO. can also be generated in the ischaemic heart by direct reduction of nitrite to NO. under the acidotic and highly reduced conditions that occur. This NO. formation is not blocked by NOS inhibitors, and with long periods of ischaemia progressing to necrosis, this mechanism of NO. formation predominates. We observe that enzyme-independent NO. generation results in myocardial injury with a loss of contractile function. The existence of this enzyme-independent mechanism of NO. formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.

A nonpeptidyl mimic of superoxide dismutase with therapeutic activity in rats.

Many human diseases are associated with the overproduction of oxygen free radicals that inflict cell damage. A manganese(II) complex with a bis(cyclohexylpyridine)-substituted macrocyclic ligand (M40403) was designed to be a functional mimic of the superoxide dismutase (SOD) enzymes that normally remove these radicals. M40403 had high catalytic SOD activity and was chemically and biologically stable in vivo. Injection of M40403 into rat models of inflammation and ischemia-reperfusion injury protected the animals against tissue damage. Such mimics may result in better clinical therapies for diseases mediated by superoxide radicals.

Phenolic Michael reaction acceptors: combined direct and indirect antioxidant defenses against electrophiles and oxidants.

The implications of oxidative stress in the pathogenesis of many chronic human diseases has led to the widely accepted view that low molecular weight antioxidants could be beneficial and postpone or even prevent these diseases. Small molecules of either plant or synthetic origins, which contain Michael acceptor functionalities (olefins or acetylenes conjugated to electron-withdrawing groups) protect against the toxicity of oxidants and electrophiles indirectly, i.e., by inducing phase 2 cytoprotective enzymes. Some of these molecules, e.g., flavonoid and curcuminoid analogues that have phenolic hydroxyl groups in addition to Michael acceptor centers, are also potent direct antioxidants, and may therefore be appropriately designated: bifunctional antioxidants. By use of spectroscopic methods we identified phenolic chalcone and bis(benzylidene)acetone analogues containing one or two Michael acceptor groups, respectively, as very efficient scavengers of two different types of radicals: (a) the nitrogen-centered 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS.+) radical cation, and (b) the oxygen-centered galvinoxyl (phenoxyl) radical. The most potent scavengers are those also bearing hydroxyl substituents on the aromatic ring(s) at the ortho-position(s). The initial reaction velocities are very rapid and concentration-dependent. In the human keratinocyte cell line HaCaT, the same compounds coordinately increase the intracellular levels of glutathione, glutathione reductase, and thioredoxin reductase. Thus, such bifunctional antioxidants could exert synergistic protective effects against oxidants and electrophiles which represent the principal biological hazards by: (i) scavenging hazardous oxidants directly and immediately; and (ii) inducing the phase 2 response to prevent and resolve the consequences of hazardous processes that are already in progress, i.e., acting indirectly, but with much more diverse and long-lasting effects.

Non-enzymatic nitric oxide synthesis in biological systems.

Nitric oxide (NO) is an important regulator of a variety of biological functions, and also has a role in the pathogenesis of cellular injury. It had been generally accepted that NO is solely generated in biological tissues by specific nitric oxide synthases (NOS) which metabolize arginine to citrulline with the formation of NO. However, NO can also be generated in tissues by either direct disproportionation or reduction of nitrite to NO under the acidic and highly reduced conditions which occur in disease states, such as ischemia. This NO formation is not blocked by NOS inhibitors and with long periods of ischemia progressing to necrosis, this mechanism of NO formation predominates. In postischemic tissues, NOS-independent NO generation has been observed to result in cellular injury with a loss of organ function. The kinetics and magnitude of nitrite disproportionation have been recently characterized and the corresponding rate law of NO formation derived. It was observed that the generation and accumulation of NO from typical nitrite concentrations found in biological tissues increases 100-fold when the pH falls from 7.4 to 5.5. It was also observed that ischemic cardiac tissue contains reducing equivalents which reduce nitrite to NO, further increasing the rate of NO formation more than 40-fold. Under these conditions, the magnitude of enzyme-independent NO generation exceeds that which can be generated by tissue concentrations of NOS. The existence of this enzyme-independent mechanism of NO formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.

Redox properties of iron-dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems.

While the Fe(2+)-dithiocarbamate complexes have been commonly used as NO traps to estimate NO production in biological systems, these complexes can undergo complex redox chemistry. Characterization of this redox chemistry is of critical importance for the use of this method as a quantitative assay of NO generation. We observe that the commonly used Fe(2+) complexes of N-methyl-D-glucamine dithiocarbamate (MGD) or diethyldithiocarbamate (DETC) are rapidly oxidized under aerobic conditions to form Fe(3+) complexes. Following exposure to NO, diamagnetic NO-Fe(3+) complexes are formed as demonstrated by the optical, electron paramagnetic resonance and gamma-resonance spectroscopy, chemiluminescence and electrochemical methods. Under anaerobic conditions the aqueous NO-Fe(3+)-MGD and lipid soluble NO-Fe(2+)-DETC complexes gradually self transform by reductive nitrosylation into paramagnetic NO-Fe(2+)-MGD complexes with yield of up to 50% and the balance is converted to Fe(3+)-MGD and nitrite. In dimethylsulfoxide this process is greatly accelerated. More efficient transformation of NO-Fe(3+)-MGD into NO-Fe(2+)-MGD (60-90% levels) was observed after addition of reducing equivalents such as ascorbate, hydroquinone or cysteine or with addition of excess Fe(2+)-MGD. With isotope labeling of the NO-Fe(3+)-MGD with (57)Fe, it was shown that these complexes donate NO to Fe(2+)-MGD. NO-Fe(3+)-MGD complexes were also formed by reversible oxidation of NO-Fe(2+)-MGD in air. The stability of NO-Fe(3+)-MGD and NO-Fe(2+)-MGD complexes increased with increasing the ratio of MGD to Fe. Thus, the iron-dithiocarbamate complexes and their NO derivatives exhibit complex redox chemistry that should be considered in their application for detection of NO in biological systems.

In vivo EPR imaging of the distribution and metabolism of nitroxide radicals in human skin.

While altered cellular free radical and redox metabolism are critical factors in many human diseases, it has not been previously possible to both measure and image these processes in humans. The development and application of electron paramagnetic resonance instrumentation capable of in vivo spectroscopy and imaging of free radicals in human skin are reported. The instrumentation uses a specially designed topical resonator and a 2.2-GHz microwave bridge. Noninvasive measurements of the distribution and metabolism of the topically applied nitroxide, (15)N-perdeuterated tempone (100 mM), in forearm skin were performed. A single broad peak due to the concentrated label at the skin surface was initially observed, followed by a sharp doublet from the diluted label that permeated the skin. The penetration of the label into the skin and its metabolic clearance were modeled using kinetic equations. It was observed that the penetration process from the skin surface into the dermis and subcutaneous regions, as well as its clearance from these regions, could be described by single exponential functions. Phantom imaging experiments using the nitroxide showed that a spatial resolution of up to 50 microm could be achieved. The skin imaging measurements showed two bands in the distribution of the label along the skin depth. The first band appeared in the outer 400 microm of the skin, the epidermis region, whereas the second band was centered at a depth of 1000 microm in the subcutaneous region with a thickness about 400 microm. These two bands decayed and merged into a single band with time. The results are important in the understanding of the permeability and metabolism of free radicals in human skin.

A tunable reentrant resonator with transverse orientation of electric field for in vivo EPR spectroscopy.

There has been a need for development of microwave resonator designs optimized to provide high sensitivity and high stability for EPR spectroscopy and imaging measurements of in vivo systems. The design and construction of a novel reentrant resonator with transversely oriented electric field (TERR) and rectangular sample opening cross section for EPR spectroscopy and imaging of in vivo biological samples, such as the whole body of mice and rats, is described. This design with its transversely oriented capacitive element enables wide and simple setting of the center frequency by trimming the dimensions of the capacitive plate over the range 100-900 MHz with unloaded Q values of approximately 1100 at 750 MHz, while the mechanical adjustment mechanism allows smooth continuous frequency tuning in the range +/-50 MHz. This orientation of the capacitive element limits the electric field based loss of resonator Q observed with large lossy samples, and it facilitates the use of capacitive coupling. Both microwave performance data and EPR measurements of aqueous samples demonstrate high sensitivity and stability of the design, which make it well suited for in vivo applications.

Development of a resonator with automatic tuning and coupling capability to minimize sample motion noise for in vivo EPR spectroscopy.

EPR spectroscopy has been applied to measure free radicals in vivo; however, respiratory, cardiac, and other movements of living animals are a major source of noise and spectral distortion. Sample motions result in changes in resonator frequency, Q, and coupling. These instabilities limit the applications that can be performed and the quality of data that can be obtained. Therefore, it is of great importance to develop resonators with automatic tuning and automatic coupling capability. We report the development of automatic tuning and automatic coupling provisions for a 750-MHz transversely oriented electric field reentrant resonator using two electronically tunable high Q hyperabrupt varactor diodes and feedback loops. In both moving phantoms and living mice, these automatic coupling control and automatic tuning control provisions resulted in an 8- to 10-fold increase in signal-to-noise ratio.

A bridged loop-gap S-band surface resonator for topical EPR spectroscopy.

The design and structure of a bridged loop-gap surface resonator developed for topical EPR spectroscopy and imaging of the distribution and metabolism of spin labels in in vivo skin is reported. The resonator is a one-loop, one-gap bridged structure. A pivoting single loop-coupling coil was used to couple the microwave power to the loop-gap resonant structure. A symmetric coupling circuit was used to achieve better shielding and minimize radiation. The frequency of the resonator can be easily adjusted by trimming the area of the capacitive foil bridge, which overlaps the gap in the cylindrical loop. The working frequency set was 2.2 GHz and the unloaded Q was 720. The B1 field of this resonator was measured and spatially mapped by three-dimensional EPR imaging. The resonator is well suited to topical measurements of large biological subjects and is readily applicable for in vivo measurements of free radicals in human skin.

Electron paramagnetic resonance imaging of the rat heart.

It has been hypothesized that free radical metabolism, oxygenation and nitric oxide generation in biological organs such as the heart may vary over the spatially defined tissue structure. To address fundamental questions regarding the role of spatially localized alterations in radical metabolism, oxygenation and nitric oxide in the pathophysiology of cellular injury during ischaemia, we have developed instrumentation optimized for 3D spatial and 3D or 4D spectral-spatial imaging of free radicals in the isolated perfused rat heart at 1.2 GHz. Using this instrumentation, high-quality 3D spectral-spatial imaging of nitroxide metabolism was performed as well as spatially localized measurements of oxygen concentrations, based on the oxygen-dependent linewidth broadening observed. In these spectral-spatial images, submillimetre resolution was observed enabling visualization of the left ventricular and right ventricular myocardium. With 3D spatial imaging using single-line labels, resolutions down to 100 to 200 microm were obtained enabling visualization of the ventricles, aortic root and proximal coronary arteries. Using metal complexes which trap nitric oxide, measurement and imaging of nitric oxide generation during ischaemia was performed. With the use of 15N isotope labelling it was possible to map the metabolic pathway of this nitric oxide generation. Thus, EPR imaging is a powerful tool which can provide unique information regarding the spatial localization of free radicals, oxygen and nitric oxide in biological organs and tissues.

Magnetic resonance study of the transmembrane nitrite diffusion.

Nitrite (NO(2)-), being a product of metabolism of both nitric oxide (NO(*)) and nitrate (NO(3)-), can accumulate in tissues and regenerate NO() by several mechanisms. The effect of NO(2)- on ischemia/reperfusion injury was also reported. Nevertheless, the mechanisms of intracellular NO(2)- accumulation are poorly understood. We suggested significant role of nitrite penetration through biological membranes in the form of undissociated nitrous acid (HNO(2)). This hypothesis has been tested using large unilamellar phosphatidylcholine liposomes and several spectroscopic techniques. HNO(2) transport across the phospholipid bilayer of liposomes facilitates proton transfer resulting in intraliposomal acidification, which was measured using pH-sensitive probes. NO(2)(-)-mediated intraliposomal acidification was confirmed by EPR spectroscopy using membrane-impermeable pH-sensitive nitroxide, AMC (2,2,5,5-tetramethyl-1-yloxy-2,5-dihydro-1H-imidazol-3-ium-4-yl)-aminomethanesulfonic acid (pK 5.25), and by (31)P NMR spectroscopy using inorganic phosphate (pK 6.9). Nitrite accumulates inside liposomes in concentration exceeding its concentration in the bulk solution, when initial transmembrane pH gradient (alkaline inside) is applied. Intraliposomal accumulation of NO(2)- was observed by direct measurement using chemiluminescence technique. Perfusion of isolated rat hearts with buffer containing 4 microM NO(2)- was performed. The nitrite concentrations in the effluent and in the tissue, measured after 1 min perfusion, were close, supporting fast penetration of the nitrite through the tissue. Measurements of the nitrite/nitrate showed that total concentration of NO(x) in myocardium increased from initial 7.8 to 24.7 microM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO(2)- and its coupling with intraliposomal acidification are discussed.

Development of chemiluminescence-based methods for specific quantitation of nitrosylated thiols.

While nitrosothiol compounds have been hypothesized to be important in the transport and function of nitric oxide (NO) in biological systems many important questions regarding their mechanism of formation and functional importance remain. In view of these fundamental questions there has been a great need for simple, sensitive, and specific methods for quantitation of nitrosothiols in biological samples. We report the development of two methods, for the measurement of nitrosothiol compounds using a chemiluminescence nitric oxide analyzer with a standard purging vessel. The first method is based on treatment with acidified solutions of potassium iodide in the presence or absence of dissolved free iodine. Quantitative release of NO occurs either from both nitrite and nitrosothiols or from nitrite alone, respectively. Subtraction of the amount of NO released without iodine from NO released in the presence of iodine allows estimation of the nitrosothiol concentration. To selectively measure nitrosothiols, we developed a redox quinone-hydroquinone alkaline reactant that selectively releases NO from nitrosothiols. This reactant quantitatively converts nitrosothiols to NO at elevated temperature, > 60 degrees C. Both methods were shown to detect nitrosothiols in biological buffers or blood plasma down to 10 nM concentration with high accuracy and reproducibility, variability less than 5%. These assays should be a useful addition to techniques used to characterize the biochemistry of NO.

Evaluation of the magnitude and rate of nitric oxide production from nitrite in biological systems.

Recently it was shown that the formation of nitric oxide (NO) is increased in biological tissues during ischemia, and that there is an enzyme-independent pathway of NO generation due to reduction of tissue nitrite under the acidic conditions which occur. To investigate the quantitative importance of this mechanism of NO generation in biological systems where pH and nitrite concentrations vary, electron paramagnetic resonance and chemiluminescence studies were performed to characterize the kinetics and magnitude of the nitrite disproportionation process. The reaction process and the corresponding rate law of NO formation from nitrite were derived. The generation and accumulation of NO from typical nitrite concentrations found in biological tissues increased 100-fold when pH decreased from normal values of 7.4 to the acidic values found in ischemic tissues, such as the heart, where pH falls to 5.5. It was also observed that ischemic heart tissue contains reducing equivalents which reduce nitrite to NO, further increasing the rate of NO formation more than 40-fold. Under these conditions the magnitude of this enzyme-independent NO generation may exceed that which can be generated by tissue concentrations of nitric oxide synthase. Thus, in ischemic tissues nitrite can be primarily a source rather than a product of NO.

Spatial mapping of nitric oxide generation in the ischemic heart using electron paramagnetic resonance imaging.

Recently, it has been shown that rat hearts subjected to global ischemia generate nitric oxide (NO) and that a significant portion of it is generated by the reduction of nitrite under the acidic and reducing conditions that occur during myocardial ischemia [Zweier, Wang, Samouilov, Kuppusamy, Nature Med. 1, 804-809 (1995)]. In the present study it is further attempted to map the spatial distributions of the NO generation in the ischemic myocardium using L-band electron paramagnetic resonance imaging. Rat hearts were loaded with 10 mM nitrite and subjected to global no-flow ischemia, during which time a series of three-dimensional spatial electron paramagnetic resonance (EPR) images of the distribution of NO were obtained using the NO trap bis(N-methyl-D-glucamine dithiocarbamate)iron(II). The images clearly showed that NO is formed throughout the myocardium. Kinetic experiments showed that maximum NO generation and trapping occur at the midmyocardium and spread out to endocardium and epicardium of the left ventricle. The magnitude of generation in the RV myocardium is four- to fivefold lower than in the LV.

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction. Evaluation of its role in nitric oxide generation in anoxic tissues.

Xanthine oxidase (XO)-catalyzed nitrite reduction with nitric oxide (NO) production has been reported to occur under anaerobic conditions, but questions remain regarding the magnitude, kinetics, and biological importance of this process. To characterize this mechanism and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism. Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. The substrate dependence of anaerobic XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. It was determined that under conditions occurring during no-flow ischemia, myocardial XO and nitrite levels are sufficient to generate NO levels comparable to those produced from nitric oxide synthase. Thus, XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions.

Measurement of nitric oxide with a solid-state-char EPR probe.

Recently, highly oxygen-sensitive solid-state paramagnetic probes have been developed from glucose chars which are capable of providing measurements of very low, millitorr, oxygen concentrations in biological cells and tissues. With the increased interest and recognition of the presence and importance of the free-radical gas nitric oxide, NO., in biology, studies were performed aimed at characterizing the effect of NO. on this probe. It was observed that NO. exerts a similar concentration-dependent broadening as does molecular oxygen, O2. In anaerobic solutions, concentrations of NO. could be detected down to 10-100 nM levels. Measurements of NO. formation in aqueous solution were performed from a pharmacological NO. donor, which produced micromolar concentrations of NO.. This technique, however, was not sufficiently sensitive to measure physiological 1-10 nM concentrations. Thus, the glucose-char EPR probe could be used to detect pharmacological levels of NO. but was not significantly affected by the low physiological levels which occur in normal biological tissues. These results indicate that, under normal physiological conditions, the linewidth alterations measured using the glucose-char EPR probe are solely caused by oxygen.

Mapping the spin-density and lineshape distribution of free radicals using 4D spectral-spatial EPR imaging.

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.

Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging.

EPR imaging has emerged as an important tool for noninvasive three-dimensional (3D) spatial mapping of free radicals in biological tissues. Spectral-spatial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be mapped. We report the development of EPR imaging instrumentation enabling 3D spatial and spectral-spatial EPR imaging of small animals. This instrumentation, along with the use of a biocompatible charcoal oximetry-probe suspension, enabled 3D spatial imaging of the gastrointestinal (GI) tract, along with mapping of oxygenation in living mice. By using these techniques, the oxygen tension was mapped at different levels of the GI tract from the stomach to the rectum. The results clearly show the presence of a marked oxygen gradient from the proximal to the distal GI tract, which decreases after respiratory arrest. This technique for in vivo mapping of oxygenation is a promising method, enabling the noninvasive imaging of oxygen within the normal GI tract. This method should be useful in determining the alterations in oxygenation associated with disease.