Kazuhiro Ichikawa

Direct Evidence for Increased Hydroxyl Radicals Originating From Superoxide in the Failing Myocardium

Experimental and clinical studies have suggested an increased production of reactive oxygen species (ROS) in the failing myocardium. The present study aimed to obtain direct evidence for increased ROS and to determine the contribution of superoxide anion (*O(2)(-)), H(2)O(2), and hydroxy radical (*OH) in failing myocardial tissue. Heart failure was produced in adult mongrel dogs by rapid ventricular pacing at 240 bpm for 4 weeks. To assess the production of ROS directly, freeze-clamped myocardial tissue homogenates were reacted with the nitroxide radical, 4-hydroxy-2,2,6, 6,-tetramethyl-piperidine-N-oxyl, and its spin signals were detected by electron spin resonance spectroscopy. The rate of electron spin resonance signal decay, proportional to *OH level, was significantly increased in heart failure, which was inhibited by the addition of dimethylthiourea (*OH scavenger) into the reaction mixture. Increased *OH in the failing heart was abolished to the same extent in the presence of desferrioxamine (iron chelator), catalase (H(2)O(2) scavenger), and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; LaMotte) (*O(2)(-) scavenger), indicating that *OH originated from H(2)O(2) and *O(2)(-). Further, *O(2)(-) produced in normal myocardium in the presence of antimycin A (mitochondrial complex III inhibitor) could reproduce the increase of H(2)O(2) and *OH seen in the failing tissue. There was a significant positive relation between myocardial ROS level and left ventricular contractile dysfunction. In conclusion, in the failing myocardium, *OH was produced as a reactive product of *O(2)(-) and H(2)O(2), which might play an important role in left ventricular failure.

Enhanced Generation of Reactive Oxygen Species in the Limb Skeletal Muscles From a Murine Infarct Model of Heart Failure

Background—The generation of reactive oxygen species (ROS) is enhanced in the failing myocardium. We hypothesized that ROS were also increased in the limb skeletal muscles in heart failure. Methods and Results—Myocardial infarction (MI) was created in mice by ligating the left coronary artery. After 4 weeks, the left ventricle was dilated and contractility was diminished by echocardiography. Left ventricular end-diastolic pressure was elevated after MI in association with an increase in lung weight/body weight and the presence of pleural effusion. The generation of ROS in the limb muscles, including the soleus and gastrocnemius muscles, which were excised after MI, was measured by electron spin resonance spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (hydroxy-TEMPO). Overall, generation was increased, but it was attenuated in the presence of dimethylthiourea or 4,5-dihydroxy-1,2-benzenedisulfonic disodium salt in the reaction mixture, indicating increased generation of hydroxyl radicals originating from superoxide anion. Thiobarbituric acid-reactive substance formation was also increased in muscles after MI. Mitochondrial complex I and III activities were both decreased after MI, which may have caused the functional uncoupling of the respiratory chain and ROS production. Antioxidant enzyme activities, including superoxide dismutase, catalase, and glutathione peroxidase, were comparable between groups. Conclusions—Skeletal muscle in post-MI heart failure expressed an increased amount of ROS in association with ROS-mediated lipid peroxidation. This supports the hypothesis that oxidative stress may cause (at least in part) skeletal muscle dysfunction in heart failure.

Simultaneous molecular imaging of redox reactions monitored by Overhauser-enhanced MRI with 14N- and 15N-labeled nitroxyl radicals

MRI has provided significant clinical utility in the diagnosis of diseases and will become a powerful tool to assess phenotypic changes in genetically engineered animals. Overhauser enhanced MRI (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity by means of the Overhauser effect. Several studies have demonstrated noninvasive assessment of reactive oxygen species generation in small animals by using low frequency electron spin resonance (ESR) spectroscopy/imaging and nitroxyl radicals. In vivo ESR signal intensities of nitroxyl radicals decrease with time after injection; and the decreases are enhanced by reactive oxygen species, generated in oxidative disease models in a site-specific manner. In this study, we show images of nitroxyl radicals with different isotopes by changing the external magnetic field for ESR irradiation between 14N and 15N nuclei in field-cycled OMRI. OMRI simultaneously obtained dual images of two individual chemical processes. Oxidation and reduction were monitored in a rate-dependent manner at nanometer scale by labeling membrane-permeable and -impermeable nitroxyl radicals with 14N and 15N nuclei. Phantom objects containing ascorbic acid-encapsulated liposomes with membrane-permeable radicals but not membrane-impermeable ones show a time-dependent decrease of the OMRI image intensity. The pharmacokinetics in mice was assessed with OMRI after radical administration. This OMRI technique with dual probes should offer significant applicability to nanometer scale molecular imaging and simultaneous assessment of independent processes in gene-modified animals. Thus, it may become a powerful tool to clarify mechanisms of disease and to monitor pharmaceutical therapy.

A platform for designing hyperpolarized magnetic resonance chemical probes

Hyperpolarization is a highly promising technique for improving the sensitivity of magnetic resonance chemical probes. Here we report [15N, D9]trimethylphenylammonium as a platform for designing a variety of hyperpolarized magnetic resonance chemical probes. The platform structure shows a remarkably long 15N spin–lattice relaxation value (816 s, 14.1 T) for retaining its hyperpolarized spin state. The extended lifetime enables the detection of the hyperpolarized 15N signal of the platform for several tens of minutes and thus overcomes the intrinsic short analysis time of hyperpolarized probes. Versatility of the platform is demonstrated by applying it to three types of hyperpolarized chemical probes: one each for sensing calcium ions, reactive oxygen species (hydrogen peroxide) and enzyme activity (carboxyl esterase). All of the designed probes achieve high sensitivity with rapid reactions and chemical shift changes, which are sufficient to allow sensitive and real-time monitoring of target molecules by 15N magnetic resonance.

In vivo ESR measurements of free radical reactions in living mice

In vivo ESR measurements were carried out to estimate free radical reactions in living mice using nitroxyl radicals as probes. The ESR signal of nitroxyl radical which was intravenously or intramuscularly injected to living female ddY mice decreased gradually by reducing to the corresponding hydroxylamine. The reduction rate was enhanced by oxidative stress, and pre-treatment of antioxidants suppressed the enhancement of signal decay. Oral administration of carbon tetrachloride enhanced signal decay in upper abdomen but not in thorax. These results indicated that free radicals, which can reduce nitroxyl radical, were produced in the upper abdomen by oral administration of carbon tetrachloride.

Nitroxide conjugate of a thermally responsive elastin‐like polypeptide for noninvasive thermometry

Hyperthermia, as an adjuvant with radiation and chemotherapy, has shown promise in the treatment of cancer. The relevant biological effects of a hyperthermia treatment are both time and temperature-dependent, creating a need for accurate thermometry. We present a novel noninvasive thermometry modality that combines a temperature responsive biopolymer, the elastin-like polypeptide (ELP), and nitroxide to produce an ELP-nitroxide conjugate. When examined with electron paramagnetic resonance (EPR) spectroscopy, the ELP-nitroxide conjugate has temperature-dependent spectral line widths whose predictive accuracy is approximately 0.3 degrees C (80 microM). We believe that the temperature-dependent changes observed in the EPR spectrum are due to the combined effect of temperature, viscosity and effective radius on the rotational correlation time of the ELP-nitroxide conjugate.

Advantageous application of a surface coil to EPR irradiation in overhauser‐enhanced MRI

The present study describes the advantageous application of a surface coil to electron paramagnetic resonance (EPR) irradiation in Overhauser‐enhanced MRI (OMRI). OMRI is a double‐resonance method for imaging free radicals based on the Overhauser effect. Proton NMR images are recorded without and with EPR irradiation of the free radical resonance, which results in a difference proton image that shows signal enhancement in spatial regions that contain the free radical. To obtain good signal enhancement in OMRI, very high RF power and a long EPR irradiation time are required. To improve sensitivity and shorten the image acquisition time, especially for localized (and topical) applications, we developed and tested a surface‐coil‐type EPR irradiation coil. Theoretical calculations and experimental data showed that EPR irradiation through the surface coil could ameliorate the localized Overhauser enhancement, which was related to the ratio of B1 surface coil/B1 volume coil in the region of interest (ROI), as expected. The increased sensitivity could also be converted into a shortened EPR irradiation time, resulting in fast data acquisition. For biomedical applications, the use of a surface coil (as opposed to a conventional volume coil) could decrease the total RF power deposition in the sample required to obtain the same Overhauser enhancement in the ROI. Magn Reson Med 57:806–811, 2007.

Whole-body kinetic image of a redox probe in mice using Overhauser-enhanced MRI.

Overhauser-enhanced MRI (OMRI) enables visualization of free radicals in animals based on dynamic nuclear polarization. Real-time data of tissue redox status gathered from kinetic images of redox-sensitive nitroxyl radical probes using OMRI provided both anatomic and physiological information. Phantom experiments demonstrated the linear correlation between the enhancement factor and the concentration of a membrane-impermeable probe, carboxy-PROXYL (3-carboxy-2,2,5,5-tetramethyl- pyrrolidine-1-oxyl). Whole-body OMRI images illustrated the in vivo kinetics of carboxy-PROXYL for 25 min. Initial distribution was observed in lung, heart, liver, and kidney, but not brain, corresponding to its minimal lipophilicity. Based on these images (pixel size, 1.33 × 1.33 mm; slice thickness, 50mm), a time-concentration curve with low coefficient of variance (<0.21) was created to assess pharmacokinetic behaviors. A biexponential curve showed a distribution phase from 1 to 10 min and an elimination phase from 15 to 25 min. The α rate constant was greater than the β rate constant in ROIs, confirming that its pharmacokinetics obeyed a two-compartment model. As a noninvasive technique, combining OMRI imaging with redox probes to monitor tissue redox status may be useful in acquiring valuable information regarding organ function for preclinical and clinical studies of oxidative diseases.

[41] Nitroxyl probes for brain research and their application to brain imaging

Publisher Summary In the chapter, various types of lipophilic nitroxyl spin probes are synthesized for the investigation of free radical reactions in the brain. Alkyl ester compounds are utilized for positron emission tomography (PET) and single photon emission computed tomography (SPECT) because of their capability of passing through the blood-brain barrier. Acetoxymethyl ester derivatives are also widely used as fluorescence probes in cultured cells. These compounds are characterized to be temporarily membrane permeable by masking their carboxyl group with ester and then to be hydrolyzed inside the cells. Ester compounds are ideal probes for brain research. The lipophilic and hydrolytic properties of these nitroxyl spin probes are characterized in relation to the tissue distribution of the probes in living animals after intravenous injection. In vivo ESR (electron spin resonance) imaging of the probes in the head of living mice has been reported. To estimate free radical reactions and its imaging in the brain of living animals, various types of lipophilic nitroxyl spin probes are synthesized. Carboxy-PROXYL acetoxymethyl ester (CxP-AM) has the potential to serve as a powerful probe for the investigation and diagnosis of free radical reactions and its imaging in the brain.

Generation of p-semiquinone radicals from chlorophenols in water during ozonation

The formation of radical species during ozonation of phenols in water was investigated with ESR spectroscopy. Ozonation of 2,3-, 2,5- and 2,6-dichlorophenol gave ESR spectra with a triplet of lines whose peaks have a height ratio of 1:2:1. The ESR parameters of the triplet were similar to those of the corresponding dichloro-p-semiquinone radical. Ozonation of 2,4,5- and 2,4,6-trichlorophenol gave the same spectra that resulted from the ozonation of 2,5- and 2,6-dichlorophenol, respectively, indicating the replacement of the chlorine in the p-position by a hydroxyl group during ozonation. The amount of the radical increased with increasing ozone concentration and was inhibited by two hydroxyl radical scavengers, mannitol and uric acid. The Fenton system, however, produced no radicals from phenols. These results indicate that an active species in addition to the hydroxyl radical participates in the formation of the p-semiquinone radical from chlorophenols during ozonation.