Kazuo Oikawa

Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging

Real-time detection of free radicals generated within the body may contribute to clarify the pathophysiological role of free radicals in disease processes. Of the techniques available for studying the generation of free radicals in biological systems, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. This article begins with a review of spin trapping detection of oxygen-centered radicals using X-band ESR spectroscopy and then describes the detection of superoxide and hydroxyl radicals by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and ESR spectroscopy in the perfusate from isolated perfused rat livers subjected to ischemia/reperfusion. This article also reviews the current status of ESR for the in vivo detection of free radicals and in vivo imaging of exogenously administered free radicals. Moreover, we show that in vivo ESR-computed tomography with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl may be useful for noninvasive anatomical imaging and also for imaging of hepatic oxidative stress in vivo.

Ex vivo measurement of tissue distribution of a nitroxide radical after intravenous injection and its in vivo imaging using a rapid scan ESR-CT system.

To establish the usefulness of ESR-CT imaging with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) in living animals, we investigated the tissue distribution of carbamoyl-PROXYL after i. v. injection. Ten minutes after injection of carbamoyl-PROXYL, its concentrations in the liver, spleen, kidney, and plasma were higher than those in the small intestine and stomach. However, the inter-organ differences in concentrations were not striking. We selected the liver as a representative organ and attempted to measure the concentration of carbamoyl-PROXYL in it after washing out all of the blood by in situ perfusion with saline. The ESR spectrum of the liver homogenate after complete blood washout revealed that the concentration of carbamoyl-PROXYL was significantly reduced. Thus, at this time, carbamoyl-PROXYL was distributed predominantly in the plasma and/or loosely attached to the surfaces of cells. We obtained high-quality ESR-CT images of the murine abdomen at a measurement time of 40 s and found that a high-intensity area of carbamoyl-PROXYL appeared in the liver and kidneys, indicating an abundant blood circulation. Although the organ specificity of carbamoyl-PROXYL was weak, we consider that ESR-CT imaging with carbamoyl-PROXYL will be a powerful new tool for non-invasive anatomic analysis of the liver and the kidneys.

Spatiotemporal Measurement of Free Radical Elimination in the Abdomen Using an In Vivo ESR-CT Imaging System

Electron spin resonance (ESR) imaging can visualize the distribution of free radicals in living systems according to their concentrations. However, the application of ESR imaging to living animals has not been well established. Using a rapid field scan L-band ESR imaging system, we have successfully obtained two-dimensional ESR projection (xz-plane projection) and three-dimensional ESR-CT (trans-axial section along the y-axis) images of the abdomen of living mice after an injection of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) into the tail vein. The in vivo two-dimensional ESR projection imaging clearly visualized the carbamoyl-PROXYL distribution and the rapid decay process in the abdomen. Because among the viscera, the liver is most abundantly associated with a blood volume, the outline of the image can be composed mainly of this organ. We therefore attempted to find whether there will be a difference in spatiotemporal dynamics of carbamoyl-PROXYL in the abdomens between the control and the mice with liver damage by two-dimensional ESR projection. In the control mice, carbamoyl-PROXYL was almost completely eliminated from the abdomen within 5 minutes after administration. On the other hand, in mice with carbon tetrachloride-damaged livers, the decay of carbamoyl-PROXYL was markedly prolonged. Even at 5 min after administration, carbamoyl-PROXYL remained clearly visible in the abdomen. In vivo three-dimensional ESR-CT imaging showed an even distribution of carbamoyl-PROXYL throughout the whole liver, which corresponded well with the images of trans-axial sections of the murine abdomen. We have succeeded in displaying two-dimensional ESR projection and three-dimensional ESR-CT images of carbamoyl-PROXYL distribution and clearance in the abdomen of a living animal. The ESR-CT imaging technique is considered to be a powerful new tool for noninvasive investigations of the in vivo spatiotemporal dynamics of free radical distribution and elimination in the organs.

Free radical imaging by electron spin resonance computed tomography in rat brain

Images of nitroxide radicals were obtained from the brains of living rats following intracarotid injection of imaging agent, [2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinylox y] (16 DS) using L-band electron spin resonance computed tomography (ESR-CT). The image patterns obtained from the 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolinyloxy injected rats showed the agent in tissues of the cranium but not within the brain. The uptake of 16 DS was found in the cortex, hippocampus, striatum, midbrain, pons medulla oblongata and cerebellum, and nuclei, mitochondria, synaptosomes of the cerebrum after intracarotid injection of 16 DS using an x-band ESR spectrometer. These results suggest that 16 DS penetrates the blood brain barrier.

Development of an L-band electron spin resonance/proton nuclear magnetic resonance imaging instrument

An imaging instrument for measuring the 2D distribution of both free radicals and protons in a single sample was developed. A common resonator was designed for this instrument that can resonate at an L-band electron spin resonance (ESR) frequency or a HF (27.7 MHz) proton nuclear magnetic resonance (NMR) frequency. This resonator enabled construction of a simple detection block for the instrument. Because the detection processes of ESR and NMR are similar, many components, including the main magnet, magnetic-field modulation coils, field gradient coils, electrical circuits, data acquisition system, computer for reconstructing images, and most of the software were shared by these two systems. By using this instrument, both ESR and NMR images of the head of a live rat were successfully produced. The free radical distribution could be positioned by superimposing the ESR image on the NMR image.

Photoacoustic spectrometric determination of trace phosphorus as molybdenum blue adsorbed on uniform anion-exchange beads

Abstract Photoacoustic spectrometry was applied to the determination of phosphorus as molybdenum blue species adsorbed on an anion-exchange resin (10 mg of uniform beads). The photoacoustic intensity of ten resin particles on a glass plate was measured at 760 nm. The calibration graph obtained was linear in the range of 0.05–0.5 μg of phosphorus. The relative standard deviation for ten determinations of 0.3 μg P was 5.0%. The detection limit was 2 ng P ml −1 .

Photoacoustic spectroscopic studies on the solid phase cell with a differential type microphone using a diode laser as radiation source

The design and applications of the solid phase photoacoustic cell with a differential type microphone are described. This cell has higher sensitivity and reproducibility relative to a cell without a differential microphone. The method has been applied to the determination of phosphate ion in water samples. The detection limit for phosphate ion concentrated from 22.5 ml of sample solution on a membrane filter as molybdophosphate-n-dodecyltrimethylammonium bromide was 3.0 ng PO(3-)(4)/ml and the coefficient of variation for five measurements at 20 ng PO(3-)(4)/ml of phosphate ion was 3% using a diode laser (30 mW, 826 nm) as radiation source. The calibration graph for phosphate ion was linear over the range 5-50 ng PO(3-)(4)/ml.

Imaging of reactive oxygen species generated in vivo

We sought to image the biodistribution of reactive oxygen species (ROS) within the living body using an in vivo electron spin resonance (ESR) imaging system using a spin probe, 1‐acetoxy‐3‐carbamoyl‐2,2,5,5‐tetramethylpyrroline (ACP) that produces ESR‐detectable nitroxide upon reaction with ROS.

Electron spin resonance imaging of rat brain

Free radical mechanisms may be involved in several neurological disorders, including Parkinson’s disease, Alzheimer’s disease, brain ischemia, Down’s syndrome, and aging (Sinet and Ceballos-Picot, 1992; Javoy-Agid, 1992; Mori et al., 1992). Thus, a diagnostic method for locating regions of pathological change related to free radicals in the brain may be of use. Ishida et al. (1992) obtained electron spin resonance - computed tomography (ESR-CT) images of the cephalic region of rats. However, they were unable to record ESR-CT images of the brain. In the present study we obtained rat brain images using an ESR-CT system with spin labels for image enhancement.

Development of Photoacoustically Detected Magnetic Resonance System and Depth Profiling of Multi-layered Paramagnetic Samples.

We have constructed an on-line system of photoacoustically detected magnetic resonance (PADMR) com-posed of an electron spin resonance (ESR) system, a photoacoustic cell and an on-line computer. PADMR spectra of multi-layer model samples containing different paramagnetic substances were measured by this system. PADMR spectra for the respective layer in the three-layered model sample could be obtained using Fourier transform (FT) correlation photoacoustic spectroscopy (PAS) technique.