Half-Life Mapping of Nitroxyl Radicals with Three-Dimensional

Jul 31, 2009 - Hiroshi Hirata, Ph.D., Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido U...
33 downloads 12 Views 3MB Size
Anal. Chem. 2009, 81, 7501–7506

Half-Life Mapping of Nitroxyl Radicals with Three-Dimensional Electron Paramagnetic Resonance Imaging at an Interval of 3.6 Seconds Hideo Sato-Akaba,†,‡ Yoko Kuwahara,§ Hirotada Fujii,§ and Hiroshi Hirata*,† Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University, Sapporo 060-0814, Japan, and School of Health Sciences, Sapporo Medical University, Sapporo 060-8556, Japan This technical note reports a continuous-wave electron paramagnetic resonance (CW-EPR) imager that can visualize the distribution of free radicals with a half-life of subminutes in three-dimensional (3D) space. A total of 46 EPR spectra under magnetic field gradients, called projections, were obtained for image reconstruction at an interval of 3.6 s. A shortened data-acquisition time was achieved with the use of analog signals that drove field gradient coils in the imager. 3D mapping of the half-lives of nitroxyl radicals (4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl) was demonstrated in their reduction reaction with ascorbic acid. Inhomogeneous half-lives were clearly mapped pixel-by-pixel in a sample tube. Visualization of the distribution of target molecules is a very powerful method for understanding physiological and pathophysiological processes in living organisms.1-3 In addition, oxidative stress in a biological system may be a key cause of several diseases, such as stroke (ischemia), epilepsy, Parkinson’s disease, and Alzheimer’s disease.4-7 The oxidative status through the reduction/oxidation reaction of nitroxyl radicals can be measured in a small rodent noninvasively.8-10 Furthermore, in chemical engineering, the visualization of chemical reactions, and particu* Corresponding author. Hiroshi Hirata, Ph.D., Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University; North 14, West 9, Kita-ku, Sapporo 060-0814, Japan. Phone and fax: +81-11-706-6762. E-mail: [email protected]. † Hokkaido University. ‡ Current address: Department of Systems Innovation, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531 Japan. § Sapporo Medical University. (1) Van der Have, F.; Vastenhouw, B.; Ramakers, R. M.; Branderhorst, W.; Krah, J. O.; Ji, C.; Staelens, S. G.; Beekman, F. J. J. Nucl. Med. 2009, 50, 599–605. (2) Gross, S.; Gammon, S. T.; Moss, B. L.; Rauch, D.; Harding, J.; Heinecke, J. W.; Ratner, L.; Piwnica-Worms, D. Nat. Med. 2009, 15, 455–461. (3) Contag, C. H.; Ross, B. D. J. Magn. Reson. Imaging 2002, 16, 378–387. (4) Fiorillo, C.; Becatti, M.; Pensalfini, A.; Cecchi, C.; Lanzilao, L.; Donzelli, G.; Nassi, N.; Giannini, L.; Borchi, E.; Nassi, P. Free Radical Biol. Med. 2008, 15, 839–846. (5) Frantseva, M. V.; Perez Velazquez, J. L.; Tsoraklidis, G.; Mendonca, A. J.; Adamchik, Y.; Mills, L. R.; Carlen, P. L.; Burnham, M. W. Neuroscience 2000, 97, 431–435. (6) Jenner, P. Ann. Neurol. 2003, 53 (Suppl. 3), S26–S38. (7) Smith, M. A.; Rottkamp, C. A.; Nunomura, A.; Raina, A. K.; Perry, G. Biochim. Biophys. Acta 2000, 1502, 139–144. 10.1021/ac901169g CCC: $40.75  2009 American Chemical Society Published on Web 07/31/2009

larly the reaction rate or distribution of specific chemicals, can provide useful information for the design of a microchannel reactor.11 To visualize the dynamics of chemical reactions within a limited time, a reasonably fast imaging method is required. Electron paramagnetic resonance (EPR) spectroscopy and imaging can be used to investigate chemical reactions involving free radicals. Sixmembered nitroxyl radicals have a short lifetime, on the order of a minute, in live animals. If six-membered nitroxyl radicals could be detected and visualized in live animals, it would be useful for studying their pharmacokinetics. In addition to five-membered nitroxyl radicals, which have a more than 10-fold longer lifetime in live animals, we could use a variety of six-membered nitroxyl radicals in small-animal experiments. To record the time-course of the distribution of free radicals with a lifetime of less than several tens of seconds, the total acquisition time in threedimensional (3D) EPR imaging should be less than a few seconds. The pulsed EPR method, as in Fourier-transform nuclear magnetic resonance (FT-NMR), can be used to rapidly obtain spectral data from subjects.12-14 However, the pulsed EPR method is still limited to free radical molecules that have longer relaxation times.15,16 In contrast, the continuous-wave (CW) EPR protocol can be applied to a variety of free radicals, without the limitation of the relaxation time of unpaired electrons. However, the longer acquisition time of CW-EPR imaging is a drawback for visualizing the distribution of free radicals with a short lifetime. The speed of data acquisition in CW-EPR imaging has been improved over (8) Kobayashi, H. P.; Watanabe, T.; Oowada, S.; Hirayama, A.; Nagase, S.; Kamibayashi, M.; Otsubo, T. J. Surg. Res. 2008, 147, 41–49. (9) Utsumi, H.; Yamada, K.; Ichikawa, K.; Sakai, K.; Kinoshita, Y.; Matsumoto, S.; Nagai, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1463–1468. (10) Kuppusamy, P.; Li, H.; Ilangovan, G.; Cardounel, A. J.; Zweier, J. L.; Yamada, K.; Krishna, M. C.; Mitchell, J. B. Cancer Res. 2002, 62, 307–312. (11) Atencia, J.; Beebe, D. J. Nature 2005, 29, 648–655. (12) Mailer, C.; Sundramoorthy, S. V.; Pelizzari, C. A.; Halpern, H. J. Magn. Reson. Med. 2006, 55, 904–912. (13) Afeworki, M.; van Dam, G. M.; Devasahayam, N.; Murugesan, R.; Cook, J.; Coffin, D. A.; Larsen, J. H.; Mitchell, J. B.; Subramanian, S.; Krishna, M. C. Magn. Reson. Med. 2000, 43, 375–382. (14) Schweiger, A.; Jeschke, G. Principles of Pulsed Electron Paramagnetic Resonance; Oxford University Press: Oxford, U.K., 2001. (15) Hyodo, F.; Matsumoto, S.; Devasahayam, N.; Dharmaraj, C.; Subramanian, S.; Mitchell, J. B.; Krishna, M. C. J. Magn. Reson. 2009, 197, 181–185. (16) Subramanian, S.; Devasahayam, N.; Murgesan, R.; Yamada, K.; Cook, J.; Taube, A.; Mitchell, J. B.; Lohman, J. A. B.; Krishna, M. C. Magn. Reson. Med. 2002, 48, 370–379.

Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

7501

the past 2 decades.17-22 A data-acquisition time of 5.8 s was recently reported for 3D CW-EPR imaging with 46 projections (spectral data under magnetic field gradients).23 The goal of this study was to demonstrate the half-life mapping of nitroxyl radicals with ascorbic acid in 3D space with an interval of a few seconds. This technical advancement practically solves the long-standing problem of a slower acquisition speed in CWEPR imaging. To measure the decay of the EPR signal intensity of nitroxyl radicals, we used a home-built 650 MHz CW-EPR imager.23,24 The total acquisition time for EPR imaging is a product of the number of projections and the acquisition time of an EPR spectrum. A decrease in both parameters directly leads to a shorter acquisition time in EPR imaging. This would enable us to measure the reduction reaction of nitroxyl radicals with a short lifetime. With this method, we were able to map the half-lives of nitroxyl radicals in a 3D subject with an interval of 3.6 s. EXPERIMENTAL SECTION Chemicals. 4-Hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl (TEMPOL) and ascorbic acid were purchased from Kanto Chemical (Tokyo, Japan) and Wako Pure Chemical Industries (Osaka, Japan), respectively. TEMPOL aqueous solution (2 mM) was prepared and used for all experiments. Phantom. A cylindrical glass bottle (5 mL) with an outer diameter of 15 mm and length of 47 mm was filled with 2 mM TEMPOL aqueous solution (4.6 mL). Ascorbic acid (100 mM, 0.28 mL) was added to the solution to start the reduction reaction of TEMPOL. The solution of ascorbic acid was injected with a syringe via a needle-accessible cap that covered the opening of the bottle, and the sample bottle was then quickly placed in the resonator of the CW-EPR imager. Data acquisition was started when the sample was set in the resonator. The EPR imager was tuned before the experiment. During tuning, the sample bottle was stably placed in the resonator for 10 min to stabilize the resonance frequency of the resonator. EPR Imager. Figure 1 shows a schematic diagram of our 650 MHz CW-EPR imager. The whole three-line spectrum of TEMPOL was used to reconstruct EPR images. Data for 46 projections were obtained under field gradients. A uniform distribution of projections was used instead of equal polar and azimuthal angles.23-25 The setup of the previously developed imager has been reported elsewhere.23 However, the improvements are briefly outlined here. Magnetic field scanning and field gradients in the X-, Y-, and Z-directions in the laboratory space depend on the voltages generated with multifunctional data-acquisition boards (PCIe-6259 (17) Demsar, F.; Walczak, T.; Morse, P. D., II.; Bacic, G.; Zolnai, Z.; Swartz, H. M. J. Magn. Reson. 1988, 76, 224–231. (18) Ishida, S.; Kumashiro, H.; Tsuchihashi, N.; Ogata, T.; Ono, M.; Kamada, H.; Yoshida, E. Phys. Med. Biol. 1989, 34, 1317–1323. (19) Alecci, M.; Colacicchi, S.; Indovina, P. L.; Momo, F.; Pavone, P.; Sotgiu, A. Magn. Reson. Imaging 1990, 8, 59–63. (20) Ishida, S.; Matsumoto, S.; Yokoyama, H.; Mori, N.; Kumashiro, H.; Tsuchihashi, N.; Ogata, T.; Yamada, M.; Ono, M.; Kitajima, T.; Kamada, H.; Yoshida, E. Magn. Reson. Imaging 1992, 10, 109–114. (21) Yokoyama, H.; Ogata, T.; Tsuchihashi, N.; Hiramatsu, M.; Mori, N. Magn. Reson. Imaging 1996, 14, 559–563. (22) Samouilov, A.; Caia, G. L.; Kesselring, E.; Petryakov, S.; Wasowicz, T.; Zweier, J. L. Magn. Reson. Med. 2007, 58, 156–166. (23) Sato-Akaba, H.; Fujii, H.; Hirata, H. Rev. Sci. Instrum. 2008, 79, 123701. (24) Sato-Akaba, H.; Fujii, H.; Hirata, H. J. Magn. Reson. 2008, 193, 191–198. (25) Ahmad, R.; Deng, Y.; Vikram, D. S.; Clymer, B.; Srinivasan, P.; Zweier, J. L.; Kuppusamy, P. J. Magn. Reson. 2007, 184, 236–245.

7502

Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

Figure 1. Diagram of the home-built 650 MHz CW-EPR imager. Analog signals generated with multifunction data-acquisition (DAQ) boards were led to bipolar power supplies. The first DAQ board generated the trigger pulse (Dev1/AO1) and the signal of field scanning (Dev1/AO0). The second DAQ board output the signals (Dev2/AO0, AO1, and AO2) for magnetic field gradients. The trigger pulse was delivered to the port of the programmable function interface (Dev2/PFI) and controlled the signals of the second DAQ board. PSD refers to a lock-in amplifier for the phase-sensitive detection of EPR spectra.

and PCIe-6251, National Instruments, Austin, TX). Analog outputs of these boards were amplified with bipolar power supplies (BP4610 and BP4620, NF Corp., Yokohama, Japan). The maximum voltages of these bipolar power supplies were ±60 V. The maximum currents for BP4610 and BP4620 were ±10 and ±20 A, respectively. The raise/fall time of switching for the maximum currents of these power supplies was 4 µs. The data-acquisition board (PCIe-6259) has a resolution of 16 bits for ±10 V and a sample rate of 1.25 MS/s. Field scanning was not controlled digitally. Instead, a combination of analog signals and bipolar power supplies was suitable for faster field scanning and setting of magnetic field gradients. The maximum current for field scanning was 10 A (BP4610), and the maximum current for the magnetic field gradient was 20 A

Figure 2. Timing chart for data acquisition in the CW-EPR imager. (a) Current for field scanning (the sequence signal for field scanning was generated by a DAQ board (PCIe-6251)), (b) trigger pulse generated by the DAQ board, (c) data-acquisition window with a period of T (50 ms), and (d) magnetic field gradients in the X-, Y-, and Z-directions. The field gradients are set for the next projection with the trigger pulse.

(BP4620). A multicoil parallel-gap resonator (MCPGR) with a sample size with an inner diameter of 22 mm and a length of 34 mm was used.26 The MCPGR consists of 14 single-turn coils and capacitors. The coils (24 mm in inner diameter) were formed with tin-plated copper wire (1.2 mm in diameter). The unloaded quality factor of the MCPGR was 685. The efficiency for generating a magnetic field was 55 µT/W1/2 at the center of the resonator when it was empty.23 Data acquisition was performed with a LabVIEW-based control program. We used National Instruments LabVIEW 8.5 on MacOS X 10.5 and an Apple Mac Pro (Dual quad-core Xeon, 2.8 GHz). Control Sequence of Field Scanning and Field Gradient. A key improvement for reducing the total acquisition time was the use of analog signals to drive the Helmholtz coil pair for field scanning and the coils for field gradients in Cartesian coordinates. Analog outputs (three channels) of a multifunctional data-acquisition board (PCIe-6259) were used for field gradients. The analog output of another data-acquisition board (PCIe-6251) was used for field scanning. Figure 2 shows the sequence of each analog output that was generated by the LabVIEW-based control program. Magnetic field gradients were set after field scanning was performed, and the trigger pulse was detected. We did not use a feedback control system for magnetic field scanning. The bipolar power supplies were operated in the constant-current mode and driven with the voltage sequence. The period of the designed sequence was 71.4 ms. This means that the total time for 46 projections, the zero-gradient spectrum, and another cycle of the sequence for setting the field gradients was 3.6 s. The signal that drives the coil for field scanning was asymmetric. At the beginning of field scanning with a ramp signal, an exponential function was added to the ramp signal to compensate for the delay of the current in the Helmholtz coil pair. This is due (26) Kawada, Y.; Hirata, H.; Fujii, H. J. Magn. Reson. 2007, 184, 29–38.

to the inductance (0.68 mH) and resistance (1.2 Ω) of the coil. The time-constant of the exponential function was set to the timeconstant of the coil for field scanning (0.57 ms). The timeconstants of the coils for magnetic field gradients in the X-, Y-, and Z-directions were 0.58, 0.50, and 0.45 ms, respectively. These time-constants were estimated from the inductance and resistance of each set of coils. The inductances and resistances of the gradient coils for the X-, Y-, and Z-directions were 1.4, 1.2, and 0.41 mH and 2.4, 2.4, and 0.91 Ω, respectively. Thus, these coils can respond to the shift in the currents that control the magnetic field gradients. A time of approximately 20 ms remained between the time windows for data acquisition (see Figure 2). In this period, the field gradients can be set, since the gradient coils have time-constants on the order of submilliseconds. Image Reconstruction. 3D image reconstruction was carried out by the filtered back-projection method.23 The reconstruction code was written in FORTRAN (Pro Fortran v10.2, Absoft Corp., Rochester Hill, MI). A Hamming filter was used to suppress noise in the process of deconvolution. The execution code for the process of 3D filtered back-projection was automatically parallelized with an Absoft Pro Fortran compiler, and the computation time was 1.2 s for 46 projections. Calculation of Half-Life. To estimate the half-lives of nitroxyl radicals at different positions in the phantom, we extracted the time-course of the signal intensities at each voxel. An exponential function was used to fit the experimental data with the leastsquares method. From the approximated exponential function, we determined the half-life of nitroxyl radicals at a given position. There were 1283 data points in 3D visualization space. Since the field-of-view (FOV) was 43.4 mm in the X-, Y-, and Z-directions, the size of a voxel was 0.34 mm. EPR Imaging of a Phantom Tube. The axis of the sample bottle was aligned to the Y-direction. The X-axis is perpendicular to the floor of the laboratory, and the Y- and Z-axes are parallel to the floor of the laboratory. The static magnetic field is parallel to the Z-direction. EPR spectra for the TEMPOL aqueous solution in the sample bottle were measured under the following conditions: rf power 11.3 dBm (13.5 mW), field scanning 7.4 mT, magnetic field modulation 0.2 mT, scan time 50 ms, and timeconstant of the lock-in amplifier 0.1 ms. In the data acquisition, 512 points were digitized per magnetic field scan with a time window of 50 ms. EPR spectra for each projection were measured under a field gradient of 50 mT/m, and 46 projections were recorded. The FOV was 7.4 mT/50 mT/m ) 148 mm. After the images were reconstructed, a data set of 150 × 150 × 150 pixels was extracted from a data set of 512 × 512 × 512 pixels (148 mm × 148 mm × 148 mm). The FOV of the 150 pixels corresponds to 43.4 mm. Finally, the data set of 150 × 150 × 150 pixels was converted into the 3D data of 128 × 128 × 128 pixels; however, the FOV was unchanged (43.4 mm). RESULTS AND DISCUSSION EPR images of Nitroxyl Radicals. We obtained 3D images of the distribution of nitroxyl radicals at an interval of 3.6 s. Figure 3 shows slice-selective images in the YZ-, XY-, and ZX-planes. Because of the initial adjustment of the imager, the first several images were incomplete. The first complete image was taken at the sixth imaging routine after a solution of ascorbic acid was Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

7503

Figure 3. Temporal changes in EPR images for nitroxyl radicals (TEMPOL) after the injection of ascorbic acid. Slice-selective 2D images in (a) the YZ-plane, (b) YX-plane, and (c) ZX-plane. The field-of-view was 43.4 mm (128 × 128 pixels). The time was started at the injection of ascorbic acid solution. The numbers following the time indicate the number of sequentially recorded images. The maximum signal intensity in the images was normalized to 255. The opening of the bottle corresponds to the left side of the images in Figure 3a,b. 7504

Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

injected into the sample bottle. The times indicated in Figure 3 were measured from the point of injection of the ascorbic acid solution. An inhomogeneous reduction reaction occurred, depending on the sample position, as shown in parts b (XY-plane) and c (ZX-plane) of Figure 3. Since the density of the solution of ascorbic acid was greater than that of the solution of nitroxyl radicals, the injected ascorbic acid solution flowed downward to the bottom of the bottle. Therefore, the early signal intensity of nitroxyl radicals was reduced at the bottom of the sample bottle. In this experiment, the EPR spectrum was not saturated under the measurement conditions. The spin system in our sample was in the range of the linear response where the signal amplitude is proportional to the square root of the incident rf power. Figure 4 shows the time-course of the reduction in signal intensities at specified positions. The signal intensities were approximated by an exponential function and a constant baseline offset. The dots represent the measured signal intensities. The lines are exponential functions fitted to the experimental data. As shown in Figure 4a, a half-life of 34.9 s was obtained from the data at position (50, 40, 63) in the data set of 128 × 128 × 128 pixels, where a set of three numbers corresponds to the data points in the X-, Y-, and Z-directions. The corners at the bottom left of each image in Figure 3 correspond to the minimum number of data points (1) for the X-, Y-, and Z-directions. As shown in Figure 4c, the half-life at position (80, 100, 63) was 182 s. For experiments in small animals such as mice, nitroxyl radicals at a concentration of approximately 100 mM are usually injected into a living mouse. For example, a 150 mM solution of 4-oxo-2,2,6,6,-tetramethyl piperidine-d16-1-15N-oxyl (15N-PDT) was injected into mice via a tail vein in ref 15. Since the volume of the solution of nitroxyl radicals was determined from the body weight (7.5 µL/g), a mouse of 25 g received approximately 0.19 mL of 150 mM nitroxyl radicals. Since the average total blood volume of a mouse is approximately 7% of the body weight, a mouse of 25 g has a blood volume of about 1.8 mL.27 When 0.1 mL of a solution of 100 mM nitroxyl radicals is intravenously injected into a living mouse, this concentration would be decreased to almost 1/20 of the initial concentration. As a result of blood circulation and metabolism in a subject mouse, the concentration of nitroxyl radicals in the mouse will further decrease. Under these circumstances, a nitroxyl radical concentration of 1 or 2 mM is reasonable for investigating the sensitivity of EPR spectroscopy and imaging. Mapping of Half-Lives. The position-dependency of the halflives of nitroxyl radicals was obtained from the data of 3D images at an interval of 3.6 s. Figure 5 shows the half-life mapping of nitroxyl radicals with ascorbic acid. These images show positions with an intensity of more than 50% of the maximum value in the initial intensity image. The distribution of the half-lives is clearly seen in the maps. Although the half-lives were uniform in the horizontally selected slice-image (Figure 4a), vertically selected images (Figure 4b,c) show a nonuniform distribution of half-lives. We achieved 3D EPR imaging with 46 projections at an interval of 3.6 s. This enables us to visualize the reduction reaction, which has a half-life of approximately 30 s, in 3D images. Previous reports (27) Fukuta, K. In The Laboratory Mouse; Hedrich, H. J., Ed.; Elsevier Academic Press: San Diego, CA, 2004; pp 543-548.

Figure 4. Time-course of the signal intensities of nitroxyl radicals after the injection of ascorbic acid solution at the given positions. Signal intensities at positions (a) (50, 40, 63), (b) (63, 63, 63), and (c) (80, 100, 63) in the 3D data space. A set of three numbers corresponds to data points in the X-, Y-, and Z-directions. The data space was 128 × 128 × 128 pixels. The half-lives at these positions were (a) 34.9, (b) 48.4, and (c) 182 s.

have described the mapping of the half-lives of nitroxyl radicals.28-30 However, this improved technique is useful for detecting free radical molecules that have a short lifetime. Previously, it was difficult to obtain such a distribution of free radicals within a few seconds. With this method, we may be able to visualize the (28) Hyodo, F.; Chuang, K. H.; Goloshevsky, A. G.; Sulima, A.; Griffiths, G. L.; Mitchell, J. B.; Koretsky, A. P.; Krishna, M. C. J. Cereb. Blood Flow Metab. 2008, 28, 1165–1174. (29) He, G.; Kutala, V. K.; Kuppusamy, P.; Zweier, J. L. Free Radical Biol. Med. 2004, 36, 665–672. (30) Hyodo, F.; Yasukawa, K.; Yamada, K.; Utsumi, H. Magn. Reson. Med. 2006, 56, 938–943.

distribution of free radicals with a short lifetime in biological subjects. The half-lives of nitroxyl radicals at specific positions were mapped in Figure 5. However, the estimated half-lives depend on the rate of the reduction reaction, the diffusion of ascorbic acid, and the fluidic movement of solutions. Our experiments did not distinguish among these influences. If more precise half-lives are required, the distribution of ascorbic acid in the solution of nitroxyl radicals should be controlled. We should be aware of this limitation regarding the mapping of half-lives of nitroxyl radicals and the overall effects on EPR signal intensities. Advantages over Existing Techniques. The speed of acquisition for 3D CW-EPR imaging has a tremendous impact on freeradical-related problems. With the present method, the acquisition time was reduced by 38% compared to the previous value (5.8 s) in CW-EPR imaging under the same conditions (46 projections). Furthermore, the sensitivity of EPR detection was not sacrificed. In CW-EPR imaging, targeted free radicals are not restricted with regard to the line-width for EPR spectra, i.e., the relaxation time of unpaired electrons. Although pulsed EPR imaging is a fast acquisition method, to date it has only been applied to free radicals that have a longer relaxation time. A kind of nitroxyl radical, 15NPDT, was successfully measured with a 300 MHz pulsed EPR spectrometer.15 With such a pulsed EPR spectrometer, a relaxation time T2* of 196 ns was recorded from the freeinduction decay of 15N-PDT. Although this progress is very important for measuring nitroxyl radicals in small-animal experiments, the detection of nitroxyl radicals with an even shorter relaxation time is still challenging due to the spectrometer recovery time. In contrast, the CW-EPR method can be applied to nitroxyl radicals with a short relaxation time. For CW-EPR spectroscopy, the line-width of an EPR absorption spectrum depends on the relaxation time of spins of unpaired electrons. However, it does not limit the applicability of the CW-EPR method to any kind of free radical. The noninvasiveness of EPR imaging is a practical consideration for in vivo animal experiments. An X-band EPR spectrometer can detect free radicals with high sensitivity. However, it is not suitable for the imaging of small animals. Overhauser-enhanced magnetic resonance imaging (OMRI) can also be used to visualize the distribution of free radicals. An acquisition time of 4.4 s has been achieved for 2D slice-selective images in OMRI (number of excitations 4, FOV 30 mm × 30 mm and 64 × 64 pixels).31 Even if a small flip-angle was used in OMRI, the total acquisition time for 3D images would be more than a few seconds. This is because the relaxation time (T2) of protons is on the order of several hundreds of milliseconds. The rapid-scan EPR method is a potentially fast acquisition approach for 3D imaging.32-35 However, rapid-scan EPR spectroscopy has not yet been demonstrated for experiments in small animals. Slice-selective EPR (31) Khramtsov, V. V.; Shet, K.; Kesselring, E.; Petryakov, S.; Sun, Z.; Zweier, J. L.; Samouilov, A. Proc. Intl. Soc. Magn. Reson. Med. 2009, 17, 4370. (32) Tseitlin, M.; Dhami, A.; Eaton, S. S.; Eaton, G. R. J. Magn. Reson. 2007, 184, 157–168. (33) Joshi, J. P.; Eaton, G. R.; Eaton, S. S. Appl. Magn. Reson. 2005, 28, 239– 249. (34) Stoner, J. W.; Szymanski, D.; Eaton, S. S.; Quine, R. W.; Rinard, G. A.; Eaton, G. R. J. Magn. Reson. 2004, 170, 127–135. (35) Joshi, J. P.; Ballard, J. R.; Rinard, G. A.; Quine, R. W.; Eaton, S. S.; Eaton, G. R. J. Magn. Reson. 2005, 175, 44–51.

Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

7505

Figure 5. Half-life mapping of nitroxyl radicals with ascorbic acid in (a) the YZ-plane, (b) YX-plane, and (c) ZX-plane. The half-lives were evaluated at each pixel and mapped. These slice-selective maps were generated at the center of the FOV, i.e., 63rd data point, in the X-, Z-, and Y-axes, respectively.

imaging is an alternative method to reduce the acquisition time.36,37 Nevertheless, further improvements are required for real applications to small animals. Limitations. The sensitivity of EPR detection is another important factor in decreasing the data-acquisition time. In CWEPR detection, a shorter duration of magnetic field scanning requires that the time-constant of a lock-in amplifier (phasesensitive detection) should be decreased to follow the response of the EPR spectra in the time domain. If EPR spectra must be averaged to achieve a reasonable signal-to-noise ratio (SNR), the data-acquisition time demonstrated here could not be achieved. The SNRs of measured spectra were 226 ± 27 when the timeconstant of the lock-in amplifier was set to 0.1 ms. The SNRs were 372 ± 68 with a time-constant of 0.3 ms. The sample was a test tube (110 mm long and 16 mm in diameter) filled with a solution of 1 mM TEMPOL. In this measurement, the SNRs of 1000 measured spectra were obtained under the following conditions: field scanning 7.0 mT, magnetic field modulation 0.2 mT, and a time of field scanning 0.1 s. The 3D CW-EPR imaging reported here is faster than the acquisition speed of OMRI. In contrast, OMRI has a better spatial resolution, since OMRI detects the signals from protons with a longer relaxation time, i.e., a narrow line-width in its spectrum. The average peak-to-peak line-width ∆Bpp of the three-line EPR spectrum for 2 mM TEMPOL solution was 0.198 mT. A magnetic field modulation of 0.2 mT was applied in the experiments. This modulation amplitude is similar to the peakto-peak line-width ∆Bpp. Since the EPR spectrum was slightly overmodulated under this measurement setting, the measured line-width was moderately broader than the intrinsic line-width of 2 mM TEMPOL solution. This broader EPR absorption led to a lower spatial resolution in this demonstration. Although it is important to study the spatial resolution in EPR imaging, the methods for resolution-enhancement were beyond the scope of this report.38-41 It is possible to apply this EPR imaging technique to small rodents such as mice with no significant modification of the (36) Sato-Akaba, H.; Abe, H.; Fujii, H.; Hirata, H. Magn. Reson. Med. 2008, 59, 885–890. (37) Devasahayam, N.; Subramanian, S.; Matsumoto, S.; Krishna, M. C. Proc. Intl. Soc. Mag. Reson. Med. 2009, 17, 4371. (38) Momo, F.; Colacicchi, S.; Sotgiu, A. Meas. Sci. Technol. 1993, 4, 60–64. (39) Hirata, H.; Wakana, M.; Susaki, H. Appl. Phys. Lett. 2006, 88, 254103. (40) Ahmad, R.; Clymer, B.; Vikram, D. S.; Deng, Y.; Hirata, H.; Zweier, J. L.; Kuppusamy, P. J. Magn. Reson. 2007, 184, 246–257. (41) Ikebata, Y.; Sato-Akaba, H.; Aoyama, T.; Fujii, H.; Itoh, K.; Hirata, H. Magn. Reson. Med. 2009, in press.

7506

Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

present instrument. In contrast, a larger magnet and coils would be required for measurements in larger animals, including humans. Larger coils usually have greater inductance and require large currents to obtain the magnetic field gradients that are needed in EPR imaging. This is a technically challenging issue in EPR imaging for larger subjects. However, our results suggest that imaging for free-radical molecules in small animals may be possible within a few seconds, although we actually demonstrated only in vitro phantom imaging. This technical advancement would extend studies on the pharmacokinetics of free radicals and the reduction/oxidation status of nitroxyl radicals in small rodents. CONCLUSIONS The total acquisition time achieved here means that CW-EPR imaging is no longer a time-consuming method for obtaining the distribution of targeted free radicals. Since 3D CW-EPR imaging can be performed in a period of 3.6 s for 46 projections, the pharmacokinetics or chemical reactions of free radical molecules on the order of subminutes can be measured. Spectral-spatial EPR imaging requires a longer acquisition time and many projections to reconstruct images of spectral information mapped in the 3D space. On the basis of our relatively fast data-acquisition technique, spectral-spatial EPR imaging should become less time-consuming. Further progress in this area should contribute to EPR imaging methods for mapping the partial pressure of oxygen and pH in small animals.42-45 ACKNOWLEDGMENT The authors are grateful to Dr. Kazuhiro Ichikawa, Kyushu University, Japan, for his helpful discussion regarding the acquisition speed of OMRI. This work was supported by SENTAN, JST.

Received for review May 29, 2009. Accepted July 17, 2009. AC901169G (42) Bratasz, A.; Pandian, R. P.; Deng, Y.; Petryakov, S.; Grecula, J. C.; Gupta, N.; Kuppusamy, P. Magn. Reson. Med. 2007, 57, 950–959. (43) Elas, M.; Bell, R.; Hleihel, D.; Barth, E. D.; McFaul, C.; Haney, C. R.; Bielanska, J.; Pustelny, K.; Ahn, K. H.; Pelizzari, C. A.; Kocherginsky, M.; Halpern, H. J. Int. J. Radiat. Oncol. Biol. Phys. 2008, 71, 542–549. (44) Matsumoto, S.; Hyodo, F.; Subramanian, S.; Devasahayam, N.; Munasinghe, J.; Hyodo, E.; Gadisetti, C.; Cook, J. A.; Mitchell, J. B.; Krishna, M. C. J. Clin. Invest. 2008, 118, 1965–1973. (45) Potapenko, D. I.; Foster, M. A.; Lurie, D. J.; Kirilyuk, I. A.; Hutchison, J. M. S.; Grigor’ev, I. A.; Bagryanskaya, E. G.; Khramtsov, V. V. J. Magn. Reson. 2006, 182, 1–11.