Skip to main content
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Jan 1.
Published in final edited form as: J Magn Reson. 2007 Oct 30;190(1):105–112. doi: 10.1016/j.jmr.2007.10.013

Monitoring Redox-Sensitive Paramagnetic Contrast Agent by EPRI, OMRI and MRI

Fuminori Hyodo 1, Ramachandran Murugesan 2, Ken-ichiro Matsumoto 1,3, Emi Hyodo 1, Sankaran Subramanian 1, James B Mitchell 1, Murali C Krishna 1,*
PMCID: PMC2258209  NIHMSID: NIHMS38886  PMID: 18006345

Abstract

A comparative study of tissue redox-status imaging using commonly used redox sensitive nitroxides has been carried out using electron paramagnetic resonance imaging (EPRI), Overhauser magnetic resonance imaging (OMRI) and conventional T1-weighted magnetic resonance imaging, MRI. Imaging studies using phantoms of different nitroxides at different concentration levels showed that EPRI and OMRI sensitivities were found to be linearly dependent on line width of nitroxides up to 2 mM, and the enhancement in MRI intensity was linear up to 5 mM. The sensitivity and resolution of EPRI and OMRI images depended significantly on the line width of the nitroxides whereas the MRI images were almost independent of EPR line width.

Reduction of the paramagnetic 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (3CP) by ascorbic acid (AsA) to the diamagnetic by hydroxylamine was monitored from a sequence of temporal images, acquired using the three imaging modalities. The decay rates determined by all the three modalities were found to be similar. However the results suggest that T1 weighted MRI can monitor the redox status, in addition to providing detailed anatomical structure in a short time. Therefore, a combination of MRI with nitroxides as metabolically responsive contrast agents can be a useful technique for the in vivo imaging probing tissue redox status.

Keywords: redox sensitive contrast agent, EPRI, MRI, OMRI, nitroxide

Introduction

A variety of intracellular molecules such as GSH, thioredoxins, NADPH, flavins, ascorbic acid (AsA), etc. contribute to the overall redox status in tissues [1]. Collectively, the reducing species determine the redox status of tumor, and are likely to influence the response of a particular tissue to oxidative stress. It is widely believed that tumor redox status is an important parameter that may be related to tumor growth and therapy [2; 3]. Hence it is of interest to develop non-invasive imaging methods to determine the in vivo redox status. In this direction, in vivo EPRI is emerging as a viable redox status imaging modality. Spatially resolved redox data, obtained using EPRI technique have shown that tumors are highly reducing and hypoxic compared to normal tissues [3; 4]. These EPRI measurements are based on the fact that the paramagnetic nitroxides are reduced to the corresponding diamagnetic one-electron reduction product (Fig. 1A), hydroxylamine in cellular incubations as well as in vivo [5-8]. It was postulated that nitroxides can accept cellular reducing equivalents, especially from the mitochondrial electron transport chain that would have otherwise gone to the reduction of molecular oxygen [9]. Hence, spatially resolved pharmacokinetics of nitroxides in an EPR experiment can report on differences that exist in various tissues which may display different redox status.

Figure 1.

Figure 1

Reversible one-electron reduction / oxidation showing the interconversion and the molecular structure of the four nitroxides, B) 15N-PDT, C) Tempone, D) 3CP and E) Tempol used in this work.

EPRI using nitroxides as a tracer was first demonstrated by Berliner and Fuji [10] and its pharmacokinetics monitored in tumor bearing animals in a subsequent paper [11]. Though EPRI is developing as an emerging imaging modality, it does not provide well-resolved morphological information, requiring the use of fiducials for a proper region of interest identification [12; 13]. Overhauser MRI (OMRI) is a double resonance technique and couples the advantages of MRI for spatial resolution with the sensitivity of EPR by making use of the Overhauser effect [14-16]. It has the potential of adding this missing anatomical information to the functional, redox or pO2 images [17]. The Overhauser enhancement depends on the line width of the paramagnetic agent, which in turn depends on oxygen concentration [18], making it possible for co-registration of the anatomical structure with the corresponding oxygen status. Using OMRI and utilizing the differences in hyperfine patterns (both position and multiplicity) it has been possible to use the infusion of isotopically differently (15N and 14N) substituted nitroxides to investigate extra- and intracellular redox status simultaneously [19].

Though the use of nitroxide as a T1-enhancing MRI contrast agent was realized early [20-23], the bio-reduction and rapid clearance have made them less useful than the metal-based Gd3+ complex contrast agents [24; 25], which had a long biological half life and high molar relaxivity, compared with the single, odd-electron containing nitroxides. However, the bio-reduction of nitroxides can be monitored to effectively probe the redox status of tissues [4; 26]. Therefore, nitroxides can play a unique dual role both as contrast agent and as redox-status reporters, with the availability of MR scanners in recent times with efficient pulse sequences such as spoiled gradient echo (SPGR) method for rapid imaging. Our study revealed that, coupled with the advantages of fast multi-slice measurement and high quality anatomical information, nitroxides as redox sensitive contrast in MRI can provide, indirectly via T1 contrast, the bio-reduction rates, and hence report on tissue redox status.

Currently three different techniques, viz, EPRI, OMRI and MRI are available for redox imaging using nitroxides. Each one of them has its own capabilities and limitations. Based on the requirement of specific application, one modality may be preferred over the other. To make an appropriate selection, it would be of interest to make a comparative assessment of the performance of each of them in monitoring the redox status. This study makes such an attempt by using four nitroxides, (15N-PDT, Tempone, 3CP and Tempol, whose structures are shown in Fig. 1 B-E, and expanded names given below under materials and methods) and evaluating the relative advantages of EPRI, OMRI and MRI.

MATERIALS AND METHODS

Chemical

15N-PDT (4-oxo-2,2,6,6-tetramethyl piperidine-d16-1-15N-oxyl) was purchased from CDN Isotopes inc (Quebec, Canada). Other nitroxides, Tempone (4-oxo-2,2,6,6-tetramethyl-1-piperidynyloxyl), 3CP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl) and Tempol (4-hydroxy-2,2,6,6,-tetramethyl-1-piperidynyloxyl) (see Fig. 1 B-D) were purchased from Sigma-Aldrich Chem. Co. (St. Louis, MO). Gd-DTPA (Magnevist) was purchased from BerLex laboratories (Wayne, Germany). Distilled, deionized water was used for all experiments. The other materials used were of analytical grade.

EPR Spectroscopy and Imaging

A seven-tube phantom, each tube (4.7 mm i.d.) containing 0, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0 mM aqueous solutions of the nitroxide contrast agents, as shown in Figure 2A, was used for performance evaluation. EPR spectra were obtained by an X-band spectrometer, and 2D spatial images perpendicular to the axes of the tubes using a 300 MHz CW EPR Imaging spectrometer [27]. These images are given in Figure 2 (left column), and the concentration-dependence of the image intensity and SNR are plotted in Figure 3A (top row). The spectrometer bridge was based on a standard hybrid T configuration with automatic frequency control capability. Signal detection was carried out by sinusoidal modulation and subsequent phase-sensitive detection. A parallel coil resonator (25 mm diameter and 25 mm length) was used for all experiments. EPR measurement conditions were as follows:

Figure 2.

Figure 2

The phantom images of four nitroxides obtained by EPRI and MRI. A) Schematics of the phantom. Additionally two tubes of 0.01 and 0.1 mM Gd-DTPA solution were added during MRI measurement. The EPRI (Left column) and MR (right column) intensity images of B) 15N-PDT, C) Tempone, D) 3CP, E) Tempol were described. EPRI conditions were follows; 18 projections were obtained every 1 min. FOV was 3.2 × 3.2 cm. Microwave frequency was 300 MHz, microwave power 2.5 mW, field modulation frequency was 13.5 kHz, time constant 0.01 s, gradient was 4.7 G/cm, sweep width was 50 Gauss. MRI: SPGE sequence (TR = 75 ms, TE = 3 ms, Flip angle = 45°, NEX = 2) was employed to observe T1 effect. Pixel resolution was 256 × 256. FOV was 3.2 × 3.2 cm. All measurements were performed at room temperature (25 ± 2 °C).

Figure 3.

Figure 3

Comparison of intensity change and SNR of nitroxides. The intensity change (left column) and SNR (right column) of nitroxides in A) EPR and B) MR images showed. The intensity was calculated for a select ROI from EPR and MR image of each nitroxides. The intensity change (%) of MRI was calculated based on the intensity of center phantom containing pure water. Number of experiments are 3.

X-band EPR

Microwave power, 1 mW; field modulation frequency, 100 kHz; field modulation amplitude 0.05 G for both 15N-PDT and Tempone; 0.24 G for 3CP; 0.38 G for Tempol; time constant, 0.003 s; sweep width, 0.5-2 G.

300 MHz EPR

Radio frequency (RF) power, 2.5 mW; field modulation frequency, 13.5 kHz; modulation amplitude 0.3 G for both 15N-PDT and Tempone; 0.7 G for 3CP; 1.2 G for Tempol; time constant, 0.01 s; sweep width, 10 G; scan time, 54 s; field gradient, 4.7 G/cm; and number of averages, 4. EPR image was reconstructed as a 128 × 128 matrix by filtered back-projection method using Shepp-Logan filter. FOV was 3.2 cm × 3.2 cm.

OMRI

A similar seven-tube phantom (4.7 mm i.d.) containing 0, 0.2, 0.5, 1, 2, 5, 10 mM 15N-PDT, 3CP and Tempol solutions (Fig. 4A) was used for OMRI experiments. OMRI imaging was performed using standard spin warp gradient echo sequence where each phase encoding step was preceded by an EPR saturation pulse to elicit the Overhauser enhancement[17]. All OMRI studies employed the following conditions: TR, 1200 ms; TE, 25 ms; TEPR, 600 ms; number of phase-encoding gradient steps, 64; NEX, 4; FOV, 3.2 cm × 3 .2 cm; slice thickness, 30 mm; scanning time, 60s; Details of the scanner are presented elsewhere [17].

Figure 4.

Figure 4

Phantom images of OMRI and MRI using wide range of nitroxide concentrations. Schematics of the 3CP, Tempol and 15N-PDT phantoms and A) OMRI and B) MRI images (left column). The phantoms contained 0.2 ∼ 10 mM nitroxides for OMRI and 0.2 ∼ 20 mM nitroxides for MRI, the signal intensity change (center column) and SNR (right column) are shown.

MRI

For MRI measurements, two other tubes (3.5 mm i.d.), containing 0.01 and 0.1 mM aqueous solutions of Gd3+-DTPA as a standard marker were additionally incorporated into the phantom (Fig. 2A). To compare with OMRI, the tubes were prepared as described in OMRI methods and two additional high concentration tubes (15, 20 mM) were added. MRI measurements were performed at 4.7 T controlled with ParaVision® 3.0.1 (Bruker BioSpin MRI GmbH, Rheinstetten, Germany) at room temperature (25 ± 2 °C). To perform T1 mapping, spin echo images were obtained using a multi-slice multi-echo (MSME) sequence with 5 different repetition times (TR: 4800, 3200, 1600, 800 and 400 ms). The scan time for T1 mapping image sets (NEX = 2) by the MSME sequence was 46 min. SPGR (also referred to as gradient echo fast imaging, GEFI) (TR = 75 ms, TE = 3 ms, Flip angle = 45°, NEX = 16) was employed to observe the T1 effect. The scan time for an image set (which included 2 slices) by the SPGR sequence was 160 s. Other typical parameters were as follows: image dimension, 256 × 256; FOV, 3.2 cm × 3.2 cm; slice thickness, 2.0 mm and number of slices, 2.

Redox imaging

The reduction of 3CP by AsA was used to demonstrate the redox imaging capability by the three techniques. The schematics of the four tube phantoms are shown in Figure 5A. Tube 1 contained PBS in the absence of 3CP. Tube 2 and 3 contained 2 mM 3CP with 5 and 10 mM AsA / phosphate buffered saline (PBS, pH 7.4), respectively. Tube 4 was filled with 2 mM 3CP without AsA. EPRI or OMRI imaging was started after addition of AsA / PBS solution (5 mM or 10 mM). Total volume of those phantoms was 500 μL. In the case of MRI experiment, since it takes time to start the measurement after preparation of sample because of shimming and tuning, 2.5 mM 3CP phantoms were initially set in the magnet before addition of AsA solution. Then 100 μL of 25 and 50 mM AsA / PBS solution was added (final AsA concentrations 5 and 10 mM) to 2.5 mM 3CP solution phantoms (400 μL, final concentration is 2 mM) through the PE-10 tube (OD = 0.63 mm) 2 min after the imaging protocol was started. The experiments were repeated three or four times. Semi-logarithmic plots of the time course of image intensity change in the region of interest (ROI) were used to compute the decay rates. The imaging conditions of EPRI, OMRI, and MRI experiments were follows; EPRI: RF, 300 MHz; microwave power, 2.5 mW; field modulation frequency, 13.5 kHz; field modulation amplitude, 0.8 G; time constant, 0.01 s; sweep width, 10 G; field gradient, 2.5 G / cm and number of averages, 2; approximate slice thickness, 25 mm (coil length) EPR image was reconstructed as a 128 × 128 matrix (FOV, 4.0 cm × 4.0 cm) by filtered back-projection method using Shepp-Logan filter. OMRI: TR, 1200 ms; TE, 25 ms; TEPR, 600 ms; number of phase-encoding gradient steps, 64; NEX, 1; FOV, 3.2 cm × 3.2 cm; slice thickness, 20 mm. MRI: TR, 75 ms; TE, 3 ms; Flip angle, 45°; NEX, 2; scan time, 20 s, image dimension, 256 × 256; FOV, s, 3.2 cm × 3.2 cm; slice thickness, 2.0 mm and number of slices, 2.

Figure 5.

Figure 5

Comparison of image intensity decay among three modalities. A) Schematic of the phantoms: Tube 1, PBS; Tube 2, 2 mM 3CP and 5 mM AsA; Tube 3, 2 mM 3CP and 10 mM AsA; Tube 4, 2 mM 3CP. Time course images and decay slops of B) EPRI, C) MRI, D) OMRI were obtained. After addition of AsA / PBS solution, the EPRI or OMRI measurements were started immediately and continuously measured up to 20 min. In the case of MRI, AsA /PBS was added using PE-10 tube 2 min after scanning was started. Therefore, time zero in the MRI experiment represents the time at addition of AsA solution. The experiments were repeated three or four times using with freshly prepared solutions. Semi-logarithmic plots of the time course of MRI signal change in the region of interest (ROI: 10 ×10 pixels) were used for decay rate calculation using imageJ software. Decay rate constants were obtained from the slope of linear portion of the decay curves.

Image Processing

The EPRI, MRI and OMRI data were analyzed using the ImageJ software package (http://rsb.info.nih.gov/ij/). T1 maps were calculated to determine relaxation times in each tube of the phantom using a plug-in (MRI analysis calculator, Karl Schmidt, HypX Laboratory, Brigham and Women’s Hospital) available in ImageJ. To obtain the relaxivity (it is the slope of relaxation rates as a function of concentration) the slope was calculated by the least squares method using relaxation rates (1/T1). To measure signal to noise ratio (SNR), 6 points of ROI in noise area were randomly measured and averaged. The signal intensity of phantom was then divided by averaged noise intensity.

Results

The x-band EPR spectrum of 15N-PDT under aerobic conditions at room temperature had the narrowest line width (0.26 ± 0.001G) followed by Tempone and 3CP were 0.40 ± 0.001 G and 1.08 ± 0.001 G, respectively. Tempol had lowest signal height in these nitroxides because of its relatively larger line width (1.50 ± 0.004 G).

Figure 2 presents the EPR and MR images of a phantom object containing several different concentrations of nitroxides. Among the EPR images, the phantom containing 2 mM 15N-PDT showed highest intensity compared with other nitroxides. Even the tube containing the lowest concentration (0.25 mM) can be readily identified in the phantom. In the case of Tempol, adjacent tubes in the phantom were not well separated because of poor image resolution (Fig. 2E). On the other hand, the MR images showed similar intensity and resolution in all nitroxides (Fig. 2, right column). The differential change in relaxation induced intensity increase as a function of nitroxide concentration could not be easily discerned by simple inspection.

The change of EPR image intensity was linear as a function of the nitroxide concentrations (R2 = 0.99 for all nitroxides). The intensities and slopes, which are related in dynamic range, were different depending on the individual nitroxides. If dynamic range is large, it shows better sensitivity in EPR detection. The slope was highest for the 15N-PDT among the nitroxides tested (Fig. 3A). Additionally, for concentration > 2 mM, the EPRI data exhibited deviation from the linear dependence (data not shown). In contrast, T1 weighted image of water protons showed similar slopes and intensity change (%) in all nitroxides (Fig. 3B). The linear dependence of MRI intensity on nitroxide concentration persisted up to 5 mM (R2 = 0.98) (Fig. 3B and 4B). Proton T1 relaxivities of all nitroxides were not significantly different (0.16 ∼ 0.18 mM-1 s-1). For a meaningful quantitative comparison, the SNR of the images was computed and plotted in the right column of Figure 3 (right column). The range of SNR in 15N-PDT was from 8 to 67. In contrast, all nitroxides showed similar SNR change from 32 to 49 in MRI. The SNR of water phantom in MRI was 34. Dynamic range of SNR in MRI was smaller compared to EPRI. It is to be noted, however, that EPRI intensity is the projection of the entire concentration of the solution in the tube corresponding to a pseudo-slice of 25 mm (the height of the solution in the tubes). On the other hand, the MRI intensity was originating from only a 2 mm thickness.

For the evaluation of OMRI, a seven tube phantom was imaged and compared with MRI (Fig. 4). The imaging experiments were extended up to 10 mM in nitroxide concentrations for OMRI and 20 mM for MRI experiments. The performance of OMRI was similar to that of EPRI, in terms of linewidth dependent sensitivity and non-linear concentration dependence above 2 mM (3CP and Tempol) or 5 mM (15N-PDT) solution. Interestingly, MRI image intensity progressively increased even up to 20 mM in nitroxide concentration.

To assess the capabilities to monitor redox reactions, the three modalities were evaluated using the redox phantom comprising of 3CP and AsA (Fig. 5). All modalities tested were capable of monitoring the redox transformation in the 3CP phantom through its reaction with AsA. After mixing of AsA solution, image intensity gradually decreased, and decay rate of intensity was dependent on AsA concentration (Fig. 5 and table 1). Approximately 19 min after the addition of AsA, image intensity in tube 3 completely disappeared in EPR and OMRI images, although MRI showed original proton images. Reduction rates were same among all these modalities, suggesting that any one of the methodology can evaluate the redox status (table 1). However, the spatial and temporal resolution available from MRI was superior compared to EPRI or OMRI.

Table 1.

Image parameters and comparison of three modalities of redox phantom experiments

EPRI OMRI MRI
Field of View (mm) 40 × 40 32 × 32 32 × 32
Image resolution (mm) > 1 0.5 0.125
Slice thisckness (mm) 25 20 2
Average 2 1 2
slice 1 3 (max 6)
Scan time (s) 54 60 20

N = 3 N = 4 N = 3
Decay rates (min-1)
5 mM AsA 0.07±0.004 0.07±0.01 0.07±0.01
10 mM AsA 0.14±0.01 0.14±0.002 0.13±0.01

No significant difference among the modalities was observed in the t test.

Discussion

With the availability of exogenous, non-toxic, bio-compatible free radical nitroxide probes, EPRI has shown its potential to provide, in a non-invasive manner, valuable information pertaining to redox status [3; 4; 28; 29]. Metabolically responsive nitroxides are useful to non-invasively delineate tissue heterogeneity differences that may exist between normal and malignant tissue with respect to redox status, using EPRI, OMRI and high-field MRI. The present study was designed to evaluate each of these modalities for their potential in redox imaging.

EPR spectral intensities were dependent on spin of nitrogen nucleus (hyperfine splitting) and line width under the conditions of identical concentration and temperature. As expected, the image of 15N-PDT, which exhibits a narrow doublet hyperfine pattern from the 15N nucleus, showed the highest intensity and superior quality image (in terms of intensity and resolution) among nitroxides tested in this paper. On the other hand, image from the Tempol phantom could not be clearly visualized by EPRI because of the relatively large line width. Deconvolution procedure can eliminate the influence of the EPR line shape giving pure spatial information [30; 31]. However, image intensity should be carefully re-normalized using signal intensities of zero gradient spectra obtained at suitable time points since the deconvolution process normalizes signal intensity. We have, however, not used any deconvolution procedures, and the images given are obtained by simple 2D filtered back-projection with a Shepp-Logan filter. In SPGR based T1-weighted MRI, the resolution is not affected by EPR line width of nitroxide. Therefore, the MR images of the four nitroxides showed similar resolution and intensity change. The image enhancement in MRI is dependent on the original T1 relaxation time and its variation by free radicals, which is related to the accessibility of the radical to water protons. In this paper, all nitroxides tested showed similar relaxivity. Image enhancement in these nitroxides should be equal in tissue having the same T1 relaxation time. On the other hand, image enhancement is affected by change of spectral shape and height in EPRI and OMRI. The near-independence of the proton relaxivity on the nature of nitroxide makes it easy to compare pharmacokinetics and redox status by MRI, whereas in OMRI and EPRI, the line width and spectral multiplicity determines the image quality in terms of resolution and dynamic range.

OMRI showed well resolved nitroxide images. The image intensity was dramatically affected by line width of the compound and experienced deep reduction in enhancement beyond 2 mM (3-CP and Tempol) or 5mM (15N-PDT), although image intensity went up in EPRI (albeit non-linear) and MRI, suggesting that OMRI enhancement is more sensitive to line broadening compared to EPRI. This is because the Overhauser enhancement is related to saturation of the EPR line which in turn depends on the line width. In contrast, the MR image intensity change was nearly linear up to 5 mM suggesting that we can use a much larger (nearly 2.5 times) nitroxide dose in MRI to improve the SNR, compared to EPRI or OMRI.

To evaluate the relative capabilities of EPRI, MRI, and OMRI using nitroxides as metabolically responsive probes, redox reactions driven by AsA to reduce the nitroxide 3CP to a diamagnetic state, was employed in all modalities. Continuous measurements of nitroxide levels examined detect similar decay rates from all three modalities (Fig. 5). The similar decay rates obtained from the phantom using MRI and EPRI, which is an accepted method in monitoring nitroxide levels validate the use of MRI for such studies. With MRI, multi-slice images were obtained in 20 s using SPGR method. This scanning allows fast pharmacokinetic imaging, besides providing detailed anatomical structure unlike in EPRI. In addition to anatomical structure, dynamic information such as blood flow, blood volume and water diffusion coefficients, etc., can be monitored by MRI and compared with redox images. Therefore, in vivo redox MR imaging using nitroxide can be a useful technique to monitor pharmacokinetics of nitroxide radicals. In particular, nitroxide such as Tempol, which has large line width and undergoes rapid in vivo reduction, will be a useful redox status-sensitive contrast agent. In this context, it is to be noted that the application of Tempol as a radioprotector to prevent alopecia is under phase I study [32].

Conclusion

EPRI and OMRI have several advantages to monitor nitroxide redox status. Dynamic ranges of image intensity in EPRI and OMRI are large compared to MRI, and spectral information leading to quantification of in vivo pO2 is an additional bonus. However, the image quality and intensity are not as good as that of proton images in MRI. On the other hand, MRI can equally effectively provide in redox status and pharmacokinetics using nitroxides with the additional advantages of well resolved anatomical images and other functional information such as blood-volume, blood flow, water diffusion coefficient etc.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reference

  • [1].Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med. 2001;30:1191–212. doi: 10.1016/s0891-5849(01)00480-4. [DOI] [PubMed] [Google Scholar]
  • [2].Kuppusamy P, Afeworki M, Shankar RA, Coffin D, Krishna MC, Hahn SM, Mitchell JB, Zweier JL. In vivo electron paramagnetic resonance imaging of tumor heterogeneity and oxygenation in a murine model. Cancer Res. 1998;58:1562–8. [PubMed] [Google Scholar]
  • [3].Kuppusamy P, Li H, Ilangovan G, Cardounel AJ, Zweier JL, Yamada K, Krishna MC, Mitchell JB. Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels. Cancer Res. 2002;62:307–12. [PubMed] [Google Scholar]
  • [4].Matsumoto K, Hyodo F, Matsumoto A, Koretsky AP, Sowers AL, Mitchell JB, Krishna MC. High-resolution mapping of tumor redox status by magnetic resonance imaging using nitroxides as redox-sensitive contrast agents. Clin Cancer Res. 2006;12:2455–62. doi: 10.1158/1078-0432.CCR-05-2747. [DOI] [PubMed] [Google Scholar]
  • [5].Krishna MC, Russo A, Mitchell JB, Goldstein S, Dafni H, Samuni A. Do nitroxide antioxidants act as scavengers of O2-. or as SOD mimics? J Biol Chem. 1996;271:26026–31. doi: 10.1074/jbc.271.42.26026. [DOI] [PubMed] [Google Scholar]
  • [6].Krishna MC, Samuni A, Taira J, Goldstein S, Mitchell JB, Russo A. Stimulation by nitroxides of catalase-like activity of hemeproteins. Kinetics and mechanism. J Biol Chem. 1996;271:26018–25. doi: 10.1074/jbc.271.42.26018. [DOI] [PubMed] [Google Scholar]
  • [7].Krishna MC, Grahame DA, Samuni A, Mitchell JB, Russo A. Oxoammonium cation intermediate in the nitroxide-catalyzed dismutation of superoxide. Proc Natl Acad Sci U S A. 1992;89:5537–41. doi: 10.1073/pnas.89.12.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Soule BP, Hyodo F, Matsumoto K, Simone NL, Cook JA, Krishna MC, Mitchell JB. The chemistry and biology of nitroxide compounds. Free Radic Biol Med. 2007;42:1632–50. doi: 10.1016/j.freeradbiomed.2007.02.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Swartz HM, Sentjurc M, Morse PD. 2nd, Cellular metabolism of water-soluble nitroxides: effect on rate of reduction of cell/nitroxide ratio, oxygen concentrations and permeability of nitroxides. Biochim Biophys Acta. 1986;888:82–90. doi: 10.1016/0167-4889(86)90073-x. [DOI] [PubMed] [Google Scholar]
  • [10].Berliner JL, Fujii H. Magnetic resonance imaging of biological specimens by electron paramagnetic resonance of nitroxide spin labels. Science. 1985;227:517–9. doi: 10.1126/science.2981437. [DOI] [PubMed] [Google Scholar]
  • [11].Berliner LJ, Fujii H, Wan XM, Lukiewicz SJ. Feasibility study of imaging a living murine tumor by electron paramagnetic resonance. Magn Reson Med. 1987;4:380–4. doi: 10.1002/mrm.1910040410. [DOI] [PubMed] [Google Scholar]
  • [12].He G, Deng Y, Li H, Kuppusamy P, Zweier JL. EPR/NMR co-imaging for anatomic registration of free-radical images. Magn Reson Med. 2002;47:571–8. doi: 10.1002/mrm.10077. [DOI] [PubMed] [Google Scholar]
  • [13].Hyodo F, Yasukawa K, Yamada K, Utsumi H. Spatially resolved time-course studies of free radical reactions through EPRI/MRI fusion technique. Magn Reson Med. doi: 10.1002/mrm.21019. In press. [DOI] [PubMed] [Google Scholar]
  • [14].Guiberteau T, Grucker D. Dynamic nuclear polarization imaging in very low magnetic fields as a noninvasive technique for oximetry. J Magn Reson. 1997;124:263–6. doi: 10.1006/jmre.1996.1055. [DOI] [PubMed] [Google Scholar]
  • [15].Liebgott T, Li H, Deng Y, Zweier JL. Proton electron double resonance imaging (PEDRI) of the isolated beating rat heart. Magn Reson Med. 2003;50:391–9. doi: 10.1002/mrm.10534. [DOI] [PubMed] [Google Scholar]
  • [16].Lurie DJ, Davies GR, Foster MA, Hutchison JM. Field-cycled PEDRI imaging of free radicals with detection at 450 mT. Magn Reson Imaging. 2005;23:175–81. doi: 10.1016/j.mri.2004.11.051. [DOI] [PubMed] [Google Scholar]
  • [17].Krishna MC, English S, Yamada K, Yoo J, Murugesan R, Devasahayam N, Cook JA, Golman K, Ardenkjaer-Larsen JH, Subramanian S, Mitchell JB. Overhauser enhanced magnetic resonance imaging for tumor oximetry: coregistration of tumor anatomy and tissue oxygen concentration. Proc Natl Acad Sci U S A. 2002;99:2216–21. doi: 10.1073/pnas.042671399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Halpern HJ, Yu C, Peric M, Barth E, Grdina DJ, Teicher BA. Oxymetry deep in tissues with low-frequency electron paramagnetic resonance. Proc Natl Acad Sci U S A. 1994;91:13047–51. doi: 10.1073/pnas.91.26.13047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Utsumi H, Yamada K, Ichikawa K, Sakai K, Kinoshita Y, Matsumoto S, Nagai M. Simultaneous molecular imaging of redox reactions monitored by Overhauser-enhanced MRI with 14N- and 15N-labeled nitroxyl radicals. Proc Natl Acad Sci U S A. 2006;103:1463–8. doi: 10.1073/pnas.0510670103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Brasch RC, London DA, Wesbey GE, Tozer TN, Nitecki DE, Williams RD, Doemeny J, Tuck LD, Lallemand DP. Work in progress: nuclear magnetic resonance study of a paramagnetic nitroxide contrast agent for enhancement of renal structures in experimental animals. Radiology. 1983;147:773–9. doi: 10.1148/radiology.147.3.6844613. [DOI] [PubMed] [Google Scholar]
  • [21].Ehman RL, Wesbey GE, Moon KL, Williams RD, McNamara MT, Couet WR, Tozer TN, Brasch RC. Enhanced MRI of tumors utilizing a new nitroxyl spin label contrast agent. Magn Reson Imaging. 1985;3:89–97. doi: 10.1016/0730-725x(85)90013-x. [DOI] [PubMed] [Google Scholar]
  • [22].Swartz HM, Chen K, Pals M, Sentjurc M, Morse PD. 2nd, Hypoxia-sensitive NMR contrast agents. Magn Reson Med. 1986;3:169–74. doi: 10.1002/mrm.1910030126. [DOI] [PubMed] [Google Scholar]
  • [23].Gallez B, Bacic G, Goda F, Jiang J, O’Hara JA, Dunn JF, Swartz HM. Use of nitroxides for assessing perfusion, oxygenation, and viability of tissues: in vivo EPR and MRI studies. Magn Reson Med. 1996;35:97–106. doi: 10.1002/mrm.1910350113. [DOI] [PubMed] [Google Scholar]
  • [24].Hayes C, Padhani AR, Leach MO. Assessing changes in tumour vascular function using dynamic contrast-enhanced magnetic resonance imaging. NMR Biomed. 2002;15:154–63. doi: 10.1002/nbm.756. [DOI] [PubMed] [Google Scholar]
  • [25].Padhani AR. Dynamic contrast-enhanced MRI in clinical oncology: current status and future directions. J Magn Reson Imaging. 2002;16:407–22. doi: 10.1002/jmri.10176. [DOI] [PubMed] [Google Scholar]
  • [26].Hyodo F, Matsumoto K, Matsumoto A, Mitchell JB, Krishna MC. Probing the Intracellular Redox Status of Tumors with Magnetic Resonance Imaging and Redox-Sensitive Contrast Agents. Cancer Res. doi: 10.1158/0008-5472.CAN-06-0879. [DOI] [PubMed] [Google Scholar]
  • [27].Koscielniak J, Devasahayam N, Moni M, Kuppusamy P, Yamada K, Mitchell JB. 300 MHz continuous wave electron paramagnetic resonance spectrometer for small animal in vivo imaging. Rev. Sci.Instrum. 2000;71:4273–81. [Google Scholar]
  • [28].Elas M, Williams BB, Parasca A, Mailer C, Pelizzari CA, Lewis MA, River JN, Karczmar GS, Barth ED, Halpern HJ. Quantitative tumor oxymetric images from 4D electron paramagnetic resonance imaging (EPRI): methodology and comparison with blood oxygen level-dependent (BOLD) MRI. Magn Reson Med. 2003;49:682–91. doi: 10.1002/mrm.10408. [DOI] [PubMed] [Google Scholar]
  • [29].Matsumoto K, Subramanian S, Devasahayam N, Aravalluvan T, Murugesan R, Cook JA, Mitchell JB, Krishna MC. Electron paramagnetic resonance imaging of tumor hypoxia: enhanced spatial and temporal resolution for in vivo pO2 determination. Magn Reson Med. 2006;55:1157–63. doi: 10.1002/mrm.20872. [DOI] [PubMed] [Google Scholar]
  • [30].Ohno K. ESR imaging: a deconvolution method for hyperfine patterns. J Magn Reson. 1982;50:145–50. [Google Scholar]
  • [31].Kuppusamy P, Chzhan M, Zweier JL. Development and optimization of three-dimensional spatial EPR imaging for biological organs and tissues. J Magn Reson B. 1995;106:122–30. doi: 10.1006/jmrb.1995.1022. [DOI] [PubMed] [Google Scholar]
  • [32].Metz JM, Smith D, Mick R, Lustig R, Mitchell J, Cherakuri M, Glatstein E, Hahn SM. A phase I study of topical Tempol for the prevention of alopecia induced by whole brain radiotherapy. Clin Cancer Res. 2004;10:6411–7. doi: 10.1158/1078-0432.CCR-04-0658. [DOI] [PubMed] [Google Scholar]

RESOURCES