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Hyperpolarized 13C-MRI of DMPO and NAC for evaluating oxidative stress in living animal

Keita Saito¹, Deepak Sail², Burchelle N. Blackman², Hellmut Merkle³, Rolf E. Swenson², James B. Mitchell¹, and Murali C. Krishna¹

¹National Cancer Institute, Bethesda, MD, United States, ²National Heart, Lung, and Blood Institute, ³National Institute of Neurological Disorder and Stroke

Synopsis

5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is used to detect reactive oxygen species in vitro, and N-acetyl-L-cysteine (NAC) is an antioxidant. We synthesized ¹³C-labeled DMPO and NAC, and investigated feasibility of hyperpolarized ¹³C-DMPO and ¹³C-NAC for evaluating oxidative stress in mice. Hyperpolarized ¹³C-DMPO and ¹³C-NAC provided a single peak at 76 ppm and 174 ppm, respectively, and the T₁ relaxation time was sufficiently long to apply them for mouse imaging. The signals ¹³C-DMPO and ¹³C-NAC were also detected in a mouse body after intravenous injection. The results showed ¹³C-DMPO and ¹³C-NAC can be applied to some disease models to evaluate oxidative stress in vivo.

Introduction

Reactive oxygen species (ROS) such as superoxide anion radical and hydroxyl radical play an important role in various physiological processes, but on the other hand they readily react with biomolecules and disrupt those functions. The excessive production of ROS is harmful to living body, and considered to be related to various diseases. Therefore, evaluation of oxidative stress in living body would be of help not only to understand the mechanisms of the diseases but also to develop prophylactic and therapeutic methods. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a spin trap agent frequently used to detect oxygen radicals in vitro with electron paramagnetic resonance spectroscopy, and N-acetyl-L-cysteine (NAC) is an antioxidant clinically used. The reaction of both compounds with ROS were well investigated in vitro studies, but the pharmacokinetics, metabolism, and the reaction of them in vivo is not elucidated in vivo. Recent development of ¹³C-MRI with hyperpolarized ¹³C-labeled compounds enabled us to detect ¹³C-labeled compounds and those metabolites in vivo. In this study, we synthesized ¹³C-labeled DMPO and NAC, and investigated feasibility of hyperpolarized ¹³C-DMPO and ¹³C-NAC to evaluate oxidative stress in living animals.

Methods

30 μL of [5-¹³C]DMPO-d9 containing 15 mM Finland-HCl or 100 μL of [1-¹³C]NAC containing 15 mM OX063 was mixed with 1.5 μL of 50 mM gadolinium chelate ProHance, polarized for 1-2 hour using a hyperpolarizer (HyperSense, Oxford Instruments), and rapidly dissolved in 4.5 mL PBS containing 100 mg/L EDTA. The hyperpolarized [5-¹³C]DMPO-d9 (60 mM) or [1-¹³C]NAC (15 mM) was intravenously injected to a mouse through a catheter placed in the tail vein of the mouse (12 μL/g body weight). ¹³C-spectra in the mouse body were acquired every 1 sec after the injection, with the flip angle of 10º. ¹³C-MRI studies were performed on a 3 T scanner (MR Solutions) using a 30 mm home-built ¹³C saddle coil.

Results

Non-labeled DMPO and NAC were hyperpolarized, and ¹³C-MR spectra of them were measured using the 3T scanner. Hyperpolarized DMPO and NAC provided a single peak at 76 ppm and 174 ppm, respectively on the ¹³C-spectrum. We synthesized [5-¹³C]DMPO-d9 and [1-¹³C]NAC. They were hyperpolarized and dissolved in PBS to estimate the T₁ relaxation time. The T₁ relaxation time of [5-¹³C]DMPO-d9 and [1-¹³C]NAC was 60 sec and 18 sec, respectively in the 3 T magnet (Figure 1). Then, ¹³C-MR spectroscopic measurements were carried out with mice. The solution of hyperpolarized [5-¹³C]DMPO-d9 or [1-¹³C]NAC was intravenously injected into the mouse, and ¹³C spectra in the mouse body were acquired every 1 sec. The signal of [5-¹³C]DMPO-d9 was detected in the mouse body for more than 100 sec after the DMPO injection, and the signal was sufficiently large for imaging. ¹³C-chemical shift imaging revealed that DMPO was distributed through the mouse body (Figure 2). The signal was higher in the chest and abdomen region, and signal from liver region was relatively small compared to the other region. The signal of [1-¹³C]NAC was also detected in the mouse body, but it was smaller than DMPO.

Conclusion

Hyperpolarized ¹³C-DMPO provided sufficient magnitude of the ¹³C signal and long T₁ to be applied to some disease models of mice. The signal of NAC was also detected in mouse body, but the T₁ was short compared to DMPO, and optimization of the experimental conditions is necessary for further animal experiments.

Acknowledgements

This study was supported by intramural research program of NCI/NIH.

References

No reference found.

Figures

Signal intensity curves of 13C-DMPO and 13C-NAC

Chemical shift image of 13C-DMPO in mouse body

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)

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