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, detection and estimation of ROS generated 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. DMPO reacts with superoxide and hydroxyl radical, and generate a relatively stable radical called a spin adduct (Figure 1). The spin adduct is easily detected by using electron paramagnetic resonance (EPR) if it’s in vitro. However, the spin adduct is rapidly reduced in tissues, and lose the EPR signal that makes it harder to detect ROS in vivo by using EPR. 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 investigated feasibility of hyperpolarized ¹³C-DMPO to detect reactive oxygen species in living animals.30 μL of ¹³C-DMPO containing 15 mM Finland-HCl and 2.5 mM gadolinium chelate ProHance was polarized for approximately 2 hour using a hyperpolarizer (HyperSense, Oxford Instruments), and rapidly dissolved in 4.5 mL PBS containing 100 mg/L EDTA. The hyperpolarized ¹³C-DMPO (60 mM) was intravenously injected through a catheter placed in the tail vein of a mouse (12 μL/g body weight). ¹³C-MRI studies were performed on a 3 T scanner (MR Solutions) using a 17 mm home-built ¹³C solenoid coil placed inside of a saddle coil for ¹H. ¹³C two-dimensional spectroscopic images were acquired 25 s after the start of the ¹³C-DMPO injection, with a 32 x 32 mm² field of view in a 8 mm coronal slice through the body, a matrix size of 16 x 16, spectral width of 3.33 kHz, repetition time of 85 ms, and flip angle of 10º. The total time required to acquire each image was 22 sec.At first, non-labeled DMPO was hyperpolarized and measured with ¹³C-MR spectroscopy. We confirmed that hyperpolarized DMPO gave us a single peak at 76 ppm on the ¹³C-spectrum even it was non-labeled DMPO. We synthesized DMPO ¹³C-labeled at C5 position, and it was deuterated to increase the T₁ relaxation times. Hyperpolarized ¹³C-DMPO provided a strong signal compared with non-labeled DMPO, and the T₁ relaxation time was 60 sec after it was dissolved in PBS. Then, ¹³C-MR spectroscopic measurements were carried out in a C3H mouse. Hyperpolarized ¹³C-DMPO was intravenously injected into the mouse, and ¹³C spectra in the mouse body were acquired every 1 sec. The signal of ¹³C-DMPO was detected in the mouse body for more than 100 sec after the ¹³C-DMPO injection (Figure 2). We also carried out ¹³C-chemical shift imaging. The obtained ¹³C image revealed that ¹³C-DMPO was distributed through the mouse body (Figure 3). The signal was higher in the chest and abdomen region, and signal from liver region was relatively small compared to the other region.Hyperpolarized ¹³C-DMPO provided sufficient magnitude of the ¹³C signal to be detected in the mouse body, and the T₁ relaxation time was relatively long. We will test this probe in some disease models such as lipopolysaccharide treated to evaluate the capability for detection of ROS in vivo.