Synthesis and evaluation of 13C-labeled 5-5-dimethyl-1-pyrroline-N-oxide aimed at in vivo detection of reactive oxygen species using hyperpolarized 13C-MRI

Abstract

Effective means to identify the role of reactive oxygen species (ROS) mediating several diseases including cancer, ischemic heart disease, stroke, Alzheimer's and other inflammatory conditions in in vivo models would be useful. The cyclic nitrone 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a spin trap frequently used to detect free radicals in vitro using Electron Paramagnetic Resonance (EPR) spectroscopy. In this study, we synthesized ¹³C-labeled DMPO for hyperpolarization by dynamic nuclear polarization, in which ¹³C NMR signal increases more than 10,000-fold. This allows in vivo ¹³C MRI to investigate the feasibility of in vivo ROS detection by the ¹³C-MRI. DMPO was ¹³C-labeled at C5 position, and deuterated to prolong the T₁ relaxation time. The overall yield achieved for 5-¹³C-DMPO-d₉ was 15%. Hyperpolarized 5-¹³C-DMPO-d₉ provided a single peak at 76 ppm in the ¹³C-spectrum, and the T₁ was 60 s in phosphate buffer making it optimal for in vivo ¹³C MRI. The buffered solution of hyperpolarized 5-¹³C-DMPO-d₉ was injected into a mouse placed in a 3 T scanner, and ¹³C-spectra were acquired every 1 s. In vivo studies showed the signal of 5-¹³C-DMPO-d₉ was detected in the mouse, and the T₁ decay of ¹³C signal of hyperpolarized 5-¹³C-DMPO-d₉ was 29 s. ¹³C-chemical shift imaging revealed that 5-¹³C-DMPO-d₉ was distributed throughout the body in a minute after the intravenous injection. A strong signal of 5-¹³C-DMPO-d₉ was detected in heart/lung and kidney, whereas the signal in liver was small compared to other organs. The results indicate hyperpolarized 5-¹³C-DMPO-d₉ provided sufficient ¹³C signal to be detected in the mouse in several organs, and can be used to detect ROS in vivo.

Keywords: DMPO: reactive oxygen species; Hyperpolarized (13)C-MRI; Spin trapping.