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 13 C-labeled DMPO for hyperpolarization by dynamic nuclear polarization, in which 13 C NMR signal increases more than 10000-fold. This allows in vivo 13 C MRI to investigate the feasibility of in vivo ROS detection by the 13 C-MRI. DMPO was 13 C-labeled at C5 position, and deuterated to prolong the T 1 relaxation time. The overall yield achieved for 5-13 C-DMPO-d 9 was 15%. Hyperpolarized 5-13 C-DMPO-d provided a single peak at 76 ppm in the 13 C-spectrum, and the T 1 was 60 s in phosphate buffer making it optimal for in vivo 13 C MRI. The buffered solution of hyperpolarized 5-13 C-DMPO-d 9 was injected into a mouse placed in a 3 T scanner, and 13 C-spectra were acquired every 1 s. In vivo studies showed the signal of 5-13 C-DMPO-d 9 was detected in the mouse, and the T 1 decay of 13 C signal of hyperpolarized 5-13 C-DMPO-d 9 was 29 s. 13 C-chemical shift imaging revealed that 5-13 C-DMPO-d 9 was distributed throughout the body in a minute after the intravenous injection. A strong signal of 5-13 C-DMPO-d 9 was detected in heart/lung and kidney, whereas the signal in liver was small compared to other organs. The results indicate hyperpolarized 5-13 C-DMPO-d 9 *

Hyperpolarized 13C Magnetic Resonance Imaging as a Tool for Imaging Tissue Redox State, Oxidative Stress, Inflammation, and Cellular Metabolism

“…HP 13 C MRI, wherein more than 10,000 times enhancement of the MRI signal of 13 C-labeled redox-sensitive metabolic tracers can be realized, has enabled real-time, noninvasive imaging of metabolism in vivo and opened new research avenues for the field of redox biology. Although more than 60 13 C metabolic tracers have been reported and attempts have been made at developing specific tracers for detecting ROS ( 60 ), to date only a few metabolic tracers have been established for monitoring tissue redox state. As such, future development of new redox-sensitive metabolic tracers and a comprehensive understanding of the correlation between metabolic alterations and the redox state are essential.…”

Challenges and Advances in the Application of Dynamic Nuclear Polarization to Liquid-State NMR Spectroscopy

“…In the spin probe technique, decay in DNP enhancement can thus be used as a quantitative indicator of ROS . Similarly, change in chemical shift of hyperpolarized spin traps upon intermediate adduct-formation with ROS provides another way to detect and quantify ROS. …”

“…89 Similarly, change in chemical shift of hyperpolarized spin traps upon intermediate adduct-formation with ROS provides another way to detect and quantify ROS. 90 3.7. Increasing the Sensitivity of Alternative NMR Methodologies and Low-Field NMR.…”

Real-Time insight into in vivo redox status utilizing hyperpolarized [1-13C] N-acetyl cysteine

“…Unfortunately, dehydroascorbic acid causes transient respiratory arrest at relatively low concentrations (10 mg kg −1 ) in mice 10 as well as pancreatic toxicity 13 , conditions which may limit its translational potential. Hyperpolarized spin trap probes based on DMPO have been developed but are limited to detecting ROS production 14 , 15 . Toxicity concerns have also been expressed for the lanthanide based redox sensitive PARACEST MRI contrast agents 16 , 17 .…”