Hyperpolarized 13C Spectroscopic Evaluation of Oxidative Stress in a Rodent Model of Steatohepatitis

Abstract

Nonalcoholic fatty liver disease (NAFLD) has become highly prevalent, now considered the most common liver disease in the western world. Approximately one-third of patients with NASH develop non-alchoholic steatohepatitis (NASH), histologically defined by lobular and portal inflammation, and accompanied by marked oxidative stress. Patients with NASH are at increased risk for cirrhosis and hepatocellular carcinoma, and diagnosis currently requires invasive biopsy. In animal models of NASH, particularly the methionine-choline deficient (MCD) model, profound changes are seen in redox enzymes and key intracellular antioxidants. To study antioxidant status in NASH non-invasively, we applied the redox probe hyperpolarized [1-¹³C] dehydroascorbic acid (HP DHA), which is reduced to Vitamin C (VitC) rapidly in the normal liver. In MCD mice, we observed a significant decrease in HP DHA to VitC conversion that accompanied hepatic fat deposition. When these animals were subsequently placed on a normal diet, resonance ratios reverted to those seen in control mice. These findings suggest that HP DHA, a potentially clinically translatable imaging agent, holds special promise in imaging NASH and other metabolic syndromes, to monitor disease progression and response to targeted therapies.

Figure 1. Imaging oxidative stress in vivo using HP [1-¹³C] DHA.

The probe is polarized using the dynamic nuclear polarization (DNP) technique, in a concentrated solution containing an unpaired electron source. Following dissolution and intravenous injection, HP [1-¹³C] DHA is transported rapidly into cells via glucose (GLUT) transporters. Enzyme mediated two-electron reduction of [1-¹³C] DHA to [1-¹³C] VitC is detected spectroscopically. This conversion depends on cellular reducing capacity, which is diminished in the setting of oxidative stress.

Figure 2. Lipid content of the liver in MCD-diet mice investigated by ¹H MRI and histologic analysis.

(a) Consistent with prior reports, a 27% decrease in total body weight was observed for MCD mice (b) Fat-water ¹H imaging revealed a 3-fold increase in hepatic fat content. (c) Representative ¹H MRI images from normal and MCD-diet mice by standard T₂-weighted and fat-water imaging. Studies were conducted on a vertical wide-bore 14T magnet. While only subtle changes were observed using the T₂-weighted sequence, dramatic hepatic steatosis was seen using the fat-water method. (d) Representative histologic sections using both hematoxylin and eosin as well as Oil Red O staining. The former shows microvesicular steatosis, while the latter shows significant lipid staining.

Figure 3. Study of control and MCD groups by HP DHA ¹³C MRSI.

(a) Representative ¹³C spectra obtained from the livers of MCD-diet mice using HP DHA at two weeks. Voxels corresponding to the liver for both C and MCD mice are juxtaposed with T₂-weighted images acquired at the time of the HP experiment. Resonances corresponding to [1-¹³C] DHA and [1-¹³C] VitC are depicted, with a clear decrease in the HP VitC to DHA ratio observed in the MCD-diet animals. (b) Scatter plots showing the metabolite ratios derived from animal experiments for the normal and MCD groups, expressed both as VitC/VitC + DHA and VitC/DHA. These ratios decreased significantly by 35% and 49% respectively (p < 0.05 for both) in the livers of MCD-diet animals relative to controls.

Figure 4. Study of recovery phase animals (MCDr group) by HP DHA.

(a) Representative ¹³C spectra obtained from the MCD group are compared to those obtained from mice first fed the MCD diet, then normal chow for 1 week. The HP DHA to VitC resonance ratios in the recovery group appear similar to those obtained for controls. (b) Bar graphs obtained for MCDr mice show significant increases in VitC/VitC + DHA and VitC/DHA ratios (p < 0.05) following return to a normal diet, which were not significantly different from baseline mice. (c) Although body weights in MCDr mice returned to normal, there was still significant hepatic steatosis (30%) seen in these animals, 2-fold that seen in the control group. (d) Mercury Orange staining showing decreased thiol content in MCD mice. Significant normalization was seen in the recovery group.

Figure 5. Model of oxidative stress imaging using HP DHA in the setting of NASH.

In MCD mice, markedly decreased HP DHA to VitC conversion was seen compared to controls. The ¹³C spectra returned to baseline following return to a normal diet, while hepatic fat content remained significantly elevated.