Fischer 344 rats (n = 9) were used for DMI experiments in healthy brain, liver, and glioma after implantation of RG2 cells. The glioma model was established as described previously (25). In short, RG2 glioma cells (American Type Culture Collection) were grown in T75 flasks using standard cell culture conditions (37°C and 5% CO2). RG2 cells were harvested, and ~1250 cells suspended in 5 μl of serum-free, low-glucose Dulbecco’s modified Eagle’s medium (DMEM) were injected in the brain of anesthetized rats.

For DMI experiments, rats were anesthetized with isoflurane using ~60% O2 and ~40% N2O delivered through a nose cone. A heating pad or heated air was used to maintain body temperature at ~37°C. Breathing, heart rate, and blood oxygenation parameters were monitored continuously using a pulse oximeter probe (MouseOx, Starr Life Sciences Corp.) on the foot.

Blood was sampled from an arterial catheter placed in the femoral artery (25). The blood sample was centrifuged (5000 rpm for 5 min), and the plasma was stored at −80°C for later processing. Plasma samples were prepared for high-resolution NMR by adding 160 μl of methanol to 80 μl of plasma to precipitate plasma proteins. This mixture was incubated at −20°C for 20 min, followed by centrifugation at 10,000 rpm for 30 min at 4°C. The supernatant was transferred to a 2-ml vial, and the methanol was evaporated under nitrogen using a TurboVap LV (Biotage) at 45°C for ~60 min. The sample was resuspended in 600 μl of a mixture of water containing phosphate buffer (100 mM), D2O (10%), sodium formate, and imidazole as a chemical shift reference and internal concentration standard, respectively.

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