These studies were conducted using the isolated canine brain perfused with blood having a normal pH, PaO2, PaCO2, glucose, and temperature and employing a high field (9.4 Tesla), wide bore (89 mm) magnet (Bruker Instruments AM-400 WB) for the NMR studies. The region of the 31P spectrum associated with Pi was subjected to nonlinear curve fitting (Peak-Fitr, Jandel Scientific) to deconvolute the peaks. We have demonstrated that the broad inorganic phosphate (Pi) peak with a chemical shift of 4.00 to 5.25 ppm can be consistently resolved into five to seven separate peaks. A Pi buffer adjusted to pH 7.40 was infused over a 15 minute period to raise the blood Pi concentration from 2 to 4 mM. This caused peaks associated with compartments at pH 7.35 (plasma) and pH 7.18 (interstitial fluid) to increase in size. We also infused 40 mg of 2-deoxyglucose (2-DG) over a 30 minute period, and continued to collect data for an additional 30 minutes following infusion. Phosphorylation of 2-DG in the brain cells resulted in 2-DG-6-P accumulation in the phosphate monoester (PME) portion of the spectrum. The 2-DG-6-P peaks were at pHs which corresponded to the three peaks in the Pi spectrum which increased dramatically in size due to high energy phosphate hydrolysis during ischemia (pH 7.05, 6.95, and 6.82). Based on partial evidence from this and other studies, we believe these peaks represent the astrocyte, neuron, and possibly the oligodendrocyte. These studies suggested that there may normally be only three cytoplasmic and two extracellular compartments in the brain tissue Pi spectrum that are defined by pH. The Pi peak falls during 2-DG infusion. By subtracting the Pi spectrum during the 2-DG infusion from the control spectrum and fitting the difference, we find that Pi disappears from the same pH compartments in which 2-DG-6-P appears. Hyperglycemia during ischemia causes pHi to fall significantly further than during normoglycemic ischemia. Surprisingly, the hyperglycemic brains maintain some high energy phosphate after 10 minutes of complete ischemia.
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