This subcontract to Project 4 will examine metabolic and physiological aspects of acidification of human melanoma xenografts that are amenable to non-invasive measurement by magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). These methods will be applied to animals supplied by Dr. Leeper's laboratory to test the hypothesis that NMR can non-invasively detect tumor specific changes in extracellular and intracellular pH (pHe and pHi, respectively) induced by hyperglycemia and by hyperglycemia plus inhibition of oxidative metabolism (with meta- iodobenzylguanidine, MiBG), and/or by hyperglycemia plus inhibition of the Na+/H+ cation exchanger (with Cariporide mesilate, HOE642) and the HCO3- /Cl-anion exchanger (with 4,4-diisothiocyanstilbene 2,2-disulphonic acid, DIDS). In addition, it is proposed that NMR can help delineate the underlying mechanism of acidification by hyperglycemia plus regulatory inhibition. To test this hypothesis, 31P MRS will be employed to measure the pHi and pHe in the tumor, in host muscle, and brain under conditions of normoglycemia, hyperglycemia plus MIBG and/or HOE642 plus DIDS. Inorganic phosphate (Pi) and 3-aminopropylphosphonate will serve as indicators of pHi and pHe, respectively. To test the hypothesis that tumor acidification under conditions of hyperglycemia and hyperglycemia plus respiratory inhibition is mediated by production of lactic acid (i.e., glycolysis), steady state lactate concentrations will be monitored on 1H MRS employing a selective multiple quantum coherence transfer pulse sequence developed by the subcontractor's laboratory to edit out the lactate resonance from interfering lipid resonances. Further insight into the mechanism of tumor acidification under conditions of hyperglycemia +/- respiratory inhibition will be obtained by measuring flux through the glycolytic pathway and through the TCA cycle from the kinetics of 13C-labeling of lactate and glutamate, respective, following infusion of [1-13C]glucose. The spatial distributions of 13C-labeled glucose and lactate in the tumor at isotope steady state (for glucose) will be imaged by 13C chemical shift imaging at constant concentrations of labeled glucose in the blood (19+/-) mM), and the vascular volume distribution will be imaged by dynamic 1H MRI using Gd-DTPA-albumin as a vascular marker. Images will be correlated with histology.
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