Isotope tracers have played an important role in determining the fate of substrates through multiple biochemical pathways in intact tissue. Virtually all of these techniques rely upon measurement of relative specific activities or fractional enrichments in individual carbon positions, for example, in a given metabolic product. We-have recently developed a NMR technique which takes advantage of 13c-13c coupling between adjacent carbons in any metabolite to provide a substantially more comprehensive analysis of the fate of any metabolic label. The method has been termed """"""""isotopomer analysis"""""""" and is applicable under a wide variety of physiological conditions, including nonsteady-state (NSS). We now proposed to use the method to examine a variety of metabolic issues in intact animals including fatty acid utilization in an in vivo pig heart model during control and postischemic periods and fatty acid, glucose, lactate, and ketone body contributions to the acetyl-CoA pools in vivo rat heart, liver, and brain in control animals, fasted animals, and in an animal model which simulates heavy exercise. For those tissues which achieve a steadystate (SS) isotopic distribution (this can be tested by comparing results from a SS and NSS isotopomer analysis of the same NMR data), the anaplerotic flux through the citric acid cycle pools relative to TCA cycle flux and the labeling pattern of the anaplerotic substrate may also be evaluated. These studies should provide a more complete, quantitative picture of the metabolic fate of various labeled subtrates in tissue and help resolve some current controversies involving fatty acid utilization in the ischemic myocardium, the direct versus indirect pathway for glycogen synthesis from glucose, and alternate sources of energy for the brain. we also propose to use these same techniques to examine the mechanism of oxygen wasting in heart muscle exposed to fatty acids using a perfused, working heart model and to investigate the possible metabolic significance of substrate channeling in tissue mitochondria under various physiological conditions.
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