Determination of acetyl-CoA enrichment in rat heart and skeletal muscle by H-1 nuclear magnetic resonance analysis of glutamate in tissue extracts

The contribution of a C-13-enriched substrate to the acetyl-CoA pool in animal tissues is typically measured by analysis of glutamate enrichment from tissue extracts. C-13 NMR analysis offers the advantages of minimal sample processing and high information content, but has a low analytical sensitivity compared to other methods of tracer analysis such as GC/MS. We present a sensitive, simple, and direct H-1 NMR measurement of glutamate C4 enrichment from tissue extracts. The method is demonstrated with heart and hindlimb muscle tissue extracts of rats infused with [2,4,6,8-C-13(4)]octanoate, a source of [2-C-13]acetyl-CoA. Glutamate C4 enrichment in extracts of individual hindlimb soleus muscles weighing approximate to 150 mg and containing approximately 0.3 mu mol of glutamate was quantified by H-1 NMR within about 40 min. Glutamate C4 enrichment measurements by H-1 NMR in heart and gastrocnemius muscle were also highly correlated with independent measurements obtained from C-13 NMR isotopomer analysis. (C) 1997 Academic Press.