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

The contribution of a ¹³C-enriched substrate to the acetyl-CoA pool in animal tissues is typically measured by analysis of glutamate enrichment from tissue extracts. ¹³C 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 ¹H NMR measurement of glutamate C4 enrichment from tissue extracts. The method is demonstrated with heart and hindlimb muscle tissue extracts of rats infused with [2,4,6,8-¹³C₄]octanoate, a source of [2-¹³C]acetyl-CoA. Glutamate C4 enrichment in extracts of individual hindlimb soleus muscles weighing ≃150 mg and containing approximately 0.3 μmol of glutamate was quantified by ¹H NMR within about 40 min. Glutamate C4 enrichment measurements by ¹H NMR in heart and gastrocnemius muscle were also highly correlated with independent measurements obtained from ¹³C NMR isotopomer analysis.