Oxidation of Intracellular and Extracellular Fatty Acids in Skeletal Muscle: Application of kinetic modeling, stable isotopes and liquid chromatography/electrospray ionization ion-trap tandem mass spectrometry technology.

Fatty acids are a major fuel for many tissues and abnormal utilization is implicated in diseases. However, tissue fatty acid oxidation has not been determined reliably in vivo. Furthermore, fatty acid oxidation has not been partitioned into intracellular and extracellular components. In this report, a one-pool model is described that enables direct quantitation of fluxes of intracellular and plasma fatty acids to mitochondria in skeletal muscle using dual stable isotopes and liquid chromatography/electrospray ionization ion-trap tandem mass spectrometry (LC/ESI-itMS2) technology. It is validated by the determination of palmitate oxidation by skeletal muscle in lean and obese rats and the regulation by insulin. Resting postabsorptive intramyocellular and plasma palmitate oxidation by gastrocnemius muscle was determined to be 3.47±0.8 and 2.06±0.5 nmol/g min in lean and 6.96±1.8 and 1.34±0.2 nmol/g min in obese rats, respectively. In obese rats, hyperinsulinemia (1 nmol/l) suppressed intramyocellular (by 59±5% to 2.88±0.3 nmol/g min P<0.05) but not plasma (1.41±0.14 nmol/g min, P>0.05) palmitate oxidation. The fractional turnover rate of palmitoylcarnitine (0.34±0.1/min vs. 0.83±0.2/min, P<0.05) was also suppressed by insulin. In obese and lean rats, there are 83% and 51%, respectively (P=0.08), of plasma fatty acids traverse triglyceride pool before being oxidized. The results demonstrated that the methodology is feasible and sensitive to metabolic alterations and thus can be used to study fatty acid utilization at tissue level in a compartmentalized manner for the firs time.