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Despite the central role of the liver in the regulation of glucose and lipid metabolism, there are currently no methods to directly assess hepatic oxidative metabolism in humans in vivo. By using a new ¹³C-labeling strategy in combination with ¹³C magnetic resonance spectroscopy, we show that rates of mitochondrial oxidation and anaplerosis in human liver can be directly determined noninvasively. Using this approach, we found the mean rates of hepatic tricarboxylic acid (TCA) cycle flux (V_TCA) and anaplerotic flux (V_ANA) to be 0.43 ± 0.04 μmol g⁻¹ min⁻¹ and 0.60 ± 0.11 μmol g⁻¹ min⁻¹, respectively, in twelve healthy, lean individuals. We also found the V_ANA/V_TCA ratio to be 1.39 ± 0.22, which is severalfold lower than recently published estimates using an indirect approach. This method will be useful for understanding the pathogenesis of nonalcoholic fatty liver disease and type 2 diabetes, as well as for …