PurposeButyrate, a short chain fatty acid, was studied as a novel hyperpolarized substrate for use in dynamic nuclear polarization enhanced magnetic resonance spectroscopy experiments, to define the pathways of short chain fatty acid and ketone body metabolism in real time.MethodsButyrate was polarized via the dynamic nuclear polarization process and subsequently dissolved to generate an injectable metabolic substrate. Metabolism was initially assessed in the isolated perfused rat heart, followed by evaluation in the in vivo rat heart.ResultsHyperpolarized butyrate was generated with a polarization level of 7% and was shown to have a T1 relaxation time of 20 s. These physical characteristics were sufficient to enable assessment of multiple steps in its metabolism, with the ketone body acetoacetate and several tricarboxylic acid cycle intermediates observed both in vitro and in vivo. Metabolite to butyrate ratios of 0.1–0.4% and 0.5–2% were observed in vitro and in vivo respectively, similar to levels previously observed with hyperpolarized [2-13C]pyruvate.ConclusionsIn this study, butyrate has been demonstrated to be a suitable hyperpolarized substrate capable of revealing multi-step metabolism in dynamic nuclear polarization experiments and providing information on the metabolism of fatty acids not currently achievable with other hyperpolarized substrates. Magn Reson Med 71:1663–1669, 2014. © 2013 Wiley Periodicals, Inc.