Ketone bodies are an important alternative source of fuel to the body during limited carbohydrate supply. Hyperpolarized acetoacetate allows a unique method for the in vivo study of ketone bodies metabolism. In this study, we compared differences in ketone bodies metabolism of the rat heart during normal feeding and after 24hr fasting.[3-13C]lithium acetoacetate was produced from [3-13C]ethyl-acetoacetate using the procedure described by Hall et al. Mixture of acetoacetate, OX063 and gadolinium was polarized using a HyperSense hyperpolarizer (Oxford instruments) for approximately 2hr. In vivo experiments were performed on a 9.4T/31cm horizontal bore MRI scanner (Agilent Technologies Inc) with a dual tuned 1H/13C cardiac coil (Rapid Inc) for RF transmission and signal reception. Animal studies were approved by A*STAR Institutional Animal Care and Use Committee. A pulse and acquire spectroscopy sequence was started immediately before the intravenous injection of 1.0-1.5mL (0.3mmol/kg body weight) of hyperpolarized acetoacetate over 3s into the anesthetized rat. The first 30s of spectral were summed and the ratios of the metabolite to substrate peaks were used for quantification.Various metabolites were detected after the delivery of the hyperpolarized [3-13C]acetoacetate (220ppm) substrate, including [1-13C]citrate (180ppm) and [1-13C]acetylcarnitine (175pm). The peak at 183ppm was likely due to the presence of unknown contaminants. From the paired student’s t-test, there is a significant increase in hyperpolarized [1-13C]citrate production when the animals are fasted for 24hours (Fig 2a). Also, a significant decrease in hyperpolarized [1-13C]acetylcarnitine formation after 24hours fasting (Fig 2b) was observed.

The increased citrate to acetoacetate ratio after fasting suggests that more ketone bodies derived acetyl-CoA enters the TCA cycle (Fig 3). In the fasted state, low blood glucose activates glucagon and together with epinephrine simulated through increased metabolic demands, they bind to G protein-coupled receptors to activate adenylate cyclase and form cyclic AMP. cAMP in turn activates protein kinase A which phosphorylates and activates hormone-sensitive lipase, thereby converting triglycerides into free fatty acids. The increased concentration of fatty acids in the blood further activates the B-oxidation pathway. Consequently, this leads to the production of ketone bodies in the liver which is then released into the bloodstream and transported to the peripheral tissues as an alternative fuel source.

A previous study by Ball et al using hyperpolarized 13C butyrate reported the lack of significance in citrate levels between the fed and fasted rat hearts. Butyrate metabolism involves both short chain fatty acid metabolism to produce acetoacetyl CoA via the actions of HCDH as well as ketone body metabolism to produce acetoacetyl CoA via SCOT1. On the other hand, the use of hyperpolarized 13C acetoacetate allows direct visualization of the contribution of ketone body metabolism to the TCA cycle via the observation of the TCA substrate citrate. It has previously been reported that ketone bodies are able to suppress cardiac fatty acid oxidation in diabetes (Hasselbaink et al 2003). As such, an increased input via ketone bodies metabolism coupled with a reduced input via fatty acid metabolism could have led to a constant citrate output.

Acetylcarnitine can act as a buffer for excess acetyl-CoA when the rate of acetyl-CoA synthesis is faster than the rate of acetyl-CoA entry into the TCA cycle through the catalytic actions of the enzyme carnitine acetyltransferase (CAT) which converts acetyl-CoA and carnitine to acetylcarnitine and CoA. In the fasted rat heart, the demand of acetyl-CoA originating from ketone bodies oxidation for use in the TCA cycle is greater than in the fed rat heart. This can result in less acetyl-CoA being converted to acetylcarnitine in the fasted rat heart (Fig 3).

A previous study by Kennedy et al reported no observable metabolic products after introduction of hyperpolarized hydroxybutyrate and acetoacetate in the mouse model. The use of a larger rat model should offer higher signal to noise ratio to allow minute quantities of metabolites to be detected.In this study, hyperpolarized acetoacetate has been shown to be able probe changes in ketone bodies metabolism in vivo in the rat heart with greater specificity than available hyperpolarized probes.