Real Time Direct Detection of β – hydroxybutyrate Production in Perfused Mice Livers Using HP DHA

Abstract

Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH) are increasingly prevalent in the United States. A diagnosis of NAFLD left untreated directly leads to the increase of NASH morbidity which is a result of over‐nutrition, poor diet, lack of exercise, and a sedentary lifestyle. However, NAFLD is typically reversible with a change in lifestyle. NAFLD is a group of common conditions that occur when excess fat accumulates in the liver of those who consume little alcohol. Changes in ketogenesis within the liver has been associated with NAFLD development in high‐fat diet models in mice, and in humans, lower ketogenesis is associated with an increase in hepatic steatosis. Since changes in ketogenesis are associated with different physiological conditions, it would be beneficial if the de novo ketone production can be measured in real‐time.Here, we report the detection of β‐hydroxybutyrate (BHB) in perfused liver using hyperpolarized [2‐13C] dihydroxyacetone (HP‐DHA) and Magnetic Resonance Spectroscopy (MRS). Dihydroxyacetone (DHA) is avidly taken up by the liver and rapidly phosphorylated into dihydroxyacetone phosphate (DHAP). DHAP is then metabolized into (Fig. 1) phosphoenolpyruvate (PEP), glycerol‐3‐phosphate (G3P) and glucose (in fasted mice), representing glycolysis, fatty acid synthesis, and gluconeogenesis, respectively. HP‐DHA injection does not drive BHB production by a mass action effect. Fasting mice show significantly increased ketone production, but a low alanine and lactate signal are observed as gluconeogenesis is increased. BHB is a compelling indicator of rapid flux of carbon skeletons towards ketones. Metabolism of DHA to BHB requires at least 11 steps with most of the metabolites in fasted mice not being observable. This establishes the interplay between pool size and observable HP MR Signal.The chemical shift of BHB makes it a prime target for imaging, as it does not overlap with other resonances. While previous research designs have been too complicated for clinical application, this work sets a foundation for a straightforward approach in measuring real‐time ketogenesis even as part of a routine MRI exam. This method, in particular, could serve as a powerful diagnostic tool for NAFLD and its transition to NASH.Support or Funding InformationNIH R01DK105346Sum spectra obtained by adding individual transients of the pseudo‐2D NMR acquisition post HP DHA injection. DHA and DHA‐hydrate resonances are not shown. Note dramatically increased BHB intensity in fasting. In the fasted state, 3‐ carbon precursors of glucose (alanine and lactate) show much reduced intensity.Figure 1