Interpreting Hyperpolarized ¹³C: Even Simple Signals Are Challenging

If sufficient signal can be acquired, ¹³C NMR is overwhelmingly the most powerful method for analysis of metabolism in biological systems ranging from cultured cells to sick patients. The introduction of hyperpolarized ¹³C dramatically expands the opportunities for ¹³C NMR in biological studies. The metabolic pathways and the physics controlling the ¹³C signal are well-understood and for this reason it would seem that acquisition of spectra and images would enable delineation of specific metabolic pathways. In the instance of products arising from a single enzyme-catalyzed reaction, HP should noninvasively detect a single reaction step. Data interpretation should be simple.

A supportive example is the appearance of HP [¹³C]bicarbonate during metabolism of [1-¹³C]pyruvate. In general, ¹³CO2 arises from [1-¹³C]pyruvate via pyruvate dehydrogenase or carboxylation of pyruvate followed by entry into the TCA cycle and decarboxylation at one or more steps. The relative activity of these pathways is tissue-specific. In the heart, by performing parallel studies with HP [1-¹³C]pyruvate and [3-¹³C]pyruvate followed by tissue extraction and high resolution spectroscopy, it was a simple matter to show that the great majority of HP [13C]bicarbonate was generated by flux through pyruvate dehydrogenase (PDH, [1]).

The same approach was used to probe the sources of HP [¹³C] bicarbonate from cancer cells [2]. A glioblastoma and hepatoma cell lines were compared. Both cell lines generated HP[¹³C]bicarbonate from [1-¹³C]pyruvate. By integration with conventional ¹³C NMR of cell extracts during metabolism of [3-¹³C]pyruvate, it was possible to show that a small amount of the HP[13C]bicarbonate arose from pyruvate carboxylation and decarboxylation in the hepatoma cells, and that all of the HP[¹³C]bicarbonate was generated via PDH in the glioblastoma cell lines. HP and conventional ¹³C NMR were again consistent and in cancer, it appears that flux through both PDH and PEPCK must be considered.

In the liver, in contrast to both cancer and the heart, pyruvate carboxylation is highly active and we hypothesized that all of the HP[¹³C]bicarbonate appearance was due to flux through PEPCK. Indeed HP[13C]bicarbonate disappeared after knockout of PEPCK in the mouse, consistent with this hypothesis [3]. However, others [4] attributed the bicarbonate signal to PDH flux, exactly like the heart. Therefore we re-examined the sources of HP[¹³C]bicarbonate in the rat liver in vivo under fed conditions (thereby suppressing gluconeogenesis and PEPCK flux) and fasting conditions (stimulating gluconeogenesis and PEPCK flux). The gold standard for PEPCK flux was conversion of the hyperpolarized [1-¹³C]pyruvate to 13C-glucose present in plasma. We found that infused HP [1-¹³C]pyruvate was not converted to plasma glucose in the fed condition but it was readily converted to plasma glucose under fasted conditions, exactly as one would expect. Surprisingly, HP [¹³C]bicarbonate followed the opposite pattern: it was only detected in fed conditions. In fasted animals where measurement of ¹³C in plasma glucose proved flux through PEPCK, HP [¹³C]bicarbonate could not be detected.

The absence of HP[¹³C]bicarbonate from the liver in vivo even when flux through PEPCK is unquestionably active is likely due to loss of polarization in the multi-step reactions involved in conversion of pyruvate to phospho-enolpyruvate. The metabolic mechanisms responsible for generating "simple" ¹³C signals such as lactate, bicarbonate and others may be complex under in vivo conditions.

References:
[1] Merritt et al. Hyperpolarized ¹³C allows a direct measure of flux through a single enzyme-catalyzed step by NMR. Proc Natl Acad Sci U S A. 2007; 104; 19773-19777. PMID: 18056642;
[2] Yang et al. Simultaneous Steady-state and Dynamic ¹³C NMR Can Differentiate Alternative Routes of Pyruvate Metabolism in Living Cancer Cells. J Biol Chem. 2014; 289: 6212-24. PMID: 24415759; PubMed Central PMCID: PMC3937686;
[3] Merritt et al. Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized ¹³C magnetic resonance. Proc Natl Acad Sci U S A. 2011; 108: 19084-9. PMID: 22065779;
[4] Lee et al. In vivo hyperpolarized carbon-13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an insulin-resistant mouse model. Hepatology. 2013; 57: 515-24. PMID: 22911492