The administration of hyperpolarized (HP) ¹³C-labeled pyruvate enables the direct measurement of real-time liver mitochondrial enzymatic activities by the incorporation of label into energy metabolite pools.^1,2,3 In order to obtain sufficient metabolite signal, hyperpolarized ¹³C pyruvate is typically infused at doses around 0.3 mmol/kg. This results in blood pyruvate levels far in excess of the normal level of 50 µM, the metabolism of the labelled by pyruvate may reflect this supraphysiological condition.^4,5 In this study we aimed to decrease the administered pyruvate dose in steps, and subsequently quantify its effect on in vivo hepatic metabolism in fed and fasted animals, using an optimized formulation of HP pyruvate with rapid automated infusion for higher sensitivity. In this work we show that the liver metabolism can be measured in vivo with HP [1-¹³C]pyruvate administered at concentrations significantly closer to the physiological levels. Polarization: Neat ¹³C-labelled pyruvic acid prepared with trityl radical (Albeda, Denmark) was polarized in a 7 T custom-built DNP polarizer at 1.00 ± 0.05 K and 196.8 GHz using a nominal output power of 55 mW. After 1.5hr, the frozen sample was rapidly dissolved with 6 ml preheated deuterated phosphate buffer (~pH 7.5). The hyperpolarized [1-¹³C]pyruvate solution was automatically transferred into a separator/infusion pump located inside the animal bore. Animal: Male Sprague Dawley rats (194±5g) were anesthetized with isoflurane. Femoral vein and artery were catheterized for substrate administration and cardiac measurements, respectively. MR acquisition: A 1mL bolus of 0.023±0.002, 0.067± 0.008 and 0.188± 0.024 mmol/kg hyperpolarized ¹³C-pyruvate was administered intervenously in 9 s to fed and overnight fasted rats. In vivo ¹³C MRS measurements were respiration and cardiac gated with a repetition time of 3 s in a 9.4T/31cm horizontal bore magnet (Varian/Magnex, USA). 30° BIR4 adiabatic RF excitation pulses with proton decoupling were applied with a custom-built quadrature ¹H/single loop ¹³C surface coil placed over the liver of the rat. Acquisitions were performed with a VNMRS spectrometer (Varian, USA). Liver tissue extracts were obtained by freeze-clamping. Data analysis: The metabolite peak integrals were quantified and normalized with total ¹³C signal obtained from summed spectra analysed using VNMRJ (Varian, USA). Peak areas of single spectra were quantified using Bayes (Washington University, St. Louis) to obtain metabolic time courses. Error bars indicate ± SEM. One-way analysis of variance (ANOVA) with a Tukey multiple comparison test was used to identify statistical differences among dose groups. Differences between groups were considered significant if p < 0.05. Fed and fasted comparison for the same dose group was independently analyzed using a Student's t-test.The metabolic conversion of hyperpolarized [1-¹³C]pyruvate into ¹³C-bicarbonate, [1-¹³C]alanine, [1-¹³C]lactate and TCA cycle intermediates, [1-¹³C]aspartate, [4-¹³C]aspartate, [1-¹³C]malate, and [4-¹³C]malate was consistently observed with all dose levels (Fig 1). The metabolite-to-total ¹³C ratios of the low dose group for fed and fasted states, respectively, were for lactate : 0.021±0.005 and 0.021±0.003; alanine: 0.029±0.005 and 0.018±0.003; bicarbonate : 0.006±0.001 and 0.003±0.001; malate-C1: 0.002±0.0004 and 0.0031±0.001; malate-C4: 0.001±0.0003 and 0.001±0.0003, aspartate-C1: 0.005±0.002 and 0.007±0.002; aspartate-C4: 0.001±0.0004 and 0.001±0.0002 (Fig. 2). A significant change in ¹³C-bicarbonate levels was observed when lowering the dose (Fig. 3). The lowest dose where TCA cycle intermediates could still be observed was 0.023±0.002 mmol/kg. We estimate that this dose results in maximum plasma levels of 0.35±0.03 mM right after injection, which is 7-fold of the normal physiological level. With its rapid distribution and metabolic conversion, the infused pyruvate likely has a smaller effect on plasma levels.4 Fasted animals showed significantly elevated lactate levels relative to alanine in all three dose groups as a result of the increased NADH / NAD+ ratio of the fasted liver. Only low dose injections lead to a significant difference for the hyperpolarized ¹³C-bicarbonate levels between fed and fasted animals (Fig 3). These observations suggest that the adaptation to supraphysiological levels can obscure feeding state-depending metabolic differences in liver. We showed the feasibility of imaging liver metabolism and TCA cycles metabolites in vivo with HP ¹³C pyruvate administered at a dose of 0.023±0.002 mmol/kg which corresponds to doses lower than is currently administered in the clinic, with plasma concentrations of at most 7-fold of the basal levels. Metabolite ratio differences across fed and fasted animals show a different metabolic response to concentration changes with a given nutritional state, and that elevated plasma substrate levels could indeed alter liver metabolism.