A low-pressure/vacuum 5 mm NMR tube (Wilmad Glass) containing 900 μL of the precursor solution was held in an oil bath maintained at 80°C. Para-enriched hydrogen gas was bubbled through the solution at 8 bar for 30 seconds. Following this, the pressure was released and the solution was extracted into a syringe for injection into the phosphate buffer solution in the magnet. The solution was injected through a 1/16 inch O.D. PTFE line, terminating in a 1/16 inch glass capillary in the detection region. This was followed by a few seconds of pushing air through the capillary to ensure efficient mixing of the reaction solution with the PBS/enzyme solution. Immediately following this, the imaging sequences were applied, and the signal was acquired every 12 s. The final volume in the 10 mm NMR tube was approximately 3 mL since some sample is lost during the transfer, mostly as droplets in the PTFE line. ). The rf pulse sequences were applied every 4 s to reveal the malate-D2 molecules that formed during that time. The spectrum of the second data point is shown for each of the sequences. For the 45° pulse experiment data, the left-half of the 4.3 ppm peak was integrated. ) A comparison between the hyperpolarized and thermal equilibrium 1 H images, with the hyperpolarized images acquired following the procedure described in the text. (e) A time series of hyperpolarized 1 H images. The receiver gain was set to 101 for hyperpolarized image acquisition and 1 for thermal equilibrium image acquisition, which gives a factor of 2 difference in signal-to-noise ratio (as discussed in the Supporting Information).