Rachel Katz-Brull

Hyperpolarized MRI is a medical imaging technology that has been used so far only in focused clinical studies. This technology enables metabolic imaging without ionizing radiation and is combined with standard‐of‐care MRI protocols. Unlike metabolic imaging performed with radiolabeled materials (e.g., PET and SPECT), hyperpolarized MRI enables differentiation between the injected metabolic substrate and its product(s). In this way, the number of imaging contrasts that may be attained is relatively large. The purpose of this review is two‐fold: to provide coherent terminology for the various contrasts obtained in HP‐MRI to facilitate further development in medicine and to highlight its potential contribution to liver nodule characterization. We reviewed 33 hyperpolarized MRI papers published from 2013 to 2024 that describe clinical studies in human subjects and patients, and characterized the metabolic contrast provided by this technology across various organs and in cancer tumors. We have further reviewed 20 preclinical studies of hyperpolarized MRI in the liver to evaluate the potential of this technology for early detection and characterization of hepatocellular carcinoma. Most clinical studies have used a single hyperpolarized substrate, namely, pyruvate, labeled with carbon‐13 at position 1 ([1‐ ¹³ C]pyruvate). Even with this single substrate, the number of possible imaging contrasts is high, making the interpretation of these images challenging. We aimed to explain these imaging contrasts and provide a consistent nomenclature for them that will serve the medical community. Preclinical MRI studies using hyperpolarized substrates suggest the potential to characterize liver nodules in patients. In summary, hyperpolarized MRI is a promising clinical imaging modality in oncology; however, the small number of patients investigated so far precludes addressing possible biological confounding factors. Further studies are required for improving tumor characterization using this technology, and studies in liver imaging aimed at early detection of HCC are warranted.

Deuterated ¹³C sites in sugars (D‐glucose and 2‐deoxy‐D‐glucose) showed 6.3‐to‐17.5‐fold higher solid‐state dynamic nuclear polarization (DNP) levels than their respective protonated sites at 3.35T. This effect was found to be unrelated to the protonation of the bath. Deuterated ¹⁵N in sites bound to exchangeable protons ([¹⁵N₂]urea) showed a 1.3‐fold higher polarization than their respective protonated sites at the same magnetic field. This relatively smaller effect was attributed to incomplete deuteration of the ¹⁵N sites due to the solvent mixture. For a ¹⁵N site that is not bound to protons or deuterons ([¹⁵N]nitrate), deuteration of the bath did not affect the polarization level. These findings suggest a phenomenon related to DNP of X‐nuclei directly bound to deuteron(s) as opposed to proton(s). It appears that direct binding to deuterons increases the solid‐state DNP polarization level of X‐nuclei which are otherwise bound to protons.

Disruption of acid–base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy, and myocardial ischemia. Previously, we have reported that the ratio of the in‐cell lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in‐cell activities were investigated under induced intracellular acidification without and with Na⁺/H⁺ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n = 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH₄Cl prepulse methodology; and (2) by combining the NH₄Cl prepulse with zoniporide. ³¹P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1‐¹³C]pyruvate was obtained using dissolution dynamic nuclear polarization. ¹³C NMR spectroscopy was used to monitor hyperpolarized [1‐¹³C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product‐selective saturating excitation approach. The intracellular acidification induced by the NH₄Cl prepulse led to reduced LDH and PDH activities (−16% and −39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH₄Cl prepulse with zoniporide led to a greater reduction in LDH activity (−29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH₄Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H⁺ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in‐cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1‐¹³C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies.

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial information for understanding the mechanism of diseases and designing treatments. In this work, we describe procedures and methodologies for studying in-cell metabolic activity in a retrogradely perfused mouse heart in real-time. The heart was isolated in situ, in conjunction with cardiac arrest to minimize the myocardial ischemia and was perfused inside a nuclear magnetic resonance (NMR) spectrometer. While in the spectrometer and under continuous perfusion, hyperpolarized [1-13C]pyruvate was administered to the heart, and the subsequent hyperpolarized [1-13C]lactate and [13C]bicarbonate production rates served to determine, in real-time, the rates of lactate dehydrogenase and pyruvate dehydrogenase production. This metabolic activity of hyperpolarized [1-13C]pyruvate was quantified with NMR spectroscopy in a model free-manner using the product selective saturating-excitations acquisition approach. 31P spectroscopy was applied in between the hyperpolarized acquisitions to monitor the cardiac energetics and pH. This system is uniquely useful for studying metabolic activity in the healthy and diseased mouse heart.

Hyperpolarized ¹⁵N sites have been found to be promising for generating long‐lived hyperpolarized states in solution, and present a promising approach for utilizing dissolution‐dynamic nuclear polarization (dDNP)‐driven hyperpolarized MRI for imaging in biology and medicine. Specifically, ¹⁵N sites with directly bound protons were shown to be useful when dissolved in D₂O. The purpose of the current study was to further characterize and increase the visibility of such ¹⁵N sites in solutions that mimic an intravenous injection during the first cardiac pass in terms of their H₂O:D₂O composition. The T₁ values of hyperpolarized ¹⁵N in [¹⁵N₂]urea and [¹⁵N]NH₄Cl demonstrated similar dependences on the H₂O:D₂O composition of the solution, with a T₁ of about 140 s in 100% D₂O, about twofold shortening in 90% and 80% D₂O, and about threefold shortening in 50% D₂O. [¹³C]urea was found to be a useful solid‐state ¹³C marker for qualitative monitoring of the ¹⁵N polarization process in a commercial pre‐clinical dDNP device. Adding trace amounts of Gd³⁺ to the polarization formulation led to higher solid‐state polarization of [¹³C]urea and to higher polarization levels of [¹⁵N₂]urea in solution.

Nitrate, the inorganic anion NO₃⁻, is found in many foods and is an endogenous mammalian metabolite, which is supplied mostly through the diet. Although much is known about the safety of sodium nitrate when given per os, methodological safety data on intravenous bolus injection of sodium nitrate to rodents are lacking. Recently, we have proposed a new use for nitrate, as a contrast agent for magnetic resonance imaging that will be metal free and leave no traces in the body and the environment further to the imaging examination. It was shown that a stable isotope-labelled analog of this ion (¹⁵NO₃⁻), in a sodium nitrate solution form and hyperpolarized state, produces a high magnetic resonance signal with prolonged visibility. Therefore, sodium nitrate was targeted for further preclinical development in this context. In the absence of methodological safety data on the potential effects of a high concentration sodium nitrate bolus intravenous injection into rodents, we carried out such an investigation in mice and rats (n = 12 of each, 6 males and 6 females in each group, altogether 24 animals). We show here that an intravenous bolus administration of sodium nitrate at a concentration of 150 mM and a dose of 51 mg/Kg does not lead to adverse effects in mice and rats. This is the first investigation of the tolerance of rodents to an intravenous injection of sodium nitrate.

3‐aminopropylphosphonate (3‐APP) is known for its use as an exogenous indicator of extracellular volume and pH in phosphorus‐31 nuclear magnetic resonance (³¹P NMR) studies. We used 3‐APP for estimating the extracellular volume in NMR studies of several ex vivo preparations including retrograde perfused mouse heart (n = 4), mouse liver slices (n = 2), xenograft breast cancer tumors (n = 7, MCF7), and rat brain slices (n = 4). In the former three preparations, the 3‐APP signal was stable in lineshape and intensity for hours and the chemical shift of the signal in the presence of the biological sample was the same as in the perfusion medium without the biological sample. However, in studies of brain slices, the 3‐APP signal appeared split into two, with an upfield component (0.7 ± 0.1 ppm to the left) increasing with time and showing a wider linewidth (66.7 ± 12.6 vs. 39.1 ± 7.6 Hz, the latter is of the perfusion medium signal). This finding suggests that 3‐APP inadvertently accumulated in brain slices, most likely as a membrane bound form. This observation limits the use of 3‐APP as an inert biochemical indicator in brain preparations and should be taken into account when using 3‐APP in vivo.

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized ¹³C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-¹³C]lactate production from hyperpolarized [1-¹³C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using ³¹P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20–77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (3) and a diameter ranging from 3 to 6 mm.

Direct and real-time monitoring of cerebral metabolism exploiting the drastic increase in sensitivity of hyperpolarized ¹³C-labeled metabolites holds the potential to report on neural activity via in-cell metabolic indicators. Here, we followed the metabolic consequences of curbing action potential generation and ATP-synthase in rat cerebrum slices, induced by tetrodotoxin and oligomycin, respectively. The results suggest that pyruvate dehydrogenase (PDH) activity in the cerebrum is 4.4-fold higher when neuronal firing is unperturbed. The PDH activity was 7.4-fold reduced in the presence of oligomycin, and served as a pharmacological control for testing the ability to determine changes to PDH activity in viable cerebrum slices. These findings may open a path towards utilization of PDH activity, observed by magnetic resonance of hyperpolarized ¹³C-labeled pyruvate, as a reporter of neural activity.

Ischemic stroke is a leading cause for neurologic disability worldwide, for which reperfusion is the only available treatment. Neuroimaging in stroke guides treatment, and therefore determines the clinical outcome. However, there are currently no imaging biomarkers for the status of the ischemic brain tissue. Such biomarkers could potentially be useful for guiding treatment in patients presenting with ischemic stroke. Hyperpolarized ¹³C MR of [1-¹³C]pyruvate is a clinically translatable method used to characterize tissue metabolism non-invasively in a relevant timescale. The aim of this study was to utilize hyperpolarized [1-¹³C]pyruvate to investigate the metabolic consequences of an ischemic insult immediately during reperfusion and upon recovery of the brain tissue. The rates of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) were quantified by monitoring the rates of [1-¹³C]lactate and [¹³C]bicarbonate production from hyperpolarized [1-¹³C]pyruvate. ³¹P NMR of the perfused brain slices showed that this system is suitable for studying ischemia and recovery following reperfusion. This was indicated by the levels of the high-energy phosphates (tissue viability) and the chemical shift of the inorganic phosphate signal (tissue pH). Acidification, which was observed during the ischemic insult, has returned to baseline level following reperfusion. The LDH/PDH activity ratio increased following ischemia, from 47.0 ± 12.7 in the control group (n = 6) to 217.4 ± 121.3 in the ischemia-reperfusion group (n = 6). Following the recovery period (ca. 1.5 h), this value had returned to its pre-ischemia (baseline) level, suggesting the LDH/PDH enzyme activity ratio may be used as a potential indicator for the status of the ischemic and recovering brain.