AIMS:A ketogenic diet (KD) can suppress cardiac carbohydrate utilization, which may adversely impact heart function. However, the reversibility of KD-induced metabolic changes is poorly understood. This study aims to characterize myocardial pyruvate dehydrogenase (PDH) flux during the transition from a prolonged KD to a normal chow diet (ND). METHODS AND RESULTS:Cardiac metabolism was longitudinally assessed in rats using hyperpolarized [1-13C]pyruvate at baseline, during a KD (2 and 5 weeks), and a subsequent ND (1, 2, 5, and 8 days) after the 5-week KD. Hyperpolarized 13C products were compared between the KD group and age-matched ND controls. In parallel, nuclear magnetic resonance isotopomer analysis of cardiac tissue with an injection of [3-13C]pyruvate and [1,2-13C2]acetate was performed along with ex vivo enzymatic analysis of PDH activity. Myocardial [13C]bicarbonate production relative to total 13C products decreased from 8.56 ± 2.29% at baseline to 0.46 ± 0.27% after 5 weeks of KD. Reverting to ND gradually restored PDH flux (8.40 ± 1.47% by Day 8) to control levels (8.69 ± 2.10%). Ex vivo NMR analysis of glutamate C4 showed reduced pyruvate contribution to acetyl-CoA during KD (4.1 ± 2.5%), which recovered upon reverting to ND (22.7 ± 1.82% vs. control: 27.6 ± 9.5%). Although PDK4 expression normalized, PDH activity remained partially impaired in the reverted group (36.80 ± 6.07 mmol NADH/min/mg) compared to controls (90.97 ± 5.40; P = 0.00007). CONCLUSION:KD-induced suppression of myocardial PDH flux is reversible, but its recovery requires significant time, with prolonged metabolic inflexibility persisting after transitioning to an ND. These findings highlight the value of in vivo assessment of cardiac PDH activity, complemented by conventional enzymatic analyses, to identify persistent metabolic inflexibility following ketogenic interventions.

Abstract Introduction: Precise tumor targeting is essential for improving the therapeutic index of cancer immunotherapies. Systemic cytokine therapies, such as interleukin-2 (IL-2), suffer from narrow therapeutic windows due to dose-limiting toxicities. While receptor engineering and prodrug approaches have been widely investigated, their application is often limited to tumor subtypes that express the corresponding targets. In contrast, tumor acidity is a universal hallmark of the microenvironment, driven by elevated glycolysis, and offers a broadly applicable opportunity for tumor-specific therapy. However, the spatial heterogeneity of tumor acidity remains poorly understood, and its therapeutic application has not been fully demonstrated. Methods: To systematically evaluate the heterogeneity of tumor acidity and its potential for targeted therapy, we employed ultra-pH-sensitive (UPS) nanoparticles that exhibit sharp ON/OFF transitions at defined pH thresholds. In 3D tumor cultures, we embedded UPS nanoprobes within extracellular matrix gels and assessed the spatial distribution of acidity at single-cell resolution. In human cancer patients, we evaluated the tumor-imaging properties of this acidity-targeting strategy and further investigated acidity patterns by analyzing UPS nanoparticle distribution in tumor tissues following intravenous injection. Building on these findings, we developed a formulation strategy to encapsulate IL-2-Fc into UPS micelles, enabling selective cytokine release in acidic microenvironments. This platform aims to provide a comprehensive understanding of spatial acidity and to test the therapeutic index of pH-activatable cytokine delivery. Results: Through this work, we identified a severe acidity phenotype in cancer, where extracellular pH drops below 5.3 due to spatially polarized lactate export. Targeting acidity allows demarcation of tumor from surrounding tissue in intraoperative imaging, even when tumors are as small as 2 mm. Spatial transcriptomic analysis of human head and neck tumors after UPS nanoprobe injection revealed that these severely acidic regions co-localize with immune-infiltrated stromal zones. These regions serve as critical entry points for acid-targeting nanosystems, linking tumor metabolism, immune cell infiltration, and immune suppression in both preclinical models and human tissues. The UPS/IL-2-Fc formulation selectively releases IL-2-Fc within these regions and demonstrates potent antitumor efficacy with markedly reduced systemic toxicity, expanding the therapeutic window by over 10-fold compared to free IL-2-Fc in preclinical models. Conclusion: This study identifies severe acidity as a spatially heterogeneous and translatable hallmark of the tumor microenvironment, enabling tumor-targeted imaging and immunotherapy. Citation Format: Qiang Feng, Jun Chen, William Hartnett, Raymundo Pantoja, Gang Huang, Isaac Chan, Baran Sumer, Jinming Gao. Severe acidity in human tumors for pH-activatable cytokine therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 283.

PURPOSE:13C MRI with hyperpolarized [1-13C]pyruvate enables noninvasive imaging of metabolic pathways. Considering the high dose of pyruvate used in hyperpolarized 13C-pyruvate studies, circulating 13C-pyruvate and the resulting 13C-lactate in plasma may influence subsequent pyruvate metabolism, yet the guidance on consecutive injections does not exist. This study is to characterize blood pyruvate and lactate dynamics following [1-13C]pyruvate injection and to evaluate the repeatability of 13C neuroimaging with consecutive pyruvate injections. METHODS:Sixteen healthy adults (mean age, 39.0 ± 15.9 years; 8 men) underwent blood sampling or MRI. Eight participants received bolus injections of non-hyperpolarized [1-13C]pyruvate following both overnight fasting and postprandial states. Blood samples were collected at baseline and up to 60 min post-injection to quantify pyruvate and lactate concentrations and 13C fractional enrichments using mass spectrometry. In parallel, eight participants underwent 13C/1H MRI that included two injections of hyperpolarized [1-13C]pyruvate with intervals of 6-55 min for time-resolved measurement of 13C-pyruvate and 13C-lactate in the brain. RESULTS:Plasma 13C-pyruvate peaked within 30 s and returned to baseline within 5-10 min, while 13C-lactate peaked at 2-3 min. Lactate 13C enrichment was higher in fasting versus fed states, and total lactate concentration in blood was unchanged by pyruvate injection. Brain 13C-lactate/pyruvate showed excellent repeatability (ICC = 0.9842), with all points within the 95% Bland-Altman limits. Voxel-wise Pearson's correlation between injections was 0.72 ± 0.22 and within-subject coefficient of variation was 17.6% ± 4.6%. CONCLUSION:Consecutive injections of hyperpolarized [1-13C]pyruvate yield repeatable blood and brain metabolic measurements in humans, supporting multi-injection protocols in 13C MRI studies.

The enzyme γ-glutamyl transferase (GGT) plays an important role in redox homeostasis and is overexpressed in many different cancers. The ability to assess GGT activity provides critical insights into cancer prognosis and pathology. Here, we present a novel imaging agent, (15N)2-DT-GG-PA, for in vivo assessment of GGT activity by hyperpolarized nitrogen-15 magnetic resonance imaging (HP 15N MRI). This 15N-labeled HP MRI probe is developed by introducing a long-lasting HP (15N)2-diazirine tag to deliver essential properties for in vivo detection of GGT activity. Our studies show a large chemical shift difference (Δδ = 6.3 ppm) between (15N)2-DT-GG-PA and its product, (15N)2-DT-PA, and long HP lifetimes of both compounds (T1 = 196 and 81 s, respectively, at 1 T). We also demonstrate the in vivo feasibility of detecting GGT activity using HP (15N)2-DT-GG-PA in rat kidneys on a clinical 3 T MRI scanner. Finally, the success of (15N)2-DT-GG-PA highlights the transformative potential of the 15N-tagging approach in designing novel HP 15N MRI probes beyond structural restrictions for 15N-isotope labeling.

Glucose and lactate are primary substrates in cerebral energy metabolism. Hyperpolarized [1-13C]pyruvate has become a powerful imaging agent for metabolic neuroimaging due to its central role in glucose and lactate metabolism, ability to cross the blood-brain barrier, and translational utility in neurological disorders. In particular, [1-13C]pyruvate enables an assessment of mitochondrial metabolism in the cerebral cortex through its conversion to [13C]bicarbonate. While it is not yet confirmed that production of [13C]bicarbonate primarily reflects neuronal metabolism, the higher affinity of neuronal transporters for lactate over pyruvate has motivated interest in hyperpolarized lactate as a more physiologic probe of neuronal metabolism. Here, we identify the predominant cellular source of [13C]bicarbonate and evaluate [1-13C]lactate as an imaging agent for neuronal metabolic imaging. Ex vivo NMR and mass spectrometry imaging of brain tissue collected after bolus injection of [U-13C3]pyruvate revealed that pyruvate dehydrogenase dominates pyruvate carboxylase in the cortex, supporting the neuronal origin of [13C]bicarbonate production. Although the bicarbonate fraction among the total 13C products in vivo was higher following hyperpolarized [1-13C]lactate injection, the signal sensitivity was markedly reduced due to lactate's shorter T1 and larger endogenous pool. Isotopomer analysis of brain tissue harvested 2 min after injection of [U-13C3]pyruvate or [U-13C3]lactate showed comparable labeling of mitochondrial intermediates. In glioma-bearing rats, in vivo imaging revealed an elevated pyruvate-to-lactate ratio within the tumor, highlighting altered redox and transport dynamics in malignancy. These findings demonstrate that both hyperpolarized [1-13C]pyruvate and [1-13C]lactate can effectively probe neuronal and glioma metabolism, although pyruvate outperforms lactate in detecting pyruvate dehydrogenase flux.

PURPOSE:Pyruvate, produced from either glucose, glycogen, or lactate, is the dominant precursor of cerebral oxidative metabolism. Pyruvate dehydrogenase (PDH) flux is a direct measure of cerebral mitochondrial function and metabolism. Detection of [13 C]bicarbonate in the brain from hyperpolarized [1-13 C]pyruvate using carbon-13 (13 C) MRI provides a unique opportunity for assessing PDH flux in vivo. This study is to assess changes in cerebral PDH flux in response to visual stimuli using in vivo 13 C MRS with hyperpolarized [1-13 C]pyruvate. METHODS:From seven sedentary adults in good general health, time-resolved [13 C]bicarbonate production was measured in the brain using 90° flip angles with minimal perturbation of its precursors, [1-13 C]pyruvate and [1-13 C]lactate, to test the hypothesis that the appearance of [13 C]bicarbonate signals in the brain reflects the metabolic changes associated with neuronal activation. With a separate group of healthy participants (n = 3), the likelihood of the bolus-injected [1-13 C]pyruvate being converted to [1-13 C]lactate prior to decarboxylation was investigated by measuring [13 C]bicarbonate production with and without [1-13 C]lactate saturation. RESULTS:In the course of visual stimulation, the measured [13 C]bicarbonate signal normalized to the total 13 C signal in the visual cortex increased by 17.1% ± 15.9% (p = 0.017), whereas no significant change was detected in [1-13 C]lactate. Proton BOLD fMRI confirmed the regional activation in the visual cortex with the stimuli. Lactate saturation decreased bicarbonate-to-pyruvate ratio by 44.4% ± 9.3% (p < 0.01). CONCLUSION:We demonstrated the utility of 13 C MRS with hyperpolarized [1-13 C]pyruvate for assessing the activation of cerebral PDH flux via the detection of [13 C]bicarbonate production.

This study demonstrates that time-resolved 13 C MR spectroscopy with the multichannel 13 C/ 1 H RF coils can be performed as an alternative to imaging for assessing pyruvate metabolism using hyperpolarized [1- 13 C]pyruvate in the human brain.

Mitochondrial oxidative phosphorylation (OXPHOS) is sensitive to a variety of biological factors, and dysregulated OXPHOS is observed during the development of numerous pathological conditions. ATP production via OXPHOS is intrinsically dependent on the availability of acetyl-coenzyme A (CoA), which can enter the tricarboxylic acid (TCA) cycle to drive the oxidative pathway. Acetyl-l-carnitine (ALCAR) is an interchangeable endogenous source of acetyl-CoA, and therefore, ALCAR-derived probes are uniquely positioned for the assessment of OXPHOS. In this report, we develop hyperpolarized (HP) [1-13C]ALCAR as a noninvasive probe to investigate cardiac TCA cycle activity in vivo. We initially synthesized the isotopically labeled substrate and demonstrated that the 13C nucleus maintained a suitable T1 value (50.1 ± 0.8 s at 3 T) and polarization levels (21.3 ± 5.3%) to execute in vivo metabolic measurements. HP [1-13C]ALCAR was employed for cardiac analyses of OXPHOS in rats under fed and fasted conditions. [5-13C]Glutamate was successfully detected, and the metabolite was used to analyze the TCA cycle activity in both nutritional states. These assessments were compared to analogous experiments with the HP [1-13C]pyruvate. Our report represents the first study to demonstrate that HP methods using [1-13C]ALCAR enable direct analyses of mitochondrial function and TCA cycle activity, which are fundamental to cardiac cell homeostasis.

PURPOSE:[13 C]Bicarbonate formation from hyperpolarized [1-13 C]pyruvate via pyruvate dehydrogenase, a key regulatory enzyme, represents the cerebral oxidation of pyruvate and the integrity of mitochondrial function. The present study is to characterize the chronology of cerebral mitochondrial metabolism during secondary injury associated with acute traumatic brain injury (TBI) by longitudinally monitoring [13 C]bicarbonate production from hyperpolarized [1-13 C]pyruvate in rodents. METHODS:Male Wistar rats were randomly assigned to undergo a controlled-cortical impact (CCI, n = 31) or sham surgery (n = 22). Seventeen of the CCI and 9 of the sham rats longitudinally underwent a 1 H/13 C-integrated MR protocol that includes a bolus injection of hyperpolarized [1-13 C]pyruvate at 0 (2 h), 1, 2, 5, and 10 days post-surgery. Separate CCI and sham rats were used for histological validation and enzyme assays. RESULTS:In addition to elevated lactate, we observed significantly reduced bicarbonate production in the injured site. Unlike the immediate appearance of hyperintensity on T2 -weighted MRI, the contrast of bicarbonate signals between the injured region and the contralateral brain peaked at 24 h post-injury, then fully recovered to the normal level at day 10. A subset of TBI rats demonstrated markedly increased bicarbonate in normal-appearing contralateral brain regions post-injury. CONCLUSION:This study demonstrates that aberrant mitochondrial metabolism occurring in acute TBI can be monitored by detecting [13 C]bicarbonate production from hyperpolarized [1-13 C]pyruvate, suggesting that [13 C]bicarbonate is a sensitive in-vivo biomarker of the secondary injury processes.

Radiation-induced heart disease is a major source of morbidity and mortality in patients receiving thoracic radiation. In this study, radiation-induced changes in cardiac metabolism is investigated using hyperpolarized [1- 13 C]pyruvate MRI in animals and patients. Myocardial bicarbonate-to-lactate ratios decreased following radiation treatments while no change was observed in the global strain, suggesting radiation-induced mitochondrial dysfunction in the heart. This translational study demonstrates clinical potential of hyperpolarized 13 C pyruvate for early and noninvasive detection of radiation-induced cardiac injury.

[ 13 C]Bicarbonate production from hyperpolarized [1- 13 C]pyruvate in the brain is directly related to pyruvate oxidation and, thus serves a potential biomarker of brain function. In this study, we assessed time-wise bicarbonate production in the visual cortex during activation with minimal perturbation of its precursors, pyruvate and lactate, using a multichannel 13 C receive array, a spectral-spatial RF pulse that fully excites bicarbonate signals, and dynamic 13 C MRS. The real-time changes of bicarbonate production in response to visual stimuli were observed in healthy volunteers.

Hydrogen peroxide (H2O2) is a type of reactive oxygen species that regulates essential biological processes. Despite the central role of H2O2 in pathophysiological states, available molecular probes for assessing H2O2 in vivo are still limited. This work develops hyperpolarized 15N-boronobenzyl-4-cyanopyridinium (15N-BBCP) as a rationally designed molecular probe for detecting H2O2. The 15N-BBCP demonstrated favorable physicochemical and biochemical properties for H2O2 detection and dynamic nuclear polarization, allowing noninvasive detection of H2O2. In particular, 15N-BBCP and the products possessed long spin-lattice relaxation times and spectrally resolvable 15N chemical shift differences. The performance of hyperpolarized 15N-BBCP was demonstrated both in vitro and in vivo with time-resolved 15N-MRS. This study highlights a promising approach to designing a reaction-based 15N-labeled molecular imaging agent for detecting oxidative stress in vivo.

Pyruvate carboxylase (PC) is an enzyme that catalyzes pyruvate carboxylation for the formation of a tricarboxylic acid (TCA) cycle intermediate, oxaloacetate. It plays a major role in the regulation of gluconeogenesis and replenishment of the TCA cycle for biosynthesis. In particular, fasting promotes gluconeogenesis from pyruvate, increasing PC flux. PC is under complex regulation by hormones, substates, and cofactors, in response to nutrient changes. Therefore, metabolic changes associated with PC need to be understood in the context of nutritional and physiological conditions. This chapter discusses currently available methods, from conventional approaches to state-of-the-art noninvasive in vivo imaging techniques, used in human and animal models to assess PC activity, particularly in fasting condition. Quantification of PC activity through in vitro or ex vivo isotopomer analysis and monitoring PC in vivo using hyperpolarized substrates are reviewed.

Noninvasive imaging with hyperpolarized (HP) pyruvate can capture in vivo cardiac metabolism. For proper quantification of the metabolites and optimization of imaging parameters, understanding MR characteristics such as T2∗ s of the HP signals is critical. This study is to measure in vivo cardiac T2∗ s of HP [1‐13C]pyruvate and the products in rodents and humans.

Abstract Background Glioblastoma remains incurable despite treatment with surgery, radiation therapy, and cytotoxic chemotherapy, prompting the search for a metabolic pathway unique to glioblastoma cells.13C MR spectroscopic imaging with hyperpolarized pyruvate can demonstrate alterations in pyruvate metabolism in these tumors. Methods Three patients with diagnostic MRI suggestive of a glioblastoma were scanned at 3 T 1–2 days prior to tumor resection using a 13C/1H dual-frequency RF coil and a 13C/1H-integrated MR protocol, which consists of a series of 1H MR sequences (T2 FLAIR, arterial spin labeling and contrast-enhanced [CE] T1) and 13C spectroscopic imaging with hyperpolarized [1-13C]pyruvate. Dynamic spiral chemical shift imaging was used for 13C data acquisition. Surgical navigation was used to correlate the locations of tissue samples submitted for histology with the changes seen on the diagnostic MR scans and the 13C spectroscopic images. Results Each tumor was histologically confirmed to be a WHO grade IV glioblastoma with isocitrate dehydrogenase wild type. Total hyperpolarized 13C signals detected near the tumor mass reflected altered tissue perfusion near the tumor. For each tumor, a hyperintense [1-13C]lactate signal was detected both within CE and T2-FLAIR regions on the 1H diagnostic images (P = .008). [13C]bicarbonate signal was maintained or decreased in the lesion but the observation was not significant (P = .3). Conclusions Prior to surgical resection, 13C MR spectroscopic imaging with hyperpolarized pyruvate reveals increased lactate production in regions of histologically confirmed glioblastoma.

The interplay between glycolysis and gluconeogenesis is central to carbohydrate metabolism. Here, we describe novel methods to assess carbohydrate metabolism using [13C]-probes derived from glycerate, a molecule whose metabolic fate in mammals remains underexplored. Isotope-based studies were conducted via NMR and mass spectrometry analyses of freeze-clamped liver tissue extracts after [2,3-13C2]glycerate infusion. The ex vivo investigations were correlated with in vivo measurements using hyperpolarized [1-13C]glycerate. Application of [13C]glycerate to N-nitrosodiethylamine (DEN)-treated rats provided further assessments of intermediary carbohydrate metabolism in hepatocellular carcinoma. This method afforded direct analyses of control versus DEN tissues, and altered ratios of 13C metabolic products as well as unique glycolysis intermediates were observed in the DEN liver/tumor. Isotopomer studies showed increased glycerate uptake and altered carbohydrate metabolism in the DEN rats.

Purpose: Noninvasive imaging with hyperpolarized (HP) pyruvate can capture in vivo cardiac metabolism. For proper quantification of the metabolites and optimization of imaging parameters, understanding MR characteristics such as T-2*s of the HP signals is critical. This study is to measure in vivo cardiac T-2*s of HP [1-C-13]pyruvate and the products in rodents and humans. Methods: A dynamic C-13 multi-echo spiral imaging sequence that acquires [C-13]bicarbonate, [1-C-13]lactate, and [1-C-13]pyruvate images in an interleaved manner was implemented for a clinical 3 Tesla system. T-2* of each metabolite was calculated from the multi-echo images by fitting the signal decay of each region of interest mono-exponentially. The performance of measuring T-2* using the sequence was first validated using a C-13 phantom and then with rodents following a bolus injection of HP [1-C-13]pyruvate. In humans, T-2* of each metabolite was calculated for left ventricle, right ventricle, and myocardium. Results: Cardiac T-2* s of HP [1-C-13]pyruvate, [1-C-13]lactate, and [C-13]bicarbonate in rodents were measured as 24.9 +/- 5.0, 16.4 +/- 4.7, and 16.9 +/- 3.4 ms, respectively. In humans, T-2* of [1-C-13]pyruvate was 108.7 +/- 22.6 ms in left ventricle and 129.4 +/- 8.9 ms in right ventricle. T-2* of [1-C-13]lactate was 40.9 +/- 8.3, 44.2 +/- 5.5, and 43.7 +/- 9.0 ms in left ventricle, right ventricle, and myocardium, respectively. T-2* of [C-13] bicarbonate in myocardium was 64.4 +/- 2.5 ms. The measurements were reproducible and consistent over time after the pyruvate injection. Conclusion: The proposed metabolite-selective multi-echo spiral imaging sequence reliably measures in vivo cardiac T-2* s of HP [1-C-13]pyruvate and products.