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Hyperpolarization Research: Unveiling Real-Time Metabolism

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by

Josh
in , , , ,

Abstract: Hyperpolarization (HP) magnetic resonance imaging (MRI) and spectroscopy (MRS) have revolutionized the field of molecular imaging by dramatically enhancing the sensitivity of nuclear magnetic resonance (NMR) for low-gamma nuclei, particularly carbon-13 ($^{13}$C). This transient yet powerful signal boost allows for the real-time, non-invasive interrogation of metabolic pathways and physiological processes in vivo at unprecedented speeds. This review synthesizes recent advancements in hyperpolarization research, detailing key methodologies, major findings across diverse biomedical applications, and outlining the persistent challenges and promising future directions that will drive its continued clinical translation. From early cancer detection and therapy response monitoring to understanding organ-specific metabolism in diabetes, cardiovascular disease, and neurological disorders, hyperpolarization is rapidly transforming our ability to visualize the dynamic metabolic landscape of living systems.

1. Introduction

Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) are invaluable tools in biomedical research and clinical diagnostics, offering excellent soft-tissue contrast and non-invasive functional information. However, conventional MRS often suffers from low sensitivity when detecting endogenous metabolites, particularly for nuclei like carbon-13 ($^{13}$C$), due to their low natural abundance and small gyromagnetic ratio. This limitation hinders the real-time study of metabolic flux, a cornerstone of understanding disease pathophysiology.

Hyperpolarization techniques overcome this sensitivity barrier by transiently enhancing the nuclear spin polarization of specific molecular probes by several orders of magnitude (typically >10,000-fold). The most prominent method, dissolution Dynamic Nuclear Polarization (dDNP), transforms a frozen solid-state sample into an injectable, physiologically compatible solution seconds before administration. This transient signal, lasting typically for tens of seconds to a few minutes, enables dynamic, real-time tracking of substrate uptake and its subsequent enzymatic conversion into metabolic products within living tissues.

The ability to non-invasively monitor metabolic fluxes in vivo offers unique insights into cellular energetics, redox states, and microenvironmental pH, which are often dysregulated in diseases such as cancer, diabetes, cardiovascular conditions, and neurodegenerative disorders. The “workhorse” probe, [1-$^{13}$C]pyruvate, has been extensively used due to its central role in cellular metabolism, rapidly converting to lactate (reflecting glycolysis), alanine (reflecting transamination), and bicarbonate (reflecting pyruvate dehydrogenase (PDH) flux and gluconeogenesis/TCA cycle activity). This review aims to provide a comprehensive overview of the current state of hyperpolarization research, highlighting the methodological innovations, diverse applications, and future potential of this transformative technology.

2. Key Methodologies

The advancements in hyperpolarization research are underpinned by sophisticated methodologies spanning probe generation, MRI/MRS acquisition, and data analysis.

2.1. Hyperpolarization Techniques

The core of HP-MRI/MRS relies on generating a high degree of nuclear spin polarization.

  • Dynamic Nuclear Polarization (DNP): This remains the most widely adopted method. It involves embedding a $^{13}$C-enriched substrate (e.g., [1-$^{13}$C]pyruvate) with a stable organic free radical (e.g., trityl radical OX063) in a glassing matrix (e.g., glycerol), cooling it to ultralow temperatures (typically 0.8-1.4 K) in a strong magnetic field (3-7 T), and irradiating it with microwaves. This process transfers the high electron polarization of the radicals to the nuclear spins. Upon sufficient polarization, the solid sample is rapidly dissolved with superheated, sterile buffer into an injectable liquid solution (e.g., Hyperpolarized Carbon (13C) MRI of the Kidneys: Basic Concept, Parameterization of hyperpolarized (13)C-bicarbonate-dissolution dynamic nuclear polarization). Recent developments include cryogen-free DNP polarizers that improve system uptime and duty cycle, achieving high and reproducible polarization levels for both $^{13}$C pyruvate ($>$30%) and $^{15}$N urea (5-6%) (e.g., Performance and reproducibility of (13)C and (15)N hyperpolarization using a cryogen-free DNP polarizer). Multi-sample/multi-nucleus probes further enhance throughput for preclinical studies (e.g., Multi-sample/multi-nucleus parallel polarization and monitoring enabled by a fluid path technology compatible cryogenic probe for dissolution dynamic nuclear polarization).
  • ParaHydrogen Induced Polarization (PHIP): This non-DNP method leverages the spin order of parahydrogen. Techniques like PHIP-Side Arm Hydrogenation (PHIP-SAH) involve hydrogenation of a precursor with parahydrogen, followed by polarization transfer and rapid hydrolysis to yield the hyperpolarized substrate (e.g., The (13)C hyperpolarized pyruvate generated by ParaHydrogen detects the response of the heart to altered metabolism in real time). PHIP offers advantages in speed and cost-effectiveness (e.g., In-vitro NMR Studies of Prostate Tumor Cell Metabolism by Means of Hyperpolarized [1-(13)C]Pyruvate Obtained Using the PHIP-SAH Method, In vivo molecular imaging of breast cancer metabolic heterogeneity using [1-13C]pyruvate-d(3) hyperpolarized by reversible exchange with parahydrogen).
  • Overhauser Dynamic Nuclear Polarization (ODNP): This liquid-state, room-temperature technique utilizes electron-nuclear spin interactions, typically with dissolved radicals, to enhance nuclear polarization. It has shown promise for small biological molecules in water, driven by intermolecular hydrogen bond dynamics (e.g., Room-temperature dynamic nuclear polarization enhanced NMR spectroscopy of small biological molecules in water).

2.2. Hyperpolarized Probes

A growing array of hyperpolarized probes enables interrogation of diverse metabolic pathways and physiological parameters:

  • Metabolic Flux Probes:
    • [1-$^{13}$C]Pyruvate: The most widely used, providing real-time flux into lactate (glycolysis, Warburg effect), alanine (transamination), and bicarbonate (TCA cycle, gluconeogenesis) (e.g., In Vivo Carbon-13 Dynamic MRS and MRSI of Normal and Fasted Rat Liver with Hyperpolarized 13C-Pyruvate, Hyperpolarized Carbon (13C) MRI of the Kidneys: Basic Concept). [2-$^{13}$C]pyruvate is used to specifically track TCA cycle intermediates like glutamate and aspartate (e.g., In vivo 13C spectroscopy in the rat brain using hyperpolarized [1-(13)C]pyruvate and [2-(13)C]pyruvate).
    • [U-$^{13}$C, $^{2}$H]Glucose: Tracks glucose metabolism into lactate and 6-phosphogluconate (PPP), with deuteration extending T1 relaxation times (e.g., Magnetic resonance imaging of tumor glycolysis using hyperpolarized 13C-labeled glucose, Measuring glucose cerebral metabolism in the healthy mouse using hyperpolarized (13)C magnetic resonance).
    • [1,4-$^{13}$C$_2$]Fumarate: A specific marker for cellular necrosis, as its conversion to malate becomes detectable when cell membranes are compromised (e.g., Hyperpolarized Carbon (13C) MRI of the Kidneys: Basic Concept, Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate).
    • [1-$^{13}$C]Lactate: Probes neuroprotection in stroke and cardiac metabolism (e.g., Evaluating the potential of hyperpolarised [1-(13)C] L-lactate as a neuroprotectant metabolic biosensor for stroke, Myocardial metabolic flexibility following ketone infusion demonstrated by hyperpolarized [2-(13)C]pyruvate MRS in pigs).
    • [1-$^{13}$C]Acetate: Investigates acetyl-CoA synthetase (ACSS) activity and renal energetic demand (e.g., In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by (13)C turnover from hyperpolarized [1-(13)C]acetate to [1-(13)C]acetylcarnitine, Hyperpolarized [1-(13)C]-acetate Renal Metabolic Clearance Rate Mapping).
    • [1,3-$^{13}$C$_2$]Acetoacetate: A ketone body probe for mitochondrial redox and $\beta$-hydroxybutyrate dehydrogenase (BDH) activity (e.g., A new hyperpolarized (13)C ketone body probe reveals an increase in acetoacetate utilization in the diabetic rat heart, In vivo investigation of hyperpolarized [1,3-(13)C(2)]acetoacetate as a metabolic probe in normal brain and in glioma).
    • [1-$^{13}$C]$\alpha$-ketobutyrate: Assesses cytoplasmic reducing capacity (e.g., Detection of early-stage NASH using non-invasive hyperpolarized (13)C metabolic imaging).
    • $\delta$-[1-$^{13}$C]gluconolactone: Probes pentose phosphate pathway (PPP) flux and activity of 6-phosphogluconolactonase (PGLS) (e.g., Hyperpolarized $\delta$-[1-(13)C]gluconolactone imaging visualizes response to TERT or GABPB1 targeting therapy for glioblastoma, Imaging 6-Phosphogluconolactonase Activity in Brain Tumors In Vivo Using Hyperpolarized $\delta$-[1-(13)C]gluconolactone).
    • [6-$^{13}$C]Arginine: Detects arginase activity in inflammatory cells like MDSCs (e.g., Detection of inflammatory cell function using (13)C magnetic resonance spectroscopy of hyperpolarized [6-(13)C]-arginine).
    • [2-$^{13}$C]methylglyoxal: Probes glyoxalase activity (e.g., Glyoxalase activity in human erythrocytes and mouse lymphoma, liver and brain probed with hyperpolarized (13)C-methylglyoxal).
  • Physiological & Redox Probes:
    • [1-$^{13}$C]Dehydroascorbate (DHA): Serves as an in vivo redox sensor, with its reduction to vitamin C indicating cellular reducing capacity and oxidative stress (e.g., Hyperpolarized Carbon (13C) MRI of the Kidneys: Basic Concept, Imaging glutathione depletion in the rat brain using ascorbate-derived hyperpolarized MR and PET probes).
    • [1-$^{13}$C]N-acetyl cysteine (NAC): Monitors glutathione redox chemistry via formation of an NAC-GSH dimer (e.g., Real-Time insight into in vivo redox status utilizing hyperpolarized [1-(13)C] N-acetyl cysteine).
    • $^{13}$C-Bicarbonate / [1,5-$^{13}$C$_2$]Z-OMPD / [$^{13}$C]Zymonic Acid: Enables non-invasive pH imaging by leveraging pH-dependent chemical shifts or equilibrium with CO$_2$ (e.g., Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate, Hyperpolarized in vivo pH imaging reveals grade-dependent acidification in prostate cancer, Simultaneous magnetic resonance imaging of pH, perfusion and renal filtration using hyperpolarized (13)C-labelled Z-OMPD, Imaging of pH in vivo using hyperpolarized (13)C-labelled zymonic acid).
  • Perfusion & Functional Probes:
    • [13C]Urea / [$^{13}$C,$^{15}$N$_2$]Urea: Used for dynamic imaging of renal function (GFR) and perfusion due to its inert metabolism and BBB impermeability (e.g., Hyperpolarized Carbon (13C) MRI of the Kidneys: Basic Concept, Translation of hyperpolarized [13C,15N2]urea MRI for novel human brain perfusion studies).
    • [13C]t-Butanol: A BBB-permeable agent for perfusion imaging (e.g., Investigating the Feasibility of In Vivo Perfusion Imaging Methods for Spinal Cord Using Hyperpolarized [(13)C]t-Butanol and [(13)C,(15)N(2)]Urea).
    • $^{129}$Xe: Evaluates lung gas exchange, interstitial barrier, and transfer to red blood cells, providing quantitative maps of pulmonary disease (e.g., Hyperpolarized Xenon MRI: Technique and Applications).
    • $^{133}$Cs: Sensitive probe for real-time monitoring of biophysical environments and transmembrane ion exchange (e.g., Rapid zero-trans kinetics of Cs(+) exchange in human erythrocytes quantified by dissolution hyperpolarized (133)Cs(+) NMR spectroscopy, Hyperpolarized (133)Cs is a sensitive probe for real-time monitoring of biophysical environments).
    • $^{29}$Si Silicon Nanoparticles: Offer background-free, long-lived signals for catheter tracking and perfusion imaging, retaining polarization for tens of minutes in vivo (e.g., Real-Time MRI-Guided Catheter Tracking Using Hyperpolarized Silicon Particles, In vivo magnetic resonance imaging of hyperpolarized silicon particles).

2.3. Imaging and Spectroscopy Techniques

HP-MRI/MRS systems are typically specialized for multinuclear capabilities and rapid acquisition:

  • Hardware: Studies are conducted on a range of MRI scanners, from preclinical (7T, 9.4T, 14.1T) to clinical (1.5T, 3T) systems. Custom-designed dual-tuned RF coils (e.g., $^{1}$H-$^{13}$C dual-tuned rat coils, surface coils, implantable arterial coils) are essential for efficient signal transmission and reception (e.g., Hyperpolarized Carbon (13C) MRI of the Kidney: Experimental Protocol, Measurement of Arterial Input Function in Hyperpolarized (13)C Studies).
  • Acquisition Sequences: The transient nature of hyperpolarization necessitates ultra-fast imaging sequences.
    • Dynamic MRS: Time-resolved spectra from a single voxel, often ECG-gated for cardiac studies, acquired every 1-3 seconds (e.g., In Vivo Carbon-13 Dynamic MRS and MRSI of Normal and Fasted Rat Liver with Hyperpolarized 13C-Pyruvate, In vivo mouse cardiac hyperpolarized magnetic resonance spectroscopy).
    • Spectroscopic Imaging (MRSI/CSI): Provides spatial maps of metabolic conversion. Variants include 2D/3D slice-selective Chemical Shift Imaging (CSI), Echo-Planar Spectroscopic Imaging (EPSI), IDEAL spiral CSI, and Free Induction Decay Chemical Shift Imaging (FIDCSI) (e.g., Hyperpolarized Carbon (13C) MRI of the Kidney: Experimental Protocol, Segmental analysis of cardiac metabolism by hyperpolarized [1-13C] pyruvate: an in-vivo 3D MRI study in pigs).
  • Acceleration Techniques: To maximize spatial and temporal resolution within the short signal window:
    • SENSE Reconstruction: Using pre-acquired $^{23}$Na sensitivity maps for $^{13}$C imaging allows for significant temporal acceleration (e.g., Enabling SENSE accelerated 2D CSI for hyperpolarized carbon-13 imaging).
    • Compressed Sensing and Local Low Rank + Sparse Reconstruction: Enable higher resolution and faster acquisitions by leveraging data sparsity (e.g., Using a local low rank plus sparse reconstruction to accelerate dynamic hyperpolarized (13)C imaging using the bSSFP sequence).
    • Multi-echo Balanced SSFP (bSSFP): Faster metabolite mapping and improved signal utilization (e.g., Fast multiecho balanced SSFP metabolite mapping of (1)H and hyperpolarized (13)C compounds).
    • Spiral CSI and Spatiotemporal Encoding: Offer sub-second acquisition times crucial for capturing rapid kinetics (e.g., In vivo application of sub-second spiral chemical shift imaging (CSI) to hyperpolarized 13C metabolic imaging: comparison with phase-encoded CSI).
  • Combined Modalities: Integrating HP-MRI with other imaging techniques provides complementary information. HyperPET, combining HP-$^{13}$C MRI with $^{18}$F-FDG PET, allows simultaneous assessment of glucose uptake and downstream glycolytic flux (e.g., Simultaneous Hyperpolarized 13C-Pyruvate MRI and 18F-FDG PET (HyperPET) in 10 Dogs with Cancer, Simultaneous hyperpolarized (13)C-pyruvate MRI and (18)F-FDG-PET in cancer (hyperPET): feasibility of a new imaging concept using a clinical PET/MRI scanner). Co-imaging with Electron Paramagnetic Resonance Imaging (EPRI) correlates tumor oxygenation and metabolism (e.g., Co-imaging of the tumor oxygenation and metabolism using electron paramagnetic resonance imaging and 13-C hyperpolarized magnetic resonance imaging before and after irradiation).

2.4. Data Analysis and Kinetic Modeling

Quantitative analysis of hyperpolarized data is complex due to the decaying signal:

  • Metabolite Ratios: Simple ratios (e.g., lactate-to-pyruvate, lactate-to-alanine) are commonly used as initial indicators of metabolic shifts (e.g., In Vivo Carbon-13 Dynamic MRS and MRSI of Normal and Fasted Rat Liver with Hyperpolarized 13C-Pyruvate).
  • Kinetic Models: Multi-compartmental models (e.g., two-site, three-site exchange models) are fitted to time-dependent metabolite signals to derive apparent rate constants (k${PL}$, k${PA}$, k$_{PB}$) that reflect enzymatic activity (e.g., Analysis Methods for Hyperpolarized Carbon (13C) MRI of the Kidney, Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy).
  • Model-Free Approaches: To circumvent challenges in measuring the arterial input function (AIF), model-free methods using the ratio of total areas under the curve (AUC) for product to substrate have been developed, demonstrating proportionality to the forward rate constant k$_{PL}$ and robustness against AIF variations (e.g., Model free approach to kinetic analysis of real-time hyperpolarized 13C magnetic resonance spectroscopy data).
  • Perfusion and Clearance Models: Bolus tracking theory and deconvolution methods are adapted to hyperpolarized signals to quantify tissue perfusion (e.g., Analysis Methods for Hyperpolarized Carbon (13C) MRI of the Kidney, Quantitative Perfusion Imaging). The Metabolic Clearance Rate (MCR) framework provides a translational approach for quantitative metabolic and perfusion information, robust to various experimental perturbations (e.g., Developing a metabolic clearance rate framework as a translational analysis approach for hyperpolarized (13)C magnetic resonance imaging).
  • pH and Thermometry Mapping: Algorithms convert chemical shift differences into pH maps (using probes like bicarbonate or zymonic acid) or temperature maps (using pyruvate/lactate chemical shifts) (e.g., Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate, Temperature dependent chemical shifts of pyruvate and lactate enable in vivo hyperpolarized (13)C MRSI thermometry).

3. Major Findings

Hyperpolarization research has yielded significant findings across a broad spectrum of biomedical applications.

3.1. Oncology: Unveiling Tumor Metabolism and Guiding Therapy

Cancer is a primary focus, given its profound metabolic reprogramming (Warburg effect).

  • Warburg Effect and Tumor Aggressiveness: Hyperpolarized [1-$^{13}$C]pyruvate imaging consistently reveals increased pyruvate-to-lactate conversion (elevated lactate/pyruvate ratios and k$_{PL}$) in various cancers, including hepatocellular carcinoma (HCC), breast cancer, prostate cancer, glioblastoma (GBM), lymphoma, and pancreatic cancer. This elevated glycolytic flux correlates with tumor aggressiveness, proliferation, and hypoxia (e.g., Hyperpolarized 13C pyruvate magnetic resonance spectroscopy for in vivo metabolic phenotyping of rat HCC, MRI with hyperpolarised [1-13C]pyruvate detects advanced pancreatic preneoplasia prior to invasive disease in a mouse model, Hyperpolarized in vivo pH imaging reveals grade-dependent acidification in prostate cancer).
  • Early Detection of Treatment Response: HP-MRI/MRS excels at detecting metabolic changes within hours or days of therapy initiation, often before any changes in tumor size are visible with conventional imaging. This has been demonstrated with chemotherapy (e.g., doxorubicin, etoposide), radiation therapy, and targeted agents (e.g., PI3K inhibitors, tyrosine kinase inhibitors like afatinib, TERT/GABPB1 inhibitors) (e.g., Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy, Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate, Evaluation of hyperpolarized [1-13C]Pyruvate Magnetic Resonance Spectroscopic Imaging for Evaluation of Early Response to Tyrosine Kinase Inhibition Therapy in Gastric Cancer). This early detection capability holds immense promise for adaptive treatment strategies.
  • Tumor Heterogeneity and Subtyping: HP-MRI can visualize intratumoral metabolic heterogeneity and differentiate cancer subtypes. For example, it can distinguish AR-dependent from AR-negative castration-resistant prostate cancer (CRPC) based on lactate levels (e.g., Androgen Receptor Signaling in Castration-Resistant Prostate Cancer Alters Hyperpolarized Pyruvate to Lactate Conversion and Lactate Levels In Vivo) or identify infiltrative GBM regions with reduced glycolysis, a feature often missed by anatomical MRI (e.g., Hyperpolarized (13)C-glucose magnetic resonance highlights reduced aerobic glycolysis in vivo in infiltrative glioblastoma).
  • Other Metabolic Pathways in Cancer:
    • Pentose Phosphate Pathway (PPP): HP $\delta$-[1-$^{13}$C]gluconolactone imaging detects PPP flux, a key pathway for NADPH production, showing reduced flux upon TERT or GABPB1 silencing in GBM (e.g., Hyperpolarized $\delta$-[1-(13)C]gluconolactone imaging visualizes response to TERT or GABPB1 targeting therapy for glioblastoma).
    • Redox Status: HP [1-$^{13}$C]dehydroascorbate (DHA) and [1-$^{13}$C]N-acetyl cysteine (NAC) probe tumor oxidative stress and glutathione status, correlating with increased glutathione levels in prostate cancer or forming NAC-GSH dimers in pancreatic tumors (e.g., Hyperpolarized [1-13C]dehydroascorbate MR spectroscopy in a murine model of prostate cancer: comparison with 18F-FDG PET, Real-Time insight into in vivo redox status utilizing hyperpolarized [1-(13)C] N-acetyl cysteine).
    • pH Imaging: HP bicarbonate and zymonic acid reveal tumor acidification, a hallmark of aggressive cancers, with high-grade prostate tumors showing significantly lower extracellular pH (e.g., Hyperpolarized in vivo pH imaging reveals grade-dependent acidification in prostate cancer, Imaging of pH in vivo using hyperpolarized (13)C-labelled zymonic acid).
  • Differentiation: HP $^{13}$C pyruvate imaging can differentiate radiation necrosis from brain tumors, with necrosis showing significantly lower lactate/pyruvate ratios due to reduced cellularity (e.g., Differentiating Radiation Necrosis from Brain Tumor Using Hyperpolarized Carbon-13 MR Metabolic Imaging).
  • Theranostics: Beyond imaging, hyperpolarized probes can have therapeutic effects. Pyruvate injection has been shown to induce transient tumor hypoxia, which can be exploited to potentiate hypoxia-activated prodrugs (HAPs) and radiotherapy, thus enabling a “theranostic” approach (e.g., Hyperpolarized MRI theranostics in cancer).

3.2. Cardiac Metabolism and Function

Hyperpolarization provides unique insights into the heart’s dynamic energy demands:

  • Feasibility and Spatial Localization: Studies in rats, mice, and pigs have demonstrated the feasibility of detecting hyperpolarized [1-$^{13}$C]pyruvate and its metabolites (lactate, alanine, bicarbonate) in the heart within seconds, even using clinical 3T scanners. Advanced imaging methods allow for segmental analysis of left ventricular metabolism, detecting changes induced by coronary occlusion (e.g., Experimental approaches to cardiac imaging with hyperpolarized [1-13c]pyruvate: a feasibility study in rats with a 3T clinical scanner, Segmental analysis of cardiac metabolism by hyperpolarized [1-13C] pyruvate: an in-vivo 3D MRI study in pigs).
  • Detecting Early Disease and Dysfunction: Hyperpolarized MRS is sensitive to changes in PDH flux, showing reductions in models of dilated cardiomyopathy even before echocardiographic signs of contractile dysfunction appear (e.g., The (13)C hyperpolarized pyruvate generated by ParaHydrogen detects the response of the heart to altered metabolism in real time). Doxorubicin-induced cardiotoxicity can be detected by decreased oxidative mitochondrial carbohydrate metabolism (reduced bicarbonate:pyruvate ratio) prior to functional decline, linking it to mitochondrial loss rather than oxidative stress (e.g., Early detection of doxorubicin-induced cardiotoxicity in rats by its cardiac metabolic signature assessed with hyperpolarized MRI).
  • Metabolic Flexibility and Substrate Utilization: HP-[2-$^{13}$C]pyruvate MRS in pigs has shown myocardial metabolic shifts in response to ketone infusion, indicating that ketones become a preferential energy substrate, suppressing glucose oxidation and increasing lactate production (e.g., Myocardial metabolic flexibility following ketone infusion demonstrated by hyperpolarized [2-(13)C]pyruvate MRS in pigs). Co-injection of pyruvate and butyrate revealed substrate competition and potential antiporting mechanisms in fasting models (e.g., Measuring changes in substrate utilization in the myocardium in response to fasting using hyperpolarized [1-(13)C]butyrate and [1-(13)C]pyruvate). A novel HP [3-$^{13}$C]acetoacetate probe revealed increased myocardial ketone body utilization in diabetic rat hearts, correlating with cardiac hypertrophy (e.g., A new hyperpolarized (13)C ketone body probe reveals an increase in acetoacetate utilization in the diabetic rat heart).
  • Sperm Metabolism: HP [1-$^{13}$C]pyruvate MRS has even been used to demonstrate active lactate fermentation and oxidative phosphorylation in motile human sperm, correlating metabolite signals with sperm concentration and progressive motility (e.g., Evidence for Rapid Oxidative Phosphorylation and Lactate Fermentation in Motile Human Sperm by Hyperpolarized (13)C Magnetic Resonance Spectroscopy).

3.3. Liver Metabolism and Disease

The liver’s central role in metabolism makes it a prime target for HP-MRS:

  • Metabolic State Differentiation: HP [1-$^{13}$C]pyruvate can distinguish metabolic states in the liver, such as differences between normal and fasted rats, reflecting perturbations in alanine aminotransferase (ALT) activity (e.g., In Vivo Carbon-13 Dynamic MRS and MRSI of Normal and Fasted Rat Liver with Hyperpolarized 13C-Pyruvate).
  • Gluconeogenesis and Pyruvate Cycling: [$^{13}$C]bicarbonate derived from HP [1-$^{13}$C]pyruvate serves as an in vivo marker of hepatic gluconeogenesis (PEPCK flux) in fasted states (e.g., [13C]bicarbonate labelled from hyperpolarized [1-13C]pyruvate is an in vivo marker of hepatic gluconeogenesis in fasted state). However, it’s not a reliable biomarker for total glucose production due to variable pyruvate cycling (e.g., Production of hyperpolarized (13)CO(2) from [1-(13)C]pyruvate in perfused liver does reflect total anaplerosis but is not a reliable biomarker of glucose production).
  • De Novo Ketogenesis: HP [2-$^{13}$C]pyruvate can detect de novo hepatic ketogenesis from carbohydrates, a process requiring upregulated PDH activity (e.g., Detecting de novo Hepatic Ketogenesis Using Hyperpolarized [2-(13)C] Pyruvate).
  • Diabetic Liver and NAFLD/NASH:
    • HP [1-$^{13}$C]pyruvate MRS reveals that ATP citrate lyase (ACLY) facilitates alanine flux and gluconeogenesis in diabetic (db/db) mouse livers, normalizing these parameters with ACLY inhibition (e.g., ACLY facilitates alanine flux in the livers of db/db mice: a hyperpolarized [1-13C]pyruvate MRS study).
    • HP [1-$^{13}$C]$\alpha$-ketobutyrate (αKB) imaging can detect diminished cytoplasmic reducing capacity in early-stage Non-alcoholic steatohepatitis (NASH), a metabolic change mediated by broad effects on intermediary metabolism (e.g., Detection of early-stage NASH using non-invasive hyperpolarized (13)C metabolic imaging).
    • HP [1-$^{13}$C]dehydroascorbate (DHA) indicates altered redox status in NASH, normalizing upon recovery despite persistent steatosis (e.g., Hyperpolarized (13)C Spectroscopic Evaluation of Oxidative Stress in a Rodent Model of Steatohepatitis).
  • Liver Fibrosis and Hepatitis: HP [1-$^{13}$C]lactate and [1-$^{13}$C]alanine levels are proposed as biomarkers for differentiating HBV-related hepatitis, liver fibrosis, and normal liver, with alanine levels particularly sensitive to fibrosis stage (e.g., In vivo Hyperpolarized Metabolic Imaging to Monitor the Progression of Hepatitis B Virus (HBV)-Related Hepatitis to Liver Fibrosis, Metabolic Changes in Different Stages of Liver Fibrosis: In vivo Hyperpolarized (13)C MR Spectroscopy and Metabolic Imaging).
  • Regional Liver Function: HP [1-$^{13}$C]pyruvate MRI can regionally quantify metabolic liver function, detecting glycolytic shifts in response to partial portal vein ligation, even before traditional regeneration markers change (e.g., Regional quantification of metabolic liver function using hyperpolarized [1-(13)C] pyruvate MRI).

3.4. Kidney Metabolism and Function

HP-MRI offers a non-invasive approach to renal physiology and pathology:

  • Renal Function and Perfusion: Metabolically inert probes like [$^{13}$C]urea and [1,5-$^{13}$C$_2$]Z-OMPD are used for dynamic imaging to quantify renal function, estimate glomerular filtration rate (GFR), and map perfusion (e.g., Hyperpolarized Carbon (13C) MRI of the Kidneys: Basic Concept, Analysis Methods for Hyperpolarized Carbon (13C) MRI of the Kidney, Simultaneous magnetic resonance imaging of pH, perfusion and renal filtration using hyperpolarized (13)C-labelled Z-OMPD). [$^{13}$C]t-butanol can also be used for perfusion (e.g., Investigating the Feasibility of In Vivo Perfusion Imaging Methods for Spinal Cord Using Hyperpolarized [(13)C]t-Butanol and [(13)C,(15)N(2)]Urea).
  • Renal Metabolism in Disease:
    • Diabetic Nephropathy: HP [1,4-$^{13}$C$_2$]fumarate imaging can detect altered renal hemodynamics and suggests apoptosis (rather than acute necrosis) as the predominant cell death mechanism in early diabetic nephropathy (e.g., Hyperpolarized [1,4-(13)C]fumarate imaging detects microvascular complications and hypoxia mediated cell death in diabetic nephropathy). Oxygen-sensitive paramagnetic agents can also quantify kidney hypoxia in diabetic mice (e.g., Dynamic nuclear polarization magnetic resonance imaging and the oxygen-sensitive paramagnetic agent OX63 provide a noninvasive quantitative evaluation of kidney hypoxia in diabetic mice).
    • Acute Kidney Injury (AKI): HP [1-$^{13}$C]pyruvate MRI detects metabolic reprogramming in renal ischemia-reperfusion injury (IRI), with early lactate-to-bicarbonate ratios correlating with long-term outcomes and injury severity (e.g., Metabolic reprogramming associated with progression of renal ischemia reperfusion injury assessed with hyperpolarized [1-(13)C]pyruvate).
    • Metabolic Drug Effects: Metformin acutely shifts healthy kidney metabolism towards anaerobic pathways (increased lactate production, reduced bicarbonate) independently of oxygen supply, an effect blunted in diabetic kidneys (e.g., Acute renal metabolic effect of metformin assessed with hyperpolarised MRI in rats).
    • Gluconeogenesis: HP [1-$^{13}$C]pyruvate-derived aspartate is identified as a promising marker for renal gluconeogenic flux (e.g., Assessment of Aspartate and Bicarbonate Produced From Hyperpolarized [1-(13)C]Pyruvate as Markers of Renal Gluconeogenesis).

3.5. Brain Metabolism and Neurological Disorders

HP-MRI faces unique challenges in the brain due to the blood-brain barrier (BBB):

  • Blood-Brain Barrier Limitations: Transport of HP [1-$^{13}$C]pyruvate across the BBB is rate-limiting for brain metabolism detection in anaesthetized animals. Metabolic signals (e.g., lactate production) are often only detectable when the BBB is disrupted by disease (e.g., brain metastasis) or experimentally permeabilized (e.g., mannitol infusion) (e.g., 13C Pyruvate Transport Across the Blood-Brain Barrier in Preclinical Hyperpolarised MRI). More lipophilic pyruvate analogues or BBB-permeable probes (e.g., [$^{13}$C]t-butanol) are being explored.
  • Stroke and Injury: HP [1-$^{13}$C]L-lactate can serve as a neuroprotectant biosensor in stroke, showing rapid, time-after-reperfusion-dependent conversion to pyruvate and bicarbonate (e.g., Evaluating the potential of hyperpolarised [1-(13)C] L-lactate as a neuroprotectant metabolic biosensor for stroke). Traumatic brain injury (TBI) causes impaired mitochondrial pyruvate metabolism and increased lactate/pyruvate ratios in the injured hemisphere (e.g., Metabolic imaging of energy metabolism in traumatic brain injury using hyperpolarized [1-(13)C]pyruvate).
  • Neuroinflammation: HP [1-$^{13}$C]pyruvate MRS can detect increased pyruvate-to-lactate conversion in MS models with neuroinflammation, detecting therapeutic responses that conventional MRI might miss (e.g., Imaging immunomodulatory treatment responses in a multiple sclerosis mouse model using hyperpolarized (13)C metabolic MRI). A murine pseudo-infection model showed long-lasting glycolytic shifts in the brain, correlating with locomotor activity and dopamine markers (e.g., Non-invasive visualisation of long-lasting brain metabolic alterations in murine pseudo-infection model using parahydrogen-polarised [1-(13)C] pyruvate MRI).
  • Neurodegeneration and Cognitive Impairment: HP [1-$^{13}$C]pyruvate MRS detected altered brain glycolysis and hypoperfusion in high-fat diet-induced diabetic mice, correlating with cognitive decline (e.g., Hyperpolarized [1-13C] pyruvate MR spectroscopy detect altered glycolysis in the brain of a cognitively impaired mouse model fed high-fat diet, Hyperpolarized [1-(13)C]Pyruvate Magnetic Resonance Spectroscopy Shows That Agmatine Increased Lactate Production in the Brain of Type 2 Diabetic Mice).
  • Glucose and Ketone Metabolism: Real-time cerebral glucose metabolism can be measured with sub-second resolution using hyperpolarized [$^{13}$C,$^{2}$H]glucose, with specific labeling improving SNR (e.g., Measuring glucose cerebral metabolism in the healthy mouse using hyperpolarized (13)C magnetic resonance). HP [1,3-$^{13}$C$_2$]acetoacetate can probe mitochondrial redox and BDH activity in glioma, revealing altered metabolism in tumors (e.g., In vivo investigation of hyperpolarized [1,3-(13)C(2)]acetoacetate as a metabolic probe in normal brain and in glioma).
  • Redox Status and Enzyme Activity: HP [1-$^{13}$C]dehydroascorbate (DHA) detects glutathione depletion in the brain (e.g., Imaging glutathione depletion in the rat brain using ascorbate-derived hyperpolarized MR and PET probes). HP $\gamma$-glutamyl-[1-$^{13}$C]glycine can non-invasively detect $\gamma$-glutamyl-transferase (GGT) upregulation in glioma, linking it to elevated GSH levels (e.g., In vivo detection of $\gamma$-glutamyl-transferase up-regulation in glioma using hyperpolarized $\gamma$-glutamyl-[1-(13)C]glycine).

3.6. Immune Cell Metabolism and Inflammation

HP-MRS offers a rapid, non-invasive way to probe immune cell metabolic adaptation:

  • Inflammation Detection: In macrophage models, HP [1-$^{13}$C]pyruvate shows a significant increase in lactate/pyruvate ratio in pro-inflammatory M1 polarized macrophages, reversible with NSAID treatment. This correlates with elevated LDH activity and NADH levels (e.g., Molecular detection of inflammation in cell models using hyperpolarized (13)C-pyruvate).
  • T-Lymphocyte Activation: HP [1-$^{13}$C]pyruvate rapidly detects increased pyruvate-to-lactate flux in activated human CD4+ T lymphocytes, correlating with LDHA upregulation (e.g., Noninvasive rapid detection of metabolic adaptation in activated human T lymphocytes by hyperpolarized (13)C magnetic resonance).
  • Arginase Activity: HP [6-$^{13}$C]arginine allows detection of arginase (ARG) activity in Myeloid-derived suppressor cells (MDSCs) via [$^{13}$C]urea production, correlating with intracellular arginase concentrations (e.g., Detection of inflammatory cell function using (13)C magnetic resonance spectroscopy of hyperpolarized [6-(13)C]-arginine).
  • CAR T Cell Metabolism: HP [U-$^{13}$C, $^{2}$H]glucose maps the dynamic metabolic plasticity of CAR T cells during expansion, showing a shift from oxidative phosphorylation to aerobic glycolysis and back, highlighting nutrient depletion bottlenecks (e.g., Mapping real-time metabolic kinetics of expanded CAR T cells using hyperpolarized (13)C-glucose and metabolomics).
  • Lung Inflammation: Ex vivo perfused lungs with bleomycin-induced inflammation show significantly increased apparent forward and reverse rate constants of LDH reaction, correlating with histological scores (e.g., Differentiating inflamed and normal lungs by the apparent reaction rate constants of lactate dehydrogenase probed by hyperpolarized (13)C labeled pyruvate).

3.7. Other Applications and General Principles

  • Brown Adipose Tissue (BAT) Activation: HP [1-$^{13}$C]pyruvate detects functional BAT in rats by increased conversion to bicarbonate and lactate upon norepinephrine stimulation, reflecting elevated carbohydrate metabolism (e.g., Noninvasive identification and assessment of functional brown adipose tissue in rodents using hyperpolarized ¹³C imaging).
  • Skeletal Muscle Function: A multi-bolus strategy with HP [1-$^{13}$C]pyruvate allows for repeated real-time measurements of glycolytic metabolism in murine skeletal muscle in response to electrical stimulation, revealing graded, non-linear metabolic responses (e.g., Hyperpolarized functional magnetic resonance of murine skeletal muscle enabled by multiple tracer-paradigm synchronizations).
  • Catheter Tracking: Hyperpolarized $^{29}$Si silicon nanoparticles enable real-time, background-free MRI-guided catheter tracking in vivo over extended durations, offering a radiation-free alternative to X-ray fluoroscopy (e.g., Real-Time MRI-Guided Catheter Tracking Using Hyperpolarized Silicon Particles).
  • In Vitro High-Throughput Analysis: Microfluidic platforms combined with dDNP-MRS imaging allow for simultaneous measurement of multiple chemical reactions from a single hyperpolarized sample, significantly increasing throughput for basic chemical and biological studies (e.g., Parallel detection of chemical reactions in a microfluidic platform using hyperpolarized nuclear magnetic resonance).

4. Challenges and Future Directions

Despite tremendous progress, several challenges remain for hyperpolarization technology to achieve widespread clinical adoption and fully realize its potential.

4.1. Technical and Methodological Hurdles

  • Signal Lifetime and Acquisition Speed: The transient nature of hyperpolarization (T1 decay typically 30-60 seconds for useful probes) remains a fundamental limitation, requiring ultra-fast acquisition sequences and limiting the total imaging time (e.g., Hyperpolarized Carbon (13C) MRI of the Kidneys: Basic Concept). Continued innovation in acquisition schemes (e.g., compressed sensing, SENSE, bSSFP variants) is crucial for improving spatial and temporal resolution simultaneously (e.g., Enabling SENSE accelerated 2D CSI for hyperpolarized carbon-13 imaging, Using a local low rank plus sparse reconstruction to accelerate dynamic hyperpolarized (13)C imaging using the bSSFP sequence).
  • Quantitative Accuracy: Accurately quantifying metabolic rates in vivo is challenging due to complex kinetic models, the need for robust arterial input function (AIF) measurements (which are difficult in humans), and confounding factors like tissue perfusion, diffusion, and partial volume effects (e.g., Analysis Methods for Hyperpolarized Carbon (13C) MRI of the Kidney, Measurement of Arterial Input Function in Hyperpolarized (13)C Studies). Model-free approaches and the Metabolic Clearance Rate (MCR) framework offer promising solutions to simplify and standardize quantification (e.g., Model free approach to kinetic analysis of real-time hyperpolarized 13C magnetic resonance spectroscopy data, Developing a metabolic clearance rate framework as a translational analysis approach for hyperpolarized (13)C magnetic resonance imaging).
  • Hyperpolarizer Throughput and Cost: Current DNP polarizers are expensive, complex, and typically prepare one sample at a time, limiting the number of studies per day. Multi-sample polarizers are being developed to address this (e.g., Multi-sample/multi-nucleus parallel polarization and monitoring enabled by a fluid path technology compatible cryogenic probe for dissolution dynamic nuclear polarization). Cost-effective alternatives like PHIP are also gaining traction (e.g., In-vitro NMR Studies of Prostate Tumor Cell Metabolism by Means of Hyperpolarized [1-(13)C]Pyruvate Obtained Using the PHIP-SAH Method).
  • Probe Development and Specificity: There is an ongoing need for novel, long-lived, and metabolically specific probes that can interrogate a wider array of metabolic pathways, redox states, and enzyme activities, or that can be targeted to specific cell types or organelles. Probes like $^{15}$N urea for perfusion, $^{133}$Cs for ion transport, and $^{29}$Si nanoparticles for unique imaging properties highlight the diversification beyond $^{13}$C (e.g., Hyperpolarized Carbon (13C) MRI of the Kidneys: Basic Concept, Hyperpolarized Xenon MRI: Technique and Applications, Real-Time MRI-Guided Catheter Tracking Using Hyperpolarized Silicon Particles).
  • Biocompatibility and Dose: Ensuring the safety and biocompatibility of hyperpolarized agents, especially their non-labeled components (e.g., radicals, solvents, catalysts), at the required doses for clinical use is paramount. While [1-$^{13}$C]pyruvate has a good safety profile, some PHIP methods may leave residual toxic components (e.g., chloroform) that require rigorous purification (e.g., In-vitro NMR Studies of Prostate Tumor Cell Metabolism by Means of Hyperpolarized [1-(13)C]Pyruvate Obtained Using the PHIP-SAH Method).

4.2. Clinical Translation and Integration

  • Regulatory Approval: Gaining FDA and other regulatory approvals for novel hyperpolarized agents is a complex and lengthy process. The first-in-human studies with hyperpolarized [1-$^{13}$C]pyruvate and [$^{13}$C,$^{15}$N$_2$]urea have demonstrated safety and feasibility, paving the way for wider clinical trials (e.g., Hyperpolarized Carbon (13C) MRI: Path to Clinical Translation in Oncology, Translation of hyperpolarized [13C,15N2]urea MRI for novel human brain perfusion studies).
  • Workflow Integration: Seamless integration of hyperpolarization technology into existing clinical workflows (from patient preparation to data acquisition and analysis) requires dedicated infrastructure, specialized training, and standardized protocols. The “HyperPET” concept, combining HP-MRI with PET in a single session, demonstrates a path toward multimodal clinical integration (e.g., Simultaneous hyperpolarized (13)C-pyruvate MRI and (18)F-FDG-PET in cancer (hyperPET): feasibility of a new imaging concept using a clinical PET/MRI scanner).
  • Clinical Utility and Comparative Effectiveness: The clinical value of HP-MRI/MRS must be definitively established in comparison to, or in combination with, existing diagnostic modalities (e.g., PET, conventional MRI). Demonstrating unique, actionable information for patient management will be key for adoption (e.g., Hyperpolarized Carbon (13C) MRI: Path to Clinical Translation in Oncology).

4.3. Future Directions

  • Multi-parametric and Multi-modal Imaging: The future will see increased integration of HP-MRI with other imaging modalities (PET, DCE-MRI, DWI, EPR) and functional readouts (pH, perfusion, oxygenation) to provide a more comprehensive picture of disease biology (e.g., Simultaneous characterization of tumor cellularity and the Warburg effect with PET, MRI and hyperpolarized (13)C-MRSI, Co-imaging of the tumor oxygenation and metabolism using electron paramagnetic resonance imaging and 13-C hyperpolarized magnetic resonance imaging before and after irradiation).
  • Advanced Image Reconstruction and AI: Leveraging artificial intelligence and deep learning for advanced image reconstruction, particularly from undersampled data, will further improve spatial-temporal resolution and accelerate acquisition, unlocking the full potential of transient hyperpolarized signals.
  • Theranostic Applications: The development of probes that can both image and modulate cellular biology, or enhance therapeutic efficacy (as seen with pyruvate-induced hypoxia), represents a burgeoning field with significant clinical promise (e.g., Hyperpolarized MRI theranostics in cancer).
  • Targeted Molecular Imaging: Engineering targeted hyperpolarized agents (e.g., conjugating probes to antibodies or specific ligands) could enable cell-specific metabolic profiling or visualization of specific enzyme activities with even greater precision.
  • Expanded Disease Applications: Continued exploration of HP-MRS/MRI in a wider range of diseases, including neurodegenerative conditions, infectious diseases, and chronic inflammatory states, will uncover new metabolic biomarkers for diagnosis, prognosis, and treatment monitoring.
  • Low-Field Imaging: Developing and validating HP-MRI/MRS at lower magnetic field strengths (e.g., 1T, 48.7 mT) could lead to more affordable, accessible, and potentially portable systems, expanding its reach (e.g., Sampling Hyperpolarized Molecules Utilizing a 1 Tesla Permanent Magnetic Field, High-resolution hyperpolarized in vivo metabolic (13)C spectroscopy at low magnetic field (48.7mT) following murine tail-vein injection).

5. Conclusion

Hyperpolarization research has ushered in a new era of metabolic imaging, offering unprecedented sensitivity and the ability to visualize real-time biochemical processes in vivo. From its foundational development in DNP and PHIP techniques to the creation of a diverse library of metabolic and physiological probes, the field has rapidly matured. Major findings span across oncology, cardiology, hepatology, nephrology, neurology, and immunology, demonstrating its power to detect early disease, monitor therapy response, and dissect complex metabolic adaptations. While challenges related to signal lifetime, quantification, throughput, and clinical translation persist, ongoing innovations in hardware, acquisition sequences, data analysis, and probe chemistry are steadily addressing these limitations. With its unique capacity to provide dynamic, non-invasive metabolic information, hyperpolarization is poised to significantly impact scientific discovery and clinical care, offering a powerful tool for personalized medicine and advancing our understanding of health and disease.

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