Single Nucleotide Polymorphism in Cell Adhesion Molecule L1 Affects Learning and Memory in a Mouse Model of Traumatic Brain Injury.

The L1 cell adhesion molecule (L1) has demonstrated a range of beneficial effects in animal models of spinal cord injury, neurodegenerative disease, and ischemia; however, the role of L1 in TBI has not been fully examined. Mutations in the L1 gene affecting the extracellular domain of this type 1 transmembrane glycoprotein have been identified in patients with L1 syndrome. These patients suffer from hydrocephalus, MASA (mental retardation, adducted thumbs, shuffling gait, aphasia) symptoms, and corpus callosum agenesis. Clinicians have observed that recovery post-traumatic brain injury (TBI) varies among the population. This variability may be explained by the genetic differences present in the general population. In this study, we utilized a novel mouse model of L1 syndrome with a mutation at aspartic acid position 201 in the extracellular domain of L1 (L1-201). We assessed the impact of this specific single nucleotide polymorphism (SNP) localized to the X-chromosome L1 gene on recovery outcomes following TBI by comparing the L1-201 mouse mutants with their wild-type littermates. We demonstrate that male L1-201 mice exhibit significantly worse learning and memory outcomes in the Morris water maze after lateral fluid percussion (LFP) injury compared to male wild-type mice and a trend to worse motor function on the rotarod. However, no significant changes were observed in markers for inflammatory responses or apoptosis after TBI.

13C MRI of hyperpolarized pyruvate at 120 µT.

Nuclear spin hyperpolarization increases the sensitivity of magnetic resonance dramatically, enabling many new applications, including real-time metabolic imaging. Parahydrogen-based signal amplification by reversible exchange (SABRE) was employed to hyperpolarize [1-13C]pyruvate and demonstrate 13C imaging in situ at 120 µT, about twice Earth's magnetic field, with two different signal amplification by reversible exchange variants: SABRE in shield enables alignment transfer to heteronuclei (SABRE-SHEATH), where hyperpolarization is transferred from parahydrogen to [1-13C]pyruvate at a magnetic field below 1 µT, and low-irradiation generates high tesla (LIGHT-SABRE), where hyperpolarization was prepared at 120 µT, avoiding magnetic field cycling. The 3-dimensional images of a phantom were obtained using a superconducting quantum interference device (SQUID) based magnetic field detector with submillimeter resolution. These 13C images demonstrate the feasibility of low-field 13C metabolic magnetic resonance imaging (MRI) of 50 mM [1-13C]pyruvate hyperpolarized by parahydrogen in reversible exchange imaged at about twice Earth's magnetic field. Using thermal 13C polarization available at 120 µT, the same experiment would have taken about 300 billion years.

Carbon-13 Radiofrequency Amplification by Stimulated Emission of Radiation of the Hyperpolarized Ketone and Hemiketal Forms of Allyl [1-13C]Pyruvate.

13C hyperpolarized pyruvate is an emerging MRI contrast agent for sensing molecular events in cancer and other diseases with aberrant metabolic pathways. This metabolic contrast agent can be produced via several hyperpolarization techniques. Despite remarkable success in research settings, widespread clinical adoption faces substantial roadblocks because the current sensing technology utilized to sense this contrast agent requires the excitation of 13C nuclear spins that also need to be synchronized with MRI field gradient pulses. Here, we demonstrate sensing of hyperpolarized allyl [1-13C]pyruvate via the stimulated emission of radiation that mitigates the requirements currently blocking broader adoption. Specifically, 13C Radiofrequency Amplification by Stimulated Emission of Radiation (13C RASER) was obtained after pairwise addition of parahydrogen to a pyruvate precursor, detected in a commercial inductive detector with a quality factor (Q) of 32 for sample concentrations as low as 0.125 M with 13C polarization of 4%. Moreover, parahydrogen-induced polarization allowed for the preparation of a mixture of ketone and hemiketal forms of hyperpolarized allyl [1-13C]pyruvate, which are separated by 10 ppm in 13C NMR spectra. This is a good model system to study the simultaneous 13C RASER signals of multiple 13C species. This system models the metabolic production of hyperpolarized [1-13C]lactate from hyperpolarized [1-13C]pyruvate, which has a similar chemical shift difference. Our results show that 13C RASER signals can be obtained from both species simultaneously when the emission threshold is exceeded for both species. On the other hand, when the emission threshold is exceeded only for one of the hyperpolarized species, 13C stimulated emission is confined to this species only, therefore enabling the background-free detection of individual hyperpolarized 13C signals. The reported results pave the way to novel sensing approaches of 13C hyperpolarized pyruvate, potentially unlocking hyperpolarized 13C MRI on virtually any MRI system─an attractive vision for the future molecular imaging and diagnostics.

Facile hyperpolarization chemistry for molecular imaging and metabolic tracking of [1-13C]pyruvate in vivo.

Hyperpolarization chemistry based on reversible exchange of parahydrogen, also known as Signal Amplification By Reversible Exchange (SABRE), is a particularly simple approach to attain high levels of nuclear spin hyperpolarization, which can enhance NMR and MRI signals by many orders of magnitude. SABRE has received significant attention in the scientific community since its inception because of its relative experimental simplicity and its broad applicability to a wide range of molecules, however in vivo detection of molecular probes hyperpolarized by SABRE has remained elusive. Here we describe a first demonstration of SABRE-hyperpolarized contrast detected in vivo, specifically using hyperpolarized [1-13C]pyruvate. Biocompatible formulations of hyperpolarized [1-13C]pyruvate in, both, methanol-water mixtures, and ethanol-water mixtures followed by dilution with saline and catalyst filtration were prepared and injected into healthy Sprague Dawley and Wistar rats. Effective hyperpolarization-catalyst removal was performed with silica filters without major losses in hyperpolarization. Metabolic conversion of pyruvate to lactate, alanine, and bicarbonate was detected in vivo. Pyruvate-hydrate was also observed as minor byproduct. Measurements were performed on the liver and kidney at 4.7 T via time-resolved spectroscopy and chemical-shift-resolved MRI. In addition, whole-body metabolic measurements were obtained using a cryogen-free 1.5 T MRI system, illustrating the utility of combining lower-cost MRI systems with simple, low-cost hyperpolarization chemistry to develop safe, and scalable molecular imaging.

Delivering Robust Proton-Only Sensing of Hyperpolarized [1,2-13C2]-Pyruvate Using Broad-Spectral-Range Nuclear Magnetic Resonance Pulse Sequences.

Hyperpolarized [1-13C]pyruvate is the leading hyperpolarized injectable contrast agent and is currently under evaluation in clinical trials for molecular imaging of metabolic diseases, including cardiovascular disease and cancer. One aspect limiting broad scalability of the technique is that hyperpolarized 13C MRI requires specialized 13C hardware and software that are not generally available on clinical MRI scanners, which employ proton-only detection. Here, we present an approach that uses pulse sequences to transfer 13C hyperpolarization to methyl protons for detection of the 13C-13C pyruvate singlet, employing proton-only excitation and detection only. The new pulse sequences are robust to the B1 and B0 magnetic field inhomogeneities. The work focuses on singlet-to-magnetization (S2M) and rotor-synchronized (R) pulses, both relying on trains of hard pulses with broad spectral width coverage designed to effectively transform hyperpolarized 13C2-singlet hyperpolarization to 1H polarization on the CH3 group of [1,2-13C2]pyruvate. This approach may enable a broader adoption of hyperpolarized MRI as a molecular imaging technique.

Small Organic Compounds Mimicking the Effector Domain of Myristoylated Alanine-Rich C-Kinase Substrate Stimulate Female-Specific Neurite Outgrowth.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a critical member of a signaling cascade that influences disease-relevant neural functions such as neural growth and plasticity. The effector domain (ED) of MARCKS interacts with the extracellular glycan polysialic acid (PSA) through the cell membrane to stimulate neurite outgrowth in cell culture. We have shown that a synthetic ED peptide improves functional recovery after spinal cord injury in female but not male mice. However, peptides themselves are unstable in therapeutic applications, so we investigated more pharmacologically relevant small organic compounds that mimic the ED peptide to maximize therapeutic potential. Using competition ELISAs, we screened small organic compound libraries to identify molecules that structurally and functionally mimic the ED peptide of MARCKS. Since we had shown sex-specific effects of MARCKS on spinal cord injury recovery, we assayed neuronal viability as well as neurite outgrowth from cultured cerebellar granule cells of female and male mice separately. We found that epigallocatechin, amiodarone, sertraline, tegaserod, and nonyloxytryptamine bind to a monoclonal antibody against the ED peptide, and compounds stimulate neurite outgrowth in cultured cerebellar granule cells of female mice only. Therefore, a search for compounds that act in males appears warranted.

Sex Differences in Immune Cell Infiltration and Hematuria in SCI-Induced Hemorrhagic Cystitis.

Rats manifest a condition called hemorrhagic cystitis after spinal cord injury (SCI). The mechanism of this condition is unknown, but it is more severe in male rats than in female rats. We assessed the role of sex regarding hemorrhagic cystitis and pathological chronic changes in the bladder. We analyzed the urine of male and female Sprague-Dawley and Fischer 344 rats after experimental spinal cord contusion, including unstained microscopic inspections of the urine, differential white blood cell counts colored by the Wright stain, and total leukocyte counts using fluorescent nuclear stains. We examined bladder histological changes in acute and chronic phases of SCI, using principal component analysis (PCA) and clustered heatmaps of Pearson correlation coefficients to interpret how measured variables correlated with each other. Male rats showed a distinct pattern of macroscopic hematuria after spinal cord injury. They had higher numbers of red blood cells with significantly more leukocytes and neutrophils than female rats, particularly hypersegmented neutrophils. The histological examination of the bladders revealed a distinct line of apoptotic umbrella cells and disrupted bladder vessels early after SCI and progressive pathological changes in multiple bladder layers in the chronic phase. Multivariate analyses indicated immune cell infiltration in the bladder, especially hypersegmented neutrophils, that correlated with red blood cell counts in male rats. Our study highlights a hitherto unreported sex difference of hematuria and pathological changes in males and females' bladders after SCI, suggesting an important role of immune cell infiltration, especially neutrophils, in SCI-induced hemorrhagic cystitis.

Spin dynamics of [1,2-13C2]pyruvate hyperpolarization by parahydrogen in reversible exchange at micro Tesla fields.

Hyperpolarization of 13C-pyruvate via Signal Amplificaton By Reversibble Exchange (SABRE) is an important recent discovery because of both the relative simplicity of hyperpolarization and the central biological relevance of pyruvate as a biomolecular probe for in vitro or in vivo studies. Here, we analyze the [1,2-13C2]pyruvate-SABRE spin system and its field dependence theoretically and experimentally. We provide first-principles analysis of the governing 4-spin dihydride-13C2 Hamiltonian and numerical spin dynamics simulations of the 7-spin dihydride-13C2-CH3 system. The analytical and the numerical results are compared to matching systematic experiments. With these methods we unravel the observed spin state mixing of singlet states and triplet states at microTesla fields and we also analyze the dynamics during transfer from micro-Tesla field to high field for detection to understand the resulting spectra from the [1,2-13C2]pyruvate-SABRE system.

13C Radiofrequency Amplification by Stimulated Emission of Radiation Threshold Sensing of Chemical Reactions.

Conventional nuclear magnetic resonance (NMR) enables detection of chemicals and their transformations by exciting nuclear spin ensembles with a radio-frequency pulse followed by detection of the precessing spins at their characteristic frequencies. The detected frequencies report on chemical reactions in real time and the signal amplitudes scale with concentrations of products and reactants. Here, we employ Radiofrequency Amplification by Stimulated Emission of Radiation (RASER), a quantum phenomenon producing coherent emission of 13C signals, to detect chemical transformations. The 13C signals are emitted by the negatively hyperpolarized biomolecules without external radio frequency pulses and without any background signal from other, nonhyperpolarized spins in the ensemble. Here, we studied the hydrolysis of hyperpolarized ethyl-[1-13C]acetate to hyperpolarized [1-13C]acetate, which was analyzed as a model system by conventional NMR and 13C RASER. The chemical transformation of 13C RASER-active species leads to complete and abrupt disappearance of reactant signals and delayed, abrupt reappearance of a frequency-shifted RASER signal without destroying 13C polarization. The experimentally observed "quantum" RASER threshold is supported by simulations.

Parahydrogen in Reversible Exchange Induces Long-Lived 15N Hyperpolarization of Anticancer Drugs Anastrozole and Letrozole.

Hyperpolarization modalities overcome the sensitivity limitations of NMR and unlock new applications. Signal amplification by reversible exchange (SABRE) is a particularly cheap, quick, and robust hyperpolarization modality. Here, we employ SABRE for simultaneous chemical exchange of parahydrogen and nitrile-containing anticancer drugs (letrozole or anastrozole) to enhance 15N polarization. Distinct substrates require unique optimal parameter sets, including temperature, magnetic field, or a shaped magnetic field profile. The fine tuning of these parameters for individual substrates is demonstrated here to maximize 15N polarization. After optimization, including the usage of pulsed µT fields, the 15N nuclei on common anticancer drugs, letrozole and anastrozole, can be polarized within 1-2 min. The hyperpolarization can exceed 10%, corresponding to 15N signal enhancement of over 280,000-fold at a clinically relevant magnetic field of 1 T. This sensitivity gain enables polarization studies at naturally abundant 15N enrichment level (0.4%). Moreover, the nitrile 15N sites enable long-lasting polarization storage with [15N]T1 over 9 min, enabling signal detection from a single hyperpolarization cycle for over 30 min.

LIGHT-SABRE Hyperpolarizes 1-13C-Pyruvate Continuously without Magnetic Field Cycling.

Nuclear spin hyperpolarization enables real-time observation of metabolism and intermolecular interactions in vivo. 1-13C-pyruvate is the leading hyperpolarized tracer currently under evaluation in several clinical trials as a promising molecular imaging agent. Still, the quest for a simple, fast, and efficient hyperpolarization technique is ongoing. Here, we describe that continuous, weak irradiation in the audio-frequency range of the 13C spin at the 121 µT magnetic field (approximately twice Earth's field) enables spin order transfer from parahydrogen to 13C magnetization of 1-13C-pyruvate. These so-called LIGHT-SABRE pulses couple nuclear spin states of parahydrogen and pyruvate via the J-coupling network of reversibly exchanging Ir-complexes. Using ∼100% parahydrogen at ambient pressure, we polarized 51 mM 1-13C-pyruvate in the presence of 5.1 mM Ir-complex continuously and repeatedly to a polarization of 1.1% averaged over free and catalyst-bound pyruvate. The experiments were conducted at -8 °C, where almost exclusively bound pyruvate was observed, corresponding to an estimated 11% polarization on bound pyruvate. The obtained hyperpolarization levels closely match those obtained via SABRE-SHEATH under otherwise identical conditions. The creation of three different types of spin orders was observed: transverse 13C magnetization along the applied magnetic field, 13C z-magnetization along the main field B 0, and 13C-1H zz-spin order. With a superconducting quantum interference device (SQUID) for detection, we found that the generated spin orders result from 1H-13C J-coupling interactions, which are not visible even with our narrow linewidth below 0.3 Hz and at -8 °C.

Efficient SABRE-SHEATH Hyperpolarization of Potent Branched-Chain-Amino-Acid Metabolic Probe [1-13C]ketoisocaproate.

Efficient 13C hyperpolarization of ketoisocaproate is demonstrated in natural isotopic abundance and [1-13C]enriched forms via SABRE-SHEATH (Signal Amplification By Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei). Parahydrogen, as the source of nuclear spin order, and ketoisocaproate undergo simultaneous chemical exchange with an Ir-IMes-based hexacoordinate complex in CD3OD. SABRE-SHEATH enables spontaneous polarization transfer from parahydrogen-derived hydrides to the 13C nucleus of transiently bound ketoisocaproate. 13C polarization values of up to 18% are achieved at the 1-13C site in 1 min in the liquid state at 30 mM substrate concentration. The efficient polarization build-up becomes possible due to favorable relaxation dynamics. Specifically, the exponential build-up time constant (14.3 ± 0.6 s) is substantially lower than the corresponding polarization decay time constant (22.8 ± 1.2 s) at the optimum polarization transfer field (0.4 microtesla) and temperature (10 °C). The experiments with natural abundance ketoisocaproate revealed polarization level on the 13C-2 site of less than 1%-i.e., one order of magnitude lower than that of the 1-13C site-which is only partially due to more-efficient relaxation dynamics in sub-microtesla fields. We rationalize the overall much lower 13C-2 polarization efficiency in part by less favorable catalyst-binding dynamics of the C-2 site. Pilot SABRE experiments at pH 4.0 (acidified sample) versus pH 6.1 (unaltered sodium [1-13C]ketoisocaproate) reveal substantial modulation of SABRE-SHEATH processes by pH, warranting future systematic pH titration studies of ketoisocaproate, as well as other structurally similar ketocarboxylate motifs including pyruvate and alpha-ketoglutarate, with the overarching goal of maximizing 13C polarization levels in these potent molecular probes. Finally, we also report on the pilot post-mortem use of HP [1-13C]ketoisocaproate in a euthanized mouse, demonstrating that SABRE-hyperpolarized 13C contrast agents hold promise for future metabolic studies.

SABRE Hyperpolarization with up to 200 bar Parahydrogen in Standard and Quickly Removable Solvents.

Parahydrogen (p-H2)-based techniques are known to drastically enhance NMR signals but are usually limited by p-H2 supply. This work reports p-H2-based SABRE hyperpolarization at p-H2 pressures of hundreds of bar, far beyond the typical ten bar currently reported in the literature. A recently designed high-pressure setup was utilized to compress p-H2 gas up to 200 bar. The measurements were conducted using a sapphire high-pressure NMR tube and a 43 MHz benchtop NMR spectrometer. In standard methanol solutions, it could be shown that the signal intensities increased with pressure until they eventually reached a plateau. A polarization of about 2%, equal to a molar polarization of 1.2 mmol L-1, could be achieved for the sample with the highest substrate concentration. While the signal plateaued, the H2 solubility increased linearly with pressure from 1 to 200 bar, indicating that p-H2 availability is not the limiting factor in signal enhancement beyond a certain pressure, depending on sample composition. Furthermore, the possibility of using liquefied ethane and compressed CO2 as removable solvents for hyperpolarization was demonstrated. The use of high pressures together with quickly removable organic/non-organic solvents represents an important breakthrough in the field of hyperpolarization, advancing SABRE as a promising tool for materials science, biophysics, and molecular imaging.

Effects of L1 adhesion molecule agonistic mimetics on signal transduction in neuronal functions.

The L1 cell adhesion molecule plays an essential role in neural development and repair. It is not only a 'lock and key' recognition molecule, but an important signal transducer that stimulates regenerative-beneficial cellular functions such as neurite outgrowth, neuronal cell migration, survival, myelination, and synapse formation. Triggering L1 functions after neurotrauma improves functional recovery. In addition, loss-of-function mutations in the L1 gene lead to the L1 syndrome, a rare, X-linked neurodevelopmental disorder with an incidence of approximately 1:30,000 in newborn males. To use L1 for beneficial functions, we screened small compound libraries for L1 agonistic mimetics that trigger L1 functions and improve conditions in animal models of neurotrauma and the L1 syndrome. To understand the mechanisms underlying these functions, it is important to gain a better understanding of L1-dependent cellular signaling that is triggered by the L1 agonistic mimetics. We tested the cell signaling features of L1 agonistic mimetics that contribute to neurite outgrowth and neuronal migration. Our findings indicates that L1 agonistic mimetics trigger the same cell signaling pathways underlying neurite outgrowth, but only the L1 mimetics tacrine, polydatin, trimebutine and honokiol trigger neuronal migration. In contrast, the mimetics crotamiton and duloxetine did not affect neuronal migration, thus limiting their use in increasing neuronal migration, leaving open the question of whether this is a desired or not desired feature in the adult.

Parahydrogen-Induced Carbon-13 Radiofrequency Amplification by Stimulated Emission of Radiation.

The feasibility of Carbon-13 Radiofrequency (RF) Amplification by Stimulated Emission of Radiation (C-13 RASER) is demonstrated on a bolus of liquid hyperpolarized ethyl [1-13 C]acetate. Hyperpolarized ethyl [1-13 C]acetate was prepared via pairwise addition of parahydrogen to vinyl [1-13 C]acetate and polarization transfer from nascent parahydrogen-derived protons to the carbon-13 nucleus via magnetic field cycling yielding C-13 nuclear spin polarization of approximately 6 %. RASER signals were detected from samples with concentration ranging from 0.12 to 1 M concentration using a non-cryogenic 1.4T NMR spectrometer equipped with a radio-frequency detection coil with a quality factor (Q) of 32 without any modifications. C-13 RASER signals were observed for several minutes on a single bolus of hyperpolarized substrate to achieve 21 mHz NMR linewidths. The feasibility of creating long-lasting C-13 RASER on biomolecular carriers opens a wide range of new opportunities for the rapidly expanding field of C-13 magnetic resonance hyperpolarization.

Triplet Photosensitized para-Hydrogen Induced Polarization.

Despite its enormous utility in structural characterization, nuclear magnetic resonance (NMR) spectroscopy is inherently limited by low spin polarization. One method to address the low polarization is para-hydrogen (p-H2) induced polarization (PHIP) which uses the singlet spin isomer of H2 to generate disparate nuclear spin populations to amplify the associated NMR signals. PHIP often relies on thermal catalysis or, more infrequently, UV-activated catalytic hydrogenation. Light-activated hydrogenation enables direct and timed control over the hyperpolarization of target substrates, critical for identifying short-lived intermediates. Here, we use an established Ir(III) triplet photosensitizer (PS) to visible light sensitize the triplet ligand-field states in the d6-transition metal dihydride Ru(CO)(PPh3)3(H)2 (1). Excitation inside a 9.4 T NMR spectrometer with the PS and a 420 nm blue LED, under 3 atm of p-H2, successfully photosensitized hyperpolarization in 1 and in a range of unsaturated substrates at and below room temperature, up to 1630-fold. In otherwise identical experimental conditions without light activation, no polarization was realized in 1 or the substrates evaluated. We believe triplet-sensitized PHIP (Trip-PHIP) represents a facile experimental means for probing triplet sensitized light activation in transition metal catalysts possessing low-lying triplet ligand-field states, providing mechanistic insight of potentially tremendous value in chemical catalysis.

Proton-Only Sensing of Hyperpolarized [1,2-13C2]Pyruvate.

Hyperpolarized MRI is emerging as a next-generation molecular imaging modality that can detect metabolic transformations in real time deep inside tissue and organs. 13C-hyperpolarized pyruvate is the leading hyperpolarized contrast agent that can probe cellular energetics in real time. Currently, hyperpolarized MRI requires specialized "multinuclear" MRI scanners that have the ability to excite and detect 13C signals. The objective of this work is the development of an approach that works on conventional (i.e., proton-only) MRI systems while taking advantage of long-lived 13C hyperpolarization. The long-lived singlet state of [1,2-13C2]pyruvate is hyperpolarized with parahydrogen in reversible exchange, and subsequently, the polarization is transferred from the 13C2 spin pair to the methyl protons of pyruvate for detection. This polarization transfer is accomplished with spin-lock induced crossing pulses that are only applied to the methyl protons yet access the hyperpolarization stored in the 13C2 singlet state. Theory and first experimental demonstrations are provided for our method, which obviates 13C excitation and detection for proton sensing of 13C-hyperpolarized pyruvate with an overall experimental-polarization transfer efficiency of ∼22% versus a theoretically predicted polarization transfer efficiency of 25%.

CHL1-Deficient and Wild-Type Male Mice do Not Differ in Locomotor Recovery from Spinal Cord Injury.

CHL1 is a close homolog of L1, a cell adhesion molecule that plays major roles in neural and tumor cell functions. We had found that young adult female mice deficient in CHL1 recovered better than their wild-type female littermates after thoracic Spinal Cord Injury (SCI). This observation was surprising, because CHL1 increases neurite outgrowth in vitro. Injury of adult mouse central and peripheral nervous systems upregulate CHL1 expression in neurons and astrocytes, consistent with CHL1's pro-active, homophilic interaction between CHL1 surface molecules in wild-type mice. After SCI, CHL1 expression was observed to increase in the glial scar, areas of axonal regrowth and remodeling of neural circuits. These observations were made only in females, and we therefore sought to analyze SCI in CHL1-deficient male mice. We now show that CHL1-deficient males did not recover better or worse than their male wild-type littermates. Primary and secondary lesion volumes were similar in the two genotypes, as seen in female mice which were studied in parallel with male mice. Assessment of peripheral leukocytes showed a significant increase in numbers of blood neutrophils at 24 h after SCI in males, but not in females. Lymphocyte numbers in mutant males increased slightly, but numbers of lymphocytes or monocytes did not differ significantly between males or females. These results indicate that CHL1-deficient males and females differ in the number of neutrophils but not lymphocytes or monocytes, suggesting that the difference between males and females is unlikely due to differences in leukocytes.

Interplay of Near-Zero-Field Dephasing, Rephasing, and Relaxation Dynamics and [1-13C]Pyruvate Polarization Transfer Efficiency in Pulsed SABRE-SHEATH.

Hyperpolarized [1-13C]pyruvate is a revolutionary molecular probe enabling ultrafast metabolic MRI scans in 1 min. This technology is now under evaluation in over 30 clinical trials, which employ dissolution Dynamic Nuclear Polarization (d-DNP) to prepare a batch of the contrast agent; however, d-DNP technology is slow and expensive. The emerging SABRE-SHEATH hyperpolarization technique enables fast (under 1 min) and robust production of hyperpolarized [1-13C]pyruvate via simultaneous chemical exchange of parahydrogen and pyruvate on IrIMes hexacoordinate complexes. Here, we study the application of microtesla pulses to investigate their effect on C-13 polarization efficiency, compared to that of conventional SABRE-SHEATH employing a static field (∼0.4 µT), to provide the matching conditions of polarization transfer from parahydrogen-derived hydrides to the 13C-1 nucleus. Our results demonstrate that using square-microtesla pulses with optimized parameters can produce 13C-1 polarization levels of up to 14.8% (when detected, averaging over all resonances), corresponding to signal enhancement by over 122,000-fold at the clinically relevant field of 1.4 T. We anticipate that our results can be directly translated to other structurally similar biomolecules such as [1-13C]α-ketoglutarate and [1-13C]α-ketoisocaproate. Moreover, other more advanced pulse shapes can potentially further boost heteronuclear polarization attainable via pulsed SABRE-SHEATH.

Catalyst-Free Aqueous Hyperpolarized [1-13C]Pyruvate Obtained by Re-Dissolution Signal Amplification by Reversible Exchange.

Despite great successes in oncology, patient outcomes are often still discouraging, and hence the diagnostic imaging paradigm is increasingly shifting toward functional imaging of the pathology to better understand individual disease biology and to personalize therapies. The dissolution Dynamic Nuclear Polarization (d-DNP) hyperpolarization method has enabled unprecedented real-time MRI sensing of metabolism and tissue pH using hyperpolarized [1-13C]pyruvate as a biosensor with great potential for diagnosis and monitoring of cancer patients. However, current d-DNP is expensive and suffers from long hyperpolarization times, posing a substantial translational roadblock. Here, we report the development of Re-Dissolution Signal Amplification By Reversible Exchange (Re-D SABRE), which relies on fast and low-cost hyperpolarization of [1-13C]pyruvate by chemical exchange with parahydrogen at microtesla magnetic fields. [1-13C]pyruvate is precipitated from catalyst-containing methanol using ethyl acetate and rapidly reconstituted in aqueous media. 13C polarization of 9 ± 1% is demonstrated after redissolution in water with residual iridium mass fraction of 8.5 ± 1.5 ppm; further improvement is anticipated via process automation. Re-D SABRE makes hyperpolarized [1-13C]pyruvate biosensor available at a fraction of the cost (<$10,000) and production time (≈1 min) of currently used techniques and makes aqueous hyperpolarized [1-13C]pyruvate "ready" for in vivo applications.