Directly Bound Deuterons Increase X-Nuclei Hyperpolarization using Dynamic Nuclear Polarization.

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

Signal enhancement of hyperpolarized 15 N sites in solution-increase in solid-state polarization at 3.35 T and prolongation of relaxation in deuterated water mixtures.

Hyperpolarized 15 N sites have been found to be promising for generating long-lived hyperpolarized states in solution, and present a promising approach for utilizing dissolution-dynamic nuclear polarization (dDNP)-driven hyperpolarized MRI for imaging in biology and medicine. Specifically, 15 N sites with directly bound protons were shown to be useful when dissolved in D2 O. The purpose of the current study was to further characterize and increase the visibility of such 15 N sites in solutions that mimic an intravenous injection during the first cardiac pass in terms of their H2 O:D2 O composition. The T1 values of hyperpolarized 15 N in [15 N2 ]urea and [15 N]NH4 Cl demonstrated similar dependences on the H2 O:D2 O composition of the solution, with a T1 of about 140 s in 100% D2 O, about twofold shortening in 90% and 80% D2 O, and about threefold shortening in 50% D2 O. [13 C]urea was found to be a useful solid-state 13 C marker for qualitative monitoring of the 15 N polarization process in a commercial pre-clinical dDNP device. Adding trace amounts of Gd3+ to the polarization formulation led to higher solid-state polarization of [13 C]urea and to higher polarization levels of [15 N2 ]urea in solution.

Correlation between lactate dehydrogenase/pyruvate dehydrogenase activities ratio and tissue pH in the perfused mouse heart: A potential noninvasive indicator of cardiac pH provided by hyperpolarized magnetic resonance.

Cardiovascular diseases account for more than 30% of all deaths worldwide and many could be ameliorated with early diagnosis. Current cardiac imaging modalities can assess blood flow, heart anatomy and mechanical function. However, for early diagnosis and improved treatment, further functional biomarkers are needed. One such functional biomarker could be the myocardium pH. Although tissue pH is already determinable via MR techniques, and has been since the early 1990s, it remains elusive to use practically. The objective of this study was to explore the possibility to evaluate cardiac pH noninvasively, using in-cell enzymatic rates of hyperpolarized [1-13 C]pyruvate metabolism (ie, moles of product produced per unit time) determined directly in real time using magnetic resonance spectroscopy in a perfused mouse heart model. As a gold standard for tissue pH we used 31 P spectroscopy and the chemical shift of the inorganic phosphate (Pi) signal. The nonhomogenous pH distribution of the perfused heart was analyzed using a multi-parametric analysis of this signal, thus taking into account the heterogeneous nature of this characteristic. As opposed to the signal ratio of hyperpolarized [13 C]bicarbonate to [13 CO2 ], which has shown correlation to pH in other studies, we investigated here the ratio of two intracellular enzymatic rates: lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), by way of determining the production rates of [1-13 C]lactate and [13 C]bicarbonate, respectively. The enzyme activities determined here are intracellular, while the pH determined using the Pi signal may contain an extracellular component, which could not be ruled out. Nevertheless, we report a strong correlation between the tissue pH and the LDH/PDH activities ratio. This work may pave the way for using the LDH/PDH activities ratio as an indicator of cardiac intracellular pH in vivo, in an MRI examination.

Hyperpolarized product selective saturating-excitations for determination of changes in metabolic reaction rates in real-time.

Investigation of hyperpolarized substrate metabolism has been showing utility in real-time determination of in-cell and in vivo enzymatic activities. Intracellular reaction rates may vary during the course of a measurement, even on the very short time scales of visibility on hyperpolarized MR, due to many factors such as the availability of the substrate and co-factors in the intracellular space. Despite this potential variation, the kinetic analysis of hyperpolarized signals typically assumes that the same rate constant (and in many cases, the same rate) applies throughout the course of the reaction as observed via the build-up and decay of the hyperpolarized signals. We demonstrate here an acquisition approach that can null the need for such an assumption and enable the detection of instantaneous changes in the rate of the reaction during an ex vivo hyperpolarized investigation, (i.e. in the course of the decay of one hyperpolarized substrate dose administered to a viable tissue sample ex vivo). This approach utilizes hyperpolarized product selective saturating-excitation pulses. Similar pulses have been previously utilized in vivo for spectroscopic imaging. However, we show here favorable consequences to kinetic rate determinations in the preparations used. We implement this acquisition strategy for studies on perfused tissue slices and develop a theory that explains why this particular approach enables the determination of changes in enzymatic rates that are monitored via the chemical conversions of hyperpolarized substrates. Real-time changes in intracellular reaction rates are demonstrated in perfused brain, liver, and xenograft breast cancer tissue slices and provide another potential differentiation parameter for tissue characterization.

The Effect of Gadolinium Doping in [13 C6 ,2 H7 ]Glucose Formulations on 13 C Dynamic Nuclear Polarization at 3.35†T.

The promise of hyperpolarized glucose as a non-radioactive imaging agent capable of reporting on multiple metabolic routes has led to recent advances in its dissolution-DNP (dDNP) driven polarization using UV-light induced radicals and trityl radicals at high field (6.7 T) and 1.1 K. However, most preclinical dDNP polarizers operate at the field of 3.35 T and 1.4-1.5 K. Minute amounts of Gd3+ complexes have shown large improvements in solid-state polarization, which can be translated to improved hyperpolarization in solution. However, this Gd3+ effect seems to depend on magnetic field strength, metal ion concentration, and sample formulation. The effect of varying Gd3+ concentrations at 3.35 T has been described for 13 C-labeled pyruvic acid and acetate. However, it has not been studied for other compounds at this field. The results presented here suggest that Gd3+ doping can lead to various concentration and temperature dependent effects on the polarization of [13 C6 ,2 H7 ]glucose, not necessarily similar to the effects observed in pyruvic acid or acetate in size or direction. The maximal polarization for [13 C6 ,2 H7 ]glucose appears to be at a Gd3+ concentration of 2 mM, when irradiating for more than 2 h at the negative maximum of the DNP intensity profile. Surprisingly, for shorter irradiation times, higher polarization levels were determined at 1.50 K compared to 1.45 K, at a [Gd3+ ]=1.3 mM. This was explained by the build-up time constant and maximum at these temperatures.

Real-time ALT and LDH activities determined in viable precision-cut mouse liver slices using hyperpolarized [1-13 C]pyruvate-Implications for studies on biopsied liver tissues.

Precision-cut liver slices (PCLS) are widely used in liver research as they provide a liver model with all liver cell types in their natural architecture. The purpose of this study was to demonstrate the use of PCLS for hyperpolarized metabolic investigation in a mouse model, for potential future application in liver biopsy cores. Fresh normal liver was harvested from six mice. 500 µm PCLS were prepared and placed in a 10 mm NMR tube in an NMR spectrometer and perfused continuously. 31 P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1-13 C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive 13 C NMR spectra were acquired immediately after the injection using both non-selective (five injections, two livers) and selective RF excitation (six injections, three livers). The 31 P spectra showed the characteristic signals of ATP, confirming the viability of the PCLS for more than 2.5 h in the spectrometer. After each of the [1-13 C]pyruvate injections, both [1-13 C]lactate and [1-13 C]alanine signals were detected. Selective RF excitation aimed at both [1-13 C]lactate and [1-13 C]alanine enabled better visualization and quantification of the metabolic activity. Using this acquisition approach only the newly formed metabolites are observed upon excitation, and their intensities relative to those of hyperpolarized pyruvate enable quantification of metabolite production rates. This rate of lactate and alanine production appeared to be constant throughout the measurement time, with alanine production about 2.3 times higher than lactate. In summary, the viability of PCLS in an NMR spectrometer was demonstrated and hyperpolarized [1-13 C]pyruvate metabolism was recorded. This study opens up the possibility of evaluating alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities in human liver biopsies, while preserving the tissue architecture and viability. In healthy, well-perfused liver slices the ratio of ALT to LDH activity is about 2.3.

Long-lived 15 N Hyperpolarization and Rapid Relaxation as a Potential Basis for Repeated First Pass Perfusion Imaging - Marked Effects of Deuteration and Temperature.

Deuteration of the exchangeable hydrogens of [15 N2 ]urea was found to prolong the T1 of the 15 N sites to more than 3 min at physiological temperatures. This significant increase in the lifetime of the hyperpolarized state of [15 N2 ]urea, compared to [13 C]urea - a pre-clinically proven perfusion agent, makes [15 N2 ]urea a promising perfusion agent. The molecular parameters that may lead to this profound effect were assessed by investigating small molecules with different molecular structures containing 15 N sites bound to labile protons and determining the hyperpolarized 15 N T1 in H2 O and D2 O. Dissolution in D2 O led to marked prolongation for all of the selected sites. In whole human blood, the T1 of [15 N2 ]urea was shortened. We present a general strategy for exploiting the markedly longer T1 outside the body and the quick decay in blood for performing multiple hyperpolarized perfusion measurements with a single hyperpolarized dose. Improved storage of the generated [15 N2 ]urea polarization prior to the contact with the blood is demonstrated using higher temperatures due to further T1 prolongation.

Clinical and functional efficacy of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis carrying the N1303K mutation.

BACKGROUND: Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) significantly improves health outcomes in people with cystic fibrosis (pwCF) carrying one or two F508del mutations. According to in vitro assays performed in FRT cells, 178 additional mutations respond to ELX/TEZ/IVA. The N1303K mutation is not included in this list of mutations. Recent in vitro data suggested that ELX/TEZ/IVA increases N1303K-CFTR activity. Based on the in vitro response, eight patients commenced treatment with ELX/TEZ/IVA. METHODS: Two homozygotes; and six compound heterozygotes N1303K/nonsense or frameshift mutation pwCF were treated off label with ELX/TEZ/IVA. Clinical data before and 8 weeks after starting treatment were prospectively collected. The response to ELX/TEZ/IVA was assessed in intestinal organoids derived from 5 study patients and an additional patient carrying N1303K that is not receiving treatment. RESULTS: Compared to the values before commencing treatment, mean forced expiratory volume in 1 second increased by 18.4 percentage points and 26.5% relative to baseline, mean BMI increased by 0.79 Kg/m2, and mean lung clearance index decreased by 3.6 points and 22.2%. There was no significant change in sweat chloride. Nasal potential difference normalized in four patients and remained abnormal in three. Results in 3D intestinal organoids and 2D nasal epithelial cultures showed a response in CFTR channel activity. CONCLUSIONS: This report supports the previously reported in vitro data, performed in human nasal and bronchial epithelial cells and intestinal organoids, that pwCF who carry the N1303K mutation have a significant clinical benefit by ELX/TEZ/IVA treatment.

Observation of glucose-6-phosphate anomeric exchange in real-time using dDNP hyperpolarised NMR.

A hyperpolarised-NMR acquisition approach that is sensitive to the process of glucose-6-phosphate anomerization is presented. Using selective depolarisation of one of the anomer's signals, it is possible to observe the replenishing of this signal due to the fast anomeric exchange of this compound. The forward to reverse reaction rate constants ratio was ca. 1.6.

Hyperpolarized [15N]nitrate as a potential long lived hyperpolarized contrast agent for MRI.

Reports on gadolinium deposits in the body and brains of adults and children who underwent contrast-enhanced MRI examinations warrant development of new, metal free, contrast agents for MRI. Nitrate is an abundant ion in mammalian biochemistry and sodium nitrate can be safely injected intravenously. We show that hyperpolarized [15N]nitrate can potentially be used as an MR tracer. The 15N site of hyperpolarized [15N]nitrate showed a T1 of more than 100 s in aqueous solutions, which was prolonged to more than 170 s below 20 °C. Capitalizing on this effect for polarization storage we obtained a visibility window of 9 min in blood. Conversion to [15N]nitrite, the bioactive reduced form of nitrate, was not observed in human blood and human saliva in this time frame. Thus, [15N]nitrate may serve as a long-lived hyperpolarized tracer for MR. Due to its ionic nature, the immediate applications appear to be perfusion and tissue retention imaging.

Determining radical penetration of lipid bilayers with new lipophilic spin traps.

Predicting the susceptibility of lipid moieties to radical attack requires a determination of the depth of radical penetration into a lipid membrane. We thus synthesized three homologous series of lipophilic spin traps--DMPO analogs 2-alkanoyl-2-methyl-1-pyrroline N-oxides (11) and PBN derivatives 4-alkoxyphenyl N-tert-butylnitrones (18) and 4-alkoxyphenyl N-admantylnitrones (20). The intercalation depth of these spin traps within the liposomal bilayer was determined via the previously reported NMR technique, which correlates the chemical shift and the micropolarity (measured in ET(30) units) experienced by the pivotal nitronyl carbon. Hydroxyl and alpha-hydroxyalkyl radicals were generated in the extraliposomal aqueous phase and the lowest depth at which a radical could be spin trapped was determined. The ESR data indicate that these radicals can exit the aqueous phase, penetrate the lipid bilayer past the head groups (ET(30)=63 kcal/mol) and the glycerol ester (ET(30)=52 kcal/mol), and pass down to an ET(30) polarity of at least 44 kcal/mol. The latter depth presumably corresponds to the upper portion of the lipid slab. It is likely, if not probable, that having come this far they can abstract the allylic/diallylic hydrogens resident in the midslab at ET(30) values of >31 kcal/mol.

Author Correction: Biochemical phosphates observed using hyperpolarized 31P in physiological aqueous solutions.

The original version of the Supplementary Information associated with this Article contained an error in Supplementary Figure 2 and Supplementary Figure 5 in which the 31P NMR spectral lines were missing. The HTML has been updated to include a corrected version of the Supplementary Information.

Real-time ex-vivo measurement of brain metabolism using hyperpolarized [1-13C]pyruvate.

The ability to directly monitor in vivo brain metabolism in real time in a matter of seconds using the dissolution dynamic nuclear polarization technology holds promise to aid the understanding of brain physiology in health and disease. However, translating the hyperpolarized signal observed in the brain to cerebral metabolic rates is not straightforward, as the observed in vivo signals reflect also the influx of metabolites produced in the body, the cerebral blood volume, and the rate of transport across the blood brain barrier. We introduce a method to study rapid metabolism of hyperpolarized substrates in the viable rat brain slices preparation, an established ex vivo model of the brain. By retrospective evaluation of tissue motion and settling from analysis of the signal of the hyperpolarized [1-13C]pyruvate precursor, the T1s of the metabolites and their rates of production can be determined. The enzymatic rates determined here are in the range of those determined previously with classical biochemical assays and are in agreement with hyperpolarized metabolite relative signal intensities observed in the rodent brain in vivo.

Higher levels of myelin phospholipids in brains of neuronal α-Synuclein transgenic mice precede myelin loss.

α-Synuclein is a protein involved in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We investigated the role of neuronal α-Syn in myelin composition and abnormalities. The phospholipid content of purified myelin was determined by 31P NMR in two mouse lines modeling PD, PrP-A53T α-Syn and Thy-1 wt-α-Syn. Significantly higher levels of phospholipids were detected in myelin purified from brains of these α-Syn transgenic mouse models than in control mice. Nevertheless, myelin ultrastructure appeared intact. To further investigate the effect of α-Syn on myelin abnormalities, we systematically analyzed the striatum, a brain region associated with neurodegeneration in PD. An age and disease-dependent loss of myelin basic protein (MBP) signal was detected by immunohistochemistry in striatal striosomes (patches). The age-dependent loss of MBP signal was associated with lower P25α levels in oligodendrocytes. In addition, we found that α-Syn inhibited oligodendrocyte maturation and the formation of membranous sheets in vitro. Based on these results we concluded that neuronal α-Syn is involved in the regulation and/or maintenance of myelin phospholipid. However, axonal hypomyelination in the PD models is evident only in progressive stages of the disease and associated with α-Syn toxicity.

Biochemical phosphates observed using hyperpolarized 31P in physiological aqueous solutions.

The dissolution-dynamic nuclear polarization technology had previously enabled nuclear magnetic resonance detection of various nuclei in a hyperpolarized state. Here, we show the hyperpolarization of 31P nuclei in important biological phosphates (inorganic phosphate and phosphocreatine) in aqueous solutions. The hyperpolarized inorganic phosphate showed an enhancement factor >11,000 (at 5.8 T, 9.3% polarization) in D2O (T1 29.4 s). Deuteration and the solution composition and pH all affected the lifetime of the hyperpolarized state. This capability opens up avenues for real-time monitoring of phosphate metabolism, distribution, and pH sensing in the live body without ionizing radiation. Immediate changes in the microenvironment pH have been detected here in a cell-free system via the chemical shift of hyperpolarized inorganic phosphate. Because the 31P nucleus is 100% naturally abundant, future studies on hyperpolarized phosphates will not require expensive isotope labeling as is usually required for hyperpolarization of other substrates.Real-time monitoring of phosphate metabolism and distribution in the live body without ionizing radiation is highly desirable. Here, the authors show dissolution-dynamic nuclear polarization technology can enable nuclear magnetic resonance detection of hyperpolarized 31P of important biological phosphates in aqueous solutions.

Defective ATP breakdown activity related to an ENTPD1 gene mutation demonstrated using 31P NMR spectroscopy.

The ecto-nucleoside triphosphate diphosphohydrolase-1 (E-NTPDase-1, CD39) enzyme is responsible for the breakdown of extracellular ATP to ADP and then to AMP by a two-step process. Defective CD39 activity has been described in a variety of medical conditions including malignancy and rheumatic diseases and has been proved to be of major diagnostic and clinical importance. Here we show for the first time that a 31P NMR spectroscopy methodology enables the quantification of these two steps in a single blood sample. We have applied this assay to determine the E-NTPDase activity on human mononuclear cells taken from two siblings affected by a stop-codon mutation in the ENTPD1 gene, their obligatory heterozygous parents, and healthy volunteers. The affected subjects presented low ATP breakdown activity, mainly expressed as low AMP production.

Transaldolase Deficiency: A New Case Expands the Phenotypic Spectrum.

Transaldolase (TALDO) deficiency has various clinical manifestations including liver dysfunction, hepatosplenomegaly, anemia, thrombocytopenia, and dysmorphic features. We report a case presenting prenatally with hyperechogenic bowel and intrauterine growth restriction. The infant was born small for gestational age, with cutis laxa and hypertrichosis. Postnatally, meconium plug was identified, complicated with intestinal obstruction necessitating laparotomy, partial resection of the intestine, and ileostomy. Liver biopsy revealed cholangiolar proliferation and portal fibrosis. He also suffered from persistent congenital thrombocytopenia requiring platelet transfusions and severe hypothyroidism with normal anatomical and structural gland responding only to the combination of T3 and T4 treatment. Neurologically, severe hypotonia and anisocoria were noted at the age of 2 months. Brain MRI was normal. Shortly after the abdominal surgery, a rapid liver failure ensued, which eventually led to his death. Specific metabolic tests ruled out glycosylation disorders, yet urine analysis using 1H NMR showed accumulation of sedoheptulose which was previously described in patients with transaldolase deficiency. Sequencing of the gene-encoding transaldolase (TALDO1) revealed a homozygous stop mutation c.669C>G; p.Tyr223*. In conclusion, we present an infant with a novel homozygous mutation in TALDO1, causing TALDO deficiency, and extend the clinical characteristics of this rare syndrome.

Application of the Steady-State Variable Nutation Angle Method for Faster Determinations of Long T 1s-An Approach Useful for the Design of Hyperpolarized MR Molecular Probes.

In the dissolution-dynamic nuclear polarization technique, molecular probes with long T 1s are preferred. 13C nuclei of small molecules with no directly bonded protons or sp(3 13)C nuclei with proton positions substituted by deuterons may fulfill this requirement. The T 1 determination of such new molecular probes is crucial for the success of the hyperpolarized observation. Although the inversion-recovery approach remained by and large the standard for T 1 measurements, we show here that the steady-state variable nutation angle approach is faster and may be better suited for the determination of relatively long T 1s in thermal equilibrium. Specifically, the T 1 of a new molecular probe, [uniformly labeled (UL)-13C6, UL-2H8]2-deoxy-d-glucose, is determined here and compared to that of [UL-13C6, UL-2H7]d-glucose.

In vitro visualization of betaine aldehyde synthesis and oxidation using hyperpolarized magnetic resonance spectroscopy.

Real-time monitoring of betaine aldehyde metabolism at high temporal resolution was accomplished using a hyperpolarized choline analog and (13)C-NMR. This represents the first observation of an aldehyde intermediate on hyperpolarized MR and opens the way for kinetic studies of oxidase/dehydrogenase enzymes in vitro and in vivo.

A hyperpolarized choline molecular probe for monitoring acetylcholine synthesis.

Choline as a reporter molecule has been investigated by in vivo magnetic resonance for almost three decades. Accumulation of choline metabolites (mainly the phosphorylated forms) had been observed in malignancy in preclinical models, ex-vivo, in vivo and in patients. The combined choline metabolite signal appears in (1) H-MRS of the brain and its relative intensity had been used as a diagnostic factor in various conditions. The advent of spin hyperpolarization methods for in vivo use has raised interest in the ability to follow the physiological metabolism of choline into acetylcholine in the brain. Here we present a stable-isotope labeled choline analog, [1,1,2,2-D(4) ,2-(13) C]choline chloride, that is suitable for this purpose. In this analog, the (13) C position showed 24% polarization in the liquid state, following DNP hyperpolarization. This nucleus also showed a long T(1) (35 s) at 11.8 T and 25 °C, which is a prerequisite for hyperpolarized studies. The chemical shift of this (13) C position differentiates choline and acetylcholine from each other and from the other water-soluble choline metabolites, namely phosphocholine and betaine. Enzymatic studies using an acetyltransferase enzyme showed the synthesis of the deuterated-acetylcholine form at thermal equilibrium conditions and in a hyperpolarized state. Analysis using a comprehensive model showed that the T(1) of the formed hyperpolarized [1,1,2,2-D(4) ,2-(13) C]acetylcholine was 34 s at 14.1 T and 37 °C. We conclude that [1,1,2,2-D(4) ,2-(13) C]choline chloride is a promising new molecular probe for hyperpolarized metabolic studies and discuss the factors related to its possible use in vivo.