investigating-cardiac-metabolism-isolated-perfused-mouse-heart-with

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial ...

Hebrew University of Jerusalem

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.  P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1- C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive  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  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- C]pyruvate injections, both [1- C]lactate and [1- C]alanine signals were detected. Selective RF excitation aimed at both [1- C]lactate and [1- 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- 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.

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

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 [N]nitrate can potentially be used as an MR tracer. The N site of hyperpolarized [N]nitrate showed a T 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 [N]nitrite, the bioactive reduced form of nitrate, was not observed in human blood and human saliva in this time frame. Thus, [N]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.

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

[1-C]pyruvate, the most widely used compound in dissolution-dynamic nuclear polarization (dDNP) magnetic resonance (MR), enables the visualization of lactate dehydrogenase (LDH) activity. This activity had been demonstrated in a wide variety of cancer models, ranging from cultured cells, to xenograft models, to human tumors in situ. Here we quantified the LDH activity in precision cut tumor slices (PCTS) of breast cancer xenografts. The Michigan Cancer Foundation-7 (MCF7) cell-line was chosen as a model for the luminal breast cancer type which is hormone responsive and is highly prevalent. The LDH activity, which was manifested as [1-C]lactate production in the tumor slices, ranged between 3.8 and 6.1 nmole/nmole adenosine tri-phosphate (ATP) in 1 min (average 4.6 ± 1.0) on three different experimental set-ups consisting of arrested vs. continuous perfusion and non-selective and selective RF pulsation schemes and combinations thereof. This rate was converted to an expected LDH activity in a mass ranging between 3.3 and 5.2 µmole/g in 1 min, using the ATP level of these tumors. This indicated the likely utility of this approach in clinical dDNP of the human breast and may be useful as guidance for treatment response assessment in a large number of tumor types and therapies ex vivo.

In-cell determination of Lactate Dehydrogenase Activity in a Luminal Breast Cancer Model ⁻  Investigation of Excised Xenograft Tumor Slices Using dDNP Hyperpolarized [1-C]pyruvate.

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.

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

A non-radioactive 2-deoxyglucose (2DG) analog has been developed here for hyperpolarized magnetic resonance investigations. The analog, [C,D]2DG, showed 13% polarization in solution (27,000-fold signal enhancement at the C site), following a dissolution-DNP hyperpolarization process. The phosphorylation of this analog by yeast hexokinase (yHK) was monitored in real-time with a temporal resolution of 1 s. We show that yHK selectively utilizes the β anomer of the 2DG analog, thus revealing a surprising anomeric specificity of this reaction. Such anomeric selectivity was not observed for the reaction of yHK or bacterial glucokinase with a hyperpolarized glucose analog. yHK is highly similar to the human HK-2, which is overexpressed in malignancy. Thus, the current finding may shed a new light on a fundamental enzyme activity which is utilized in the most widespread molecular imaging technology for cancer detection - positron-emission tomography with F-2DG.

[C,D]2-deoxyglucose phosphorylation by hexokinase shows selectivity for the β-anomer.

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 Gd complexes have shown large improvements in solid-state polarization, which can be translated to improved hyperpolarization in solution. However, this Gd effect seems to depend on magnetic field strength, metal ion concentration, and sample formulation. The effect of varying Gd concentrations at 3.35 T has been described for  C-labeled pyruvic acid and acetate. However, it has not been studied for other compounds at this field. The results presented here suggest that Gd doping can lead to various concentration and temperature dependent effects on the polarization of [ C , H ]glucose, not necessarily similar to the effects observed in pyruvic acid or acetate in size or direction. The maximal polarization for [ C , H ]glucose appears to be at a Gd 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 [Gd ]=1.3 mM. This was explained by the build-up time constant and maximum at these temperatures.

The Effect of Gadolinium Doping in [ C , H ]Glucose Formulations on  C Dynamic Nuclear Polarization at 3.35 T.

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- 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  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 [ C]bicarbonate to [ CO ], 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- C]lactate and [ 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.

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.

The ischemic penumbra in stroke is not clearly defined by today's available imaging tools. This study aimed to develop a model system and noninvasive biomarkers of ischemic brain tissue for an examination that might potentially be performed in humans, very quickly, in the course of stroke triage. Perfused rat brain slices were used as a model system and  P spectroscopy verified that the slices were able to recover from an ischemic insult of about 3.5 min of perfusion arrest. This was indicated as a return to physiological pH and adenosine triphosphate levels. Instantaneous changes in lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) activities were monitored and quantified by the metabolic conversions of hyperpolarized [1- C]pyruvate to [1- C]lactate and [ C]bicarbonate, respectively, using  C spectroscopy. In a control group (n = 8), hyperpolarized [1- C]pyruvate was administered during continuous perfusion of the slices. In the ischemia group (n = 5), the perfusion was arrested 30 s prior to administration of hyperpolarized [1- C]pyruvate and perfusion was not resumed throughout the measurement time (approximately 3.5 min). Following about 110 s of the ischemic insult, LDH activity increased by 80.4 ± 13.5% and PDH activity decreased by 47.8 ± 25.3%. In the control group, the mean LDH/PDH ratio was 16.6 ± 3.3, and in the ischemia group, the LDH/PDH ratio reached an average value of 38.7 ± 16.9. The results suggest that monitoring the activity of LDH and PDH, and their relative activities, using hyperpolarized [1- C]pyruvate, could serve as an imaging biomarker to characterize the changes in the ischemic penumbra.

The metabolic representation of ischemia in rat brain slices: A hyperpolarized  C magnetic resonance study.

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

Curbing action potential generation or ATP-synthase leads to a decrease in in-cell pyruvate dehydrogenase activity in rat cerebrum slices.

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

Accumulation of 3-aminopropylphosphonate in the ex vivo brain observed by phosphorus-31 nuclear magnetic resonance.

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  1.6.

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

Hyperpolarized  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,  N sites with directly bound protons were shown to be useful when dissolved in D O. The purpose of the current study was to further characterize and increase the visibility of such  N sites in solutions that mimic an intravenous injection during the first cardiac pass in terms of their H O:D O composition. The T values of hyperpolarized  N in [ N ]urea and [ N]NH Cl demonstrated similar dependences on the H O:D O composition of the solution, with a T of about 140 s in 100% D O, about twofold shortening in 90% and 80% D O, and about threefold shortening in 50% D O. [ C]urea was found to be a useful solid-state  C marker for qualitative monitoring of the  N polarization process in a commercial pre-clinical dDNP device. Adding trace amounts of Gd to the polarization formulation led to higher solid-state polarization of [ C]urea and to higher polarization levels of [ N ]urea in solution.

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

We report the case of a 65 year old female patient, presenting with a combination of bilateral hearing loss, otalgia, and hyperacusis. Pure tone audiometry revealed mixed bilateral hearing loss. Conventional cranial imaging tests failed to show a significant brain pathology, but fat-suppressed T1-weighted gadolinium-enhanced magnetic resonance imaging scan displayed a diffuse infiltrative skull base process, extending from the nasopharynx to the jugular fossa, and encasing the internal carotid artery. The latter findings, besides elevated inflammatory markers and a positive perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA) led to the diagnosis of ANCA-associated vasculitis. Additional disease manifestations sequentially appeared, including a right peripheral nerve palsy, aortitis, hepatitis, peripheral neuropathy, and uveitis. Therapy with corticosteroids, azathioprine, and then cyclophosphamide brought no evident benefit, but rituximab led to impressive clinical and radiologic improvement.

Article	Year
Real-time ALT and LDH activities determined in viable precision-cut mouse liver slices using hyperpolarized [1- C]pyruvate-Implications for studies on biopsied liver tissues. NMR in biomedicine\| PubMed ID: 30575159	2019
Hyperpolarized [N]nitrate as a potential long lived hyperpolarized contrast agent for MRI. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 30660069	2019
In-cell determination of Lactate Dehydrogenase Activity in a Luminal Breast Cancer Model ⁻ Investigation of Excised Xenograft Tumor Slices Using dDNP Hyperpolarized [1-C]pyruvate. Sensors (Basel, Switzerland)\| PubMed ID: 31060334	2019
Hyperpolarized product selective saturating-excitations for determination of changes in metabolic reaction rates in real-time. NMR in biomedicine\| PubMed ID: 31793111	2020
[C,D]2-deoxyglucose phosphorylation by hexokinase shows selectivity for the β-anomer. Scientific reports\| PubMed ID: 31873121	2019
The Effect of Gadolinium Doping in [ C , H ]Glucose Formulations on C Dynamic Nuclear Polarization at 3.35 T. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 31922367	2020
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 hyper... NMR in biomedicine\| PubMed ID: 33258527	2021
The metabolic representation of ischemia in rat brain slices: A hyperpolarized C magnetic resonance study. NMR in biomedicine\| PubMed ID: 33774865	2021
Curbing action potential generation or ATP-synthase leads to a decrease in in-cell pyruvate dehydrogenase activity in rat cerebrum slices. Scientific reports\| PubMed ID: 33986346	2021
Accumulation of 3-aminopropylphosphonate in the ex vivo brain observed by phosphorus-31 nuclear magnetic resonance. NMR in biomedicine\| PubMed ID: 35229366	2022
Observation of glucose-6-phosphate anomeric exchange in real-time using dDNP hyperpolarised NMR. RSC advances\| PubMed ID: 35519178	2020
Signal enhancement of hyperpolarized N sites in solution-increase in solid-state polarization at 3.35 T and prolongation of relaxation in deuterated water mixtures. NMR in biomedicine\| PubMed ID: 35704397	2022
ANCA-associated refractory vasculitis with multiple systemic involvement: A rare case report. International journal of rheumatic diseases\| PubMed ID: 36599652	2023

J. Moshe Gomori

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