Real-time influence of intracellular acidification and Na+ /H+ exchanger inhibition on in-cell pyruvate metabolism in the perfused mouse heart: A 31 P-NMR and hyperpolarized 13 C-NMR study.

Disruption of acid-base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy, and myocardial ischemia. Previously, we have reported that the ratio of the in-cell lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in-cell activities were investigated under induced intracellular acidification without and with Na+ /H+ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n = 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH4 Cl prepulse methodology; and (2) by combining the NH4 Cl prepulse with zoniporide. 31 P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1-13 C]pyruvate was obtained using dissolution dynamic nuclear polarization. 13 C NMR spectroscopy was used to monitor hyperpolarized [1-13 C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product-selective saturating excitation approach. The intracellular acidification induced by the NH4 Cl prepulse led to reduced LDH and PDH activities (-16% and -39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH4 Cl prepulse with zoniporide led to a greater reduction in LDH activity (-29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH4 Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H+ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in-cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1-13 C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies.

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.

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 information for understanding the mechanism of diseases and designing treatments. In this work, we describe procedures and methodologies for studying in-cell metabolic activity in a retrogradely perfused mouse heart in real-time. The heart was isolated in situ, in conjunction with cardiac arrest to minimize the myocardial ischemia and was perfused inside a nuclear magnetic resonance (NMR) spectrometer. While in the spectrometer and under continuous perfusion, hyperpolarized [1-13C]pyruvate was administered to the heart, and the subsequent hyperpolarized [1-13C]lactate and [13C]bicarbonate production rates served to determine, in real-time, the rates of lactate dehydrogenase and pyruvate dehydrogenase production. This metabolic activity of hyperpolarized [1-13C]pyruvate was quantified with NMR spectroscopy in a model free-manner using the product selective saturating-excitations acquisition approach. 31P spectroscopy was applied in between the hyperpolarized acquisitions to monitor the cardiac energetics and pH. This system is uniquely useful for studying metabolic activity in the healthy and diseased mouse heart.

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.

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

3-aminopropylphosphonate (3-APP) is known for its use as an exogenous indicator of extracellular volume and pH in phosphorus-31 nuclear magnetic resonance (31 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.

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

Direct and real-time monitoring of cerebral metabolism exploiting the drastic increase in sensitivity of hyperpolarized 13C-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 13C-labeled pyruvate, as a reporter of neural activity.

[TREATMENT OF A PATIENT IN A TERMINAL CONDITION].

INTRODUCTION: It is generally understood that Jewish law requires every effort to be made to extend the life of a terminally ill patient using routine treatments, whether he is conscious or not, and whether he wants his life prolonged or is opposed to it. The "Law for Patients Wishing to Die" proposes this approach with slight variations. This article discusses the patient who wishes to die from a Jewish viewpoint, illustrating that this is not the only Jewish approach. The role of the doctor is to cure and not to extend a life of suffering in any case. If the doctor cannot cure him, he should respect the wishes of the patient who does not wish to continue to suffer, by stopping life-prolonging treatment and providing only pain-reducing treatment. This law is correct not only in cases where the patient is aware of his situation but also in cases where the patient is comatose and has no realistic chance of returning to life. Ideas from the Talmud, from the Shulchan Aruch, and from the rulings of many poskim (rabbis) confirm that Judaism places great emphasis on personal autonomy and on limiting medical intervention to situations of need. The authors of the article call for the law to be changed in this respect.

LDH and PDH Activities in the Ischemic Brain and the Effect of Reperfusion-An Ex Vivo MR Study in Rat Brain Slices Using Hyperpolarized [1-13C]Pyruvate.

Ischemic stroke is a leading cause for neurologic disability worldwide, for which reperfusion is the only available treatment. Neuroimaging in stroke guides treatment, and therefore determines the clinical outcome. However, there are currently no imaging biomarkers for the status of the ischemic brain tissue. Such biomarkers could potentially be useful for guiding treatment in patients presenting with ischemic stroke. Hyperpolarized 13C MR of [1-13C]pyruvate is a clinically translatable method used to characterize tissue metabolism non-invasively in a relevant timescale. The aim of this study was to utilize hyperpolarized [1-13C]pyruvate to investigate the metabolic consequences of an ischemic insult immediately during reperfusion and upon recovery of the brain tissue. The rates of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) were quantified by monitoring the rates of [1-13C]lactate and [13C]bicarbonate production from hyperpolarized [1-13C]pyruvate. 31P NMR of the perfused brain slices showed that this system is suitable for studying ischemia and recovery following reperfusion. This was indicated by the levels of the high-energy phosphates (tissue viability) and the chemical shift of the inorganic phosphate signal (tissue pH). Acidification, which was observed during the ischemic insult, has returned to baseline level following reperfusion. The LDH/PDH activity ratio increased following ischemia, from 47.0 ± 12.7 in the control group (n = 6) to 217.4 ± 121.3 in the ischemia-reperfusion group (n = 6). Following the recovery period (ca. 1.5 h), this value had returned to its pre-ischemia (baseline) level, suggesting the LDH/PDH enzyme activity ratio may be used as a potential indicator for the status of the ischemic and recovering brain.

The metabolic representation of ischemia in rat brain slices: A hyperpolarized 13 C magnetic resonance study.

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 31 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 [1-13 C]pyruvate to [1-13 C]lactate and [13 C]bicarbonate, respectively, using 13 C spectroscopy. In a control group (n = 8), hyperpolarized [1-13 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 [1-13 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 [1-13 C]pyruvate, could serve as an imaging biomarker to characterize the changes in the ischemic penumbra.

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.

In-cell determination of Lactate Dehydrogenase Activity in a Luminal Breast Cancer Model ⁻ ex vivo Investigation of Excised Xenograft Tumor Slices Using dDNP Hyperpolarized [1-13C]pyruvate.

[1-13C]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 [1-13C]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.

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.