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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

The Wohl Institute for Translational Medicine

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.

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.

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 C MR of [1-C]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-C]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-C]lactate and [C]bicarbonate production from hyperpolarized [1-C]pyruvate. P 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 ( = 6) to 217.4 ± 121.3 in the ischemia-reperfusion group ( = 6). Following the recovery period ( 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.

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-C]Pyruvate.

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.

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-C]lactate production from hyperpolarized [1-C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20-77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (<3 mm) and a diameter ranging from 3 to 6 mm.

Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids-A New Method to Characterize the Metabolism of Brain Organoids?

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.

Gal Sapir

Department of Radiology,
Hadassah Medical Organization and Faculty of Medicine,
Department of Radiology, Hadassah Medical Organization and Faculty of Medicine

Hebrew University of Jerusalem

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

Gal Sapir

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Radiology,Hadassah Medical Organization and Faculty of Medicine,Department of Radiology, Hadassah Medical Organization and Faculty of Medicine

Hebrew University of Jerusalem

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

Department of Radiology,
Hadassah Medical Organization and Faculty of Medicine,
Department of Radiology, Hadassah Medical Organization and Faculty of Medicine

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