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.

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.

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.