Matthew E. Merritt

Numerical solution of the Bloch equations provides insights into the optimum design of PARACEST agents for MRI.

Paramagnetic lanthanide complexes that display unusually slow water exchange between an inner sphere coordination site and bulk water may serve as a new class of MRI contrast agents with the use of chemical exchange saturation transfer (CEST) techniques. To aid in the design of paramagnetic CEST agents for reporting important biological indices in MRI measurements, we formulated a theoretical framework based on the modified Bloch equations that relates the chemical properties of a CEST agent (e.g., water exchange rates and bound water chemical shifts) and various NMR parameters (e.g., relaxation rates and applied B1 field) to the measured CEST effect. Numerical solutions of this formulation for complex exchanging systems were readily obtained without algebraic manipulation or simplification. For paramagnetic CEST agents of the type used here, the CEST effect is relatively insensitive to the bound proton relaxation times, but requires a sufficiently large applied B1 field to highly saturate the Ln3+‐bound water protons. This in turn requires paramagnetic complexes with large Ln3+‐bound water chemical shifts to avoid direct excitation of the exchanging bulk water protons. Although increasing the exchange rate of the bound protons enhances the CEST effect, this also causes exchange broadening and increases the B1 required for saturation. For a given B1, there is an optimal exchange rate that results in a maximal CEST effect. This numerical approach, which was formulated for a three‐pool case, was incorporated into a MATLAB nonlinear least‐square optimization routine, and the results were in excellent agreement with experimental Z‐spectra obtained with an aqueous solution of a paramagnetic CEST agent containing two different types of bound protons (bound water and amide protons). Magn Reson Med 53:790–799, 2005.

Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR

13C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing 13C nuclei now offers the potential to measure metabolic fluxes through select enzyme-catalyzed steps with substantially improved sensitivity. Here, we investigated the metabolism of hyperpolarized [1-13C1]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved 13CO2, the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of ≈1 s along with H13CO3−, the hydrated form of 13CO2 generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-13C1]pyruvate, production of 13CO2 and H13CO3− was suppressed by ≈90%, whereas the signal from [1-13C1]lactate was enhanced. In separate experiments, it was shown that O2 consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of 13CO2 and H13CO3− from [1-13C1]pyruvate does not reflect production of CO2 in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively.

Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Mitochondrial metabolism mediates oxidative stress and inflammation in fatty liver

Mitochondria are critical for respiration in all tissues; however, in liver, these organelles also accommodate high-capacity anaplerotic/cataplerotic pathways that are essential to gluconeogenesis and other biosynthetic activities. During nonalcoholic fatty liver disease (NAFLD), mitochondria also produce ROS that damage hepatocytes, trigger inflammation, and contribute to insulin resistance. Here, we provide several lines of evidence indicating that induction of biosynthesis through hepatic anaplerotic/cataplerotic pathways is energetically backed by elevated oxidative metabolism and hence contributes to oxidative stress and inflammation during NAFLD. First, in murine livers, elevation of fatty acid delivery not only induced oxidative metabolism, but also amplified anaplerosis/cataplerosis and caused a proportional rise in oxidative stress and inflammation. Second, loss of anaplerosis/cataplerosis via genetic knockdown of phosphoenolpyruvate carboxykinase 1 (Pck1) prevented fatty acid-induced rise in oxidative flux, oxidative stress, and inflammation. Flux appeared to be regulated by redox state, energy charge, and metabolite concentration, which may also amplify antioxidant pathways. Third, preventing elevated oxidative metabolism with metformin also normalized hepatic anaplerosis/cataplerosis and reduced markers of inflammation. Finally, independent histological grades in human NAFLD biopsies were proportional to oxidative flux. Thus, hepatic oxidative stress and inflammation are associated with elevated oxidative metabolism during an obesogenic diet, and this link may be provoked by increased work through anabolic pathways.

Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13C magnetic resonance

In the heart, detection of hyperpolarized [13C]bicarbonate and 13CO2 by magnetic resonance (MR) after administration of hyperpolarized [1-13C]pyruvate is caused exclusively by oxidative decarboxylation of pyruvate via the pyruvate dehydrogenase complex (PDH). However, liver mitochondria possess alternative anabolic pathways accessible by [1-13C]pyruvate, which may allow a wider diagnostic range for hyperpolarized MR compared with other tissue. Metabolism of hyperpolarized [1-13C]pyruvate in the tricarboxylic acid (TCA) cycle was monitored in the isolated perfused liver from fed and fasted mice. Hyperpolarized [1-13C]pyruvate was rapidly converted to [1-13C]lactate, [1-13C]alanine, [1-13C]malate, [4-13C]malate, [1-13C]aspartate, [4-13C]aspartate, and [13C]bicarbonate. Livers from fasted animals had increased lactate:alanine, consistent with elevated NADH:NAD+. The appearance of asymmetrically enriched malate and aspartate indicated high rates of anaplerotic pyruvate carboxylase activity and incomplete equilibration with fumarate. Hyperpolarized [13C]bicarbonate was also detected, consistent with multiple mechanisms, including cataplerotic decarboxylation of [4-13C]oxaloacetate via phosphoenolpyruvate carboxykinase (PEPCK), forward TCA cycle flux of [4-13C]oxaloacetate to generate 13CO2 at isocitrate dehydrogenase, or decarboxylation of [1-13C]pyruvate by PDH. Isotopomer analysis of liver glutamate confirmed that anaplerosis was sevenfold greater than flux through PDH. In addition, signal from [4-13C]malate and [4-13C]aspartate was markedly blunted and signal from [13C]bicarbonate was completely abolished in livers from PEPCK KO mice, indicating that the major pathway for entry of hyperpolarized [1-13C]pyruvate into the hepatic TCA cycle is via pyruvate carboxylase, and that cataplerotic flux through PEPCK is the primary source of [13C]bicarbonate. We conclude that MR detection of hyperpolarized TCA intermediates and bicarbonate is diagnostic of pyruvate carboxylase and PEPCK flux in the liver.

Hyperpolarized Magnetic Resonance as a Sensitive Detector of Metabolic Function

Hyperpolarized magnetic resonance allows for noninvasive measurements of biochemical reactions in vivo. Although this technique provides a unique tool for assaying enzymatic activities in intact organs, the scope of its application is still elusive for the wider scientific community. The purpose of this review is to provide key principles and parameters to guide the researcher interested in adopting this technology to address a biochemical, biomedical, or medical issue. It is presented in the form of a compendium containing the underlying essential physical concepts as well as suggestions to help assess the potential of the technique within the framework of specific research environments. Explicit examples are used to illustrate the power as well as the limitations of hyperpolarized magnetic resonance.

Comparison of kinetic models for analysis of pyruvate‐to‐lactate exchange by hyperpolarized 13C NMR

The activity of specific enzyme‐catalyzed reactions may be detected in vivo by 13 C NMR of hyperpolarized (HP) substrates. The signals from HP substrates and products, acquired over time, have been fitted to a number of different mathematical models to determine fluxes, but these models have not been critically compared. In this study, two‐pool and three‐pool first‐order models were constructed to measure flux through lactate dehydrogenase in isolated glioblastoma cells by NMR detection of lactate and pyruvate following the addition of HP [1‐13C]pyruvate. Mass spectrometry (MS) was used to independently monitor 13 C enrichment in intra‐ and extracellular lactate. Six models were evaluated using time‐dependent pyruvate C2 and lactate C1 HP NMR data acquired by the use of selective excitation pulses, plus 13 C enrichment data from intracellular and extracellular lactate measured by MS. A three‐pool bidirectional model provided the most accurate description of pyruvate metabolism in these cells. With computed values for T1 of pyruvate and lactate, as well as the effect of pulsing, the initial flux through lactate dehydrogenase was well determined by both the two‐pool bidirectional and unidirectional models when only HP data were available. The three‐pool model was necessary to fit the combined data from both MS and HP, but the simpler two‐pool exchange model was sufficient to determine the 13 C lactate concentration when the lactate appearance was measured only by HP. Copyright

Inhibition of carbohydrate oxidation during the first minute of reperfusion after brief ischemia: NMR detection of hyperpolarized 13CO2 and H13CO3-.

Isolated rat hearts were studied by 31P NMR and 13C NMR. Hyperpolarized [1‐13C]pyruvate was supplied to control normoxic hearts and production of [1‐13C]lactate, [1‐13C]alanine, 13CO2 and H13CO  −3 was monitored with 1‐s temporal resolution. Hearts were also subjected to 10 min of global ischemia followed by reperfusion. Developed pressure, heart rate, oxygen consumption, [ATP], [phosphocreatine], and pH recovered within 3 min after the ischemic period. During the first 90 s of reperfusion, [1‐13C]alanine and [1‐13C]lactate appeared rapidly, demonstrating metabolism of pyruvate through two enzymes largely confined to the cytosol, alanine aminotransferase, and lactate dehydrogenase. 13CO2 and H13CO  −3 were not detected. Late after ischemia and reperfusion, the products of pyruvate dehydrogenase, 13CO2 and H13CO  −3 were easily detected. Using this multinuclear NMR approach, we established that during the first 90 s of reperfusion PDH flux is essentially zero and recovers within 20 min in reversibly‐injured myocardium. Magn Reson Med 60:1029–1036, 2008.

Impact of Gd3+ on DNP of [1-13C]Pyruvate Doped with Trityl OX063, BDPA, or 4-Oxo-TEMPO

Hyperpolarized [1-(13)C]pyruvate has become an important diagnostic tracer of normal and aberrant cellular metabolism for in vitro and in vivo NMR spectroscopy (MRS) and imaging (MRI). In pursuit of achieving high NMR signal enhancements in dynamic nuclear polarization (DNP) experiments, we have performed an extensive investigation of the influence of Gd(3+) doping, a parameter previously reported to improve hyperpolarized NMR signals, on the DNP of this compound. [1-(13)C]Pyruvate samples were doped with varying amounts of Gd(3+) and fixed optimal concentrations of free radical polarizing agents commonly used in fast dissolution DNP: trityl OX063 (15 mM), 4-oxo-TEMPO (40 mM), and BDPA (40 mM). In general, we have observed three regions of interest, namely, (i) a monotonic increase in DNP-enhanced nuclear polarization P(dnp) upon increasing the Gd(3+) concentration until a certain threshold concentration c(1) (1-2 mM) is reached, (ii) a region of roughly constant maximum P(dnp) from c(1) until a concentration threshold c(2) (4-5 mM), and (iii) a monotonic decrease in P(dnp) at Gd(3+) concentration c > c(2). Of the three free radical polarizing agents used, trityl OX063 gave the best response to Gd(3+) doping, with a 300% increase in the solid-state nuclear polarization, whereas addition of the optimum Gd(3+) concentration on BDPA and 4-oxo-TEMPO-doped samples only yielded a relatively modest 5-20% increase in the base DNP-enhanced polarization. The increase in P(dnp) due to Gd(3+) doping is ascribed to the decrease in the electronic spin-lattice relaxation T(1e) of the free radical electrons, which plays a role in achieving lower spin temperature T(s) of the nuclear Zeeman system. These results are discussed qualitatively in terms of the spin temperature model of DNP.

DNP by Thermal Mixing under Optimized Conditions Yields >60 000-fold Enhancement of 89Y NMR Signal

Hyperpolarized (89)Y complexes are attractive NMR spectroscopy and MR imaging probes due to the exceptionally long spin-lattice relaxation time (T(1) ≈ 10 min) of the (89)Y nucleus. However, in vivo imaging of (89)Y has not yet been realized because of the low NMR signal enhancement levels previously achieved for this ultra low-γ(n) nucleus. Here, we report liquid-state (89)Y NMR signal enhancements over 60,000 times the thermal signal at 298 K in a 9.4 T magnet, achieved after the dynamic nuclear polarization (DNP) of Y(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) samples at 3.35 T and 1.4 K. The (89)Y DNP was shown to proceed by thermal mixing and the liquid state (89)Y NMR signal enhancement was maximized by (i) establishing the optimal microwave irradiation frequency, (ii) optimizing the glassing matrix, (iii) choosing a radical with negligible inhomogeneous line broadening contribution to the ESR linewidth, and (iv) addition of an electron T(1e) relaxation agent. The highest enhancements were achieved using a trityl OX063 radical combined with a gadolinium relaxation agent in water-glycerol matrix. Co-polarization of (89)YDOTA and sodium [1-(13)C]pyruvate showed that both (89)Y and (13)C nuclear species acquired the same spin temperature, consistent with thermal mixing theory of DNP. This methodology may be applicable for the optimization of DNP of other low-γ(n) nuclei.