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

Abstract

C NMR is a powerful tool for monitoring metabolic fluxes in vivo.The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing 13 C 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 [1-13 C1]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved 13 CO2, the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of Ϸ1 s along with H 13 CO 3 ؊ , the carbon dioxide ͉ heart ͉ hyperpolarization ͉ NMR spectroscopy ͉ pyruvate N oninvasive measures of flux through specific enzymecatalyzed reactions remain an important goal in physiology and clinical medicine. Standard radionuclide imaging methods do not provide information about individual reactions because the measured signal represents the weighted sum of the tracer plus the biochemical products produced by tissue. 13 C NMR spectroscopy is much more powerful in this regard because it can easily differentiate between specific 13 C-labeled products of biochemical reactions (1). The sensitivity enhancement gained by hyperpolarization of 13 C nuclei (2) offers the possibility of using noninvasive 13 C NMR spectroscopy and imaging to measure f luxes through individual enzyme-catalyzed reactions.[1-13 C 1 ]Pyruvate, for example, is a substrate that is avidly metabolized in most tissues. The individual metabolic products of this tracer, [1-13 C 1 ]lactate, [1-13 C 1 ]alanine, and H 13 CO 3 Ϫ , can be separately detected in vivo because of the inherent chemical shift dispersion of 13 C NMR (3). Because decarboxylation of [1-13 C 1 ]pyruvate via pyruvate dehydrogenase (PDH) must produce 13 CO 2 , the appearance of H 13 CO 3 Ϫ potentially directly reflects flux through PDH. A substantially reduced H 13 CO 3 Ϫ signal in hearts after coronary occlusion and reflow was previously attributed to an effect of transient ischemia on the tricarboxylic acid (TCA) cycle (3). However, it is known that the heart switches rapidly among a wide variety of substrates to supply acetyl-CoA (4, 5), and because fats and ketones are not metabolized via PDH the rate of production of H 13 CO 3 Ϫ from [1-13 C 1 ]pyruvate in vivo should be sensitive to the availability of other substrates.Noninvasive detection of flux through PDH would unquestionably be of value in understanding potentially high-impact therapies for heart disease. Pharmacological and metabolic interventions that would be expected to increase flux through PDH have been examined since the 1960s with the goal of protecting ischemic myocardium (6-9) or improving function in the failing heart (10, 11). However, because of the complex interconnections between fatty acid and carbohydrate oxidation, it has been difficult to separate effects of metabolism through a specific reaction, PDH, from effects on oxygen consumption, myocardial efficiency, and overa...

Direct noninvasive estimation of myocardial tricarboxylic acid cycle flux in vivo using hyperpolarized 13C magnetic resonance

“…The technique provides unique insights into important questions regarding myocardial metabolism, substrate preference, pharmacological intervention, and the ability to differentiate between normal and pathological metabolism [22][23][24][25][26][27][28][29][30][31][32][33]. Pyruvate is by far the most widely used hyperpolarized 13 C substrate and it can be served as a probe for mitochondrial oxidative flux [34].…”

Metabolic imaging of acute and chronic infarction in the perfused rat heart using hyperpolarised [1‐13C]pyruvate

“…With the recent enhancements in the MR sensitivity of the 13 C nucleus achieved using dynamic nuclear polarisation (DNP) (1), it is now possible to complement structural data using MRI with global metabolic information in the myocardium (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Work with several in vivo animal models has combined DNP with different imaging techniques (18)(19)(20)(21)(22), including chemical shift imaging (CSI) (23), to obtain metabolic maps of the heart using hyperpolarised [1-13 C]pyruvate (2,20,(24)(25)(26)(27).…”

“…The aim of the work described here was to develop a combined DNP and CSI protocol for application in the isolated perfused rat heart to generate metabolic images of hyperpolarised [1][2][3][4][5][6][7][8][9][10][11][12][13] (5,13). It should be possible to generate maps of these metabolites in the isolated perfused rat heart and to use the metabolic images of [1- 13 C]pyruvate as a marker of tissue perfusion, [1][2][3][4][5][6][7][8][9][10][11][12][13] C]lactate as a marker of anaerobic metabolism and [ 13 C]bicarbonate as a marker of aerobic metabolism.…”

“…It should be possible to generate maps of these metabolites in the isolated perfused rat heart and to use the metabolic images of [1- 13 C]pyruvate as a marker of tissue perfusion, [1][2][3][4][5][6][7][8][9][10][11][12][13] C]lactate as a marker of anaerobic metabolism and [ 13 C]bicarbonate as a marker of aerobic metabolism.…”

A referenceless Nyquist ghost correction workflow for echo planar imaging of hyperpolarized [1‐13C]pyruvate and [1‐13C]lactate

“…For example, the imaging of hyperpolarized [1‐ 13 C]pyruvate and its downstream metabolic products has been used in cancer research to assess treatment response6, 7 and disease progression,8, 9 and in other tissues, such as heart10, 11 and kidney,12 to assess tissue function. The technique has now been translated to the clinic with studies in prostate cancer13 and in heart 14…”