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Glyoxalase Activity in Human Erythrocytes and Mouse Lymphoma, Liver and Brain Probed with Hyperpolarized C-13-Methylglyoxal Dmitry Shishmarev, Philip W

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Glyoxalase Activity in Human Erythrocytes and Mouse Lymphoma, Liver and Brain Probed with Hyperpolarized C-13-Methylglyoxal Dmitry Shishmarev, Philip W Glyoxalase activity in human erythrocytes and mouse lymphoma, liver and brain probed with hyperpolarized C-13-methylglyoxal Dmitry Shishmarev, Philip W. Kuchel, Guilhem Pages, Alan J. Wright, Richard L. Hesketh, Felix Kreis, Kevin M. Brindle To cite this version: Dmitry Shishmarev, Philip W. Kuchel, Guilhem Pages, Alan J. Wright, Richard L. Hesketh, et al.. Glyoxalase activity in human erythrocytes and mouse lymphoma, liver and brain probed with hyperpolarized C-13-methylglyoxal. Communications Biology, Nature Publishing Group, 2018, 1, 10.1038/s42003-018-0241-1. hal-02624539 HAL Id: hal-02624539 https://hal.inrae.fr/hal-02624539 Submitted on 26 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License ARTICLE https://doi.org/10.1038/s42003-018-0241-1 OPEN Glyoxalase activity in human erythrocytes and mouse lymphoma, liver and brain probed with hyperpolarized 13C-methylglyoxal Dmitry Shishmarev 1, Philip W. Kuchel2, Guilhem Pagès 3, Alan J. Wright 4, Richard L. Hesketh4, 1234567890():,; Felix Kreis 4 & Kevin M. Brindle 4 Methylglyoxal is a faulty metabolite. It is a ubiquitous by-product of glucose and amino acid metabolism that spontaneously reacts with proximal amino groups in proteins and nucleic acids, leading to impairment of their function. The glyoxalase pathway evolved early in phylogeny to bring about rapid catabolism of methylglyoxal, and an understanding of the role of methylglyoxal and the glyoxalases in many diseases is beginning to emerge. Metabolic processing of methylglyoxal is very rapid in vivo and thus notoriously difficult to detect and quantify. Here we show that 13C nuclei in labeled methylglyoxal can be hyperpolarized using dynamic nuclear polarization, providing 13C nuclear magnetic resonance signal enhancements in the solution state close to 5,000-fold. We demonstrate the applications of this probe of metabolism for kinetic characterization of the glyoxalase system in isolated cells as well as mouse brain, liver and lymphoma in vivo. 1 The Australian National University, John Curtin School of Medical Research, Canberra, ACT, Australia. 2 The University of Sydney, School of Life and Environmental Sciences, Sydney, NSW, Australia. 3 INRA, AgroResonance – UR370 Qualité des Produits Animaux, F-63122, Saint Genès Champanelle, France. 4 University of Cambridge, Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Cambridge, UK. Correspondence and requests for materials should be addressed to P.W.K. (email: [email protected]) COMMUNICATIONS BIOLOGY | (2018) 1:232 | https://doi.org/10.1038/s42003-018-0241-1 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-018-0241-1 ethylglyoxal (MeGx) is formed both inside cells1–3 and Hyperpolarization led to high spectral signal-to-noise ratios for all in blood plasma4–6, primarily as a by-product of gly- the MeGx species (~200:1 for MGMH), despite the [2-13C]MeGx M 7,8 9 colysis and amino acid metabolism , with a total concentration in the sample of only 1.3 mM. In comparison, a daily production estimated at 0.2–1.2 g in the adult human8,10.As signal-to-noise ratio of ~12:1 was achieved for MGMH in 13C-NMR 13 a ketoaldehyde, MeGx is highly reactive and spontaneously spectra of non-hyperpolarized ~40 mM [2- C]MeGx in D2O, modifies side chain amino groups in proteins and nucleic acids, recorded from 64 transients in 4 min (Supplementary Figure 3). leading to impairment of their function and degradation11. Initial Thus, the DNP signal enhancement was ~5000. catabolism of MeGx occurs via the glyoxalase pathway12,13, which The MGMH resonance declined rapidly due to glyoxalase- consists of glyoxalase I (Glo1, enzyme commission number mediated flux and longitudinal relaxation, whereas the MGBH 4.4.1.5, lactoylglutathione lyase) and glyoxalase II (Glo2, enzyme resonance declined more slowly despite a similar T1 (see below commission number 3.1.2.6, hydroxyacylglutathione hydrolase), for estimates of these parameter values), primarily because which in red blood cells (RBCs) have a maximum flux capacity MGMH is the intermediate that feeds rapidly onto the free ~100 times that of glycolysis14,15. The pathway requires reduced ketoaldehyde (MG) and the rest of the pathway, while exchange glutathione (GSH) as a co-substrate and converts MeGx into D- between the two hydrated species is relatively slower. The smaller lactate (D-Lac) (as opposed to the L-lactate produced by glycolysis resonances from the ketoaldehyde and hemithioacetal (HTA) in higher organisms16), which is then further metabolized in persisted in the spectra (Fig. 2b), consistent with continuing peroxisomes17. Understanding the fate of MeGx in biology and its regeneration of these minor forms during the flow of 13C label role in disease development is beginning to emerge18. Additional from MGMH/MGBH via MG/HTA to SLG and D-Lac (Fig. 1). functions of MeGx and the glyoxalase pathway in cell physiology SLG was prominent initially (at ~2 s) and declined thereafter, remain largely unresolved. consistent with a very fast build-up catalyzed by Glo1, followed The kinetics of MeGx catabolism has been studied previously by removal in the reaction catalyzed by Glo2, with concurrent 1 in dilute lysates of RBCs using H nuclear magnetic resonance loss of signal due to T1 relaxation. (NMR) spectroscopy15. However, due to poor intrinsic sensitivity The emergence of D-Lac occurred more slowly than for SLG, as of NMR and the need to accumulate each NMR spectrum for ~4 expected for a metabolite that is further downstream. A similar min, the glyoxalase reactions appeared to be too fast to study in pattern of labeling was evident in RBC lysates (Supplementary intact cells and tissues (going to completion in <1 min). We Figure 5) and in suspensions of murine lymphoma EL4 cells demonstrate here, using rapid dissolution dynamic nuclear (Supplementary Figure 6). polarization (RD-DNP)19, that it is possible to monitor the glyoxalase pathway non-invasively in whole cells and tissues, on Quantification of glyoxalase kinetics in whole RBCs. Figure 2c the sub-minute timescale. Using hyperpolarization, we achieve shows the temporal evolution of 13C-labeled MGMH, MGBH, ~5,000-fold enhancements in NMR sensitivity, which allows SLG and D-Lac, obtained upon injection of four different amounts detection and quantification of the glyoxalase reactions in RBCs of [2-13C]MeGx (final concentrations of 1.05, 2.1, 4.7 and 9.4 and mouse tissues in vivo. mM). The time at which the maximum D-Lac peak intensity occurred varied only slightly with changes in the concentration of 13 Results [2- C]MeGx. The extent of reaction, indicated by the relative Synthesis of 13C-labeled methylglyoxal. Two isotopomers of 13C- maximum amplitude of the D-Lac signal, was highest for the labeled MeGx, [2-13C]MeGx and [1,3-13C]MeGx, were synthesized lowest starting concentration of MeGx. Also, at higher substrate from [2-13C]acetone and [1,3-13C]acetone, respectively. The syn- concentrations the glyoxalase system became partially saturated. thetic procedure was adapted from a method for radiolabeling The MGMH signal showed an apparent single-exponential decay, 20 13 as expected for the main substrate species, while MGBH showed MeGx and described in Methods. Since the CnucleusattheC2 13 position in MeGx does not have any attached protons (Fig. 1), it has the appearance of a bi-exponential decay, consistent with C label flowing through MGMH prior to yielding the other two a much larger NMR longitudinal relaxation time T1 and hence longer nuclear polarization lifetime than the C1 or C3 carbons. products. SLG was formed so fast that its maximum amplitude Consequently, we observed better spectral quality in RD-DNP time occurred shortly after MeGx addition; while D-Lac showed the 13 rise-and-fall profile expected for a product at the end of a reaction courses obtained with hyperpolarized [2- C]MeGx and therefore 21–24 focused on this isotopomer. (Results obtained with [1,3-13C]MeGx sequence, as has previously been observed . The rate constants describing 13C label flux were estimated by are shown in Supplementary Figure 1.) The purity of the synthe- fi sized [2-13C]MeGx was confirmed by one-dimensional (1D) 1H iterative tting of the following (continuous) differential (Supplementary Figure 2), 1D 13C (Supplementary Figure 3) and equations, which were integrated numerically, to the signal two-dimensional (2D) 1H-13C HMBC (Supplementary Figure 4) intensities in Fig. 2c: 1 à NMR spectra. MeGx is spontaneously hydrated (Fig. 1)and Hand d½MGBH 1 à à à 13 fi ¼À ½ÀMGBH k ½þMGBH kÀ ½MGMH CNMRspectracon rmed that it exists as three interchanging (on dt TMGBH 1 1 the sub-minute timescale) forms in aqueous solution, viz., free keto- 1 aldehyde (MG), the monohydrate (MGMH), and the bishydrate à (MGBH), which at 37 oC were in the ratio 1:71:28 (keto-aldehyde: d½MGMH 1 à à ¼À ½ÀMGMH k ½MGMH MGMH:MGBH). MGMH Glo1 dt T1 À ½þà ½Ã kÀ1 MGMH k1 MGBH Detection of methylglyoxal catabolism in whole cells. A solu- tion of hyperpolarized [2-13C]MeGx was injected into an RBC ½Ã suspension that was thermally pre-equilibrated at 37 °C inside the d SLG ¼À 1 ½Àà ½þà ½Ã 13 SLG SLG kGlo2 SLG kGlo1 MGMH NMR spectrometer bore, and a series of 1D C NMR spectra (1 s dt T1 per spectrum) was acquired using a small flip angle (~4°) exci- tation pulse.
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