Redox Molecular Imaging Using Human-Applicable DNP-MRI

doi: 10.1016/j.freeradbiomed.2015.10.051 doi: xxxxx

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shifra Sela1, Ronit Geron1, Galina Shapiro1, Elisheva Milo1, Kimberly Bjugstad, 2, Raphael Bar-Or2, David Bar-Or2, and Batya Kristal1 1 Galilee Medical Center, Nahariya, Israel, 2Swedish Medical Center, Englewood, Colorado, USA A session of hemodialysis (HD) is accompanied by an increase in oxidative stress (OS). Measures of OS could serve as a good model for evaluating in vivo oxidation and provide a way to validate new therapeutic platforms, provided the measure of OS is sensitive enough to pick up treatment induced changes. Oxidation-reduction potential (ORP), a global measure of all oxidant and antioxidant activity, was compared to two other measures of OS to determine if pre-treatment with oral pomegranate juice (PJ) could reduce the OS induced by HD. Methods: 15 hemodialysis (HD) patients with and without PJ were studied. Measures of OS included: ORP, advanced oxidation protein products (AOPP), and myeloperoxidase (MPO). OS was determined in blood samples drawn before and after a dialysis session. All samples were frozen at -80oC until analyzed. Results: All three OS measures found increases in oxidative stress post-dialysis (p< 0.05): ORP (mV) 174.3±15 (pre) to 196.0±17.1(post); AOPP(μM) 175.8±69.0 (pre) to 232.1±93.0 (post); MPO (ng/ml) 6.6±4.9 (pre) to 15.1±11.7(post); PJ treatment caused a decrease in the differences pre to post dialysis, ORP measures revealed a significantly smaller increase post-dialysis after PJ treatment, while there were no more significant differences between pre to post values in MPO and AOPP. Further analyses revealed that longer dialysis vintage (> 40 months) had higher ORP values both pre and post-dialysis compared to shorter dialysis vintage (<40 months), and that PJ had its greatest reducing effect in the longer dialysis vintage. Conclusions: ORP is a sensitive method to identify slight changes in OS. By measuring ORP, it was possible to observe reductions in OS after dialysis as a result of pre-treatment with PJ. This effect was most prominent in the longer vintage patients. ORP provided a robust indicator of OS that could be used to identify the benefits of intervention like PJ, for use in HD. doi: 10.1016/j.freeradbiomed.2015.10.052 doi: xxxxx

in several microscopic fields of view to an identical perturbation and observed a striking degree of heterogeneity of fluorescence ratios from individual cells. The expression level of the probe and phase within the cell cycle were each examined as potential contributors to the observed heterogeneity. Higher ratiometric responses correlated with greater expression levels of the probe, and phase in the cell cycle was also shown to influence the magnitude of response. To aid in the interpretation of experimental observations, we incorporated the reaction of the reduced probe with peroxide and the reactions of the oxidized probe with glutathione and glutaredoxin into a larger kinetic model of peroxide metabolism. The predictions of the kinetic model suggest possible explanations for the experimental observations. This work highlights the importance of a systems-level approach to understanding the output of genetically encoded sensors that function via redox reactions involving thiol and disulfide groups.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.053 18 5HGR[0ROHFXODU,PDJLQJRI)UHH5DGLFDOV8VLQJWKH 6DPSOH5RWDWLQJ7\SH7'1305,6\VWHP

Tomoko Tahira1, Ryoma Kobayashi1, Tatsuya Naganuma2, Fuminori Hyodo1, and Hideo Utsumi1 1 Innovation Center for Medical Redox Navigation, Kyushu University, Japan, 2Japan REDOX Limited, Japan The dynamic nuclear polarization magnetic resonance imaging (DNP-MRI) is a new imaging method to detect free radical species in vivo. In this method, the electron spins of radicals are excited by ESR irradiation and utilized to enhance the NMR signal of protons. We have developed sample rotation type DNP-MRI system where the resonator carrying the sample is rotated between 20 mT arc-shaped magnet for ESR excitation and 1.5 T magnet for MR detection. This magnetic field cycling enables high-resolution redox imaging of model organisms. We examined conditions for sensitive and high resolution imaging using the phantoms containing nitroxyl radical, 3-carbamoyl-PROXYL (3CmP). The images of radical were obtained in 1 mm slices with space resolution of approximately 0.2 mm. We then tried to detect free radical intermediates produced from endogenous molecules, such as CoQ10 and FAD (Hyodo et al., Anal. Chem. 2014) and succeeded in visualization of these radicals. Compared with static DNP-MRI system previously used, this field cycling DNP-MRI system has advantage of improved resolution.

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doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.054

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Hadley D. Sikes1 1 Massachusetts Institute of Technology, USA

Fluorescent, genetically encoded sensors of hydrogen peroxide have enabled visualization of perturbations to the intracellular level of this signaling molecule with sub-cellular and temporal resolution. Ratiometric sensors hold the additional promise of meaningful quantification of intracellular hydrogen peroxide levels as a function of time, a longstanding goal in the field of redox signaling. To date, studies that have connected the magnitudes of observed ratios with peroxide concentrations have either examined suspensions of cells or small numbers of adherent cells (~10). In the present work, we examined the response of all cells

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Hideo Utsumi1, Yuji Okubo2, Toshiki Masumizu2, Hidenori Kajiwara3, Atsushi Iikura3, Ryoma Kobayasi1, Tomoko Tahira1, and Fuminori Hyodo1 1 Kyushu University, Japan, 2MEIKO Co., LTD., Japan, 3Fuji Electric Co., Ltd., Japan Free radical imaging with ESRI have been utilized in life science but not so widely as MRI due to its low spatiotemporal resolution. DNP-MRI (dynamic nuclear polarization magnetic resonance imaging), a new imaging method for free radical species in vivo, was first reported by Lurie, et al. (1987). In this method, the

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electron spins of radicals are excited by ESR irradiation and the enhancement of the NMR signal is induced by DNP. We developed a DNP-MR system (magnet rotation type) applicable to the human hand. To secure the stability and safety of the system, the magnets (5mT) for ESR and (0.3T) for MRI are stored in the jacket. MR Imaging were carried out with the gradient echo sequence, 2-10sec/cycle of magnets rotation, with/without 1.3-6.7sec of ESR excitation. Using this system, the clear images of the phantoms containing nitroxyl radical were obtained with and without ESR irradiation. We then tried to image the redox molecule intermediate produced from FAD and succeeded in visualization of this intermediate. doi: 10.1016/j.freeradbiomed.2015.10.055 doi: xxxxx

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Thomas Joost van 't Erve1, Fred Lih1, Casey Jelsema1, Maria Kadiiska1, Leesa Deterding1, Thomas Eling1, and Ronald Mason1 1 NIH/NIEHS, USA Background: Oxidative stress is characterized by the excessive oxidation of biomolecules in diseases and environmental exposures. However, accurate quantitation of this is not straightforward. The best accepted biomarker of oxidative stress in human specimens is the lipid oxidation product 8-isoprostaglandin FĮ (8-iso-PGFĮ). Generation of 8-iso-PGFĮ has been attributed exclusively to non-enzymatic or chemical lipid peroxidation (CLP). However, significant enzymatic generation of 8-iso-PGFĮ could occur via the prostaglandin-H-synthases (PGHS) in vivo. Ignoring the contribution of this pathway could have led to widespread over-quantitation of oxidative stress in many studies. Method: Both sources of 8-iso-PGFĮ can be simultaneously distinguished by measuring the concentration of 8-iso-PGFĮ as well as prostaglandin FĮ (PGFĮ) with HPLC/MS/MS and calculating the 8-iso-PGFĮ / PGFĮ ratio. We investigated this approach in two well-known animal models of oxidative stress, namely exposure to carbon tetrachloride (CCl4) or lipopolysaccharide (LPS). Results: Upon exposure to a 120 mg/kg dose of CCl4, the contribution of CLP accounted for 55.6 ± 19.4 % of measured 8iso-PGFĮ, whereas in the 1200 mg/kg dose, CLP was the predominant source of 8-iso-PGFĮ (86.6 ± 8.0 % of total). Conversely, exposure to 0.5 mg/kg LPS was characterized by a significant increase in the contribution of enzymatic lipid peroxidation by PGHS as well as CLP (59.5 ± 7.0 and 40.5 ± 14.0 % of total). Conclusion: A quantitative method to measure excessive lipid oxidation was developed and investigated in vivo. The distribution of the contributions is dependent on the exposure agent as well as the dose. The 8-iso-PGFĮ / PGFĮ ratio accurately quantitates the source of 8-iso-PGFĮ and provides unbiased data to study oxidative stress in human specimens. A custom, user-friendly program to perform the calculations required for this method can be downloaded free of charge.

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.056

21 8VLQJ4XDQWLWDWLYH5HGR[%LRORJ\WR$FKLHYHD0RUH 8QLYHUVDODQG*OREDO%LRHQHUJHWLF3KHQRW\SH$QDO\VLV Brett A Wagner1, Claire M Doskey1, Juan Du1, Joseph J Cullen1, and Garry R Buettner1 1 The University of Iowa, USA Cellular bioenergetics is intimately linked with the basic biology of health and disease. Deviations from the norm in bioenergetics have been identified as contributing, even major, factors in metabolic syndromes, e.g. obesity and diabetes. Abnormal bioenergetics is also suspected to be a modifying factor in cancer, heart disease, aging and other human health issues. Therefore, quantitative bioenergetic profiling of in vitro and ex vivo samples is essential to better understand the role that cellular energy metabolism has on human health and to identify actionable therapeutic maneuvers. Technologies such as those used in Seahorse metabolic flux analysis allow bioenergetic profiling of in vitro and ex vivo samples by simultaneously measuring both oxidative and glycolytic metabolism in real time ³%LRHQHUJHWLF SKHQRW\SH DQDO\VLV´ LV D UHFHQWO\ GHYHORSHG DQDO\WLFDO PHWKRG that assesses the basal glycolytic and oxidative metabolism of cells, as well as changes under stress. This type of analysis allows a systematic approach to describe phenotypic changes in cellular bioenergetics. We propose that this type of analysis can be further leveraged to a more universal metric and yield more LQIRUPDWLRQ LI WKH UHVXOWV IURP WKH ³ELRHQHUJHWLFV SKHQRW\SH´ analysis were to be expressed on a mole per cell (or mole per cell second, as appropriate) basis. With this, more quantitative comparisons can be made between cell types and cellular bioenergetics. This approach integrates more easily with modeling of biological processes, resulting in more information about these fundamental chemical and physical processes. Here we present example results from bioenergetic profiling as well as methods that can be employed tomorrow in all laboratories that allow a more quantitative approach to comparative analysis. Supported NIH grants R01 CA169046, R01 CA184051, R21 CA137230, P30 CA086862, R01 GM073929, P42 ES013661 and VA Merit Award 1101BX001318)

doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.057 22 6SLQ7UDSSLQJZLWK)OXRUHVFHQFH'HWHFWLRQ1RYHO 0HWKRGIRU6SHFLILFDQG6HQVLWLYH)OXRULPHWULF 'HWHFWLRQRI6XSHUR[LGH5DGLFDO$QLRQ

Jacek Zielonka1, Monika Zielonka1, Micael Hardy1,2, Olivier Ouari2, and Balaraman Kalyanaraman1 1 Medical College of Wisconsin, USA, 2Aix Marseille Université, France Superoxide radical anion (O2Ɣ-) is the primary reactive oxygen species (ROS) formed in cells via one-electron reduction of molecular oxygen. Further reactions of superoxide lead to formation of hydrogen peroxide and/or peroxynitrite, which in turn can lead to other forms of ROS. Reliable detection of superoxide is, therefore, of high importance for any study of the involvement of ROS in biological processes. Specific and sensitive detection of O2Ɣ-, however, still remains a challenge. Here, we propose a new approach for the detection of superoxide radical anion by combination of the spin trapping of O2Ɣ- with fluorescence detection of the spin adduct by reacting it in situ with fluorogenic boronate-based probe. Previously, superoxide spin trapping with cyclic nitrones has been carried out in combination

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