Medical Hypotheses 82 (2014) 627–630

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Medical Hypotheses

journal homepage: www.elsevier .com/locate /mehy

EPR spectroscopy as a predictive tool for the assessment of marginaldonor livers perfused on a normothermic ex vivo perfusion circuit

http://dx.doi.org/10.1016/j.mehy.2014.02.0250306-9877/Published by Elsevier Ltd.

⇑ Corresponding author at: Division of Transplantation, The Ohio State UniversityWexner Medical Center, 395 W 12th Ave. Suite, Columbus, OH 43210, United States.Tel.: +1 (614) 293 3212; fax: +1 (614) 293 4541.

E-mail address: Sylvester.Black@osumc.edu (S.M. Black).

Sylvester M. Black a,d,⇑, Bryan A. Whitson b,d, Murugesan Velayutham c,d

a Division of Transplantation, Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, OH 43210, United Statesb Division of Cardiac Surgery, Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, OH 43210, United Statesc Center for Biomedical EPR Spectroscopy and Imaging and Department of Anesthesiology, The Ohio State University Wexner Medical Center, Columbus, OH 43210, United Statesd The Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Laboratory, The Ohio State University Wexner Medical Center, Columbus, OH 43210,United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 October 2013Accepted 24 February 2014

Liver transplantation is a highly successful treatment for end-stage liver disease. While liver transplan-tation is often the only effective treatment for cirrhosis there is a critical shortage of donor organs, lead-ing to death of many potential recipients on the waiting list. Marginal liver grafts are increasingly beingused in an attempt to increase the number of donor livers utilized for transplantation. Marginal donorlivers often have complications and worse outcomes for recipients receiving these types of transplant.The ability to predict the outcome with the use of marginal grafts is difficult and often imprecise leadingdecreased use of potentially suitable grafts. The development and maturation of normothermic ex vivoperfusion as a platform for the assessment of donor organs presents an opportunity to increase the num-ber of usable donor livers available for transplantation. Furthermore, direct measurement of reactive oxy-gen species (ROS) present in the donor liver on an ex vivo perfusion circuit by electron paramagneticresonance (EPR) spectroscopy would allow for precise real-time quantification of donor organ injury.The combination normothermic ex vivo liver perfusion with EPR spectroscopy could therefore presenta powerful platform to increase the number of donor organs utilized for transplantation.

Published by Elsevier Ltd.

Introduction

The success of liver transplantation for the treatment of end-stage liver disease is one of the more remarkable stories in modernmedicine. Many patients who were once considered untreatablehave been helped with liver transplantation. Organ transplantationis a young specialty with transplantation beginning in earnest inthe 1960’s and really achieving the results that we would equatewith successful modern practice in the 1980’s. Transplantationhowever has become a victim of its own success in that the supplyof suitable donor organs is far outstripped by the number of poten-tial recipients on the waiting list. Furthermore the quality of donororgans has generally decreased as the donor population has aged,becoming more obese and having been affected by diseases suchas diabetes, hypertension and atherosclerosis [1,2]. There has beena movement in the field of transplantation to increase the numberof donor organs by expanding the donor pool [1,3]. This occurs by

multiple mechanisms such as increasing the number of donors andgeneral awareness of organ donation, using marginal or extendedcriteria donors, and utilizing living donation. Despite the effortsto make donor organs more available the deficit persists and pa-tients continue to die on the waiting list before they can receivea transplant.

One of the important strategies for increasing the number oftransplants performed and decreasing the death on the waiting listwould be to make maximal use of the available donor organs fortransplantation. The concept being that a number of organs frompotential donors are discarded or considered not suitable for trans-plantation [3]. Furthermore, a percentage of organs from high riskdonors are, in fact, utilized and the outcomes in many cases are lessthan optimal with a high incidence of primary non-function, bileduct complications [4] and aggressive hepatitis C recurrence as inthe case of some non-heart beating donors (DCD) liver transplants[5,6]. As we begin to utilize more marginal organs, these issues willbecome more prevalent. Normothermic ex vivo liver perfusion asan experimental strategy attempts to provide a solution to forassessment of organs at normal physiology and provides a poten-tial platform for repair of suboptimal organs [7,8]. Normothermicex vivo perfusion is not a new technique or concept but was

Fig. 1. 1-Hydroxy-3-carboxymethyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) reactswith superoxide radical (O2

��) and peroxynitrite (ONOO�) to form 3-carboxymethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy radical (CM�).

628 S.M. Black et al. / Medical Hypotheses 82 (2014) 627–630

envisioned in the 1930’s by Alexis Carrel and Charles Lindbergh asa method to indefinitely preserve organs outside of the body forassessment of function, repair and potential transplantation intowaiting recipients [9,10]. Technological limitations and practicalconsiderations have limited the application of this technique inmodern transplantation. However with advancements in pumptechnology, perfusates, surgical technique, and biologicalknowledge there has been a recent re-evaluation of this technologyand a consideration for potential application to the field oftransplantation.

Rationale

Currently, ex vivo organ perfusion has been clinically imple-mented in the field of lung transplantation, where the Universityof Toronto group has had great success in demonstrating the effi-cacy and feasibility of this technique [11]. Normothermic ex vivoliver perfusion however, has taken longer to implement with clin-ical trials currently underway in Europe to evaluate this technol-ogy. There have been a number of notable achievements in thelaboratory, including 72 h of normothermic ex vivo porcine liverperfusion, which maintained excellent viability in the donor organover that time period [12,13]. The liver unlike the lung and many ofthe other organs that have been perfused, is strictly a metabolic or-gan with a high oxygen demand. Assessment of the function andviability of the donor organ in liver transplantation has been chal-lenging and has traditionally relied on indirect laboratory mea-surements such ALT/AST, bilirubin, INR/PT and histology in thedonor prior to procurement. However, normothermic ex vivo liverperfusion presents the opportunity to directly evaluate the organoutside of the body for extended periods of time. The implicationsof this technology are that direct assessment of the organ becomespossible. With the appropriate tools, one could make a very de-tailed analysis of the metabolic state of the organ and predict organfunction on reperfusion in the recipient.

3461 3486Magnetic Fiel

A

B

aN

Fig. 2. Room temperature EPR spectra of CM�. All the reactions were performed in phosgenerated by xanthine and xanthine oxidase oxidizes CMH to CM�. Spectrum A: CMH (1mL). EPR spectra were recorded using quartz flat cell at room temperature with a Bruker Ea TM110 cavity. EPR instrument parameters used were: microwave frequency 9.778 GHz,30 s, and time constant 82 ms. The observed isotropic hyperfine splitting constant is aN

There is strong evidence that reactive oxygen species (ROS) areimportant mediators of ischemia reperfusion injury in liver trans-plantation [14]. Partial reduction of molecular oxygen, results inthe formation of ROS, which include the superoxide radical (O2

��),hydrogen peroxide (H2O2) and the hydroxyl radical (HO�) [15,16].Under normal physiologic conditions 1–2% of the oxygen con-sumed is converted into ROS [17]. ROS are produced as a byproductof cellular respiration and are important in many cell-signalingcascades [18] . Under periods of increased physiologic stress, suchas would occur in ischemia with disordered cellular respiration,there is a dramatic increase in the production of ROS. The increasedproduction of ROS leads to peroxidation of membrane lipids, pro-tein and DNA and eventually to cellular dysfunction and death.This oxidative stress is defined as the imbalance of pro- and anti-oxidants in a biologic system in favor of the former. Furthermore,the level of ROS is greatly accelerated with the sudden reintroduc-tion of oxygen at the onset of reperfusion of a transplanted organ[19]. Hence, the restoration of blood flow, following hepatic ische-mia, can result in significant injury and organ dysfunction.

Real time quantification of highly reactive, short-lived ROS hasproven difficult in complex biologic systems. Electron paramagnetic

3511d (Gauss) phate buffer saline (pH = 7.4) containing 50 lM desferoxamine. Superoxide radicalmM). Spectrum B: CMH (1 mM), xanthine (0.1 mM), and xanthine oxidase (15 mU/SP 300E spectrometer operating at X-band with 100-kHz modulation frequency andmodulation amplitude 1 G, microwave power 20 mW, number of scans 1, scan time= 16.1 G.

Fig. 3. Proposed experiments utilizing brain dead donor (DBD) or non heart beatingdonors (DCD) in pigs. The DCD group would wait a period of 60 min to maximizeischemic injury prior to procurement, whereas the DBD group would proceedimmediately with organ procurement minimizing ischemia. EPR spectroscopywould be conducted upon placement of the donor organ in the ex vivo circuit. UW:university of Wisconsin. ROS: reactive oxygen species.

S.M. Black et al. / Medical Hypotheses 82 (2014) 627–630 629

resonance (EPR) spectroscopy is a method that allows for quantifi-cation of ROS by utilizing a spin probe which reacts with ROS to gen-erate a radical with a longer half-life that is detectable on a time

Fig. 4. Normothermic ex vivo liver perfusion circuit with introduction of the spin prspectroscopy from the vena cava. The spin probe would be introduced and collected at

scale that can be measured [20]. The EPR spin probe 1-hydroxy-3-carboxymethyl-2,2,5,5-tetramethyl-pyrrolidine (CMH, EPR silent)is oxidized by the superoxide radical (O2

��) and peroxynitrite(ONOO�) to 3-carboxymethyl-2,2,5,5-tetramethyl-pyrrolidinyloxyradical (CM�, EPR active) as shown in Fig. 1 [21]. The O2

�� generatedby the xanthine and xanthine oxidase system, oxidizes CMH toCM� and the corresponding EPR spectrum is shown in Fig. 2. TheEPR signals formed are proportional to the amount of ROS produced[22,23]. Thus EPR, when used with a known reference compound al-lows for the quantification of ROS levels in a biological system[16,22,23].

Presentation of the hypothesis

Ischemia reperfusion injury of the donor liver is the majormechanism behind the dysfunction and non-function upon reper-fusion of the liver in the recipient. Buildup of ROS secondary toanaerobic cellular respiration that occurs during donor organprocurement and static cold storage is the mediator of ischemiareperfusion injury.

Hypothesis

EPR spectroscopy would provide a platform for dynamic assess-ment of donor liver viability and metabolism on a normothermicex vivo perfusion circuit, allowing for determination of organ suit-ability prior to transplantation. Furthermore, determination of ROSlevels after many ex vivo perfusions would allow for the creation ofa redox index, which would further facilitate identification of or-gans associated with good outcomes following transplantation.

Supportive observations for this hypothesis and futuredirections

Normothermic ex vivo liver perfusion is a technique, which incontrast to static cold storage or hypothermic machine perfusionseeks to maintain normal organ physiology and cellular metabo-lism. In the laboratory normothermic ex vivo liver perfusion hassuccessfully maintained liver viability and metabolism for as long

obe in the hepatic artery and portal vein with collection of the sample for EPRvarious time points to quantify ROS accumulation.

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as 72 h [12,13]. Experimental ex vivo liver perfusion has demon-strated the ability to mitigate ATP loss sustained during long peri-ods of warm and cold ischemia, reverse liver injury and preservebile duct integrity [24].

Assessment of organ injury is often indirect such as measuringserum transaminase levels in the donor, which can give a mislead-ing impression as to how the organ will function in the recipient,especially after prolonged cold storage. EPR spectroscopy has pro-ven accurate in quantifying the redox status of tissues and organssubjected to an ischemic insult [19,22,25]. EPR spectroscopy hasalso been used for quantification of ROS in an experimental modelof rodent pancreas transplantation, which was consistent withconventional markers or tissue damage such as lactate and ATP[26].

The hypothesis could be tested utilizing a porcine model,whereby brain dead donors with minimal ischemia would be com-pared with cardiac death or non-heart beating donors (DCD) with60 min of warm ischemia prior to normothermic ex vivo liver per-fusion (Fig. 3). Donor livers would be procured in the standardmanner after a cold preservative flush with University of Wiscon-sin solution. The donor livers would be placed on ice for a periodof 4 h and then would be connected to an ex vivo normothermiccircuit and perfused with heparinized blood for a period of 4 h(or longer). The spin probe CMH would be introduced at each timepoint into the hepatic artery and portal vein and collected from thevena cava (Fig. 4). Samples would be collected at various timepoints from 30 s after reperfusion for the duration of the experi-ment. One would expect that the DCD donor livers would demon-strate significant ROS accumulation and this would correlate withtraditional markers of heptic injury, such as AST/ALT. As the directmediators of the ischemia reperfusion injury one would expectthat the ROS increase would occur prior to any increase in tradi-tional markers of hepatic injury such as AST/ALT, alkaline phospha-tase, and LDH. The EPR spectroscopy would provide an earlyindicator with likely predictive value for subsequent liver trans-plantation as it would directly measure the mediator of the ische-mia reperfusion injury. Furthermore, with a large number ofexperiments it may be possible to develop a redox index for predic-tion of organ performance prior to transplantation to help optimizeand improve transplant outcomes.

Conclusions

Detailed assessment of donor liver metabolism and injurywould be possible with the combination of ex vivo liver perfusionand EPR spectroscopy. Evaluation of the redox state of a donor or-gan on a normothermic ex vivo liver perfusion circuit would theo-retically allow for the mitigation of ROS buildup limitingreperfusion injury in the recipient upon transplantation. Further-more organs significantly damaged with dysfunctional oxygenmetabolism and high ROS concentrations may be deemed unsuit-able for transplantation and discarded, thus preventing primarynon-function in the recipient. EPR spectroscopy when used in com-bination with normothermic ex vivo liver perfusion could repre-sent a powerful tool that would allow for accurate organassessment and potentially improve liver transplant outcomes.

Conflict of Interest

The authors have no conflicts of interest to disclose.

References

[1] Sela N, Croome KP, Chandok N, Marotta P, Wall W. The changing donorcharacteristics in liver transplantation over the last 10 years in Canada. LiverTranspl. 2013.

[2] Orman ES, Barritt ASt, Wheeler SB, Hayashi PH. Declining liver utilization fortransplantation in the United States and the impact of donation after cardiacdeath. Liver Transpl. 2013;19:59–68.

[3] Vrochides D, Metrakos P. Moving toward the utilization of all donated livergrafts. The ‘‘b-list’’ concept. Hippokratia 2012;16:312–6.

[4] Seehofer D, Eurich D, Veltzke-Schlieker W, Neuhaus P. Biliary complicationsafter liver transplantation: old problems and new challenges. Am. J. Transpl.2013;13:253–65.

[5] Ciria R, Briceno J, Rufian S, Luque A, Lopez-Cillero P. Donation after cardiacdeath: where, when, and how? Transpl. Proc. 2012;44:1470–4.

[6] Ozhathil DK, Li YF, Smith JK, Tseng JF, Saidi RF, et al. Impact of center volumeon outcomes of increased-risk liver transplants. Liver Transpl.2011;17:1191–9.

[7] Brockmann J, Reddy S, Coussios C, Pigott D, Guirriero D, et al. Normothermicperfusion: a new paradigm for organ preservation. Ann. Surg. 2009;250:1–6.

[8] Monbaliu D, Brassil J. Machine perfusion of the liver: past, present and future.Curr. Opin. Organ Transpl. 2010;15:160–6.

[9] Carrel A, Lindbergh CA. The culture of whole organs. Science 1935;81:621–3.[10] Carrel A. On the permanent life of tissues outside of the organism. J. Exp. Med.

1912;15:516–28.[11] Wigfield CH, Cypel M, Yeung J, Waddell T, Alex C, et al. Successful emergent

lung transplantation after remote ex vivo perfusion optimization andtransportation of donor lungs. Am. J. Transpl. 2012;12:2838–44.

[12] St Peter SD, Imber CJ, Lopez I, Hughes D, Friend PJ. Extended preservation ofnon-heart-beating donor livers with normothermic machine perfusion. Br. J.Surg. 2002;89:609–16.

[13] Rees MA, Butler AJ, Chavez-Cartaya G, Wight DG, Casey ND, et al. Prolongedfunction of extracorporeal hDAF transgenic pig livers perfused with humanblood. Transplantation 2002;73:1194–202.

[14] Zhang W, Wang M, Xie HY, Zhou L, Meng XQ, et al. Role of reactive oxygenspecies in mediating hepatic ischemia-reperfusion injury and its therapeuticapplications in liver transplantation. Transpl. Proc. 2007;39:1332–7.

[15] Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, et al. Free radicals andantioxidants in normal physiological functions and human disease. Int. J.Biochem. Cell Biol. 2007;39:44–84.

[16] Velayutham M, Hemann C, Zweier JL. Removal of H(2)O(2) and generation ofsuperoxide radical: role of cytochrome c and NADH. Free Radic. Biol. Med.2011;51:160–70.

[17] O’Rourke B, Cortassa S, Aon MA. Mitochondrial ion channels: gatekeepers oflife and death. Physiology (Bethesda) 2005;20:303–15.

[18] Finkel T. Signal transduction by reactive oxygen species. J. Cell Biol.2011;194:7–15.

[19] Zweier JL, Talukder MA. The role of oxidants and free radicals in reperfusioninjury. Cardiovasc. Res. 2006;70:181–90.

[20] Oettl K, Dikalov S, Freisleben HJ, Mlekusch W, Reibnegger G. Spin trappingstudy of antioxidant properties of neopterin and 7,8-dihydroneopterin.Biochem. Biophys. Res. Commun. 1997;234:774–8.

[21] Fink B, Dikalov S, Bassenge E. A new approach for extracellular spin trapping ofnitroglycerin-induced superoxide radicals both in vitro and in vivo. Free Radic.Biol. Med. 2000;28:121–8.

[22] Mrakic-Sposta S, Gussoni M, Montorsi M, Porcelli S, Vezzoli A. Assessment of astandardized ROS production profile in humans by electron paramagneticresonance. Oxid. Med. Cell Longev. 2012;2012:973927.

[23] Kohr MJ, Wang H, Wheeler DG, Velayutham M, Zweier JL, et al. Targeting ofphospholamban by peroxynitrite decreases beta-adrenergic stimulation incardiomyocytes. Cardiovasc. Res. 2008;77:353–61.

[24] Boehnert MU, Yeung JC, Bazerbachi F, Knaak JM, Selzner N, et al.Normothermic acellular ex vivo liver perfusion reduces liver and bile ductinjury of pig livers retrieved after cardiac death. Am. J. Transpl.2013;13:1441–9.

[25] West MB, Rokosh G, Obal D, Velayutham M, Xuan YT, et al. Cardiac myocyte-specific expression of inducible nitric oxide synthase protects againstischemia/reperfusion injury by preventing mitochondrial permeabilitytransition. Circulation 2008;118:1970–8.

[26] Neeff HP, von Dobschuetz E, Sommer O, Hopt UT, Drognitz O. In vivoquantification of oxygen-free radical release in experimental pancreastransplantation. Transpl. Int. 2008;21:1081–9.