Tryptophan Metabolism Via the Kynurenine Pathway: Implications for Graft Optimization During Machine Perfusion

Journal of Clinical Medicine

Article Tryptophan Metabolism via the Kynurenine Pathway: Implications for Graft Optimization during Machine Perfusion

Anna Zhang 1,2, Cailah Carroll 1,3, Siavash Raigani 1,3,4 , Negin Karimian 1,3, Viola Huang 1,3, Sonal Nagpal 1,3, Irene Beijert 1,5, Robert J. Porte 5, Martin Yarmush 1,3,6, Korkut Uygun 1,3 and Heidi Yeh 4,*

1 Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA; [email protected] (A.Z.); [email protected] (C.C.); [email protected] (S.R.); [email protected] (N.K.); [email protected] (V.H.); [email protected] (S.N.); [email protected] (I.B.); [email protected] (M.Y.); [email protected] (K.U.) 2 Tufts University School of Medicine, Boston, MA 02111, USA 3 Shriners Hospital for Children, Boston, MA 02114, USA 4 Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA 5 Division of Hepatobiliary Surgery and Liver Transplantation, University Medical Center Groningen, 9700 Groningen, The Netherlands; [email protected] 6 Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854, USA * Correspondence: [email protected]; Tel.: +1-617-726-3664; Fax: +1-617-643-4579

Received: 8 May 2020; Accepted: 10 June 2020; Published: 15 June 2020

Abstract: Access to liver transplantation continues to be hindered by the severe organ shortage. Extended-criteria donor livers could be used to expand the donor pool but are prone to ischemia- reperfusion injury (IRI) and post-transplant graft dysfunction. Ex situ machine perfusion may be used as a platform to rehabilitate discarded or extended-criteria livers prior to transplantation, though there is a lack of data guiding the utilization of different perfusion modalities and therapeutics. Since amino acid derivatives involved in inflammatory and antioxidant pathways are critical in IRI, we analyzed differences in amino acid metabolism in seven discarded non-steatotic human livers during normothermic- (NMP) and subnormothermic-machine perfusion (SNMP) using data from untargeted metabolomic profiling. We found notable differences in tryptophan, histamine, and glutathione metabolism. Greater tryptophan metabolism via the kynurenine pathway during NMP was indicated by significantly higher kynurenine and kynurenate tissue concentrations compared to pre-perfusion levels. Livers undergoing SNMP demonstrated impaired glutathione synthesis indicated by depletion of reduced and oxidized glutathione tissue concentrations. Notably, ATP and energy charge ratios were greater in livers during SNMP compared to NMP. Given these findings, several targeted therapeutic interventions are proposed to mitigate IRI during liver machine perfusion and optimize marginal liver grafts during SNMP and NMP.

Keywords: kynurenine; tryptophan; histamine; glutathione; machine perfusion; liver transplant; metabolomics; normothermic; subnormothermic; ex situ perfusion

1. Introduction Liver transplantation (LT) remains the only deﬁnitive cure for end-stage liver diseases. However, the number of patients waiting for LT continues to increase due to the shortage of organs while waitlist

J. Clin. Med. 2020, 9, 1864; doi:10.3390/jcm9061864 www.mdpi.com/journal/jcm J. Clin. Med. 2020, 9, 1864 2 of 14 mortality remains high [1]. One solution to remedy the severe organ shortage is to improve the utilization of extended-criteria donor (ECD) livers. The current limitation to the widespread use of ECD livers is the increased severity of post-transplant ischemia-reperfusion injury (IRI), especially as a result of warm ischemia during donation after circulatory death (DCD). As a result, many ECD livers are discarded due to concerns for early allograft dysfunction or primary nonfunction. Machine perfusion (MP) devices have emerged as a promising platform for both viability assessment and delivery of novel therapeutics aimed at rehabilitating suboptimal grafts for transplantation [2]. Despite the advent of machine perfusion, the debate continues regarding the best practices. Currently, most transplant centers use either normothermic machine perfusion (NMP) at 37 ◦C[3] or hypothermic oxygenated perfusion (HOPE) at 2–10 ◦C[4,5]. Controlled oxygenated rewarming (COR) after static cold storage has also demonstrated improved graft function in animal and human trials [6–8]. Subnormothermic machine perfusion (SNMP) at 20–25 ◦C[4,9], however, remains largely experimental. Many physicians envision a future where MP is used to improve the function of each donor liver to achieve maximal graft utilization and minimize discard rates [10,11], and one potential method to improve graft function is targeting metabolism. While numerous studies have demonstrated potential metabolic targets in the liver, renal, and intestinal IRI response [12–16], few studies have examined the metabolic changes that occur in the human liver during transplantation [12,17–20] and fewer still during ex situ machine perfusion [4,21]. This is a critical area of research since the machine perfusion platform is the only setting for liver-specific adjunct therapies prior to transplantation. To address this knowledge gap, we sought to assess the metabolomic profiles of discarded human livers without significant steatosis (<20% macrosteatosis) during SNMP and NMP. We hypothesized was that metabolic changes taking place in the liver driven by different perfusion modalities would identify potentially harmful and beneficial processes, which can be used to optimize future perfusion outcomes by mitigating IRI. These insights provide valuable knowledge that can be used to generate new hypotheses and design therapeutic interventions to salvage or rehabilitate discarded or ECD livers for transplantation.

2. Materials and Methods

2.1. Donor Livers Seven human donor livers with <20% macrosteatosis that were declined for transplant were obtained through New England Donor Services (NEDS). Livers with evidence of cirrhosis, signiﬁcant steatosis, or major trauma were excluded. Table1 describes the reason each organ was declined for transplantation. The Massachusetts General Hospital Institutional Review Board (IRB) and NEDS approved this study (No. 2011P001496). No organs were procured from prisoners and no vulnerable populations were included in this study.

Table 1. Reasons for the discarding of donor livers.

Group Liver # Reason for Discard 1 No appropriate recipient, maximum cold ischemic time exceeded NMP 2 DCD with prolonged WIT 3 DCD with prolonged WIT, history of alcohol abuse 1 DCD with prolonged WIT 2 DCD in donor >50 years of age SNMP 3 DCD with prolonged WIT 4 DCD with prolonged WIT Abbreviations: NMP, normothermic machine perfusion; SNMP, subnormothermic machine perfusion; DCD, donation after circulatory death; WIT, warm ischemic time. J. Clin. Med. 2020, 9, 1864 3 of 14

2.2. Procurement of Grafts Procurement techniques based on donation after brain death (DBD) and donation after cardiac death (DCD) followed standard methods [9]. Donor livers were flushed in situ with cold University of Wisconsin (UW) preservation solution. Warm ischemic time (WIT) was defined as the period between extubation and in situ cold flush. Cold ischemic time (CIT) was defined as the period between in situ cold flush and initiation of machine perfusion. Livers arrived in the laboratory under static cold storage and underwent back table preparation according to previously described methods [22].

2.3. Machine Perfusion Livers underwent three hours of either NMP or SNMP using the Liver Assist device (Organ Assist, Groningen, Netherlands). Previous studies have shown the liver reaches a steady-state within this time frame with regards to perfusion dynamics, biomarkers, and energy capacity [9,23]. Clinically, viability assessment is also performed within this time frame [24]. The temperatures of the NMP and SNMP circuits were maintained between 35–37 ◦C and 20–22 ◦C, respectively. Perfusate composition has been previously described [21] but is notably diﬀerent between the groups given the addition of a hemoglobin-based oxygen carrier, HBOC-201 (HbO2 Therapeutics LLC, Souderton, PA, USA) in the NMP group. Detailed perfusate compositions are provided in the Supplementary Materials (Table S1). SNMP livers were perfused at a mean hepatic arterial pressure (HAP) of 30–60 mmHg and a portal venous pressure (PVP) of 3–7 mmHg. NMP livers were perfused at a HAP of 60–70 mmHg and a PVP of 6–8 mmHg. Detailed methods for sample collection have been previously described [21].

2.4. Energy Cofactor Analysis Wedge liver biopsies collected hourly were frozen, pulverized in liquid nitrogen, and analyzed for metabolic cofactors. The targeted multiple reaction monitoring analysis was performed at the principle research institution on a Sciex TripleTOF 6600 Quadruple Time-of-Flight system. Metabolite extraction was conducted according to a previously described protocol [25]. Concentrations of hepatic adenosine mono-, di-, and triphosphate (AMP, ADP, and ATP) were quantiﬁed. Energy charge was calculated as: [ATP + ADP*0.5] / [ATP + ADP + AMP].

2.5. Untargeted Metabolomic Analysis Liver biopsies were further analyzed by Metabolon, Inc. (Durham, NC, USA) for 1600 known compounds. Analyses were performed using ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). Detailed methods and statistical approaches have been previously described [21].

2.6. Statistical Analysis Demographic and perfusion data are presented as the median interquartile range (IQR), ± unless otherwise specified, with statistical significance defined as p < 0.05. Wilcoxon’s rank-sum (Mann–Whitney U) test and Fischer’s exact test were used for continuous and categorical comparisons, respectively. A random intercept mixed model with a categorical effect of time was used to analyze repeated measures data. Stata 15.1 (StataCorp, College Station, TX, USA) was used to perform statistical analyses and Prism 8 (GraphPad, San Diego, CA, USA) was used to create graphics. Statistical analysis for untargeted metabolomic profiling has been previously described [21]. Fold changes of metabolites are presented as the mean with statistical significance defined as p 0.05. ≤ J. Clin. Med. 2020, 9, 1864 4 of 14

3. Results

3.1. PerfusionJ. Clin. Med. and 2020 Functional, 9, x FOR PEER Parameters REVIEW 4 of 15 Baseline characteristics of the NMP and SNMP livers are provided in Table2. Arterial and 3. Results portal flow rates were comparable between the two groups (Figure1a,b). NMP livers demonstrated significantly3.1. Perfusion higher arterial and Functional resistance Parameters after 60 min of perfusion compared to the SNMP group (Figure1c). Portal resistanceBaseline tended characteristics to be higher of the in NMP the NMPand SNMP group, livers but are only provided reached in Table significance 2. Arterial afterand portal 180 min of perfusionflow (Figure rates1 d).were comparable between the two groups (Figure 1a,b). NMP livers demonstrated significantly higher arterial resistance after 60 min of perfusion compared to the SNMP group (Figure 1c). Portal resistanceTable 2. Donortended demographicsto be higher in ofthe SNMP NMP group, and NMP but non-steatoticonly reached significance livers. after 180 min of perfusion (Figure 1d). NMP (n = 3) SNMP (n = 4) p Value Table 2. Donor demographics of SNMP and NMP non-steatotic livers. Age (years) 44 (28–60) 49 (33.5–52) 0.64 Gender (male) 2NMP (67%) (n = 3) SNMP 4 (100%) (n = 4) p Value 0.43 Age (years) 44 (28–60) 49 (33.5–52) 0.64 2 BMIGender (kg/m (male)) 24.7 (16.9–32.5) 2 (67%) 28.2 4 (26.1–32.1) (100%) 0.43 0.64 DCD RecoveryBMI (kg/m2) 24.7 2 (67%) (16.9–32.5) 28.2 4 (26.1–32.1) (100%) 0.64 0.43 WITDCD (min) Recovery 34 (33–35) 2 (67%) 30 4 (20–33)(100%) 0.43 0.14 WIT (min) 34 (33–35) 30 (20–33) 0.14 CIT (min)CIT (min) 690 690 (360–930) (360–930) 754 754 (685.5–861)(685.5–861) 0.48 0.48 LiverLiver weight weight (g) (g)1350 1350 (1300–2200) (1300–2200) 2139 2139 (1646–2614)(1646–2614) 0.28 0.28 ContinuousContinuous variables presentedvariables aspresented median withas median interquartile with ranges. interquartile Abbreviations: ranges. NMP,Abbreviations: normothermic NMP, machine perfusion;normothermic SNMP, subnormothermic machine perfusion; machine SNMP, perfusion; subnormothermic BMI, body mass machine index; DCD,perfusion; donation BMI, afterbody cardiac mass death; WIT, warmindex; ischemic DCD, time; donation CIT, coldafter ischemiccardiac death; time. WIT, warm ischemic time; CIT, cold ischemic time.

a. Arterial Flow Rate b. Portal Flow Rate

400 2500

2000 300 1500 200

mL/hr mL/hr 1000 SNMP 100 NMP 500

0 0 30 60 90 120 150 180 30 60 90 120 150 180 Time (min) Time (min)

c. Arterial Resistance d. Portal Resistance

0.6 0.010 * -1 -1

mL 0.4 mL 1 ***** 1 0.005 min min 1 1 0.2 mmHg mmHg

0.0 0.000 30 60 90 120 150 180 30 60 90 120 150 180 Time (min) Time (min)

Figure 1. Hemodynamics of discarded human non-steatotic livers during SNMP vs. NMP. (a) Arterial and (b) portalFigure flows1. Hemodynamics were similar of discarded between human groups non-steatoti duringc threelivers during hours SNMP of perfusion. vs. NMP. (a) Both Arterial NMP and and (b) portal flows were similar between groups during three hours of perfusion. Both NMP and SNMP livers demonstrate a steady increase in arterial flow. (c) Arterial resistance was significantly SNMP livers demonstrate a steady increase in arterial flow. (c) Arterial resistance was significantly higher inhigher the NMP in the groupNMP group after after the the initiation initiation of of perfusion.perfusion. (d) (d Portal) Portal resistance resistance tended tended to be higher to be in higher in the NMPthe NMP group group but but only only reached reached significancesignificance after after 180 180 min min of perfusion. of perfusion. * indicates * indicates p < 0.05 forp < 0.05 for comparisonscomparisons using using Wilcoxon’s Wilcoxon’s rank-sum rank-sum test.test. Abbreviations: SNMP, SNMP, subnormothermic subnormothermic machine machine perfusion;perfusion; NMP, normothermic NMP, normothermic machine machine perfusion. perfusion.

Perfusate glucose levels were comparable between the two groups but varied widely in the NMP group (Figure2a). Venous lactate levels were comparable between the groups at the initiation of J. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 15

J. Clin. Med. 2020, 9, 1864 5 of 14 Perfusate glucose levels were comparable between the two groups but varied widely in the NMP group (Figure 2a). Venous lactate levels were comparable between the groups at the initiation of perfusion but were cleared at a signiﬁcantly faster rate in the NMP group, as expected (Figure2b). perfusion but were cleared at a significantly faster rate in the NMP group, as expected (Figure 2b). ALT levelsALT were levels comparable were comparable between between the groups the groups at 60 at and 60 and 180 180 min min of perfusion,of perfusion, though though within-groupwithin- variancegroup was largevariance (Figure was large2c). Bile(Figure was 2c). produced Bile was produced in consistent in consistent volumes volumes during during each each hour hour of of SNMP comparedSNMP to increasing compared to volumes increasing during volumes NMP during (Figure NMP2 (Figured). 2d).

a. Venous Glucose b. Venous Lactate

1000 SNMP 10 NMP 800 * * 600 * 5

mg/dL 400 mg/dL

200

0 0 Pre 30 60 90 120 150 180 30 60 90 120 150 180 Time (min) Time (min)

c. ALT d. Bile Production 4000 SNMP 10 NMP 8 3000 6 U/L 2000

mL/hr 4

1000 2

0 0 60 180 60 180 60 120 180 60 120 180 Time (min) Time (min)

Figure 2. Biomarkers of liver function during SNMP vs. NMP. (a) Venous (perfusate) glucose levels were similarFigure between 2. Biomarkers the two of liver groups function throughout during SNMP perfusion, vs. NMP. though (a) Venous variance (perfusate) was greater glucose inlevels the NMP group. (wereb) Venous similar between lactate wasthe two rapidly groups clearedthroughout during perfusion, NMP though compared variance to was a slowergreater in decline the NMP during group. (b) Venous lactate was rapidly cleared during NMP compared to a slower decline during SNMP. (c) Alanine aminotransferase (ALT) levels were similar between groups at 60 and 180 min of SNMP. (c) Alanine aminotransferase (ALT) levels were similar between groups at 60 and 180 min of perfusion.perfusion. (d) Bile (d) production Bile production increased increased at at each each hour hour ofof NMPNMP but but was was statistically statistically similar similar between between groups. *groups. indicates * indicatesp < 0.05 p < for0.05 Wilcoxon’s for Wilcoxon’s rank-sum rank-sum test. test. Abbreviations: SNMP SNMP,, subnormothermic subnormothermic machinemachine perfusion; perfusion; NMP, NMP, normothermic normothermic machine machine perfusion. perfusion.

3.2. Greater3.2. ATPGreater Conservation ATP Conservation and Energyand Energy Charge Charge Ratios Ratios during during SNMP SNMP ComparedCompared to pre-perfusion to pre-perfusion levels, levels, ATP:ADP ATP:ADP ratiosratios increased at ateach each hour hour of SNMP of SNMP but only but only reached signiﬁcancereached significance at 60 min at 60 (Figure min (Figure3a). ATP:ADP 3a). ATP:ADP ratios ratios increased increased signiﬁcantly significantly at at each each hourhour of NMP, NMP, though absolute ratio values were smaller compared to the SNMP group. A similar trend was though absoluteseen with ratioATP:AMP values ratio were (Figure smaller 3b) and compared energy charge to the(Figure SNMP 3c). group. A similar trend was seen with ATP:AMPJ. Clin. Med. 2020 ratio, 9, x FOR (Figure PEER 3REVIEWb) and energy charge (Figure3c). 6 of 15

a. ATP:ADP Ratio b. ATP:AMP Ratio c. Energy Charge 1.0 * 1.0 0.6 SNMP * * 0.8 0.8 NMP 0.4 0.6 0.6 * 0.4 0.4 * 0.2 0.2 ** 0.2 *

0.0 0.0 0.0 Pre 60 120 180 Pre 60 120 180 Pre 60 120 180 Pre 60 120 180 Pre 60 120 180 Pre 60 120 180 Time (min) Time (min) Time (min)

Figure 3. Energy cofactor changes during SNMP vs. NMP. (a) ATP:ADP, (b) ATP:AMP, and (c) energy Figure 3. Energy cofactor changes during SNMP vs. NMP. (a) ATP:ADP, (b) ATP:AMP, and (c) energy chargecharge ratios ratios were were quantitatively quantitatively much much higher higher inin thethe SNMPSNMP group group after after initiation initiation of perfusion, of perfusion, but but with large variance.with large variance. Livers undergoing Livers undergoing NMP NMP demonstrate demonstrate a consistent a consistent increase increase inin energyenergy cofactor, cofactor, though absolutethough values absolute were values lower were compared lower compared to SNMP to indicating SNMP indicating the more the activemore active metabolic metabolic state state at physiologicat temperatures.physiologic * indicatestemperatures.p < 0.05* indicates for the p random< 0.05 for interceptthe random mixed intercep model.t mixed Abbreviations: model. Abbreviations: ATP,adenosine triphosphate;ATP, adenosine ADP, adenosine triphosphate; diphosphate; ADP, adenosine AMP, adenosinediphosphate; monophosphate; AMP, adenosine SNMP,monophosphate; subnormothermic machineSNMP, perfusion; subnormothermic NMP, normothermic machine perfusion; machine NMP, perfusion.normothermic machine perfusion. 3.3. Greater Tryptophan Metabolism in Livers during NMP The kynurenine pathway generates nicotinamide adenine dinucleotide (NAD+) from tryptophan, in addition to several other notable metabolites (Figure 4a) [26]. Tryptophan levels decreased significantly in SNMP livers (fold change range 0.55–0.65, p ≤ 0.05 for all) but showed no significant change in NMP livers (fold change range 0.80–1.02, p not significant) (Figure 4b). Kynurenine fold change ratios decreased non-significantly during SNMP but increased during NMP (fold change range 1.95–2.27, 0.05 < p < 0.10 at 60 and 120 min, p not significant at 180 min) (Figure 4c). Kynurenate fold change ratios increased significantly in SNMP livers (fold change range 1.85– 3.57, p ≤ 0.05 at 120 and 180 min) and to an even larger degree in NMP livers (fold change range 10.03– 17.16, p ≤ 0.05 for all) (Figure 4d). The fold change ratios of tryptophan metabolites largely increased during NMP compared to SNMP. The one exception to this pattern was indole-3-carboxylic acid, which decreased during NMP (Figure 4e). No significant changes were seen in NAD+ levels during NMP or SNMP (Supplementary Materials, Figure S1).

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3.3. Greater Tryptophan Metabolism in Livers during NMP The kynurenine pathway generates nicotinamide adenine dinucleotide (NAD+) from tryptophan, in addition to several other notable metabolites (Figure4a) [ 26]. Tryptophan levels decreased significantly in SNMP livers (fold change range 0.55–0.65, p 0.05 for all) but showed no significant ≤ change in NMP livers (fold change range 0.80–1.02, p not significant) (Figure4b). Kynurenine fold change ratios decreased non-significantly during SNMP but increased during NMP (fold change range 1.95–2.27, 0.05 < p < 0.10 at 60 and 120 min, p not significant at 180 min) (Figure4c). Kynurenate fold change ratios increased significantly in SNMP livers (fold change range 1.85–3.57, p 0.05 at 120 and ≤ 180 min) and to an even larger degree in NMP livers (fold change range 10.03–17.16, p 0.05 for ≤ all) (Figure4d). The fold change ratios of tryptophan metabolites largely increased during NMP compared to SNMP. The one exception to this pattern was indole-3-carboxylic acid, which decreased duringJ. NMPClin. Med. (Figure 2020, 9, x4 FORe). PEER No significantREVIEW changes were seen in NAD + levels during NMP7 of 15 or SNMP (Supplementary Materials, Figure S1).

a. Tryptophan Kynurenate Xanthurenic Acid PLP PLP N-formylkynurenine Kynurenine 3-Hydroxykynurenine PLP PLP Anthranilic acid 3-Hydroxyanthranilic Acid

2-Amino-3-carboxymuconic acid-6-semialdehyde

Quinolinate 2-Aminomuconic-6-semialdehyde b. Tryptophan 1.5 Picolinate Acetyl CoA SNMP NAD+ NMP 1.0 Tryptophan Metabolism ** ** ** e. 0.5 SNMP NMP Fold Change 5 0.0 tryptophan 0.65 0.56 0.55 1.01 1.02 0.80 :0 :0 :0 :0 0 0 0 60 2 80:0 60 2 80: 4 T 1 T 1 T T1 T T1 N-acetyltryptophan 0.58 0.42 0.41 2623.20 2315.00 2238.60 3 c. Kynurenine C-glycosyltryptophan 0.93 0.92 0.90 0.94 0.97 0.85 2.5 * 2 NMP * 2.0 tryptophan betaine 0.85 0.85 0.78 1.89 2.14 2.22 SNMP 1 1.5 kynurenine 0.55 0.54 0.53 2.05 2.27 1.95 1.0 0 Fold Change Fold 0.5 N-acetylkynurenine 1.00 1.00 1.00 11.22 6.52 6.85

0.0 kynurenate 1.85 2.87 3.57 10.03 12.64 17.16 :0 :0 0 :0 :0 0 0 0: 0 0 0:0 6 2 8 6 2 8 T 1 T 1 T1 T T1 T N-formylanthranilic acid 1.00 0.94 1.01 2.36 1.83 1.74 d. Kynurenate 20 picolinate 0.97 1.02 1.08 2.54 2.09 1.82 ** NMP 15 SNMP ** indolelactate 1.11 1.71 2.09 4.49 6.84 8.25 ** 10 indole-3-carboxylic acid 0.68 0.77 0.82 0.46 0.52 0.66

Fold Change Fold ** 5 ** 3-indoxyl sulfate 1.11 1.12 1.04 3.88 3.34 2.21 0 0 0 0 0 0 0 0: 0: 0: 0: :0 :0 :0 :0 :0 60: 2 8 60: 2 8 0 0 T 1 1 T 1 6 2 80:0 T T T T1 T60 T 1 1 T120 T180 T T Figure 4. Tryptophan metabolism via the kynurenine pathway during SNMP vs. NMP. (a) Tryptophan metabolismFigure via4. theTryptophan kynurenine metabolism pathway. via PLPthe iskynurenine a necessary pathway cofactor during for severalSNMP stepsvs. NMP. in the (a) pathway. Tryptophan metabolism via the kynurenine pathway. PLP is a necessary cofactor for several steps in (b) SNMP livers demonstrate a signiﬁcant decrease in liver tryptophan concentrations compared to the pathway. (b) SNMP livers demonstrate a significant decrease in liver tryptophan concentrations NMP livers.compared However, to NMP NMPlivers. liversHowever, demonstrate NMP livers ademonstrate signiﬁcant a increasesignificant in increase tissue (inc) tissue kynurenine (c) and (d) kynurenatekynurenine compared and (d) kynurenate to the SNMP compared group. to the (SNMPe) Heatmap group. (e) of Heatmap metabolites of metabolites involved involved in tryptophan metabolitesin tryptophan analyzed metabolites in the untargetedanalyzed in the metabolomic untargeted metabolomic analysis. analysis. ** indicates ** indicatesp 0.05 p ≤ 0.05 and and * indicates ≤ 0.05 < p* dioxygenase; tryptophan 2,3-dioxygenase; KAT, kynurenine KAT, kynurenine aminotransferase; KMO,aminotransferase; kynurenine 3-monooxygenase; KMO, kynurenine PLP,3-monooxygenase; pyridoxal 5-phosphate, PLP, pyridoxal NAD 5-phosphate,+, nicotinamide NAD+, adenine nicotinamide adenine nucleotide; SNMP, subnormothermic machine perfusion; NMP, normothermic nucleotide; SNMP, subnormothermic machine perfusion; NMP, normothermic machine perfusion; machine perfusion; x-axis represents fold change at 60, 120, and 180 min compared to pre-perfusion x-axis representsconcentrations. fold change at 60, 120, and 180 min compared to pre-perfusion concentrations.

BiologicallyBiologically active active vitamin vitamin B6 B6 exists exists asas six interconvertible interconvertible compounds: compounds: pyridoxine, pyridoxine, pyridoxine pyridoxine 5-phosphate,5-phosphate, pyridoxamine, pyridoxamine, pyridoxamine pyridoxamine 5-phosphate, 5-phosphate, pyridoxal,pyridoxal, andand pyridoxalpyridoxal 5-phosphate 5-phosphate (PLP). PLP is(PLP). a necessary PLP is a coenzyme necessary coenzyme for several for steps several of steps tryptophan of tryptophan metabolism metabolism via via the the kynurenine kynurenine pathway pathway and is synthesized from pyridoxal at the expense of one ATP molecule (Figure 5a) [27]. Pyridoxal levels increased significantly in both temperature modalities, while PLP increased significantly only during SNMP (fold change range 1.94–2.4, p ≤ 0.05 for all) (Figure 5b,c).

J. Clin. Med. 2020, 9, 1864 7 of 14

J.and Clin. is Med. synthesized 2020, 9, x FOR from PEER pyridoxal REVIEW at the expense of one ATP molecule (Figure5a) [ 27]. Pyridoxal8 of 15 levels increased signiﬁcantly in both temperature modalities, while PLP increased signiﬁcantly only J. Clin. Med. 2020, 9, x FOR PEER REVIEW Pyridoxal kinase 8 of 15 during SNMP (fold changea. Pyridoxal range + ATP 1.94–2.4, p 0.05 for all)Pyridoxal (Figure 5-phosphate5b,c). + ADP Pyridoxal≤ phosphatase Pyridoxal kinase b. a. PyridoxalPyridoxal + ATP c. Pyridoxal Pyridoxal5-phosphate 5-phosphate + ADP 3 Pyridoxal phosphatase 3 ** ** SNMP ** b. Pyridoxal c. Pyridoxal 5-phosphate ** ** ** NMP ** 3 3 ** 2 ** 2 ** ** SNMP ** ** ** ** NMP ** ** 2 ** 2 1 1 Fold Change Fold Change Fold

1 1 Fold Change Fold Change Fold 0 0 :0 :0 0 0 :0 :0 0 0 0:0 0:0 0: 0:0 0 6 2 8 2 8 20: 80 2 T 0 1 1 T60:0 0 T60:0 T6 180:0 T T T1 T1 T1 T1 T1 T :0 :0 0 0 :0 :0 0 0 0:0 0:0 0: 0:0 0 6 2 8 2 8 20: 80 2 T 1 1 T60:0 T60:0 T6 180:0 T T T1 T1 T1 T1 T1 T Figure 5. Vitamin B6 metabolism during SNMP vs. NMP. (a) Pyridoxal 5-phosphate (PLP) is Figure 5. Vitamin B6 metabolism during SNMP vs. NMP. (a) Pyridoxal 5-phosphate (PLP) is generated generatedFigure from5. Vitamin pyridoxal B6 metabolism in a reversible during reactionSNMP vs. requiring NMP. (a) ATP.Pyridoxal SNMP 5-phosphate livers demonstrate (PLP) is a fromgenerated pyridoxal from in a reversiblepyridoxal reactionin a reversible requiring reaction ATP. requiring SNMP livers ATP. demonstrate SNMP livers a signiﬁcantdemonstrate increase a significant increase in both (b) pyridoxal and (c) PLP levels during perfusion, whereas NMP livers in bothsignificant (b) pyridoxal increase and in both (c) PLP(b) pyridoxal levels during and (c) perfusion, PLP levels whereasduring perfusion, NMP livers whereas only NMP demonstrate livers a only demonstrate a significant increase in pyridoxal. ** indicates p ≤ 0.05 and * indicates 0.05 < p < 0.10. signiﬁcantonly demonstrate increase in a significant pyridoxal. increase ** indicates in pyridoxal.p 0.05 ** indicates and * indicates p ≤ 0.05 and 0.05 * indicates< p < 0.10. 0.05 Abbreviations: < p < 0.10. ≤ Abbreviations:ATP,Abbreviations: adenosine ATP, triphosphate; ATP, adenosine adenosine ADP, triphosphate; triphosphate; adenosine ADP, diphosphate; adenosineadenosine PLP, diphosphate; diphosphate; pyridoxal PLP, 5-phosphate; PLP, pyridoxal pyridoxal 5- SNMP, 5- phosphate;subnormothermicphosphate; SNMP, SNMP, machinesubnormothermic subnormothermic perfusion; machine NMP,machine normothermic perfusion;ion; NMP, NMP, machine normothermic normothermic perfusion; machine machine x-axis perfusion; represents perfusion; x- fold x- axischange representsaxis at represents 60, 120, fold andfold change 180change min at at60, compared 60, 120, 120, and and to 18 18 pre-perfusion00 minmin compared concentrations. to to pre-pe pre-perfusionrfusion concentrations. concentrations.

3.4. Greater Greater3.4. Greater Histamine Histamine Histamine Reduction Reduction Reduction in in Livers Livers during during SNMP Histamine,Histamine, formed formed formed from from from its its amino amino acidacid precurso precursorprecursorr histidine,histidine, is is a pro-inflammatorya pro-inflammatorypro-inflammatory mediator mediator involvedinvolved in in many in many diverse diverse inflammatory inflammatory inflammatory and andand immune immune responsesresponses [28]. [28]. [28 The]. The The liver liver liver is also is is also an important an important location of excess histamine clearance [29]. No significant changes were seen in histidine in livers location of excess histamine cleara clearancence [29]. [29]. No No significant significant changes changes were were seen seen in histidine in livers undergoing NMP or SNMP (Figure 6a). However, histamine levels decreased in both groups, though undergoing NMP or SNMP (Figure 66a).a). However, histaminehistamine levelslevels decreaseddecreased inin bothboth groups,groups, thoughthough to a much larger degree during SNMP (fold change range 0.12–0.24, p ≤ 0.05 for all) (Figure 6b). to a much larger degree during SNMP (fold change range 0.12–0.24, pp ≤ 0.050.05 for for all) all) (Figure (Figure 66b).b). Similarly, most histidine metabolites demonstrated an overall decrease during≤ SNMP compared to Similarly,NMP (Figure most histidine 6c). metabolites demonstrated an overall decrease during SNMP compared to NMP (Figure 66c).c). a. Histidine c. Histidine Metabolism SNMP 1.5 SNMP NMP a. Histidine NMP Histidine Metabolism c. 5 SNMP histidine 0.92 1.08 1.07 1.12 1.17 1.03 1.5 SNMP NMP 1.0 NMP 4 1-methylhistidine 0.76 0.68 0.65 0.97 1.49 1.24 5 histidine 0.92 1.08 1.07 1.12 1.17 1.03 3 1.0 0.5 3-methylhistidine 0.50 0.43 0.45 1.45 1.54 1.48 4 Fold Change 1-methylhistidine 0.76 0.68 0.65 0.97 1.49 1.24 N-acetylhistidine 0.85 1.12 1.18 1.18 1.85 2.29 2 3 0.0 3-methylhistidine 0.50 0.43 0.45 1.45 1.54 1.48 0.5 0.77 0.70 0.60 1.02 0.98 1.00 Fold Change N-acetyl-1-methylhistidine 1 :0 :0 0 0:0 0:0 6 8 2 T T60:0 180 2 T120:0 T1 T1 T N-acetylhistidine 0.85 1.12 1.18 1.18 1.85 2.29 hydantoin-5-propionic acid 0.63 0.62 0.73 0.75 1.30 1.71 0 0.0 N-acetyl-1-methylhistidineimidazole propionate 0.790.77 0.860.70 1.01 0.60 1.07 1.021.58 0.981.76 1.00 1 :0 :0 0 0:0 0:0 6 8 2 T T60:0 180 b. T120:0 T1 HistamineT1 T hydantoin-5-propionicformiminoglutamate acid 0.620.63 5.080.62 6.37 0.73 6.29 0.753.49 1.306.28 1.71 0

1.0 imidazoleimidazole propionate lactate 0.730.79 0.710.86 0.69 1.01 1.02 1.071.21 1.581.33 1.76

b. 0.8 Histamine carnosine 0.65 0.69 0.71 0.43 0.38 0.35 formiminoglutamate 0.62 5.08 6.37 6.29 3.49 6.28 * 0.6 histamine 0.24 0.12 0.23 0.60 0.78 0.73 1.0 imidazole lactate 0.73 0.71 0.69 1.02 1.21 1.33 0.4 1-methylhistamine 0.58 0.53 0.56 0.55 0.40 0.48

Fold Change ** ** 0.8 carnosine 0.65 0.69 0.71 0.43 0.38 0.35 0.2 ** * 1-methylimidazoleacetate 4.45 5.23 4.78 2.91 3.12 2.98 0.6 0.0 histamine 0.24 0.12 0.23 0.60 0.78 0.73 2.59 4.62 4.07 3.02 4.37 2.77 :0 :0 :0 :0 4-imidazoleacetate 0 0 0 0 6 0.4 T60:0 T T12 T18 T120:0 T18 1-methylhistamine 0.58 0.53 0.56 0.55 0.40 0.48

Fold Change ** ** :0 :0 :0 0 0:0 0:0 0:0 2 8 2 0.2 ** T60 T60 18 1-methylimidazoleacetate 4.45T1 5.23T1 4.78 2.91T1 T3.12 2.98 0.0 2.59 4.62 4.07 3.02 4.37 2.77 :0 :0 :0 :0 4-imidazoleacetate 0 0 0 0 6 T60:0 T T12 T18 T120:0 T18 :0 :0 :0 0 0:0 0:0 0:0 Figure 6. Histidine metabolism during SNMP vs. NMP. (a) Histidine2 8 concentrations2 are unchanged T60 T60 18 T1 T1 T1 T during SNMP and NMP, whereas (b) histamine tissue concentrations are significantly decreased Figureduring 6. Histidine SNMP. (c) metabolism Heatmap showing during SNMPconcentration vs. NMP. fold ( achanges) Histidine of histidine concentrations metabolites are during unchanged FigureduringSNMP 6. SNMP Histidine and and NMP. NMP, metabolism ** whereasindicates during( bp) ≤ histamine 0.05 SNMP and tissuevs.* indicates NMP. concentrations (a) 0.05 Histidine < p < are 0.10. concentrations signiﬁcantly Abbreviations: decreased are SNMP,unchanged during duringSNMP.subnormothermic (SNMPc) Heatmap and NMP, showing machine whereas perfusion; concentration (b) NMP,histamine fold normotherm changes tissueic concentrations of machine histidine perfusion; metabolites are x-axis significantly represents during SNMPdecreased fold and NMP.change ** indicates at 60, 120,p and0.05 180 and min * compar indicatesed to 0.05 pre-perfusion< p < 0.10. concentrations. Abbreviations: SNMP, subnormothermic during SNMP. (c) Heatmap≤ showing concentration fold changes of histidine metabolites during SNMPmachine and perfusion; NMP. ** NMP, indicates normothermic p ≤ 0.05 machineand * indicates perfusion; 0.05 x-axis < p represents < 0.10. Abbreviations: fold change at SNMP, 60, 120, 3.5. Decreased Antioxidant Capacity in Livers during SNMP subnormothermicand 180 min compared machine to pre-perfusion perfusion; NMP, concentrations. normothermic machine perfusion; x-axis represents fold change at 60, 120, and 180 min compared to pre-perfusion concentrations.

3.5. Decreased Antioxidant Capacity in Livers during SNMP

J. Clin. Med. 2020, 9, x FOR PEER REVIEW 9 of 15

Four metabolites involved in antioxidant pathways were further examined: taurine, N- acetylcysteine (N-Ac), reduced glutathione (GSH), and oxidized glutathione (GSSG). Taurine tissue concentrations decreased significantly in SNMP livers (fold change range 0.69–0.77, p ≤ 0.05 for all) butJ. Clin. only Med. at2020 180, 9 min, 1864 in NMP livers (fold change 0.77, p ≤ 0.05) (Figure 7a). Tissue concentrations of8 of N- 14 Ac, an essential cysteine donor for glutathione synthesis [30], increased in both groups, although to a3.5. greater Decreased extent Antioxidant and with Capacitystatistical in significance Livers during in SNMP the NMP livers (fold change range 6.41–9.65, p ≤ 0.05 for all) compared to SNMP (fold change range 1.33–1.34, p not significant) (Figure 7b). GlutathioneFour metabolites is the involved most abundant in antioxidant thiol pathways antioxidant were in further mammalian examined: tissues taurine, and N-acetylcysteineis composed of the(N-Ac), amino reduced acids cysteine, glutathione glutamic (GSH), acid, and and oxidized glycine glutathione [31]. Both (GSSG).GSH and Taurine GSSG tissue concentrations decreased significantly in SNMP livers (fold change range 0.69–0.77, p 0.05 for all) but only at 180 decreased significantly in SNMP livers indicating catabolic activity (GSH≤ fold change range 0.04– min in NMP livers (fold change 0.77, p 0.05) (Figure7a). Tissue concentrations of N-Ac, an essential 0.21, p ≤ 0.05 for all; GSSG fold change≤ range 0.01–0.23, p ≤ 0.05 for all) (Figure 7c). NMP livers also demonstratedcysteine donor fordecreasing glutathione tissue synthesis concentrations [30], increased of GSH in both and groups, GSSG, although though to to a greatera lesser extent degree and with statistical significance in the NMP livers (fold change range 6.41–9.65, p 0.05 for all) compared compared to the SNMP livers. In addition, tissue concentration comparisons≤ to pre-perfusion levels into the SNMP NMP (fold group change did rangenot reach 1.33–1.34, significancep not significant)(Figure 7d). (Figure 7b).

a. Taurine SNMP b. N-acetylcysteine 1.0 NMP 10 **

0.8 ** ** ** ** ** ** 0.6 5 0.4 Fold Change Fold Change Fold 0.2

0.0 0 0 :0 :0 :0 :0 :0 :0 :0 :0 :0 0: 0 0 0 2 80:0 T6 1 T6 180:0 T60 120 T60 T T18 T120 T T T180 T120 T1

c. SNMP Glutathione d. NMP Glutathione 1.0 Reduced 1.0 Reduced Oxidized Oxidized 0.8 0.8

0.6 0.6

0.4 0.4 ** Fold Change Fold ** Change Fold 0.2 0.2 ** ** ** ** 0.0 0.0 :0 :0 :0 :0 0 0:0 20 80 60 20: 8 T60 T 1 T1 T1 T T1 Figure 7. Oxidative stress metabolites during SNMP vs. NMP. (a) Taurine levels demonstrate a Figure 7. Oxidative stress metabolites during SNMP vs. NMP. (a) Taurine levels demonstrate a small small but significant decrease during SNMP. (b) N-acetylcysteine levels are significantly increased but significant decrease during SNMP. (b) N-acetylcysteine levels are significantly increased during during NMP. However, oxidized and reduced glutathione tissue concentrations are decreased in livers NMP. However, oxidized and reduced glutathione tissue concentrations are decreased in livers undergoing (c) SNMP and (d) NMP. Glutathione levels are significantly depleted during SNMP, but do undergoingnot reach statistical (c) SNMP significance and (d) NMP. during Glutathione NMP. ** levels indicates are significantlyp 0.05 and depleted * indicates during 0.05 SNMP,< p < 0.10. but ≤ doAbbreviations: not reach statistical SNMP, subnormothermicsignificance during machine NMP. ** perfusion; indicates NMP, p ≤ 0.05 normothermic and * indicates machine 0.05 < perfusion; p < 0.10. Abbreviations:x-axis represents SNMP, fold change subnormothermic at 60, 120, and 180machin min comparede perfusion; to pre-perfusion NMP, normothermic concentrations. machine perfusion; x-axis represents fold change at 60, 120, and 180 min compared to pre-perfusion concentrations.Glutathione is the most abundant thiol antioxidant in mammalian tissues and is composed of the amino acids cysteine, glutamic acid, and glycine [31]. Both GSH and GSSG tissue concentrations 3.6.decreased Bile Acid significantly Metabolism in in SNMP Livers liversduring indicating SNMP and catabolic NMP activity (GSH fold change range 0.04–0.21, p 0.05 for all; GSSG fold change range 0.01–0.23, p 0.05 for all) (Figure7c). NMP livers also ≤ ≤ demonstratedCholesterol decreasing and choline tissue concentrations are essential ofsubstrates GSH and for GSSG, bile thoughacid synthesis. to a lesser Cholesterol degree compared levels remainedto the SNMP stable livers. during In addition, SNMP but tissue demonstrated concentration a small, comparisons significant to pre-perfusion increase during levels the in first the 2 NMP h of group did not reach significance (Figure7d). NMP. Choline levels increased significantly in both groups. With respect to primary bile acids, NMP livers3.6. Bile showed Acid Metabolism a significant in Liversincrease during in taurocholate SNMP and NMP(fold change range 1.61–1.95, 0.05 < p < 0.10 at 60 min, Cholesterolp ≤ 0.05 at 120 and andcholine 180 min) are and essential an increase substrates in glycocholate for bile sulfate, acid synthesis.though without Cholesterol significance. levels remained stable during SNMP but demonstrated a small, significant increase during the first 2 h of NMP. Choline levels increased significantly in both groups. With respect to primary bile acids, NMP livers showed a significant increase in taurocholate (fold change range 1.61–1.95, 0.05 < p < 0.10 at 60 min, p 0.05 at 120 and 180 min) and an increase in glycocholate sulfate, ≤ J. Clin. Med. 2020, 9, 1864 9 of 14 though without significance. SNMP livers demonstrated decreasing cholate levels that reached significance at 120 min and decreasing chenodeoxycholate levels that reached significance after 120 min (Supplementary Materials, Figure S2a–c).

4. Discussion The study hypothesis was that physiologic changes taking place in the liver induced by normothermic versus subnormothermic perfusion modalities would identify metabolic processes, which could be leveraged to optimize future perfusion outcomes by mitigating IRI. We found that livers undergoing NMP demonstrated significantly greater tryptophan metabolism via the kynurenine pathway, which has significant clinical implications and therapeutic value. Livers undergoing SNMP showed a greater reduction in histamine, suggesting decreased inflammatory signaling, and impaired glutathione generation. In addition, SNMP livers demonstrated increased ATP and energy charge ratios compared to NMP livers. In light of these findings, several therapeutic strategies were identified for adjunct delivery during machine perfusion studies to optimize grafts for transplant. One major finding of this study is the greater metabolism of tryptophan (TRP) via the kynurenine pathway (KP) in the NMP group with subsequently increased tissue concentrations of kynurenine (KYN) and kynurenate (KYNA). The difference in tryptophan metabolism during NMP compared to SNMP represents a clinically significant finding, as two studies found higher levels of KYN in transplanted livers that experienced primary nonfunction compared to livers with normal function [17,20]. Though KYNA levels were not reported, this highlights the therapeutic potential of this metabolic pathway. The activity of kynurenine aminotransferase (KAT), which converts KYN to KYNA [32], is temperature dependent and may explain why livers undergoing SNMP produced less KYNA. KAT exists as four isozymes (KAT I, II, III, and IV) in humans and rodents [32]. Both human and murine KAT II demonstrate maximum activity near 50 ◦C[33,34]. Decreased KAT activity may be a disadvantage of SNMP as KYN harbors pro-oxidant properties while KYNA has antioxidant properties [35]. Reassuringly, there was no significant accumulation of KYN during SNMP. Greater tryptophan metabolism during NMP is further significant as the liver oversees 90% of TRP catabolism via the KP, which generates both pro- and anti-inflammatory signaling metabolites [36,37]. Two rate-limiting enzymes catalyze the conversion of TRP to N-formylkynurenine in the first step of the KP: tryptophan 2, 3-dioxygenase (TDO) and indolamine-2,3-dioxygenase (IDO). Although IDO is mainly expressed in extrahepatic tissues, it is also found in Kupffer cells, the resident macrophages in the liver involved in IRI [38]. Interferon-gamma (INF-γ) induced expression of IDO in Kupffer cells has been shown to promote immune tolerance via T cell apoptosis [39]. Similarly, greater TDO activity may also offer a potential immunosuppressive advantage as TRP catabolism by TDO has been suggested to play a role in immune tolerance of allogeneic liver transplantation by inhibiting T cell proliferation [40]. Therefore, one can consider enhancing TDO activity in SNMP livers to reap these immunosuppressive benefits. There are numerous studied positive regulators of TDO including glucocorticoids, TRP, and heme [36]. Upregulated TDO activity via glucocorticoid administration as part of routine post-transplant immunosuppressive therapy has been speculated to play a role in liver graft tolerance [40]. TRP supplementation, however, has been shown to negate TDO-mediated inhibition of T cell proliferation and IFN-γ production [40]. In this study, a synthetic hemoglobin oxygen carrier was used during NMP but not SNMP. As a result, a greater availability of heme may have contributed to the increased tryptophan metabolism seen in livers undergoing NMP. Another potential enzyme target is kynurenine 3-monooxygenase (KMO), which catalyzes the conversion of KYN to 3-hydroxykynurenine (3-OHK), a pro-oxidant, neurotoxic metabolite that can induce endothelial cell apoptosis [35]. Compared to wild type, KMO knockout mice demonstrate decreased 3-OHK levels but increased KYN, KYNA, and anthranilic acid (AA) in the liver [41]. Furthermore, KMO inhibition has been shown to enhance KAT activity [42], which may be particularly beneficial during SNMP given lower functional activity of KAT at subnormothermic temperatures [34,35]. Shunting the metabolism of KYN from 3-OHK by KMO inhibition to KYNA may J. Clin. Med. 2020, 9, 1864 10 of 14 also be advantageous given the anti-inflammatory and immunosuppressive properties of KYNA [35]. Interestingly, there was no difference in NAD+ content between knockout and wild-type mice [41], suggesting NAD+ production is preserved in the setting of KMO inhibition. With respect to histidine metabolism, livers undergoing SNMP demonstrated significantly lower tissue histamine concentrations than NMP livers, suggesting a potential reduction in the histamine-mediated inflammatory response during SNMP. Notably, a clinical study of transplanted livers using metabolomics found higher tissue histidine concentrations in grafts that experienced early allograft dysfunction compared to those with immediate function [18]. In the liver, histamine has been associated with biliary injury and hepatic fibrosis [43–45]. Porcine portal veins during hepatic IRI have demonstrated up to fourfold elevations in histamine [46]. In rat hepatocytes subjected to IRI, histamine reduced cell growth, enhanced oxidative stress, and promoted apoptosis [47]. Thus, livers undergoing NMP may benefit from histamine antagonism to prevent histamine release through degranulation. In the aforementioned study involving rat hepatocytes, the histamine H2 receptor antagonist cimetidine reduced the oxidative stress, apoptosis, and poor cell growth associated with histamine [47]. Cimetidine has also been shown to inhibit P450 activity, reducing subsequent endogenous production of reactive oxygen species [48]. Intrahepatic mast cells reside in the portal tracts and sinusoids of normal livers, though in low numbers [49]. Increased mast cell populations have been observed in liver pathologies, including fibrosis, nonalcoholic fatty liver disease, chronic hepatitis C, and hepatocellular carcinoma [50,51]. Doxantrazole and sodium cromoglycate are mast cell stabilizers with coincidental radical scavenging abilities [52], although their effects on hepatic IRI are not known. Investigating such H2 antagonists and mast cell stabilizers on histamine-associated inflammation in NMP livers may be a worthwhile future pursuit. Finally, the higher ATP and lower glutathione concentrations seen during SNMP mirror the findings from a similar study of machine perfused steatotic livers [21]. While the higher energy charge ratios are attributed to the lower metabolic activity at subnormothermic temperatures, the depletion of glutathione is likely due to the inability to expend ATP needed to resynthesize glutathione after its breakdown. Similarly, several clinical studies have demonstrated a correlation between higher concentrations of glutathione metabolites and early allograft dysfunction [17,18]. Therefore, supplementation of the circulating perfusate during SNMP with exogenous glutathione could be another potential therapy to improve a DCD graft’s ability to tolerate oxidative stress during transplantation. There were several limitations to this study. First, sample sizes were small in the two perfusion groups. As this research relied on discarded human donor livers, there was limited availability of this precious resource and inherent variability in donor characteristics. To address the small sample size, multiple comparison corrections and false-discovery rates were taken into account for the untargeted metabolomic profiling. As a result, the analysis was able to demonstrate statistically and clinically significant differences between the two groups. In addition, the NMP group included one DBD liver whereas the SNMP group was comprised of only DCD livers. Future studies should examine DCD and DBD livers separately as they have been shown to exhibit different metabolomic profiles at the end of the static cold storage period [17]. To the best of our effort, donor demographics were otherwise similar. Livers with other skewing factors such as fibrosis or significant steatosis were excluded. Lastly, the decision to use a synthetic hemoglobin-based oxygen carrier instead of packed red blood cells (PRBC) was made in recognition of the possibility that PRBC may interfere with metabolic reactions and induce unwanted immunological responses. Compared to pooled PRBC gathered from several donors, HBOC-201 is also an immunologically inert oxygen carrier [53].

5. Conclusions Non-steatotic livers undergoing normothermic versus subnormothermic machine perfusion differ broadly in their metabolomic profiles especially in terms of amino acid metabolism. Targeted enzyme inhibition and metabolite supplementation during machine perfusion offer potentially therapeutic methods for optimization of these metabolic pathways to mitigate IRI, allowing for the expansion J. Clin. Med. 2020, 9, 1864 11 of 14 of the donor pool to include livers that would have otherwise been deemed unsuitable for human transplantation.

Supplementary Materials: The following are available online at http://www.mdpi.com/2077-0383/9/6/1864/s1, Table S1: Perfusate Compositions, Figure S1: NAD+ content in livers during SNMP vs. NMP, Figure S2: Bile acid composition during SNMP vs. NMP, Dataset S1: Complete untargeted metabolomic profile dataset. Author Contributions: Conceptualization, M.Y., K.U., and H.Y.; methodology, S.R., N.K., V.H., K.U., and H.Y.; software, A.Z., C.C., and S.R.; validation, S.R., K.U., and H.Y.; formal analysis, A.Z., C.C., S.R., N.K., V.H., S.N., and I.B.; investigation, A.Z., C.C., S.R., N.K., V.H., S.N., and I.B.; resources, R.J.P., M.Y., K.U., and H.Y.; data curation, A.Z., C.C., S.R., N.K., V.H., S.N., and I.B.; writing—original draft preparation, A.Z., C.C., and S.R.; writing—review and editing, S.R., R.J.P., M.Y., K.U., and H.Y.; visualization, A.Z., C.C., and S.R.; supervision, R.J.P., M.Y., K.U., and H.Y.; project administration, K.U.; funding acquisition, M.Y., K.U., and H.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the US National Institutes of Health (grants R01DK096075, R01DK107875) is gratefully acknowledged. Acknowledgments: We would like to acknowledge the Mass Spectrometry Core at Shriners Hospital for Children for processing our samples. The hemoglobin-based oxygen carrier, HBOC-201, was kindly provided by HbO2 Therapeutics. Finally, we would like to thank the donors, their families, and New England Donor Services (NEDS) for making this work possible. Conflicts of Interest: Uygun is the inventor of pending patents relevant to this study (WO/2011/002926; WO/2011/35223) and has a provisional patent application relevant to this study (MGH 22743). Uygun has a financial interest in Organ Solutions, a company focused on developing organ preservation technology. Uygun’s interests are managed by the MGH and Partners HealthCare in accordance with their conflict of interest policies.

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