Oxygenation during hypothermic rat liver preservation: An in vitro slice study to demonstrate beneficial or toxic oxygenation effects

Authors


Abstract

Hypothermic machine perfusion (HMP) of abdominal organs is shown to be superior compared to cold storage. However, the question remains if oxygenation is required during preservation as oxygen is essential for energy resynthesis but also generates toxic reactive oxygen species (ROS). To determine if oxygenation should be used during HMP, urea-synthesis rate, adenosine triphosphate (ATP), and generation of ROS were studied in an in vitro model, modeling ischemia-reperfusion injury. Furthermore, expression of uncoupling protein-2 (UCP-2) mRNA was assessed since UCP-2 is a potentially protective protein against ROS. Rat liver slices were preserved for 0, 24, and 48 hr in University of Wisconsin machine perfusion solution (UW-MP) with 0%, 21%, or 95% oxygen at 0-4°C and reperfused for 24 hours. In the 0% and 95% groups, an increase of ROS was found after cold storage in UW-MP. After slice reperfusion, only the 0% oxygen group showed higher levels. The 0% group showed a lower urea-synthesis rate as well as lower ATP levels. mRNA upregulation of UCP-2 was, in contrast to kidney mRNA studies, not observed. In conclusion, oxygenation of UW-MP gave better results. This study also shows that ROS formation occurs during hypothermic preservation and the liver is not protected by UCP-2. We conclude that saturation of UW-MP with 21% oxygen allows optimal preservation results. (Liver Transpl 2005;11:1403–1411.)

One of the key factors that remains crucial for early graft function and long-term outcome after transplantation is the efficacy of organ preservation. Eminent contributions to improve organ preservation were made by Belzer et al.,1 using hypothermic machine perfusion (HMP), and later by Collins et al.,2 who developed static cold storage to preserve donor kidneys.3 Nowadays, the question is raised as to whether the conventional cold-storage technique should be reverted to HMP preservation, to allow the inclusion of non-heart-beating, older, and marginal donors in the donor pool.3 In the past, a number of transplantation centers have reported the beneficial effects of HMP with kidneys.4, 5 Due to its success in increasing the kidney donor pool and prolonging cold-storage times, continuous perfusion preservation has regained interest by liver teams as well. The first successful experiments were already documented by the Wisconsin group in 1986, first by D'Alessandro et al.6 and later by Pienaar et al.7 Both managed to preserve and transplant good-quality canine livers after 72-hour HMP in a dog model and demonstrated that machine preservation of the liver is feasible.

Despite kidney preservation using HMP being used since the 1960s, successful application of HMP in the liver encounters several obstacles. One key question that has remained is whether oxygenation of the preservation solution is a prerequisite for effective preservation and maintenance of organ viability. Since the first use of experimental HMP of the liver, this aspect of oxygenation has been neglected and is often only briefly described as “one of the components of the HMP set-up.” Oxygenation of the preservation solution, however, does require substantial attention since oxygen is necessary for energy resynthesis, but then it could also result in an increase of toxic reactive oxygen species (ROS). Previously, Yamamoto et al.8 described the use of oxygenation during hypothermic continuous perfusion. More recently, Dutkowski et al.9, 10 suggested that oxygen should be included during preservation; however, oxygen saturation levels were not reported. This group showed that an increased partial oxygen pressure during preservation at 0-4°C resulted in a reduction of lipid peroxidation during reperfusion,11 in contrast to other reports that have found an increase in toxic ROS during cold oxygenated preservation for both kidney and liver.12–14 Unfortunately, it remains unclear if an increase in ROS is due to oxygen itself, to the dynamic character of HMP, or to both.

In respect to ROS generation during HMP, determination of uncoupling protein-2 (UCP-2) can play a role. It has been suggested that UCP-2 can protect kidneys from ischemia-reperfusion injury and formation of ROS15, 16 since UCP-2 is a protein facilitating a shift of hydrogen ions from the mitochondrial inner-membrane space toward the mitochondrial matrix. This dissipation of the proton gradient acts as a protective mechanism against an increase in ROS formation.17, 18 On the other hand, UCP-2 also reduces the proton motive force necessary to generate adenosine triphosphate (ATP) and prevents efficient ATP generation. It is known that fatty livers, regenerating livers, and livers exposed to endotoxins show an increase in UCP-2 levels, possibly as a protective mechanism against mitochondrial ROS formation.16, 19, 20 The question remains, nevertheless, whether UCP-2 is protective against ischemia-reperfusion injury of the liver.

The goal of this study was to determine the need for oxygenation during cold preservation. We have used an in vitro model to assess the optimal oxygen tension, without inducing ROS formation due to shear stress at endothelial cells, as it might occur during HMP.21 Also, the potentially protective protein UCP-2 is studied.

Abbreviations

HMP, hypothermic machine perfusion; ROS, reactive oxygen species; UCP-2, (mitochondrial) uncoupling protein-2; ATP = adenosine triphosphate; UW-MP, University of Wisconsin machine perfusion solution; WME, Williams medium E; AST, aspartate amino transferase; ALT, alanine amino transferase; LDH, lactate dehydrogenase; TBARS, thiobarbituric acid reactive substances; GSH, reduced glutathione; GSSG, oxidized glutathione; GSHt, total glutathione.

Materials and Methods

Eight adult male Wistar rats (250-350 g) were used in each group. All animals received care in compliance with the guidelines of the local Animal Care and Use Committee following National Institutes of Health guidelines.

Hepatectomy

First, inhalation anesthesia was induced with isoflurane and a gas mixture of nitrous oxygen (66%) and oxygen (33%). Next, through a transverse abdominal incision, the portal vein and infrahepatic lower caval vein were identified and cleared from surrounding tissue. One milliliter of 0.9% NaCl with 500 iE/mL of heparin was administered. Then, the infrahepatic caval vein was ligated, followed by a portal perfusion with cold University of Wisconsin machine perfusion solution (UW-MP) using a 10-mL syringe with a 6 FG cannula (Portex, Kent, UK). UW-MP was prepared using the original recipe for liver machine preservation.

Preparation of Liver Slices

The precision-cut liver slice model was used to allow the measurement of injury parameters and liver function at several time points in a single liver (Table 1). This model allowed a reduction in laboratory animals and still allows a comprehensive analysis of data.22, 23 After hepatectomy, livers were cold-stored in UW-MP until slices were prepared. Briefly, random cores of 8 mm diameter were made from the liver lobes; these cores were preserved in cold UW-MP prior to starting the slicing procedure, which was performed in cold modified University of Wisconsin solution (Table 1). Next, cores were cut into slices of approximately 300 μm thickness in a Brendel/Vitron tissue slicer (Vitron, Tucson, AZ).24, 25 Slices weighed approximately 12-15 mg, to allow diffusion of nutrients and oxygen through the entire slice.24

Table 1. Schematic View of Cold Preservation and Warm Incubation Time Periods of Rat Liver Slices
  1. Black bars, preservation for 24 or 48 hours; gray bars, 24-hour warm incubation; O, sampling of either UW-MP or WME and slices.

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Cold Preservation

The preservation period started at the moment the hepatectomy was completed and when livers were perfused with ice-cold UW-MP. The temperature was maintained at 0-4°C (Frigomix; Salm en Kipp BV, Breukelen, The Netherlands). The liver slices were divided in three groups: UW-MP desaturated with nitrogen (0% oxygen, group A), UW-MP saturated with ambient air (21% oxygen, group B), and UW-MP saturated with 95% oxygen (carbogen-gas, group C). Saturation of UW-MP was maintained throughout the experiment using the appropriate and continuously refreshing gaseous phase above the cold preservation solution. The normally used oxygen saturation of UW was 21%, 95% oxygen saturation was chosen to test if high oxygen levels could result in an increase in ROS formation and UCP-2 mRNA, and 0% was used as control. The (de)oxygenated solutions were corrected to a pH of 7.4. Slices were preserved for 0, 24, or 48 hours in 3.2 mL UW-MP supplemented with 3 mmol/L fresh glutathione.26 Sterile 6-well plates were used with one slice per well, placed in a reciprocating water bath (GFL 1083; Burgwedel, Germany).

Incubation

To simulate reperfusion, liver slices were incubated in oxygenated Williams medium E (WME) at 37°C for a period of 24 hours. Each slice was submerged in 3.2 mL WME in a sterile 6-well plate on a reciprocal shaker (GFL 3006) at a frequency of 90/min.27 The WME with L-Glutamax (32551-020; Invitrogen, Breda, The Netherlands) was supplemented with 25 mmol/L D-glucose (G 6138; Sigma, Zwijndrecht, The Netherlands), 50 μg/mL gentamicin (Invitrogen, 15750-037), and insulin 100 iE/L (Humuline; Lilly, Nieuwegein, The Netherlands) saturated with 95% O2:5% CO2 for the duration of the experiment.28, 29

Cellular Integrity

Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) (Mega; Merck, Amsterdam, The Netherlands) were determined in WME to assess the amount of cellular injury after cold preservation–warm incubation. Samples were collected after completing 24-hour warm incubation.

Cellular Energy

ATP levels were measured using the ATP Bioluminescence assay kit CLS II (Roche Diagnostics, Mannheim, Germany): liver slices were homogenized with a sonificator in 70% ethanol and 2 mmol/L ethylenediaminetetraacetic acid and stored at −80°C until measurement. Luciferase (50 μL) was added to 50 μL supernatant of diluted samples (100 mmol/L phosphate buffer, pH 7.6-8.0), followed by measurement in a luminescence apparatus (Lucy-1; Anthos Labtec Instruments, Salzburg, Germany). ATP levels were expressed per milligram protein. The Bio-Rad (Munich, Germany) protein microphotospectrometric assay was used at 620 nm.

Hepatocyte Function

Metabolic capacity requiring ATP and effective oxidative phosphorylation was determined by calculating the urea synthesis rate.30 After 22-hours warm incubation, NH4 + and L-ornithine were added to WME to final concentrations of 1 mmol/L and 0.1 mmol/L, respectively. Urea levels were measured at 22 and 24 hours to allow calculation of the urea synthesis rate over that period. The difference between urea at 22 and 24 hours warm incubation reflects the metabolic function of the liver. The urea concentration after 22-hour incubation is an indication of the degradation of proteins.

ROS Formation

Thiobarbituric acid-reactive substances (TBARS) were chosen to indicate an increase in lipid peroxidation after cold preservation and after warm incubation31 and measured in UW-MP and WME. Malondialdehyde binds to thiobarbituric acid. The formed TBARS were extracted in a butanol layer and measured with a fluorescence spectrophotometer at 530/590 nm (FL 600; Baun de Ronde, Abcoude, The Netherlands). An oxidant, 2.5 mmol/L tert-butyl hydroperoxide (all chemicals from Sigma), was used as a positive control. Hydrogen peroxide was measured in WME, using the Amplex Red assay (A-22188; Molecular Probes, Leiden, The Netherlands). 10-Acetyl-3,7-dihydroxyphenoxazine reacts with H2O2 in the presence of horseradish peroxidase and produces resorufin. Resorufin was measured using fluorescence spectrophotometry at 530/590 nm.

Intracellular Oxygen Radical Scavenger

The ratio between reduced glutathione and oxidized glutathione (GSH/GSSG ratio) was determined, using kinetic photospectrometry.32 The total GSH content, i.e., the total amount of reduced and oxidized glutathione (GSHt), was determined in UW-MP and WME, using oxidation of GSH to GSSG in the presence of 5,5-dithio-bis-nitrobenzoic acid at 30°C, generating 5-thio-bis-nitrobenzoic acid, which was measured at 405 nm (EL 808, Baun de Ronde). GSSG is subsequently reduced to GSH by glutathione reductase and reduced nicotinamide adenine dinucleotide phosphate, to allow, again, oxidation of GSH to GSSG. Samples were stored at −80°C with and without 2-venylpyridine, allowing the measurement of GSHt and GSSG (all chemicals from Sigma). The rate at which 5-thio-bis-nitrobenzoic acid is formed is a marker of the GSH and GSSG content of the solution. The experimental values were compared to levels determined in normal rat liver tissue harvested during the procurement procedure.

Reverse-Transcriptase Polymerase Chain Reaction on UCP-2

The reverse-transcriptase polymerase chain reaction technique was used to detect changes in UCP-2 mRNA levels after warm incubation. Total tissue RNA was isolated from one slice, using Trizol reagent (Invitrogen). RNA (1 μg) was used to form cDNA to the total amount of 20 μL (Invitrogen). cDNA (2 μL) was amplified using UCP-2 and β-actin primers: UCP-2 sense 5′-TAA AGG TCC GCT TCC AGG C-3′, UCP-2 antisense 5′-CGT CTT GAC CAC ATC AAC GG-3′, β-actin sense 5′-AAC ACC CCA GCC ATG TAC G-3′ and β-actin antisense 5′-ATG TCA CGC ACG ATT TCC C-3′ in 45 μL primer mix, also containing H2O (Promega, Leiden, The Netherlands), polymerase chain reaction buffer, dNTCPs, MgCl, and Taq polymerase (Invitrogen). Twenty-eight cycles were used with a hot start for 3 minutes at 94°C, followed by 94°C denaturation for 40 seconds, annealment at 59°C for 40 seconds, and elongation at 72°C for 40 seconds. The 301 bp product of UCP-2 and the 254-bp product of β-actin were separated with a 2% agarose (Hispanagar, Burgos, Spain) gel containing 0.01% ethidium bromide (Sigma). Imagemaster (Amersham Biosciences, Roosendaal, The Netherlands) was used to quantify the UCP-2 and β-actin bands. UCP-2 results were corrected with an internal control and divided by β-actin results, and for fresh nontreated slices, the UCP-2/β-actin ratio was considered to be 1.

Statistical Analysis

Each experiment was performed with eight livers using slices in triplicate. Results are means ± standard error of the mean. One-way analysis of variance was used for comparison within and between the three groups, with Bonferroni's correction for multiple comparisons. Pearson's correlation was used to compare markers. A P value <0.05 was considered to be statistically significant.

Results

Livers were harvested within 12 min after the abdominal incision. The initial wash-out with UW-MP macroscopically showed a complete distribution of the preservation solution. Livers were cold-stored in UW-MP on melting ice and kept cold at all times. The partial oxygen pressures reached using nitrogen saturation and carbogen-gas saturation were 8.9 ± 0.3 and 98.0 ± 1.2 kPa, respectively.

Cellular Integrity

Aminotransferases and LDH were measured in UW-MP. After 24-hour cold preservation, group A, with 0% oxygen saturation, showed higher concentrations, 123.9 ± 14.7 U/L for AST, 137.0 ± 18.4 U/L for ALT, and 1,590.0 ± 189.2 U/L for LDH compared to both groups B (47.1 ± 8.3, 52.8 ± 11.6, 600.5 ± 85.3, respectively) and C (44.3 ± 12.3, 35.6 ± 10.0, 474.0 ± 153.6, respectively). After 48-hour cold preservation, a similar pattern was observed with slightly higher concentrations: group A, 141.0 ± 7.4 U/L for AST, 151.9 ± 8.7 U/L for ALT, and 1,948.0 ± 75.3 U/L for LDH, showed higher concentrations compared to groups B (67.8 ± 14.6, 69.1 ± 15.5, 842.4 ± 160.3, respectively) and C (59.8 ± 15.5, 51.9 ± 13.6, 684.0 ± 210.0, respectively). A different pattern was found after warm incubation of the slices. No differences were found between the three experimental groups, which all showed an increasing trend after 48-hour cold preservation (Table 2). Furthermore, only group C (95% oxygen saturation) showed a statistically significant increase in ALT and LDH concentrations between 24- and 48-hour cold preservation and warm incubation (Table 2).

Table 2. Enzyme Release, Glutathione, and H2O2, Measured After 24-Hour Warm Incubation of Rat Liver Slices in Oxygenated WME
 Parameter0-hour preservation (mean ± SEM)24-hour preservation (mean ± SEM)48-hour preservation (mean ± SEM)
  • *

    P < 0.05 vs. group A,

  • #

    P < 0.05 vs 24 hours cold preservation.

Group A (0%)AST (U/L) 74.4 ± 23.184.5 ± 15.1
 ALT (U/L) 10.8 ± 2.310.8 ± 1.8
 LDH (U/L) 17.0 ± 4.446.5 ± 25.3
 GSHt 30.8 ± 11.82.7 ± 0.8
 H2O2 (μmol/L) 1.17 ± 0.211.44 ± 0.27
Group B (21%)AST (U/L)42.0 ± 7.958.5 ± 16.378.5 ± 14.2
 ALT (U/L)11.4 ± 1.29.1 ± 1.412.6 ± 1.0
 LDH (U/L)30.1 ± 11.016.8 ± 6.473.6 ± 31.7
 GSHt98.1 ± 3.874.1 ± 7.8* 51.0 ± 9.9*
 H2O2 (μmol/L)* 0.53 ± 0.060.79 ± 0.271.09 ± 0.20
Group C (95%)AST (U/L) 54.9 ± 13.470.9 ± 14.6
 ALT (U/L) 6.8 ± 1.113.1 ± 1.9#
 LDH (U/L) 9.1 ± 3.460.9 ± 21.5#
 GSHt 71.9 ± 10.7* 36.7 ± 7.6*
 H2O2 (μmol/L) 0.81 ± 0.201.13 ± 0.17
Normal tissueGSHt223.6 ± 25.2  
Positive controlGSHt0.5 ± 0.1  

Cellular Energy

ATP reflects the energy content of the liver slice immediately after warm incubation. Increasing the preservation time resulted in a decrease in ATP concentration. Group B (21%) showed a decrease of 39% in ATP after 24 hr cold preservation and a significant decrease of 60% after 48-hour cold preservation compared to liver slices reperfused without preservation. Due to the slicing procedure, a mean preservation time of 51 minutes was necessary for the control measurements. Comparison of groups A, B, and C regardless of preservation times showed a significantly higher (P < 0.01) ATP concentration for groups B and C compared to group A. ATP levels after 24-hour cold preservation and warm incubation were significantly higher for group C, 1.11 ± 0.28 pmol/μg protein, vs. groups A and B, 0.30 ± 0.14 and 1.03 ± 0.16 pmol/μg protein, respectively; normal fresh livers show a concentration of 4.9 ± 0.4 pmol/μg protein. ATP levels after 48-hour cold preservation did not reveal any significant differences (Fig. 1).

Figure 1.

ATP concentration (mean ± standard error of the mean) after cold preservation and warm incubation of rat liver slices.White bars, group A (0%); black bars, group B (21%); and gray bars, group C (95%). *P< 0.05 vs. groups B and C. The 0-hour preserved slice served as reference value to 24- and 48-hour preserved slices.

Hepatocyte Function

Hepatocyte function was determined by stimulating the hepatocytes to convert ammonia to urea. To define the rate of synthesis, a fixed time period of 2 hours was chosen. The level of urea was already indicative of hepatocyte function at 22 hours as it reflects protein catabolism. A significant increase of urea was found between groups A and B and between groups A and C. After 24 hr cold preservation and 24 hr warm incubation, the urea levels reached 0.22 ± 0.04 mmol/L in group A, 0.39 ± 0.02 mmol/L in group B, and 0.37 ± 0.04 mmol/L in group C. After 48-hour cold preservation and 24-hour warm incubation, these concentrations were 0.14 ± 0.08, 0.29 ± 0.03 and 0.35 ± 0.05 mmol/L, respectively. The urea synthesis rate showed a similar pattern between the three groups (Fig. 2). In addition, this functional parameter revealed a significant decrease when preservation time increased. Between livers preserved for 0 hours and those preserved for 24 hours, the urea synthesis rate slightly decreased during warm incubation from 1.64 ± 0.21 μmol/L/min to 1.42 ± 0.12 μmol/L/min. A significant decrease was found between 0 hours and 48 hours preserved slices as 48-hour cold preservation resulted in a urea synthesis rate of 1.13 ± 0.12 μmol/L/min during warm incubation.

Figure 2.

Urea synthesis rate (mean ± standard error of the mean) after cold preservation and warm incubation of rat liver slices. White bars, group A (0%); black bars, group B (21%); and gray bars, group C (95%). *P< 0.05 vs. groups B and C. The 0-hour preserved slices served as reference value to 24- and 48-hour preserved slices.

ROS formation

Measurement of TBARS and hydrogen peroxide allowed detection of ROS. The TBARS-positive control showed 6.04 ± 2.4 μm. TBARS levels in UW-MP were higher for groups A and C compared to group B (Fig. 3). TBARS were also measured after warm incubation. During warm incubation, another pattern was found, showing high TBARS levels in group A. Twenty-four–hour cold preservation followed by 24-hour warm incubation showed 3.26 ± 0.59 μmol/L TBARS in group A vs. 0.77 ± 0.15 in group B and 0.70 ± 0.27 in group C. The 48-hour preserved and 24-hour reperfused slices showed TBARS levels of 3.32 ± 0.33 in group A, 1.03 ± 0.25 in group B and 1.50 ± 0.54 in group C. The results with hydrogen peroxide showed a similar pattern (Table 2).

Figure 3.

TBARS (mean ± standard error of the mean) after cold preservation and warm incubation of rat liver slices. White bars, group A (0%); black bars, group B (21%); and gray bars, group C (95%). *P< 0.05 vs. groups B and C. The 0-hour preserved slices served as reference value to 24- and 48-hour preserved slices.

Intracellular Oxygen Radical Scavenger

The GSH/GSSG ratio demonstrates the capacity of the cell to overcome cellular injury due to oxygen radical formation. A decrease in the ratio was observed between normal hepatic tissue and preserved as well as reperfused tissue in group B. Normal levels (25.6 ± 4.9) significantly decreased to a ratio of 10.2 ± 2.4 at 0-hour cold preservation and 24-hour warm incubation. No changes in the GSH/GSSG ratio were observed when the preservation time increased to 24- and 48-hour (8.6 ± 1.6 and 4.9 ± 0.5, respectively). Neither were differences observed between the experimental groups. GSHt decreased after warm incubation in group B after 0, 24, and 48 hours cold preservation in comparison to nonpreserved normal tissue. GSHt levels were lower in group A compared to groups B and C (Table 2).

UCP-2 mRNA

The UCP-2 data showed a decrease of UCP-2 mRNA when the preservation time increased from 0 to 24 and 48 hours. No differences were found between the three groups. Results are expressed as the ratio UCP-2/β-actin. For 0-hour cold preservation, a ratio of 1 was chosen. Twenty-four–hour cold preservation resulted in 0.83 ± 0.24, 1.02 ± 0.34, and 0.92 ± 0.23 for groups A, B, and C, respectively. Forty-eight–hour cold preservation showed significantly lower levels of 0.62 ± 0.24, 0.55 ± 0.21, and 0.70 ± 0.20, respectively (Fig. 4). UCP-2 expression correlated with ATP content of the slices (P < 0.05).

Figure 4.

UCP-2/β-actin (mean ± standard error of the mean) after cold preservation and warm incubation of rat liver slices. White bars, group A (0%); black bars, group B (21%); and gray bars, group C (95%). *P< 0.05 vs. 0-hour cold preservation. Fresh nontreated liver slices served as reference value (i.e., a ratio of 1) to 0, 24, and 48 hour preserved and 24-hour reperfused slices.

Discussion

The necessity for oxygenation of the preservation solution with its potential beneficial as well as detrimental effects during continuous perfusion has never been thoroughly investigated for HMP of abdominal organs. Most groups have simply used some kind of oxygenation of the preservation solution without any explanation of why oxygen is required during hypothermic organ perfusion.8–10, 33, 34 Oxygenation during cold preservation, however, could be a double-edged sword: additional oxygen might support aerobic metabolism in the cold but is also potentially harmful since an increase in toxic ROS could occur as well.12, 13

To improve organ preservation, especially since nowadays more older, marginal, and even non-heart-beating donors are used, other methods of preservation have to be considered. In the first part of our study, we have shown that oxygenation during cold preservation is necessary to maintain cellular energy and function and prevent cellular injury. Due to the limited number of experiments, we used the one-way analysis of variance statistical model. It should be kept in mind that a more appropriate multilevel design is preferable; this model, however, requires significantly more experimental numbers and thus laboratory animals. In the past, it has been documented that dynamic preservation without additional oxygen prevents a significant loss of cellular ATP.35 We considered ATP content of the liver as an important parameter to assess the effects of oxygenation of the preservation solution and expected high levels in the 21% and 95% groups. Our results showed indeed that ATP content was significantly higher in the 21% and 95% oxygen saturation groups, which is beneficial for essential hepatic functions such as detoxification of ammonia to urea. The ATP-dependent urea synthesis rate showed a similar pattern as the ATP concentrations, with better results for 21% and 95% oxygenation. ATP metabolism and urea synthesis thus indicate the beneficial effects of oxygenation during hypothermic preservation. In addition, livers preserved without oxygen showed AST, ALT, and LDH levels that were significantly higher and probably related to a decreased energy state preventing homeostasis of the cell.

In the second part of our study, we focused on the toxic effects of ROS. The formation of ROS, such as superoxide anions, hydroxyl radicals, and hydrogen peroxide, has long been considered to contribute to cellular injury during the reperfusion phase but not during cold preservation.36, 37 Some reports suggest that oxygen radicals are formed during reperfusion as well as during cold preservation12, 13, 38 and justify its use during cold storage. Also, several reports showed beneficial effects of ROS scavengers in preservation solutions.3, 26, 39 The mechanism explaining effective ROS scavenging during cold preservation is probably the capacity of some UW components to chelate iron, although suppression of ROS does not necessarily result in an improvement of graft outcome during reperfusion.37 In our experiments, livers preserved in UW-MP saturated with 95% oxygen (group C) showed that indeed the expected ROS formation occurred during preservation using high partial oxygen pressures. Following preservation in anoxic conditions, lipid peroxidation and hydrogen peroxide production reached higher levels during warm incubation compared to livers preserved in UW-MP saturated with 21% or 95% oxygen. In parallel, we observed an increase in cellular injury with a concurrent inferior liver outcome after preservation in deoxygenated UW-MP compared to preservation in oxygenated UW-MP. Thus, an increase in oxygen tension during preservation does not per se imply an increase in harmful ROS formation during warm incubation. This fact is probably due to hepatocyte defense mechanisms including naturally occurring ROS scavengers such as GSH. The ratio of GSH to GSSG did not confirm this hypothesis as we were not able to demonstrate a decrease in the intracellular GSH/GSSG ratio. We did find, however, a decrease in GSHt, indicating that GSH was indeed oxidized to GSSG and subsequently diffused across cellular membranes to be missed by our intracellular measurements.40 GSH measurements in the cold preservation and warm incubation solutions were inaccurate due to high GSH levels already present in these solutions and not used in the analyses.

Impairment of liver function following preservation is in part attributed to impaired oxidative phosphorylation. In ischemia-reperfusion injury, a decrease in ATP synthesis can be explained by a reduced function of the mitochondrial reduced nicotinamide adenine dinucleotide–oxidoreductase complex (of the electron transport chain) but also by an increase in ROS.18, 41, 42 The first mechanism prevents optimal generation of an electrochemical gradient across the inner mitochondrial membrane, and since the driving force for oxidative phosphorylation decreases, ATP synthase will be impaired. The second mechanism is an increase in mitochondrial ROS that is injurious to mitochondrial function41 and may lead to an increase in UCP-2.43 Prevention of mitochondrial ROS formation is achieved by lowering the electrochemical gradient by uncoupling proteins. Up- or downregulation of UCP-2 is a mechanism of balance, can be protective when mildly increased, and can result in a decrease in mitochondrial ROS formation.44 Yoshida et al.16 demonstrated a 2.8-fold upregulation of UCP-2 mRNA after warm ischemia and reperfusion in kidneys. It remains unknown whether UCP-2 mRNA upregulation is followed by UCP-2 protein upregulation after warm or cold ischemia-reperfusion injury of the liver. Although UCP-2 is normally not expressed by hepatocytes, it can be induced in fatty livers, regenerating livers, and livers exposed to endotoxins.16, 19, 20 Also, in normal liver, nonparenchymal cells are known to express UCP-2. The question of whether UCP-2 is protective in ischemia-reperfusion of the liver remains. The uncoupling protein mRNA levels we found correlated with a decrease in ATP content; thus, the UCP-2 levels appear to reflect liver viability, as does the ATP content,45 and are probably not protective in cold ischemia-reperfusion injury of the liver and not beneficial in respect to HMP.

In summary, to improve hypothermic liver preservation, oxygenation of the preservation solution is mandatory. In this study, we have demonstrated better results with 21% and 95% oxygenation compared to preservation of the liver without oxygenation. In addition, we showed that toxic ROS are generated not only during warm incubation but also during cold preservation in UW-MP. Oxygen saturation with 21% allowed optimal results. The potentially protective mitochondrial protein UCP-2 was not upregulated after cold ischemia and warm incubation of the liver.

Acknowledgements

The authors thank Ing P.J. Ottens for the molecular analysis and R.E. Nap for his advise concerning the statistical analysis of the experimental data.

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