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How much Oxidative Stress Exists Without the Liver? Wieviel oxidativer Stress entsteht ohne die Leber?
Authors
C. Thiel1, T. Katt1, M. Schenk1, C. Grasshoff2, M. H. Morgalla3, A. Peter4, A. Königsrainer1, K. Thiel1
Affiliations
1
3 4
Department of General, Visceral and Transplant Surgery, University Hospital Tuebingen Department of Anesthesiology, University Hospital Tuebingen Department or Neurosurgery, University Hospital Tuebingen Division of Endocrinology, Diabetology, Angiology, Nephrology, Pathobiochemistry and Clinical Chemistry, Department of Internal Medicine, University Hospital Tuebingen
Schlüsselwörter
Zusammenfassung
Abstract
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Hintergrund: Oxidativer Stress (OS) stellt einen wichtigen pathogenetischen Faktor beim akuten Leberversagen und bei chronischen Lebererkrankungen dar. In der vorliegenden Studie wurde OS und der antioxidative Status bei einem anhepatischen Schweinemodell gemessen, um herauszufinden, ob die Leber selbst eine entscheidende Rolle bei der Entstehung von OS spielt. Methode: 6 Schweine wurden hepatektomiert, die Kontrollgruppe bestand aus 5 Schweinen. OS und antioxidativer Status wurden bestimmt, indem die Plasmakonzentrationen von Malondialdehyd (MDA), Xanthinoxidase (XO), Superoxiddismutase (SOD) und der eisenreduzierenden antioxidativen Kapazität (FRAP) zu Beginn des Experiments, postoperativ sowie 8 und 16 Stunden nach Hepatektomie gemessen wurden. Ergebnisse: Erhöhte MDA-Konzentrationen wurden bei den anhepatischen Tieren postoperativ (p < 0,02) und nach 8 Stunden (p < 0,003) im Vergleich zur Kontrolle beobachtet. Die XO-Aktivität nahm postoperativ zu (p < 0,03 vs. Kontrolle), kehrte jedoch im weiteren Verlauf auf Normalwerte zurück. Es fanden sich keine Veränderungen der SOD-Werte in beiden Gruppen. FRAPBestimmungen nahmen signifikant zu gegenüber der Kontrolle (p < 0,015). Es bestand eine positive Korrelation zwischen MDA- und FRAP-Werten (Spearman’s ρ = 0,62; p < 0,0001). Schlussfolgerung: Diese Ergebnisse zeigen, dass das Auftreten von OS durch eine Hepatektomie nicht verhindert werden kann, da sowohl die Produktion als auch die Regulation auch außerhalb der Leber stattfinden.
Background: Oxidative stress (OS) represents an important pathogenetic factor of acute liver failure and chronic liver diseases. To elucidate whether the liver itself is a major source of OS, the present study was performed to assess OS and antioxidant status in an anhepatic porcine model. Methods: Six pigs underwent a total hepatectomy, five pigs were sham operated. OS and antioxidant status were evaluated by measuring plasma concentrations of malondialdehyde (MDA), xanthine oxidase (XO), superoxide dismutase (SOD) and the ferric reducing ability of plasma (FRAP). They were sampled at the start of the experiment, immediately after surgery, and then at 8 and 16 hours post hepatectomy. Results: Increased concentrations of MDA were observed in anhepatic pigs postoperatively (p < 0.02) and 8 hours after hepatectomy (p < 0.003) compared to controls. XO activity increased soon after hepatectomy (22.6 ± 5.4 mU/L versus 3.3 ± 2.1 mU/ L in sham animals, p < 0.03) but returned to normal values in the further course. SOD levels did not change during the observational period in both groups. FRAP values rose significantly in the anhepatic animals compared to control (p < 0.015). A significant positive correlation was observed between MDA levels and FRAP levels (Spearman’s ρ = 0.62; p < 0.0001). Conclusions: These findings show that hepatectomy does not completely prevent the occurrence of OS because the production and regulation of OS are also located outside the liver.
Introduction
patocytes. The necrotic liver can cause cardiovascular shock, acute renal failure and respiratory failure which results in a life-threatening condition, described as “toxic liver syndrome” [1]. Total
● Leber ● akutes Leberversagen ● Hepatektomie ● oxidativer Stress " " "
Key words
● liver ● acute hepatic failure ● hepatectomy ● oxidative stress " " " "
received accepted
30.10.2013 12.12.2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1356362 Z Gastroenterol 2014; 52: 43–49 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0044-2771
Correspondence Dr. Karolin Thiel Department of General, Visceral and Transplant Surgery, University Hospital Tuebingen Hoppe-Seyler-Str. 3 72076 Tuebingen Germany Tel.: ++ 49/70 71/2 98 66 00 Fax: ++ 49/70 71/29 49 34 Karolin.Thiel@med. uni-tuebingen.de
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Acute liver failure (ALF) is characterized by a sudden and extensive necrosis and/or apoptosis of he-
Thiel C et al. How much Oxidative … Z Gastroenterol 2014; 52: 43–49
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hepatectomy of the toxic liver has been described as a successful bridge therapy before transplantation [2]. It is well-known that removal of the necrotic liver improves hemodynamics by eliminating the source of vasoactive agents and cytokines [3], but it remains unclear if a total hepatectomy may avoid the amount of OS. On the one hand necrotic hepatocytes are seen as a source of massive oxidative stress [4]. On the other hand it has been shown that mitochondrial oxidant stress precedes cell necrosis as identified by the permeability increase of the cell membrane and cell content release [5]. The origin and amount of OS remains undefined but may be an important target for new therapeutic approaches. Most previous studies investigating the effect of OS on liver injuries focus on chronic liver diseases [6 – 9], only a few studies examined the role of OS in the pathogenesis of acute liver failure [10 – 12]. Lipid peroxidation (LPO) was detectable in ALF and in all chronic liver diseases studied [6 – 8], suggesting that OS plays a crucial role in the pathogenesis [13]. Although OS has been shown to occur in every liver disease, the origin and clinical relevance of OS in particular in ALF remains unknown. Up to now it is unclear how much the liver itself contributes to oxidative stress in acute liver failure and if a total hepatectomy may eliminate the amount of OS. Furthermore it is not known how much the remaining organism accounts to the production of OS. Therefore the aim of the study was to evaluate the occurrence of oxidative stress and antioxidant levels in an anhepatic animal model. Since OS is an important pathogenetic factor of acute liver failure, the genesis of OS without any liver left is of main interest. In addition, any surgical trauma is known to cause OS. Our second aim was to investigate postoperative changes of redox parameters after a large operation in terms of a hepatectomy.
Materials and Methods !
Animal model The study was performed in 11 female domestic pigs weighing 37 ± 2 kg. After approval by the institutional review board for animal experiments, 6 pigs underwent total hepatectomy; a sham group consisted of 5 pigs. Animal care and all investigations were performed according to the international principles governing research on animals and under the supervision of a veterinarian, who set the guidelines for minimizing pigs suffering.
Premedication and anesthesia Intramuscular premedication consisted of atropine 0.1 % (0.05 mg/ kg), ketamine (14 mg/kg), azaperone (2 mg/kg) and midazolam (0.5 mg/kg). Body temperature was maintained at 38.5° C with a warming blanket. Preliminary procedures including intubation, ventilation and anesthesia during surgery have been described previously in detail [14]. In brief, continuous infusion of ketamine (15 mg/kg/h), fentanyl (0.02 mg/kg/h) and midazolam (0.9 mg/kg/h) was administered to maintain anesthesia during the experiment. Pigs were ventilated with a pressure controlled ventilation modus (Siemens KION SC 9000 XL anesthesia system; Siemens Medical Solutions Inc., Solna, Sweden). Arterial blood gas analysis (ABL 800, Radiometer Copenhagen, Denmark) was performed hourly and ventilation was adjusted accordingly.
Thiel C et al. How much Oxidative … Z Gastroenterol 2014; 52: 43–49
Surgical procedure Animals were kept under standard laboratory conditions. They fasted preoperatively and received an antibiotic prophylaxis of 2 g ceftriaxone (Rocephin®, Hoffmann-La Roche, Basel, Switzerland). Study animals and sham animals received the following catheters and probes: The jugular veins (Multi-Lumen Central Venous Catheter, Arrow International, Reading, PA, USA) and the internal carotid artery (Leadercath, Vygon, Écouen, France) were instrumented to measure central venous and arterial pressure. Following a parietofrontal cranial trepanation, a probe was inserted in the frontal brain parenchyma to measure intracranial pressure and brain temperature (Camino® MPM-1 monitor, Integra Neurosciences, Plainsboro, NJ, USA). The abdominal cavity was entered through a midline abdominal incision and a urinary catheter (Gentle-FloTM, Tyco Healthcare, Tullamore, Ireland) was placed. In 5 sham animals the abdominal wall was now closed with a running suture (Silkam®2, silk, braided, coated, nonabsorbable), the other 6 pigs from the study groups underwent total hepatectomy. The surgical procedure of the total hepatectomy using a Y-shaped bypass (Uni-Graft® K DV, Denkendorf, Germany) between the infrahepatic vena cava and the portal vein to the suprahepatic vena cava was performed as previously described [15]. After surgery animals remained under general anesthesia until death, receiving a standardized intensive care management as described recently [14]. During the experiment sodium chloride solution 0.9 %, hydroxyethylstarch 6 % (Voluven® HES 130/0.4, Fresenius, Bad Homburg, Germany) and Norepinephrine were infused, adjusted for mean arterial and central venous pressure. Continuously monitoring throughout surgery and the postoperative observation period included electrocardiogram, mean arterial pressure, central venous pressure, intracranial pressure, oxygen saturation and core body temperature. Urinary output, hemoglobin, hematocrit and lactate, serum electrolytes, acidbase balance, blood gases and blood glucose levels were monitored hourly and immediately corrected as required. Sham animals were euthanized at the end of the observation period with a single intravenous bolus of 10 mL T 61 (Intervet, Unterschleißheim, Germany).
Laboratory Analysis All blood samples were obtained from the internal carotid catheter preoperatively, postoperatively as well as 8 hours and 16 hours after hepatectomy. Sample analysis was conducted within 1 hour of collection at each time point and was performed by the certified central laboratories of the University Hospital Tuebingen (Division of Endocrinology, Diabetology, Angiology, Nephrology, Pathobiochemistry and Clinical Chemistry, Department of Internal Medicine, University Hospital Tuebingen, Germany). Albumin, total plasma protein and bilirubin concentrations were performed on the ADVIA 1800 Clinical Chemistry analyzer. Uric Acid analysis was performed using a commercial kid (Infinity, Uric Acid Liquid Stable Reagent, Thermo, Victoria, Australia).
Measurement of oxidative stress and antioxidant levels Arterial blood samples were immediately centrifuged at 3000 g for 10 min. Plasma samples were stored at – 80 °C until they were analyzed.
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man’s ρ-Test. Results and figures are reported as mean ± standard error of mean (SEM).
Analysis of malondialdehyde (MDA), xanthine oxidase (XO) and superoxide dismutase (SOD) MDA plasma concentrations were detected using the OxiSelect TBARS assay kit (Cell Biolabs, San Diego, USA). XO activity was quantified by using the Xanthine Oxidase Assay Kit (Cayman Chemical Company, Michigan, USA). SOD assaying was performed by utilizing the 19 160 SOD determination kit (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). All assays were performed according to the manufacturer's instructions.
Results Preoperative mean MDA concentrations did not differ significantly between both groups (10.6 ± 1.5 µmol/L in anhepatic ani" Fig. 1a). Directly mals vs. 8.7 ± 2.2 µmol/L in sham animals) (● after hepatectomy plasma concentration of MDA started to increase steadily (postoperatively: 11.6 ± 1 µmol/L in anhepatic animals vs. 8.3 ± 0.5 µmol/L in sham animals, p < 0.02; 8 hours after hepatectomy: 14.5 ± 0.8 µmol/L in anhepatic animals vs. 8.6 ± 1.3 µmol/L in sham animals, p < 0.003; 16 hours after hepatectomy: 20.1 ± 4.1 µmol/L in anhepatic animals vs. 9.6 ± 0.9 µmol/L in sham animals, p: n.s.). The pattern of change in MDA levels is " Fig. 1a. shown in ● Mean XO activities were not significantly different preoperatively between both groups (14.6 ± 3 mU/L in anhepatic animals vs. " Fig. 1b). Postoperatively a sig11.4 ± 3.8 mU/L in sham animals) (● nificant increase of XO activity was observed in the acute liver failure group compared to a decline of XO activity in the sham group (22.6 ± ;5.4 mU/L versus 3.3 ± 2.1 mU/L, p < 0.03. In the further course XO activities of the anhepatic animals declined steadily to 10.1 ± 1.8 mU/L (12.5 ± 5.3 mU/L in sham animals, p: n.s.) 8 hours
FRAP was measured according to the method of Benzie and Strain [16]. In brief, FRAP reagent was prepared as a mixture of acetate buffer (pH 3.6), TPTZ (2,4,6-tripyridyl-s-trazine) and ferric chloride (10:1:1). 300 µl FRAP reagent was warmed to 37°C; 10 µl of blood plasma and 30 µl of water were then added. Absorbance readings were measured at 520 nm after 6 min. Results were calibrated against a ferrous sulfate standard solution.
Statistical Analysis Mean values of the selected variables determined before and after hepatectomy were compared by ANOVA t-Test, (JMP® 9.0, SAS Institute, Cary, NC, USA). A p value < 0.05 was considered significant. Nonlinear correlations were evaluated by the Spear-
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Fig. 1 The course of malondialdehyd a, xanthine oxidase b, superoxide dismutase c and ferric reducing ability of plasma d preoperatively, postoperatively and 8 hours and 16 hours after hepatectomy. All values are given as mean ± SEM. ■ indicate anhepatic pigs, ● indicate sham operated pigs, * p < 0.05.
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Analysis of the Ferric Reducing Ability of Plasma (FRAP)
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after hepatectomy until a value of 6.3 ± 4.1 mU/L 16 hours after hepatectomy, which was comparable to sham XO activity after 16 hours (3.5 ± 2.1 mU/L p: n.s.). No significant differences in the enzyme activity of SOD were observed preoperatively between both groups (39 ± 5 U/mL in anhepatic animals vs. 34.8 ± 3.3 U/mL in sham animals) and SOD ac" Fig. 1c). tivity did not change during the observational period (● Preoperative mean FRAP values were not significantly different between the groups (82.3 ± 11.9 µmol Fe 2 +/L in anhepatic pigs vs. 87 ± 5.5 µmol Fe 2 +/L in sham animals). FRAP values rose significantly during anhepaty (postoperatively: 144.5 ± 21.8 µmol Fe 2 +/L in anhepatic animals vs. 76 ± 15.3 µmol Fe 2 +/L in sham animals, p < 0.04; 8 hours after hepatectomy: 139 ± 7 µmol Fe 2 +/L in anhepatic animals vs. 72.2 ± 7.8 µmol Fe 2 +/L in sham animals, p < 0.0001; 16 hours after hepatectomy: 219.2 ± 40.1 µmol Fe 2 +/L in anhepatic animals vs. 73.6 ± 22.8 µmol Fe 2 +/L in sham " Fig. 1 d). animals, p < 0.015) (● Preoperatively and postoperatively total bilirubin values were not significantly different in both groups (pre: 0.05 ± 0.03 mg/dl, post: 0.18 ± 0.02 mg/dl in anhepatic animals vs. pre: 0.07 ± 0.03 mg/dl, " Fig. 2a). 8 h and 16 h post: 0.15 ± 0.06 mg/dl in sham animals) (● after intervention total bilirubin levels significantly differed between anhepatic and sham group (after 8h: 0.65 ± 0.06 mg/dl in the anhepatic group versus 0.08 ± 0.02 mg/dl in the sham group,
p < 0.0001 and after 16h: 0.65 ± 0.19 mg/dl in the anhepatic group " Fig. 2a). versus 0.04 ± 0.02 mg/dl in the sham group p < 0.018) (● Total bilirubin levels correlated positively to MDA concentrations " Fig. 3a) and to FRAP levels (Spearman’s ρ = 0.53; p = 0.0007) (● (Spearman’s ρ = 0.77; p < 0.0001). No correlation was found between the levels of FRAP and either albumin or total protein. A significant positive correlation was observed between MDA concentrations as marker for oxidative stress and the antioxidant capacity of plasma, as reflected by the FRAP levels (Spearman’s " Fig. 3b). ρ = 0.62; p < 0.0001) (● Uric acid concentrations were below the detection limit of 0.5 mg/dL. Under standardized intensive care therapy concentration of hemoglobin remained stabile (> 8 g/dl), methemoglobin concentration stayed beyond 0.7 % and serum electrolytes, acidbase balance, blood gases as well as blood glucose were maintained within the physiological range during the whole observational period. Anhepatic animals showed an increase of lactate concentration from 1.5 ± 0.3 mmol/l preoperatively up to 14 ± 4 mmol/l after 16 hours, while lactate values of sham animals " Fig. 2b). Restayed at 0.7 ± 0.2 mmol/l during the experiment (● nal function impaired after hepatectomy and after 16 hours creatinine concentrations differed significantly between both groups (pre: 1.2 ± 0.2 mg/dl, after 16 hours: 2.2 ± 0.3 mg/dl in anhepatic animals vs. pre: 1 ± 0.4 mg/dl, after 16 hours: 1.2 ± 0.3 mg/dl in
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Fig. 2 The course of Total Bilirubin a, Lactate b, Creatinine c and INR d preoperatively, postoperatively and 8 hours and 16 hours after hepatectomy. All values are given as mean ± SEM. ■ indicate anhepatic pigs, ● indicate sham operated pigs, * p < 0.05.
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Fig. 4 The course of Intracranial Pressure (ICP) a and Mean Arterial Pressure b preoperatively, postoperatively and 4, 8,12 and 16 hours after hepatectomy. All values are given as mean ± SEM. ■ indicate anhepatic pigs, ● indicate sham operated pigs, * p < 0.05.
" Fig. 2c). A significant sham animals; pre: p: n.s.; post: p = 0.04) (● positive correlation was observed between creatinine and MDA concentrations (Spearman’s ρ = 0.65; p < 0.0001) and between creatinine and FRAP values (Spearman’s ρ = 0.40; p < 0.017). Only a slight increase in INR as a marker of coagulation was observed until 16 hours after hepatectomy and did not differ between anhepatic and sham animals (pre: 0.9 ± 0.02, after 16 hours: 1.3 ± 0.1 mg/dl in anhepatic animals vs. pre: 0.9 ± 0 mg/dl, after 16 hours: 1.5 ± 0.1 mg/dl in sham animals; pre/post: p: n.s.) " Fig. 2 d). (● Anhepatic pigs stayed hemodynamically stable until conclusion of " Fig. 4b). Intracranial pressure increased 8 hours the protocol (● after hepatectomy and differed significantly from sham animals after 12 and 16 hours (pre: 16 ± 4 mmHg, after 12 hours: 25 ± 1 mmHg, after 16 hours: 31 ± 5 mmHg in anhepatic animals vs. pre: 15 ± 4 mmHg, after 12 hours: 18 ± 1 mmHg after 16 hours: 18 ± 2 mmHg in sham animals; pre/post: p: n.s.; after 12h: p = " Fig. 4a). 0.017; after 16 h: p < 0.5) (●
Discussion !
Hitherto it remains unclear if a total hepatectomy may avoid the occurrence of oxidative stress in ALF. Hence it is necessary to assess the amount of OS produced from the remaining organism without any liver tissue left. Therefore the present study was performed to evaluate oxidative stress and the antioxidant capacity in an anhepatic pig model. After total hepatectomy a markedly increase of the oxidative marker MDA and an augmentation of FRAP as marker of the antioxidant capacity were observed. XO activity increased soon after hepatectomy, but returned to normal activity in the further course. SOD activity did not change during the observational period. Elevated lipid peroxidation products have been interpreted to reflect the destruction of hepatocytes in patients with acute liver failure [4]. In our study we observed an increase of LPO although no functional liver tissue remained. Thus it must be questioned what might be the source of lipid peroxidation. The accumulation
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MDA ( μmol/L)
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of bile acids in serum is discussed as a cause of LPO [17]. We found a positive correlation of total bilirubin concentration and MDA levels in our study. Elevated FRAP values might be expounded as an adaptive response to oxidative stress. Further support for this causal connection is reflected by a significant positive correlation between MDA and FRAP. Increase in antioxidant capacity as a possible sign of a compensatory phenomenon has been observed also in other liver diseases [6, 18]. Though these findings are in contrast to the study of Bhatia et al. who observed lower FRAP levels among acute liver failure patients compared with healthy controls as well as a significant negative correlation between the TBARS and the FRAP levels [4]. The observation period started when patients with ALF were admitted to the intensive care unit. At that time the levels of antioxidant reserves may be exhausted by severe illness already. Impairment of antioxidant defense system is descript in the context of chronic diseases as a result of chronically elevated ROS [19, 20]. FRAP values consist of uric acid, ascorbic acid, α-tocopherol, protein and bilirubin [16]. Therefore hyperproteinemia and hyperbilirubinemia might be responsible for elevated FRAP levels. In the present study no correlation was found between the levels of FRAP and either albumin or total protein. Hence, increased FRAP levels are not caused by protein. In our study a positive correlation between bilirubin concentration and FRAP level was seen. But bilirubin only contributes 5 % to FRAP values of plasma. Uric acid is estimated to contribute around 60 % to the FRAP values in human fresh plasma [16]. Due to the enzyme uricase, which catalyses the conversion of uric acid concentrations to allantoin in nonprimate mammals, physiologic serum uric acid concentrations in pigs are 5 – 6-fold lower than in humans [21]. FRAP levels in our animal study are considerably lower than in human studies [19, 20, 22]. However uric acid concentrations were below the detection limit of 0.5 mg/dL and therefore did not cause an increase of FRAP levels. Consequently the source of the augmentation of FRAP levels after hepatectomy remains unclear. No significant differences of SOD enzyme activity were observed during the course of the study. Bhatia et al. found significantly higher SOD activity at baseline among ALF patients than healthy controls and increased SOD activity was interpreted as an adaptive response against the prevalent oxidative stress during ALF [4]. Hepatectomy in healthy animals produces an acute damage and therefore a gradual adaptation of the organism with a compensatory antioxidative agent production may not occur. XO activity increased directly after hepatectomy, but returned to normal activity in the further course. XO is known to be disseminated throughout various organs including, liver, intestine, lung, kidney, heart and brain as well as the plasma, with highest levels being detected in liver and intestine [23]. Our results indicate a key role of the liver for circulating XO activity. In the anhepatic course no compensatory increase in XO levels induced by other organs like the intestine occurs. Hepatectomy as a large surgical procedure per se might lead to oxidative stress. It is well-known that any form of trauma, including surgery leads to oxidative stress. The main organs that are affected by oxidative stress during abdominal surgery are liver, intestine and peritoneum [24]. However, MDA and SOD concentrations as well as FRAP levels did not change during hepatectomy in our study, which indicates that the oxidative damage was rather minor. XO activity increased directly after hepatectomy, which is the only indicator of oxidative stress due to surgical trauma. The animals used in our study were young and healthy animals and for that reason might be able to maintain their redox status
Thiel C et al. How much Oxidative … Z Gastroenterol 2014; 52: 43–49
better. This goes along with the observation that young children are less vulnerable to oxidative stress induced by fine particulate pollution than elderly [25]. It could be questioned if a total hepatectomy as a large surgical procedure might lead to a delayed increase in biomarkers of oxidative stress. A review concerning oxidative stress after abdominal surgery describes a return of the markers of oxidative stress to normal ranges in the early postoperative period after abdominal surgery [24]. In our study we observed similar MDA concentrations and similar FRAP levels pre-and postoperatively. The increase of MDA concentrations and FRAP levels started 8 hours after hepatectomy and did not return to normal ranges in the further course. Therefore it seems unlikely that surgical trauma is responsible for these elevated markers. The results of the present study demonstrate that OS it not only produced outside the liver, the regulation is also not exclusively liver-dependent. Progressive liver insufficiency leads to multiple organ failure with deterioration of renal and pulmonary function and encephalopathy. These secondary affected organs might be involved in the production and regulation of OS [26 – 28]. In addition OS is known to play a role in intestinal injury like intestinal congestion caused by increased portal back pressure or hypoxia during the anhepatic phase in liver transplantation [29]. Hepatectomy in our study was performed by lateral vascular clamping of only one third of the vessels to prevent serious congestion of abdominal organs. The autopsy of the animals showed no macroscopically signs of intestinal congestion. Hepatectomy removes the main organ for detoxification. Toxic metabolic such as the accumulation of bile acids may cause the production of LPO [17]. We found a positive correlation of total bilirubin concentration and MDA levels in our study. Although OS has been shown to occur in every liver disease, attempts to therapeutically reduce oxidative stress using antioxidant vitamins and agents with antioxidant effect as for example zinc, herbal drugs and silymarin have failed to improve the outcome of patients [30]. The only promising results were observed for the use of vitamin E for non-alcoholic steatohepatitis (NASH) [31]. One of the major difficulties is that the exact mechanisms of actions of the therapeutically used antioxidants are not known [30]. In conclusion, our study demonstrates that lipid peroxidation products are not necessarily originating in the liver and an increase of the antioxidant defense could be observed without the presence of any functional liver tissue. These findings show that hepatectomy does not completely prevent the occurrence of OS because the production and regulation of LPO are also located outside the liver.
Acknowledgments !
The authors thank T. O. Greiner, A. Stolz and M. Seitzer for their excellent veterinarian and technical assistance and Jörg Glatzle for his kind contribution to the preparation of the manuscript.
References 01 Ringe B, Lubbe N, Kuse E et al. Total hepatectomy and liver transplantation as two-stage procedure. Ann Surg 1993; 218 (1): 3 – 9 02 Ringe B, Pichlmayr R, Lubbe N et al. Total hepatectomy as temporary approach to acute hepatic or primary graft failure. Transplant Proc 1988; 20 (1): 552 – 557
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03 Arora H, Thekkekandam J, Tesche L et al. Long-term survival after 67 hours of anhepatic state due to primary liver allograft nonfunction. Liver Transpl 2010; 16 (12): 1428 – 1433 04 Bhatia V, Bhardwaj P, Elikkottil J et al. A 7-day profile of oxidative stress and antioxidant status in patients with acute liver failure. Hepatol Int 2008; 2 (4): 465 – 470 05 Bajt ML, Knight TR, Lemasters JJ et al. Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes: protection by Nacetyl cysteine. Toxicol Sci 2004; 80 (2): 343 – 349 06 Bhardwaj P, Madan K, Thareja S et al. Comparative redox status in alcoholic liver disease and nonalcoholic fatty liver disease. Hepatol Int 2008; 2 (2): 202 – 208 07 Nagasaka H, Takayanagi M, Tsukahara H. Children's toxicology from bench to bed–Liver Injury (3): Oxidative stress and anti-oxidant systems in liver of patients with Wilson disease. J Toxicol Sci 2009; 34 (Suppl 2): SP229 – SP236 08 Madill J, Arendt BM, Aghdassi E et al. Hepatic lipid peroxidation and antioxidant micronutrients in hepatitis virus C liver recipients with and without disease recurrence. Transplant Proc 2009; 41 (9): 3800 – 3805 09 Svegliati-Baroni G, De Minicis S, Marzioni M. Hepatic fibrogenesis in response to chronic liver injury: novel insights on the role of cell-to-cell interaction and transition. Liver Int 2008; 28 (8): 1052 – 1064 10 Shapiro H, Ashkenazi M, Weizman N et al. Curcumin ameliorates acute thioacetamide-induced hepatotoxicity. J Gastroenterol Hepatol 2006; 21 (2): 358 – 366 11 Ritter C, Reinke A, Andrades M et al. Protective effect of N-acetylcysteine and deferoxamine on carbon tetrachloride-induced acute hepatic failure in rats. Crit Care Med 2004; 32 (10): 2079 – 2083 12 Kusmic C, Boggi U, Bellini R et al. Oxidative stress in fulminant hepatic failure: comparison of two pig models with and without liver necrosis. Hepatogastroenterology 2001; 48 (39): 762 – 769 13 Paradis V, Kollinger M, Fabre M et al. In situ detection of lipid peroxidation by-products in chronic liver diseases. Hepatology 1997; 26 (1): 135 – 142 14 Thiel C, Thiel K, Etspueler A et al. Standardized intensive care unit management in an anhepatic pig model: new standards for analyzing liver support systems. Crit Care 2010; 14 (4): R138 15 Knubben K, Thiel C, Schenk M et al. A new surgical model for hepatectomy in pigs. Eur Surg Res 2008; 40 (1): 41 – 46 16 Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 1996; 239 (1): 70 – 76 17 Fabris C, Pirisi M, Panozzo MP et al. Intensity of inflammatory damage and serum lipid peroxide concentrations in liver disease. J Clin Pathol 1993; 46 (4): 364 – 367
18 Notas G, Miliaraki N, Kampa M et al. Patients with primary biliary cirrhosis have increased serum total antioxidant capacity measured with the crocin bleaching assay. World J Gastroenterol 2005; 11 (27): 4194 – 4198 19 Verlaan M, Roelofs HM, van Schaik A et al. Assessment of oxidative stress in chronic pancreatitis patients. World J Gastroenterol 2006; 12 (35): 5705 – 5710 20 Karajibani M, Hashemi M, Montazerifar F et al. The status of glutathione peroxidase, superoxide dismutase, vitamins A, C, E and malondialdehyde in patients with cardiovascular disease in Zahedan, Southeast Iran. J Nutr Sci Vitaminol (Tokyo) 2009; 55 (4): 309 – 316 21 Terkeltaub R. Learning how and when to employ uricase as bridge therapy in refractory gout. J Rheumatol 2007; 34 (10): 1955 – 1958 22 Staruchova M, Collins AR, Volkovova K et al. Occupational exposure to mineral fibres. Biomarkers of oxidative damage and antioxidant defence and associations with DNA damage and repair. Mutagenesis 2008; 23 (4): 249 – 260 23 Pacher P, Nivorozhkin A, Szabo C. Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol Rev 2006; 58 (1): 87 – 114 24 Arsalani-Zadeh R, Ullah S, Khan S et al. Oxidative stress in laparoscopic versus open abdominal surgery: a systematic review. J Surg Res 2011; 169 (1): e59 – e68 25 Kim K, Park EY, Lee KH et al. Differential oxidative stress response in young children and the elderly following exposure to PM(2.5). Environ Health Prev Med 2009; 14 (1): 60 – 66 26 Wang Z, Holthoff JH, Seely KA et al. Development of oxidative stress in the peritubular capillary microenvironment mediates sepsis-induced renal microcirculatory failure and acute kidney injury. Am J Pathol 2012; 180 (2): 505 – 516 27 Rahman I, MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J 2000; 16 (3): 534 – 554 28 Bosoi CR, Yang X, Huynh J et al. Systemic oxidative stress is implicated in the pathogenesis of brain edema in rats with chronic liver failure. Free Radic Biol Med 2012; 52 (7): 1228 – 1235 29 Ge M, Chi X, Zhang A et al. Intestinal NF-E2-related factor-2 expression and antioxidant activity changes in rats undergoing orthotopic liver autotransplantation. Oncol Lett 2013; 6 (5): 1307 – 1312 30 Singal AK, Jampana SC, Weinman SA. Antioxidants as therapeutic agents for liver disease. Liver Int 2011; 31 (10): 1432 – 1448 31 Sanyal AJ, Chalasani N, Kowdley KV et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med 2010; 362 (18): 1675 – 1685
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How much Oxidative Stress Exists Without the Liver? Wieviel oxidativer Stress entsteht ohne die Leber?
Authors
C. Thiel1, T. Katt1, M. Schenk1, C. Grasshoff2, M. H. Morgalla3, A. Peter4, A. Königsrainer1, K. Thiel1
Affiliations
1
3 4
Department of General, Visceral and Transplant Surgery, University Hospital Tuebingen Department of Anesthesiology, University Hospital Tuebingen Department or Neurosurgery, University Hospital Tuebingen Division of Endocrinology, Diabetology, Angiology, Nephrology, Pathobiochemistry and Clinical Chemistry, Department of Internal Medicine, University Hospital Tuebingen
Schlüsselwörter
Zusammenfassung
Abstract
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Hintergrund: Oxidativer Stress (OS) stellt einen wichtigen pathogenetischen Faktor beim akuten Leberversagen und bei chronischen Lebererkrankungen dar. In der vorliegenden Studie wurde OS und der antioxidative Status bei einem anhepatischen Schweinemodell gemessen, um herauszufinden, ob die Leber selbst eine entscheidende Rolle bei der Entstehung von OS spielt. Methode: 6 Schweine wurden hepatektomiert, die Kontrollgruppe bestand aus 5 Schweinen. OS und antioxidativer Status wurden bestimmt, indem die Plasmakonzentrationen von Malondialdehyd (MDA), Xanthinoxidase (XO), Superoxiddismutase (SOD) und der eisenreduzierenden antioxidativen Kapazität (FRAP) zu Beginn des Experiments, postoperativ sowie 8 und 16 Stunden nach Hepatektomie gemessen wurden. Ergebnisse: Erhöhte MDA-Konzentrationen wurden bei den anhepatischen Tieren postoperativ (p < 0,02) und nach 8 Stunden (p < 0,003) im Vergleich zur Kontrolle beobachtet. Die XO-Aktivität nahm postoperativ zu (p < 0,03 vs. Kontrolle), kehrte jedoch im weiteren Verlauf auf Normalwerte zurück. Es fanden sich keine Veränderungen der SOD-Werte in beiden Gruppen. FRAPBestimmungen nahmen signifikant zu gegenüber der Kontrolle (p < 0,015). Es bestand eine positive Korrelation zwischen MDA- und FRAP-Werten (Spearman’s ρ = 0,62; p < 0,0001). Schlussfolgerung: Diese Ergebnisse zeigen, dass das Auftreten von OS durch eine Hepatektomie nicht verhindert werden kann, da sowohl die Produktion als auch die Regulation auch außerhalb der Leber stattfinden.
Background: Oxidative stress (OS) represents an important pathogenetic factor of acute liver failure and chronic liver diseases. To elucidate whether the liver itself is a major source of OS, the present study was performed to assess OS and antioxidant status in an anhepatic porcine model. Methods: Six pigs underwent a total hepatectomy, five pigs were sham operated. OS and antioxidant status were evaluated by measuring plasma concentrations of malondialdehyde (MDA), xanthine oxidase (XO), superoxide dismutase (SOD) and the ferric reducing ability of plasma (FRAP). They were sampled at the start of the experiment, immediately after surgery, and then at 8 and 16 hours post hepatectomy. Results: Increased concentrations of MDA were observed in anhepatic pigs postoperatively (p < 0.02) and 8 hours after hepatectomy (p < 0.003) compared to controls. XO activity increased soon after hepatectomy (22.6 ± 5.4 mU/L versus 3.3 ± 2.1 mU/ L in sham animals, p < 0.03) but returned to normal values in the further course. SOD levels did not change during the observational period in both groups. FRAP values rose significantly in the anhepatic animals compared to control (p < 0.015). A significant positive correlation was observed between MDA levels and FRAP levels (Spearman’s ρ = 0.62; p < 0.0001). Conclusions: These findings show that hepatectomy does not completely prevent the occurrence of OS because the production and regulation of OS are also located outside the liver.
Introduction
patocytes. The necrotic liver can cause cardiovascular shock, acute renal failure and respiratory failure which results in a life-threatening condition, described as “toxic liver syndrome” [1]. Total
● Leber ● akutes Leberversagen ● Hepatektomie ● oxidativer Stress " " "
Key words
● liver ● acute hepatic failure ● hepatectomy ● oxidative stress " " " "
received accepted
30.10.2013 12.12.2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1356362 Z Gastroenterol 2014; 52: 43–49 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0044-2771
Correspondence Dr. Karolin Thiel Department of General, Visceral and Transplant Surgery, University Hospital Tuebingen Hoppe-Seyler-Str. 3 72076 Tuebingen Germany Tel.: ++ 49/70 71/2 98 66 00 Fax: ++ 49/70 71/29 49 34 Karolin.Thiel@med. uni-tuebingen.de
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Acute liver failure (ALF) is characterized by a sudden and extensive necrosis and/or apoptosis of he-
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hepatectomy of the toxic liver has been described as a successful bridge therapy before transplantation [2]. It is well-known that removal of the necrotic liver improves hemodynamics by eliminating the source of vasoactive agents and cytokines [3], but it remains unclear if a total hepatectomy may avoid the amount of OS. On the one hand necrotic hepatocytes are seen as a source of massive oxidative stress [4]. On the other hand it has been shown that mitochondrial oxidant stress precedes cell necrosis as identified by the permeability increase of the cell membrane and cell content release [5]. The origin and amount of OS remains undefined but may be an important target for new therapeutic approaches. Most previous studies investigating the effect of OS on liver injuries focus on chronic liver diseases [6 – 9], only a few studies examined the role of OS in the pathogenesis of acute liver failure [10 – 12]. Lipid peroxidation (LPO) was detectable in ALF and in all chronic liver diseases studied [6 – 8], suggesting that OS plays a crucial role in the pathogenesis [13]. Although OS has been shown to occur in every liver disease, the origin and clinical relevance of OS in particular in ALF remains unknown. Up to now it is unclear how much the liver itself contributes to oxidative stress in acute liver failure and if a total hepatectomy may eliminate the amount of OS. Furthermore it is not known how much the remaining organism accounts to the production of OS. Therefore the aim of the study was to evaluate the occurrence of oxidative stress and antioxidant levels in an anhepatic animal model. Since OS is an important pathogenetic factor of acute liver failure, the genesis of OS without any liver left is of main interest. In addition, any surgical trauma is known to cause OS. Our second aim was to investigate postoperative changes of redox parameters after a large operation in terms of a hepatectomy.
Materials and Methods !
Animal model The study was performed in 11 female domestic pigs weighing 37 ± 2 kg. After approval by the institutional review board for animal experiments, 6 pigs underwent total hepatectomy; a sham group consisted of 5 pigs. Animal care and all investigations were performed according to the international principles governing research on animals and under the supervision of a veterinarian, who set the guidelines for minimizing pigs suffering.
Premedication and anesthesia Intramuscular premedication consisted of atropine 0.1 % (0.05 mg/ kg), ketamine (14 mg/kg), azaperone (2 mg/kg) and midazolam (0.5 mg/kg). Body temperature was maintained at 38.5° C with a warming blanket. Preliminary procedures including intubation, ventilation and anesthesia during surgery have been described previously in detail [14]. In brief, continuous infusion of ketamine (15 mg/kg/h), fentanyl (0.02 mg/kg/h) and midazolam (0.9 mg/kg/h) was administered to maintain anesthesia during the experiment. Pigs were ventilated with a pressure controlled ventilation modus (Siemens KION SC 9000 XL anesthesia system; Siemens Medical Solutions Inc., Solna, Sweden). Arterial blood gas analysis (ABL 800, Radiometer Copenhagen, Denmark) was performed hourly and ventilation was adjusted accordingly.
Thiel C et al. How much Oxidative … Z Gastroenterol 2014; 52: 43–49
Surgical procedure Animals were kept under standard laboratory conditions. They fasted preoperatively and received an antibiotic prophylaxis of 2 g ceftriaxone (Rocephin®, Hoffmann-La Roche, Basel, Switzerland). Study animals and sham animals received the following catheters and probes: The jugular veins (Multi-Lumen Central Venous Catheter, Arrow International, Reading, PA, USA) and the internal carotid artery (Leadercath, Vygon, Écouen, France) were instrumented to measure central venous and arterial pressure. Following a parietofrontal cranial trepanation, a probe was inserted in the frontal brain parenchyma to measure intracranial pressure and brain temperature (Camino® MPM-1 monitor, Integra Neurosciences, Plainsboro, NJ, USA). The abdominal cavity was entered through a midline abdominal incision and a urinary catheter (Gentle-FloTM, Tyco Healthcare, Tullamore, Ireland) was placed. In 5 sham animals the abdominal wall was now closed with a running suture (Silkam®2, silk, braided, coated, nonabsorbable), the other 6 pigs from the study groups underwent total hepatectomy. The surgical procedure of the total hepatectomy using a Y-shaped bypass (Uni-Graft® K DV, Denkendorf, Germany) between the infrahepatic vena cava and the portal vein to the suprahepatic vena cava was performed as previously described [15]. After surgery animals remained under general anesthesia until death, receiving a standardized intensive care management as described recently [14]. During the experiment sodium chloride solution 0.9 %, hydroxyethylstarch 6 % (Voluven® HES 130/0.4, Fresenius, Bad Homburg, Germany) and Norepinephrine were infused, adjusted for mean arterial and central venous pressure. Continuously monitoring throughout surgery and the postoperative observation period included electrocardiogram, mean arterial pressure, central venous pressure, intracranial pressure, oxygen saturation and core body temperature. Urinary output, hemoglobin, hematocrit and lactate, serum electrolytes, acidbase balance, blood gases and blood glucose levels were monitored hourly and immediately corrected as required. Sham animals were euthanized at the end of the observation period with a single intravenous bolus of 10 mL T 61 (Intervet, Unterschleißheim, Germany).
Laboratory Analysis All blood samples were obtained from the internal carotid catheter preoperatively, postoperatively as well as 8 hours and 16 hours after hepatectomy. Sample analysis was conducted within 1 hour of collection at each time point and was performed by the certified central laboratories of the University Hospital Tuebingen (Division of Endocrinology, Diabetology, Angiology, Nephrology, Pathobiochemistry and Clinical Chemistry, Department of Internal Medicine, University Hospital Tuebingen, Germany). Albumin, total plasma protein and bilirubin concentrations were performed on the ADVIA 1800 Clinical Chemistry analyzer. Uric Acid analysis was performed using a commercial kid (Infinity, Uric Acid Liquid Stable Reagent, Thermo, Victoria, Australia).
Measurement of oxidative stress and antioxidant levels Arterial blood samples were immediately centrifuged at 3000 g for 10 min. Plasma samples were stored at – 80 °C until they were analyzed.
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man’s ρ-Test. Results and figures are reported as mean ± standard error of mean (SEM).
Analysis of malondialdehyde (MDA), xanthine oxidase (XO) and superoxide dismutase (SOD) MDA plasma concentrations were detected using the OxiSelect TBARS assay kit (Cell Biolabs, San Diego, USA). XO activity was quantified by using the Xanthine Oxidase Assay Kit (Cayman Chemical Company, Michigan, USA). SOD assaying was performed by utilizing the 19 160 SOD determination kit (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). All assays were performed according to the manufacturer's instructions.
Results Preoperative mean MDA concentrations did not differ significantly between both groups (10.6 ± 1.5 µmol/L in anhepatic ani" Fig. 1a). Directly mals vs. 8.7 ± 2.2 µmol/L in sham animals) (● after hepatectomy plasma concentration of MDA started to increase steadily (postoperatively: 11.6 ± 1 µmol/L in anhepatic animals vs. 8.3 ± 0.5 µmol/L in sham animals, p < 0.02; 8 hours after hepatectomy: 14.5 ± 0.8 µmol/L in anhepatic animals vs. 8.6 ± 1.3 µmol/L in sham animals, p < 0.003; 16 hours after hepatectomy: 20.1 ± 4.1 µmol/L in anhepatic animals vs. 9.6 ± 0.9 µmol/L in sham animals, p: n.s.). The pattern of change in MDA levels is " Fig. 1a. shown in ● Mean XO activities were not significantly different preoperatively between both groups (14.6 ± 3 mU/L in anhepatic animals vs. " Fig. 1b). Postoperatively a sig11.4 ± 3.8 mU/L in sham animals) (● nificant increase of XO activity was observed in the acute liver failure group compared to a decline of XO activity in the sham group (22.6 ± ;5.4 mU/L versus 3.3 ± 2.1 mU/L, p < 0.03. In the further course XO activities of the anhepatic animals declined steadily to 10.1 ± 1.8 mU/L (12.5 ± 5.3 mU/L in sham animals, p: n.s.) 8 hours
FRAP was measured according to the method of Benzie and Strain [16]. In brief, FRAP reagent was prepared as a mixture of acetate buffer (pH 3.6), TPTZ (2,4,6-tripyridyl-s-trazine) and ferric chloride (10:1:1). 300 µl FRAP reagent was warmed to 37°C; 10 µl of blood plasma and 30 µl of water were then added. Absorbance readings were measured at 520 nm after 6 min. Results were calibrated against a ferrous sulfate standard solution.
Statistical Analysis Mean values of the selected variables determined before and after hepatectomy were compared by ANOVA t-Test, (JMP® 9.0, SAS Institute, Cary, NC, USA). A p value < 0.05 was considered significant. Nonlinear correlations were evaluated by the Spear-
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Fig. 1 The course of malondialdehyd a, xanthine oxidase b, superoxide dismutase c and ferric reducing ability of plasma d preoperatively, postoperatively and 8 hours and 16 hours after hepatectomy. All values are given as mean ± SEM. ■ indicate anhepatic pigs, ● indicate sham operated pigs, * p < 0.05.
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Analysis of the Ferric Reducing Ability of Plasma (FRAP)
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after hepatectomy until a value of 6.3 ± 4.1 mU/L 16 hours after hepatectomy, which was comparable to sham XO activity after 16 hours (3.5 ± 2.1 mU/L p: n.s.). No significant differences in the enzyme activity of SOD were observed preoperatively between both groups (39 ± 5 U/mL in anhepatic animals vs. 34.8 ± 3.3 U/mL in sham animals) and SOD ac" Fig. 1c). tivity did not change during the observational period (● Preoperative mean FRAP values were not significantly different between the groups (82.3 ± 11.9 µmol Fe 2 +/L in anhepatic pigs vs. 87 ± 5.5 µmol Fe 2 +/L in sham animals). FRAP values rose significantly during anhepaty (postoperatively: 144.5 ± 21.8 µmol Fe 2 +/L in anhepatic animals vs. 76 ± 15.3 µmol Fe 2 +/L in sham animals, p < 0.04; 8 hours after hepatectomy: 139 ± 7 µmol Fe 2 +/L in anhepatic animals vs. 72.2 ± 7.8 µmol Fe 2 +/L in sham animals, p < 0.0001; 16 hours after hepatectomy: 219.2 ± 40.1 µmol Fe 2 +/L in anhepatic animals vs. 73.6 ± 22.8 µmol Fe 2 +/L in sham " Fig. 1 d). animals, p < 0.015) (● Preoperatively and postoperatively total bilirubin values were not significantly different in both groups (pre: 0.05 ± 0.03 mg/dl, post: 0.18 ± 0.02 mg/dl in anhepatic animals vs. pre: 0.07 ± 0.03 mg/dl, " Fig. 2a). 8 h and 16 h post: 0.15 ± 0.06 mg/dl in sham animals) (● after intervention total bilirubin levels significantly differed between anhepatic and sham group (after 8h: 0.65 ± 0.06 mg/dl in the anhepatic group versus 0.08 ± 0.02 mg/dl in the sham group,
p < 0.0001 and after 16h: 0.65 ± 0.19 mg/dl in the anhepatic group " Fig. 2a). versus 0.04 ± 0.02 mg/dl in the sham group p < 0.018) (● Total bilirubin levels correlated positively to MDA concentrations " Fig. 3a) and to FRAP levels (Spearman’s ρ = 0.53; p = 0.0007) (● (Spearman’s ρ = 0.77; p < 0.0001). No correlation was found between the levels of FRAP and either albumin or total protein. A significant positive correlation was observed between MDA concentrations as marker for oxidative stress and the antioxidant capacity of plasma, as reflected by the FRAP levels (Spearman’s " Fig. 3b). ρ = 0.62; p < 0.0001) (● Uric acid concentrations were below the detection limit of 0.5 mg/dL. Under standardized intensive care therapy concentration of hemoglobin remained stabile (> 8 g/dl), methemoglobin concentration stayed beyond 0.7 % and serum electrolytes, acidbase balance, blood gases as well as blood glucose were maintained within the physiological range during the whole observational period. Anhepatic animals showed an increase of lactate concentration from 1.5 ± 0.3 mmol/l preoperatively up to 14 ± 4 mmol/l after 16 hours, while lactate values of sham animals " Fig. 2b). Restayed at 0.7 ± 0.2 mmol/l during the experiment (● nal function impaired after hepatectomy and after 16 hours creatinine concentrations differed significantly between both groups (pre: 1.2 ± 0.2 mg/dl, after 16 hours: 2.2 ± 0.3 mg/dl in anhepatic animals vs. pre: 1 ± 0.4 mg/dl, after 16 hours: 1.2 ± 0.3 mg/dl in
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Fig. 2 The course of Total Bilirubin a, Lactate b, Creatinine c and INR d preoperatively, postoperatively and 8 hours and 16 hours after hepatectomy. All values are given as mean ± SEM. ■ indicate anhepatic pigs, ● indicate sham operated pigs, * p < 0.05.
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Fig. 4 The course of Intracranial Pressure (ICP) a and Mean Arterial Pressure b preoperatively, postoperatively and 4, 8,12 and 16 hours after hepatectomy. All values are given as mean ± SEM. ■ indicate anhepatic pigs, ● indicate sham operated pigs, * p < 0.05.
" Fig. 2c). A significant sham animals; pre: p: n.s.; post: p = 0.04) (● positive correlation was observed between creatinine and MDA concentrations (Spearman’s ρ = 0.65; p < 0.0001) and between creatinine and FRAP values (Spearman’s ρ = 0.40; p < 0.017). Only a slight increase in INR as a marker of coagulation was observed until 16 hours after hepatectomy and did not differ between anhepatic and sham animals (pre: 0.9 ± 0.02, after 16 hours: 1.3 ± 0.1 mg/dl in anhepatic animals vs. pre: 0.9 ± 0 mg/dl, after 16 hours: 1.5 ± 0.1 mg/dl in sham animals; pre/post: p: n.s.) " Fig. 2 d). (● Anhepatic pigs stayed hemodynamically stable until conclusion of " Fig. 4b). Intracranial pressure increased 8 hours the protocol (● after hepatectomy and differed significantly from sham animals after 12 and 16 hours (pre: 16 ± 4 mmHg, after 12 hours: 25 ± 1 mmHg, after 16 hours: 31 ± 5 mmHg in anhepatic animals vs. pre: 15 ± 4 mmHg, after 12 hours: 18 ± 1 mmHg after 16 hours: 18 ± 2 mmHg in sham animals; pre/post: p: n.s.; after 12h: p = " Fig. 4a). 0.017; after 16 h: p < 0.5) (●
Discussion !
Hitherto it remains unclear if a total hepatectomy may avoid the occurrence of oxidative stress in ALF. Hence it is necessary to assess the amount of OS produced from the remaining organism without any liver tissue left. Therefore the present study was performed to evaluate oxidative stress and the antioxidant capacity in an anhepatic pig model. After total hepatectomy a markedly increase of the oxidative marker MDA and an augmentation of FRAP as marker of the antioxidant capacity were observed. XO activity increased soon after hepatectomy, but returned to normal activity in the further course. SOD activity did not change during the observational period. Elevated lipid peroxidation products have been interpreted to reflect the destruction of hepatocytes in patients with acute liver failure [4]. In our study we observed an increase of LPO although no functional liver tissue remained. Thus it must be questioned what might be the source of lipid peroxidation. The accumulation
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of bile acids in serum is discussed as a cause of LPO [17]. We found a positive correlation of total bilirubin concentration and MDA levels in our study. Elevated FRAP values might be expounded as an adaptive response to oxidative stress. Further support for this causal connection is reflected by a significant positive correlation between MDA and FRAP. Increase in antioxidant capacity as a possible sign of a compensatory phenomenon has been observed also in other liver diseases [6, 18]. Though these findings are in contrast to the study of Bhatia et al. who observed lower FRAP levels among acute liver failure patients compared with healthy controls as well as a significant negative correlation between the TBARS and the FRAP levels [4]. The observation period started when patients with ALF were admitted to the intensive care unit. At that time the levels of antioxidant reserves may be exhausted by severe illness already. Impairment of antioxidant defense system is descript in the context of chronic diseases as a result of chronically elevated ROS [19, 20]. FRAP values consist of uric acid, ascorbic acid, α-tocopherol, protein and bilirubin [16]. Therefore hyperproteinemia and hyperbilirubinemia might be responsible for elevated FRAP levels. In the present study no correlation was found between the levels of FRAP and either albumin or total protein. Hence, increased FRAP levels are not caused by protein. In our study a positive correlation between bilirubin concentration and FRAP level was seen. But bilirubin only contributes 5 % to FRAP values of plasma. Uric acid is estimated to contribute around 60 % to the FRAP values in human fresh plasma [16]. Due to the enzyme uricase, which catalyses the conversion of uric acid concentrations to allantoin in nonprimate mammals, physiologic serum uric acid concentrations in pigs are 5 – 6-fold lower than in humans [21]. FRAP levels in our animal study are considerably lower than in human studies [19, 20, 22]. However uric acid concentrations were below the detection limit of 0.5 mg/dL and therefore did not cause an increase of FRAP levels. Consequently the source of the augmentation of FRAP levels after hepatectomy remains unclear. No significant differences of SOD enzyme activity were observed during the course of the study. Bhatia et al. found significantly higher SOD activity at baseline among ALF patients than healthy controls and increased SOD activity was interpreted as an adaptive response against the prevalent oxidative stress during ALF [4]. Hepatectomy in healthy animals produces an acute damage and therefore a gradual adaptation of the organism with a compensatory antioxidative agent production may not occur. XO activity increased directly after hepatectomy, but returned to normal activity in the further course. XO is known to be disseminated throughout various organs including, liver, intestine, lung, kidney, heart and brain as well as the plasma, with highest levels being detected in liver and intestine [23]. Our results indicate a key role of the liver for circulating XO activity. In the anhepatic course no compensatory increase in XO levels induced by other organs like the intestine occurs. Hepatectomy as a large surgical procedure per se might lead to oxidative stress. It is well-known that any form of trauma, including surgery leads to oxidative stress. The main organs that are affected by oxidative stress during abdominal surgery are liver, intestine and peritoneum [24]. However, MDA and SOD concentrations as well as FRAP levels did not change during hepatectomy in our study, which indicates that the oxidative damage was rather minor. XO activity increased directly after hepatectomy, which is the only indicator of oxidative stress due to surgical trauma. The animals used in our study were young and healthy animals and for that reason might be able to maintain their redox status
Thiel C et al. How much Oxidative … Z Gastroenterol 2014; 52: 43–49
better. This goes along with the observation that young children are less vulnerable to oxidative stress induced by fine particulate pollution than elderly [25]. It could be questioned if a total hepatectomy as a large surgical procedure might lead to a delayed increase in biomarkers of oxidative stress. A review concerning oxidative stress after abdominal surgery describes a return of the markers of oxidative stress to normal ranges in the early postoperative period after abdominal surgery [24]. In our study we observed similar MDA concentrations and similar FRAP levels pre-and postoperatively. The increase of MDA concentrations and FRAP levels started 8 hours after hepatectomy and did not return to normal ranges in the further course. Therefore it seems unlikely that surgical trauma is responsible for these elevated markers. The results of the present study demonstrate that OS it not only produced outside the liver, the regulation is also not exclusively liver-dependent. Progressive liver insufficiency leads to multiple organ failure with deterioration of renal and pulmonary function and encephalopathy. These secondary affected organs might be involved in the production and regulation of OS [26 – 28]. In addition OS is known to play a role in intestinal injury like intestinal congestion caused by increased portal back pressure or hypoxia during the anhepatic phase in liver transplantation [29]. Hepatectomy in our study was performed by lateral vascular clamping of only one third of the vessels to prevent serious congestion of abdominal organs. The autopsy of the animals showed no macroscopically signs of intestinal congestion. Hepatectomy removes the main organ for detoxification. Toxic metabolic such as the accumulation of bile acids may cause the production of LPO [17]. We found a positive correlation of total bilirubin concentration and MDA levels in our study. Although OS has been shown to occur in every liver disease, attempts to therapeutically reduce oxidative stress using antioxidant vitamins and agents with antioxidant effect as for example zinc, herbal drugs and silymarin have failed to improve the outcome of patients [30]. The only promising results were observed for the use of vitamin E for non-alcoholic steatohepatitis (NASH) [31]. One of the major difficulties is that the exact mechanisms of actions of the therapeutically used antioxidants are not known [30]. In conclusion, our study demonstrates that lipid peroxidation products are not necessarily originating in the liver and an increase of the antioxidant defense could be observed without the presence of any functional liver tissue. These findings show that hepatectomy does not completely prevent the occurrence of OS because the production and regulation of LPO are also located outside the liver.
Acknowledgments !
The authors thank T. O. Greiner, A. Stolz and M. Seitzer for their excellent veterinarian and technical assistance and Jörg Glatzle for his kind contribution to the preparation of the manuscript.
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