The Nutritional Status Modulates Preservation-Reperfusion Injury in Rat Fatty Liver

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  • The Nutritional Status Modulates Preservation-Reperfusion Injury in Rati

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    Journal of Surgical Research 127, 190196 (2005)doi:10.1016/j.jss.2005.02.018

    002 2ile PG infusion had no effect. Glucose supplemen-tion did not affect portal pressure, which, in con-st, was reduced in livers receiving PG. Finally, allatments lowered oxidative injury.Conclusions. Preservation injury in fatty liver iseatly related to nutritional status. Energetic sub-ate supplementation may represent a clinically fea-

    blood flow is greatly reduced after transplantation offatty livers as the result of the narrowed sinusoidallumen, which is compressed by the swollen fat-loadedhepatocytes and partially or completely obstructed bycell debris and platelets and leukocytes adherent to theendothelium [13]. Thus, it is not surprising that sub-stances with vasodilator and antiinflammatory proper-ties, such as prostaglandins, have been found to beprotective in animal models of fatty liver transplanta-tion [4, 5].

    However, other evidences indicate that hepatocyte

    To whom correspondence and reprint requests should be ad-ssed at Dipartimento di Medicina Interna, Cardioangiologia,atologia, University of Bologna, Policlinico SantOrsola, Via Al-toni 15, 40138 Bologna, Italy. E-mail:

    1902-4804/05 $30.00005 Elsevier Inc. All rights reserved.Fatty L

    olo Caraceni,*,,1 Marco Domenicali,*, Anna Maria PAlessandro Principe,*, Giuseppe Palasciano,

    *Department of Internal Medicine, Cardioangiology, and Hepatologpplied Research (C.R.B.A.), University of Bologna, Bologna, Italy; an

    of Bari, Ba

    Submitted for publicat

    Background. Microcirculation disturbances are es-ntial factors of preservation injury in fatty liver.wever, hepatocyte injury is alsomarkedly excessivefatty liver resulting, at least in part, from energytabolism impairment and oxidative stress. Thus,is study aimed to determine whether nutritional sta-s influences preservation injury in fatty liver andether energetic substrate supplementation, alonewith a vasodilator, is protective.Materials and methods. Normal or fatty livers in-ced by a choline-deficient diet were isolated fromand fasted rats, preserved in University of Wiscon-solution at 4C for 18 h, and then reperfused with

    ebsHenseleit solution at 37C for 120 min. Fastedts with fatty liver were also treated as follows: (1)ucose supplementation: rats had access to a glucoselution for 18 h prior procurement; (2) ProstaglandinG): alprostadil was continuously infused duringperfusion; (3) Combined treatment: Glucose supple-ntation PG.Results. Fasting-induced liver injury was signifi-ntly greater in fatty than normal liver. In fatty liversm fasted rats, all treatments reduced the alanineinotransaminase release. Hepatic oxygen consump-n improved in the glucose and glucose PG groups,ver

    osa,*, Elisabetta Maiolini,* Ignazio Grattagliano,ranco Trevisani,* and Mauro Bernardi*,

    University of Bologna, Bologna, Italy; Center for BiomedicalDepartment of Internal Medicine and Public Health, UniversityItaly

    October 28, 2004

    le protective strategy and a multistep approachding vasodilators could offer further benefit by act-g on different pathogenetic mechanisms. 2005evier Inc. All rights reserved.

    Key Words: fatty liver; experimental liver transplan-tion; preservation injury; reperfusion injury; nutri-nal status; prostaglandins.


    Fatty liver is the most frequent alteration of the livernd in the general population and its prevalence

    aches about 25% of the potential donors for ortho-pic liver transplantation and living donor liver trans-antation. The presence of fatty degeneration reducese tolerance of the liver to the ischemia-reperfusionjury invariably associated with these surgical proce-res, leading to higher mortality and postoperativemplication rates. Thus, albeit the persistent short-e of donor organs, most centers do not transplanters with moderate-massive fatty infiltration [1].Impaired microcirculation has been proposed as they event in the pathogenesis of preservation-perfusion injury in steatotic livers [1]. The sinusoidal

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    191CARACENI ET AL.: PRESERVATION INJURY IN FATTY LIVERcold preservation compared to that observed in nor-al organs [5]. Several potential events have beenggested as mediators of this parenchymal cell injurycluding oxidative stress [6], mitochondrial dysfunc-n [7], and energy metabolism impairment [8].The preexisting nutritional status is a major deter-inant of hepatocyte injury associated with ischemia-perfusion [9]. In normal livers, it is universally ac-pted that fasting exacerbates warm ischemic injury, 10]. Less homogenous results have been obtained ifrmal livers are exposed to cold ischemia; when fast-g is prolonged up to 4 days, the starved livers appearpable of tolerating the transplant procedure betteran do livers obtained from fed donors [11, 12]. Nev-theless, preharvesting nutritional repletion by glu-se supplementation improves survival and livernction in both fasted and fed animals [13, 14].Conversely, the data on the effect of nutritional sta-s in fatty livers subjected to ischemia-reperfusion arery limited. We previously reported that the preexist-g nutritional status of the animal adversely affectsrm ischemia-reperfusion injury in fatty livers much

    ore than it does in normal livers [15]. A preliminaryport suggested that fasting rats with fatty liver for 4ys reduces early reperfusion injury after cold isch-ia likely by suppressing Kupffer cell function [16].wever, long-term starvation is not applicable in the

    tting of clinical transplantation and the effect of aorter period of fasting has never been tested thus, no clear data exist on the relation between fattyer, preservation injury, and nutritional status.Therefore, the aim of this study was to compare in aodel of isolated perfused rat liver the influence ofimal starvation on cold ischemia-reperfusion injuryperienced by normal and fatty liver induced by aoline-deficient diet. Furthermore, since the alter-ion of the nutritional status is a common condition oftential donors staying in intensive care units forveral days and the beneficial effect of a nutritionalpport has been already demonstrated in normal liv-s exposed to cold ischemia [13, 14], experiments wereo designed to determine whether administration ofergetic substrates, alone or with a vasodilator, isotective against preservation injury in fatty liverm fasted rats.


    Animals and Induction of Fatty Liver

    Male Sprague Dawley rats (Charles River, Calco, LC, Italy),ighing 150175 g, were allowed to acclimate to the animal quar-s and were given free access to a standard chow diet and water foreek. Then, rats were fed a choline-deficient diet (Dyets Inc.,

    thlehem, PA) for an additional 5 days. With this procedure, theer of rats fed the choline-deficient diet presented a moderateatosis, predominantly macrovesicular and involving about 50-60%h an increased molar percentage of palmitic and oleic acids as iturs in fatty liver developing in humans for an excessive dietaryake of carbohydrates [17]. Prior to sacrifice, the animal weights similar in the two groups ranging between 230 and 280 g.All procedures involving rats were conducted according to theidelines for the care and use of laboratory animals approved by ourtitution.

    Procurement and Preservation of the Liver

    The rats were anesthetized with enflurane (Abbott Laboratories,icago, IL); the abdomen was opened with a midline incision andportal vein was cannulated with a 16-G angiocath. The liver was

    shed out with 20 ml of the University of Wisconsin (UW) solution4C and then immediately removed. After 18 h of preservation inUW solution at 4C, all livers were exposed to room temperature

    a Petri dish for 15 min prior to reperfusion to mimic the period ofarming during surgical implantation in vivo. The organs weren rinsed with 10 ml of a KrebsHenseleit bicarbonate (KHB)ution and connected to the perfusion circuit.

    Isolated Perfused Rat Liver Model

    The livers were perfused in a non-recirculating fashion by using aistaltic pump for 120 min at 37C with a KHB buffer containingM glucose and 0.5% serum bovine albumin within a thermostat-

    lly controlled plexiglas cabinet designed to perform two perfusionsultaneously (Ditta Disa, Milan, Italy) [8]. The KHB solution wasosed to a gas mixture of 95% O2 and 5% CO2 in a pressuredmbrane oxygenator, which generated a partial pressure of O2ater than 500 mm Hg. The organs were initially perfused at a flowe of 0.5 ml/min/g liver and then increased progressively up to 3.0/min/g liver in a 5-min period to avoid barotrauma. The portalssure was continuously monitored through a three-way stopcockced before the portal vein cannula and expressed as cm H2O.mples were taken from a portal inflow port and from the effluentfusate for measurement of the alanine aminotransaminase (ALT)el and oxygen consumption. In most of the experiments, liversue was also collected for the biochemical determinations beforelantation (basal conditions) and at the end of reperfusion.

    Study Design

    The day before the experiment, both the rats with fatty liver andrats with normal liver were further divided as follows: (a) rats

    h free access to food and water until the time of surgery (fedups) (n 68); and (b) rats with access only to water for the 18 hor surgery (fasted groups) (n 8).Besides the group receiving no treatment, fasted rats with fattyer were also subjected to three different treatment modalities andordingly divided: (1) Glucose supplemented group: fasted rats hade access to a 40% glucose oral solution for the 18 h prior procure-nt (n 8); (2) Prostaglandin (PG) group: the livers explanted fromted rats were infused with alprostadil (Prostin VR, Upjohn-armacia, Puurs, Belgium), a PGE1 analog, during the 120 min oferfusion at the rate of 1 g/kg rat/min (n 8); (3) Combinedatment group: fasted rats had access to a 40% glucose oral solutionthe 18 h prior procurement and the explanted livers were reper-ed with alprostadil during the 120 min of reperfusion at the rateg/kg rat/min (n 6). The experimental design is summarized in

    . 1.

    Liver Oxygen Consumption

    The perfusate was sampled from a portal inflow port and from antflow port during reperfusion to determine the oxygen concentra-n with a blood gas analyzer (ABL 5, ABL Instruments, Copenha-

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    192 JOURNAL OF SURGICAL RESEARCH: VOL. 127, NO. 2, AUGUST 2005, Denmark). Rates of oxygen consumption were calculated fromuent minus effluent concentration differences, liver wet weight,

    d flow rate.

    Biochemical Methods

    T Release

    The effluent perfusate from the liver was collected during reper-ion for measurement of the ALT levels using a commerciallyilable kit test (Sigma-Aldrich, Milan, Italy).


    The preischemic hepatic glycogen content was quantified usingmethod of Keppler and Decker [18] and expressed as mg/g DNA.

    e liver DNA concentration was measured according the method ofneider and Greco [19].

    iobarbituric Acid-Reactive Substances (TBARs)

    Approximately 100 mg of liver tissue was homogenized in 5 vol of0 mM KCl, 50 mM Tris HCl, and 10 mM EDTA (pH 7.4) containing2% (w/v) butylated hydroxytoluene. Then, the homogenate wascipitated with 10% trichloroacetic acid and the supernatant wasubated at 100C for 45 min with an equal volume of 0.67% thio-rbituric acid. After cooling, the supernatant was extracted with 1of n-butanol and the absorption peak was measured at 532 nm].

    otein Carbonyl (PC)

    Aliquots of liver tissue homogenates containing approximately 2of proteins were incubated for 60 min at room temperature withl of 2% dinitrophenylhydrazine (DNPH) in 2 N HCl or 1 ml of 2Cl as control blank. Next, proteins were precipitated with 200 l

    50% TCA and washed three times with 1:1 (w:v) ethanol:ethylac-te and three times with 10% TCA. The final precipitate was solved6 M guanidine and the spectrum of the DNPH versus HCl controlss followed at 350375 nm [21]. The concentration of carbonylups was then calculated using 21.5 nM1 cm1 as the extinctionfficient for aliphatic hydrazones.

    tal Protein

    Total protein concentration in liver homogenate was determinedthe method of Lowry [22]. The protein concentration expressed asprotein/g of liver homogenate did not differ significantly betweenexperimental groups.

    IG. 1. Flow diagram of the experimental design. PG alpros-il during reperfusion.All chemicals were purchased from Sigma-Aldrich.

    Statistical Analysis

    The values of the ALT release, oxygen consumption, and portalssure resulted normally distributed as assessed by thelmogorovSmirnov test and were reported as mean SD. Theference between groups was calculated by using a two-wayOVA for repeated samples. The data obtained for the liver glyco-content and the oxidative parameters did not result normally

    tributed and were reported as median range. Statistically sig-cant differences between the two groups were determined by thelcoxon rank sum test. Statistical analysis was performed by run-g the SPSS Version 10.0 for Windows package on a personalputer. A P value less than 0.05 was considered to be significant.


    Preischemic Hepatic Glycogen Content

    Under fed conditions, the preischemic hepatic con-nt of glycogen was significantly lower in fatty thanrmal rats (median: 1.62; range: 1.002.10 mg/gA versus median: 1.07; range: 0.501.59 mg/gA; P 0.05). As expected, in all of the experimental

    oups exposed to 18 h of fasting, the glycogen storesre almost completely depleted. Nutritional supple-

    entation of fasted rats with fatty liver restored ap-oximately 60% of the glycogen stores measured un-r fed conditions in the group supplemented with oralcose alone (median: 0.62; range: 0.580.69 mg/gA) and in the group supplemented with glucose andated with PG (median: 0.63; range 0.520.72 mg/gA) (Fig. 2).

    Effect of Fasting on Preservation-Reperfusion Injury inNormal and Fatty Liver

    Under fed conditions, as expected, the ALT releases greater in fatty than in normal livers throughoutof the reperfusion time (at 30 min: 236 71 versus

    IG. 2. Preischemic hepatic glycogen content in the differenterimental groups (n 46). G: glucose supplementation prior

    rvesting; PG: alprostadil during reperfusion. Fatty fed versus nor-l fed P 0.05. Fasted versus fed...


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