Original Contribution

MINOR ROLE OF OXIDATIVE STRESS DURING INTERMEDIATE PHASEOF ACUTE PANCREATITIS IN RATS

PETER KRUSE,* M ARY E. ANDERSON,†‡,1 and STEFFEN LOFT§

*Department of Pharmacology, University of Copenhagen, Copenhagen, Denmark;†Department of Biochemistry, CornellUniversity Medical College, New York, NY, USA;‡Department of Microbiology and Molecular Cell Sciences, The University of

Memphis, Memphis, TN, USA; and§Institute of Public Health, University of Copenhagen, Copenhagen, Denmark

(Received30 August2000;Accepted26 October2000)

Abstract—Reactive oxygen species have been implicated in the pathogenesis of acute pancreatitis. Few studies havefocused on the loss of endogenous antioxidants and molecular oxidative damage. Two acute pancreatitis models in rats;taurocholate (3% intraductal infusion) and cerulein (10mg/kg/h), were used to study markers of oxidative stress:Glutathione, ascorbic acid, and their oxidized forms (glutathione disulfide and dehydroascorbic acid), malondialdehyde,and 4-hydroxynoneal in plasma and pancreas, as well as 7-hydro-8-oxo-29-deoxyguanosine in pancreas. In both models,pancreatic glutathione depleted by 36–46% and pancreatic ascorbic acid depleted by 36–40% (p , .05). In thetaurocholate model, plasma glutathione was depleted by 34% (p , .05), but there were no significant changes inplasma ascorbic acid or in plasma and pancreas dehydroascorbic acid, malondialdehyde, and 4-hydroxynoneal, and nosignificant changes in the pancreas glutathione disulfide/glutathione ratio. While pancreas glutathione disulfide/glutathione ratio increased in the cerulein model, there were no significant changes in plasma glutathione, plasma, orpancreas ascorbic acid, dehydroascorbic acid, 4-hydroxynoneal, and malondialdehyde, or in pancreas 7-hydro-8-oxo-29-deoxyguanosine. Reactive oxygen species have a minor role in the intermediate stages of pancreatitismodels. © 2001 Elsevier Science Inc.

Keywords—Acute pancreatitis, Animal models, Oxidative stress, Glutathione, Ascorbic acid, 8-oxodG, Free radicals

INTRODUCTION

Experimental studies by Sanfey [1] suggested a possibleinvolvement of reactive oxygen species in acute pancre-atitis. This notion has been further investigated by theuse of three main experimental settings: (i) direct mea-surement of reactive oxygen species with electron spinresonance or chemiluminescence techniques [2,3]; (ii)intervention with scavenger treatment primarily by ad-ministration of pretreatments [4,5]; and (iii) use of mark-ers of oxidative stress, such as antioxidant levels andlipid peroxidation levels [6–8].

Although many approaches have been used to study theinvolvement of reactive oxygen species in acute pancreati-tis, the first approach, direct detection of reactive oxygenspecies in vivo, still have technical problems. For example,

electron spin resonance techniques need advanced and ex-pensive machinery and require the use of spin trappingagents that may interfere with biological systems beingstudied. Moreover, secondary molecular and cellular eventsinvolved in the pathogenesis are not addressed with thosemodels. In the second approach, the scavenger interventionstudies rely on the specificity of the administered scavenger.Thus, to obtain clear results, the scavenger should have noother biological effect than the specific scavenging one onthe treated animal; however, this is difficult to achieve inpractice. The third approach, and perhaps the best way todetermine an involvement of reactive oxygen species, as-sesses the loss of endogenous antioxidants and the increasein markers of oxidative damage, such as lipid peroxidation.

Few human clinical studies are available. Heightenedoxidative stress was shown in one study assessing superox-ide radicals, ascorbic acid, dehydroascorbic acid, tocoph-erol, and lipid peroxidation [9]. But in a human model ofERCP-induced acute pancreatitis, the prophylactic use of ascavenger sodium selenite did not show any beneficial

Address correspondence to: Peter Kruse, M.D., Ph.D., University ofCopenhagen, Department of Pharmacology, Stavangergade 3, 3. th,DK-2100 Copenhagen, Denmark; E-Mail: peterkruse@dadlnet.dk.

1Visiting guest professor.

Free Radical Biology & Medicine, Vol. 30, No. 3, pp. 309–317, 2001Copyright © 2001 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/01/$–see front matter

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effect on the clinical outcome [10]. In a recent and exten-sive review, the available data on oxidative stress in acutepancreatitis from experimental models and from clinicalstudies have been compared. Free radicals seem to beinvolved, although efficient antioxidant treatment to estab-lished acute pancreatitis has proven useless [11]. Therefore,the current study seeks to deepen our understanding of therole, if any, of oxidative stress in acute pancreatitis. Severeacute pancreatitis has a known high mortality and a lack oftherapeutic modalities. This suggests that more understand-ing about what happens during the intermediate timecourse, that is, the clinical relevant time where the acutepancreatitis is just established, may lead to sufficient ther-apy.

The two physiologically most important nonproteinantioxidants are glutathione and ascorbic acid [12,13].Glutathione is a major contributor to the intracellularreducing environment and acts as a scavenger of hydro-gen peroxide and other peroxides [14]. Ascorbic acid, orvitamin C, found in the cytosol, plasma, and other bodyfluids, [15] can scavenge oxygen-, nitrogen- and sulphur-centered radicals [16]. Glutathione metabolism is closelyrelated to the metabolism of ascorbic acid, and bothsubstances can spare the other [17,18]. When the levelsof reactive oxygen species exceed the capacity of thedefense systems, oxidative stress occurs with possibledamage to cellular components such as lipids, proteins,and DNA [19]. Markers of lipid peroxidation includemalondialdehyde and 4-hydroxynoneal, [20] whereas7-hydro-8-oxo-29-deoxyguanosine (8-oxodG) is the mostcommonly used marker of DNA oxidation [21]. Thesemarkers are increased by oxidative stress in the pancreas[22] and are found in the human pancreas [23].

In order to assess the role of oxidative stress in acutepancreatitis we monitored several markers in two modelsin rats. Taurocholate induces a fast developing necrotiz-ing acute pancreatitis and cerulein induces a more slowlyprogressing edematous acute pancreatitis [24,25]. Theworking hypothesis was that induction of acute pancre-atitis would lead to an intermediate depletion of ascorbicacid and glutathione, a simultaneous rise in the oxidizedforms ascorbic acid and glutathione, followed by anincrease in malondialdehyde and 4-hydroxynoneal inboth plasma and in tissue. Furthermore, in the ceruleinmodel we expected an increase of 8-oxodG levels in thepancreas over time.

MATERIALS AND METHODS

Animals, anesthesia and analgesia

Adult male Wistar rats (Pan: WIST) were housed oneto a cage (Makrololen, Type III) on aspen bedding(Tapvei). They were fed standard pelleted rat diet (Al-

tromin 1314, Copenhagen, Denmark), subjected to reg-ular 12 h light-dark cycles, the air was changed 12–14times/h, the room temperature was 21–23°C, and therelative humidity was 45–70%. The rats were fasted for12 h before the operation, but had free access to water.The animals were anaesthetized with halothane/N2O/O2

(1.5%/50%/50%). At the end of each operation the ani-mals received buprenorphine subcutaneously (Anorfin,GEA, Copenhagen, Denmark); 0.2 mg/kg body weight,repeatedly if necessary, to relieve postoperative pain.During anesthesia the rats were placed supinely on aheating pad (body temperature: 38.06 0.5°C) beforeinduction of acute pancreatitis. The experimental proce-dures employed conform to the principles and practice ofthe Danish law regulating experiments on animals:Lovom dyreforsogdated June 30, 1993. As a member of theEuropean Union, Denmark is bound by Directive 86/609/EEC dated November 24, 1986.

Induction of acute pancreatitis

Taurocholate model. Through a midline incision, thepancreatico biliary duct was cannulated transduodenally(24 G Neoflon, Ohmeda, Sweden). A micro vascularclamp (20 g/mm2 clamp pressure) was placed on the ductat the hilum of the liver. A microtube clamp (ModifiedAcland clamp, TC-1, S & T, Switzerland) was placedaround the cannula and the wall of the duct, close to theduodenum, to prevent back flow. Taurocholate 3% inisotonic saline (0.1 ml/100 g body weight, taurocholicacid, sodium salt, Sigma T-0750), was infused into thepancreatico biliary duct by means of a pressure con-trolled infusion pump. This microprocessor controlledpump was equipped with an actuator unit carrying a 1 mlsyringe, a connection to a Baxter Uniflow pressure trans-ducer and a tube system connecting the syringe with theinfusion catheter. The pump allowed infusion of tauro-cholate into the pancreatico biliary duct with a steadymaximal pressure of 28 cm H2O corresponding to phys-iologic pressure. The cannula and the clamps were re-moved, thus leaving the pancreatico biliary duct intact.The duodenal perforation was closed with a 6-0-nylonpurse string suture and the abdominal wall with a 4-0-nylon suture.

Cerulein model. Cerulein was purchased from SigmaChemicals, St. Louis, MO, USA (C-9026) and dissolvedin physiological saline adjusted to pH 7.4 with ammo-nium hydroxide 0.01 M. For administration, a 24 hmini-osmotic pump (Alzet, CA, USA, Model 2001D)was attached to a polyethylene tube with an inner diam-eter of 0.38 mm and an outer diameter of 0.76 mm(Tygon, Norton Plastics, Akron, OH, USA) and weighedto control correct output. The pumps were filled using

310 P. KRUSE et al.

sterile techniques and presoaked in physiologic saline 3 hbefore insertion. Under anesthesia a prefilled and pre-soaked pump was placed subcutaneously in the midcapu-lar region and the catheter was tunneled subcutaneouslyand inserted approximately 2 cm into the vein with thetip placed just above the heart. The skin incisions wereclosed using nylon 4-0 suture.

Experimental design

Taurocholate model. Sixty male Wistar rats, weighing216–265 g, were randomly allocated to three groups: (i)Controls: sham operated, that is, laparotomy followed bycanullation of the pancreatico biliary duct without anyinfusion; (ii) Saline controls: As in (i), but with pressurecontrolled injection of saline into pancreatico biliaryduct; and (iii) Taurocholate: As in (i), but with pressurecontrolled injection of 3% taurocholate into the pancre-atico biliary duct. At 0, 2, 4, and 6 h for the controls, andat 2, 4, and 6 h for the saline controls and the tauro-cholate group, six rats from each group were sacrificedfor sampling of blood and tissue. Due to surgical com-plications (perforation of the pancreatico biliary duct andleakage from the duodenal puncture), two rats in thecontrol group, one at 0 and one at 6 h, were excludedbefore data processing.

Cerulein model. Forty-three male Wistar rats, weighing232–275 g, were randomized into two groups: (i) Con-trols: IV infusion of saline; (ii) Cerulein: IV infusion ofcerulein (10mg/kg/h). At 0, 4, 6, and 12 h for the controlgroup and at 4, 6 and 12 h for the cerulein group, six ratsfrom each group were sacrificed for sampling of bloodand tissue. The tubing fell out of one rat in the ceruleingroup; thus, a new rat was added to this group beforedata processing. For measuring base line values of 8-ox-odG, an additional seven rats were added to the controlgroup (at 0 h). Thus for the assessment of 8-oxodG, thecontrol group consisted of 9, 4, 3, and 6 rats at the timepoints 0, 4, 6, and 12 h respectively, and the ceruleingroup consisted of 5, 4, and 6 rats at the time points 4, 6,and 12 h respectively.

Sample collection. Blood was drawn from the left ven-tricle of the heart in vacuum glass containers with EDTAor heparin (Venoject). The samples were quickly centri-fuged (10,0003 g, 1.5 min, 4°C), and plasma wasderivatized according to analyses described below andimmediately stored at minus 70°C. The pancreas wasquickly removed, freed from fat and lymph nodes, rinsedin ice-cold physiologic saline, blotted dry, and freezeclamped in liquid nitrogen. The whole pancreas was thenpowdered using a mortar and pestle on dry ice andimmediately stored at minus 70°C.

Laboratory determinations

Glutathione/glutathione disulfide. EDTA-treated plasmawas quickly acidified with 5-sulfosalicylic acid 10%(w/v) containing 0.3 mM diethylenetriamine-dipentace-tic acid (2 parts plasma and 1 part sulfosalicylic acid),vortexed well, and centrifuged (10,0003 g, 5 min, 4°C).The supernatant was divided in two portions (one wasderivatized with 2-vinylpyridine for glutathione disulfidemeasurement), and both portions were immediatelystored at minus 70°C. Pancreatic powder was homoge-nized in 5-sulfosalicylic acid 5% (w/v) containing 0.3mM diethylenetriamine-dipentacetic acid using an icedteflon Potter-Elvehjem homogenizer (0.2 g tissue/ml).After centrifugation (10,0003 g, 10 min, 4°C), thesupernatant was collected, divided in two portions (onewas derivatized with 2-vinylpyridine for glutathione di-sulfide measurement), and both were immediately storedat minus 70°C. Plasma and pancreatic total glutathione,that is, glutathione1 glutathione disulfide, and glutathi-one disulfide (both in glutathione equivalents) were de-termined spectrophotometrically (UV-2100, UV-VIS,Shimadzu Corp., Kyoto, Japan) using the 5,59-dithio-bis(2-nitrobenzoic acid), DTNB-glutathione disulfide re-ductase recycling assay for glutathione, and for glutathi-one disulfide using 2-vinylpyridine for the glutathionedisulfide determination [26].

Ascorbic acid/dehydroascorbic acid. EDTA-treatedplasma was quickly stabilized with precooled 10% (w/v)metaphosphoric acid, centrifuged (40003 g, 10 min,4°C) and stored at270°C. Pancreatic powder was ho-mogenized with a glass/glass homogenizer in pre-cooled5% metaphosphoric acid (0.05 g tissue/ml), centrifuged(4000 3 g, 10 min, 4°C) and stored at minus 70°C.Ascorbic acid and dehydroascorbic acid were measuredby HPLC [27].

Malondialdehyde and 4-hydroxynoneal. EDTA-treatedplasma was immediately stored at minus 70°C. Pancre-atic powder was homogenized in ice-cold 20 mM Tris-HCL buffer, pH 7.4 with a Potter-Elvehjem homogenizer(0.1 g tissue/ml), centrifuged (30003 g, 10 min, 4°C)and the supernatant was stored at minus 70°C. Malondi-aldehyde and 4-hydroxynoneal was measured spectro-photometrically using a commercial kit (Bioxytech LPO-586, Oxis International, Portland, OR, USA); achromogenic agent 1-methyl-2 phenylindole, reacts withmalondialdehyde and/or 4-hydroxynoneal to produce astable chromophore with maximal absorbance at 586 nm(UV-2100, UV-VIS, Shimadzu Corp.).

8-oxodG and deoxyguanosine. Pancreas tissue was ho-mogenized in HEPES buffer (5 mM HEPES, 90 mM

311Minor role of oxidative stress during intermediate phase of acute pancreatitis

sucrose, 250 mM manitol, pH 7.4). The amount of 8-ox-odG and deoxyguanosine were measured using HPLCwith an electrochemical detector and UV after DNAextraction and enzymatic hydrolysis as previously de-scribed [28].

Pancreatitis indicators. Plasma was analyzed for lipaseanda-amylase activities by two commercial kits (LipaseMPR1 and a-amylase EPS, Boehringer/Mannheim,Mannheim, Germany) with a recording spectrophotom-eter (UV-2100, UV-VIS, Shimadzu Corp.). Peritonealexudate was carefully collected by absorbent cotton andweighed.

Pancreatic protein content. Protein in pancreas was mea-sured by using the Lowry method with bovine serumalbumin as standard [29].

Statistical analysis

The data was expressed as means6 SD. To approx-imate a normal distribution, a log10-transformation wasperformed. Homogeneity of variances was tested usingLevenes Test. Differences between the groups weretested using a two-way analysis of variance (2-way-ANOVA); In the taurocholate model differences be-tween Controls (2–6 h). Saline (2–6 h) and Taurocholate(2–6 h) and in the cerulein model differences betweenControls (4–12 h) and Cerulein (4–12 h) were testedusing 2-way-ANOVA in a complete design. Correlationwas expressed by the correlation coefficient (Pearsonr).In all instances, ap-value less than .05 was consideredstatistically significant. All data handling and statisticswere performed using the statistical software packageSTATISTICA 5.0 for Windows, StatSoft, Inc., 1996.

RESULTS

Taurocholate model

All 60 rats survived the observation period. All therats in the taurocholate group developed macroscopicand enzymatic signs of acute pancreatitis with significantincreases in plasmaa-amylase and lipase activities, aswell as significant increases in peritoneal exudate within2 h (p , .05, Table 1). Protein concentration (mg/gtissue) of the pancreas decreased in the taurocholategroup to 0.74–0.88 times that of the control group (p ,.001).

Markers of oxidative stress in the taurocholate modelare shown in Table 2. Plasma glutathione in the tauro-cholate group decreased significantly during the 6 hobservation time (0.66 times that of controls and 0.78times that of saline controls,p 5 .04), while there wasa trend to increases in pancreatic-glutathione disulfide(as a percentage of total glutathione) in the taurocholategroup (p 5 .06). Total glutathione in the pancreas,when related to wet weight, was significantly lower inthe taurocholate group (p , .001).After correcting forthe substantial edema by expressing the total glutathionein the pancreas per mg of protein, the taurocholate groupstill had significantly lower glutathione values than thecontrol group (p 5 .004), forexample, at 2 h the levelwas 0.74 times that of controls.

Total plasma ascorbic acid was not significantly dif-ferent in the taurocholate group compared with the othertwo groups (p 5 .14). Plasma dehydroascorbic acid, asa percentage of total ascorbic acid, did not show signif-icant differences between the groups (p 5 .34, data notshown). Total ascorbic acid in the pancreas, per g wetweight, was significantly lower in the taurocholate groupcompared with the other two groups (p 5 .011), forexample, at 6 h the levels were 0.76 and 0.75 times thatof no infusion controls and saline controls, respectively.

Table 1. Taurocholate Model: Pancreatitis Markers

CON group SAL group TAU group

P0 h 2 h 4 h 6 h 2 h 4 h 6 h 2 h 4 h 6 h

P-Lipase(U/l 3 100)

0.046 0.02 0.156 0.18 0.096 0.08 0.136 0.13 0.316 0.40 0.136 0.12 0.586 0.81 446 30 606 31 746 27 †

P-amylase(U/l 3 1000)

3.76 0.2 4.06 2.0 3.86 1.3 3.56 1.1 6.16 3.2 4.76 1.6 8.06 2.9 106 2.9 176 7.7 216 6.1 †

Peritoneal exudate(g)

0.26 0.1 0.96 0.2 1.66 2.6 0.56 0.3 0.76 0.2 0.56 0.3 1.36 1.2 2.26 0.9 3.46 2.5 1.86 0.9 †

Pan-proteinconcentration(mg/g)

1556 18 1346 26 1646 18 1656 24 1436 37 1706 16 1466 27 1236 25 1226 16 1296 22 ‡

Results are given as means6 SD. CON5 Controls; SAL5 Saline Controls; TAU5 Taurocholate; P5 plasma; Pan5 pancreas. Differencesbetween groups: Controls (2–6 h); Saline (2–6 h), and Taurocholate (2–6 h) were tested by 2-way ANOVA with significance levels as indicated:† p , .05, ‡ p , .001. n 5 5 in control 0 h and 6 h,n 5 6 in the rest of the groups.

312 P. KRUSE et al.

The pancreas dehydroascorbic acid, as a percentage oftotal ascorbic acid, showed no significant differencesbetween the groups (p 5 .07, data not shown).

Malondialdehyde and 4-hydroxynoneal in plasma didnot differ significantly in the taurocholate group com-pared with the other two groups (p 5 .08 andp 5 .31,respectively, data not shown). Malondialdehyde1 4-hy-droxynoneal in pancreatic tissue per mg protein wassignificantly lower in the taurocholate group when com-pared to the other two groups (p 5 .021).

Correlation between antioxidants was done by pool-ing the time points of the taurocholate groups and thecontrols att 5 0 h (n 5 23). Inverse correlation wasfound between plasma-ascorbic acid and glutathione inthe pancreas (r 5 20.51, p , .05) and betweenpancreas glutathione levels per mg protein and glutathi-one disulfide ratio in pancreas (r 5 20.67, p , .05).No correlations were found between any of the otherantioxidants in the taurocholate group.

Cerulein model

All 43 rats survived the observation period. In thecerulein group, all rats developed macroscopic and en-

zymatic signs of acute pancreatitis with significant in-creases in plasmaa-amylase and lipase activities inplasma, as well as in peritoneal exudate within 2 h (p ,.0001).Pancreas protein concentration decreased signif-icantly in the cerulein group (e.g., to 0.56 times at 12 h)compared with the control group (p , .0001,Table 3).

Markers of oxidative stress in the cerulein model areshown in Table 4. Plasma-total-glutathione was slightlylower, but not significantly different in the cerulein groupcompared with the control group (p 5 .06). However,pancreatic-glutathione disulfide (as a percentage of totalglutathione) increased 1.9 times in the cerulein group at4 h and 3.4 times at 12 h when compared with the controlgroup (p , .0001). Total-glutathione in pancreas tis-sue, when related to wet weight, was significantly lowerin the cerulein group compared with the control group( p , .0001); 0.34 times that of controls at 4 h. Whenpancreatic total glutathione levels were related to mg ofprotein, the values in the cerulein group were still sig-nificantly lower than the values in the control group( p , .0001);0.54 times and 0.71 times that of controlsat 4 and 6 h, respectively.

Plasma-total-ascorbic acid decreased significantly

Table 2. Taurocholate Model: Markers of Oxidative Stress

CON group SAL group TAU group

P0 h 2 h 4 h 6 h 2 h 4 h 6 h 2 h 4 h 6 h

P-total GSH (mM) 15.96 3.1 17.26 6.9 18.96 5.3 18.96 5.7 17.66 2.1 15.16 3.2 15.96 2.0 14.76 3.5 13.46 5.1 12.66 2.5 †

Pan-GSSG(% of total GSH)

3.06 1.0 2.46 0.9 1.96 0.6 2.06 0.3 1.96 0.9 2.86 2.1 1.76 0.5 3.26 1.8 4.66 3.9 2.26 0.4 ns

Pan-total GSH(mmol/g tissue)

2.06 0.2 1.56 0.3 1.96 0.4 2.06 0.3 1.76 0.5 1.46 0.3 1.56 0.5 0.96 0.3 0.96 0.4 1.26 0.3 ‡

Pan-total GSH(nmol/mg protein)

13.06 1.4 11.06 2.4 11.36 1.8 12.16 2.7 11.66 2.5 8.46 2.2 10.06 3.0 8.26 3.4 7.76 3.0 9.16 2.4 †

P-total AA (mM) 85 6 8 1006 40 936 18 1086 50 1246 31 1276 54 1036 33 1396 28 1226 21 976 24 nsPan-total AA

(nmol/g tissue)3326 108 3466 84 3946 93 4166 71 3846 64 4026 33 3896 92 3426 111 3036 93 3126 59 †

Pan-MDA 1 4HNE(pmol/mg protein)

2026 65 2806 36 2506 130 3456 160 2496 31 2266 162 3006 45 1636 126 996 22 2446 21 †

Results are given as means6 SD. CON5 Controls; SAL5 Saline controls; TAU5 Taurocholate; P5 plasma; Pan5 pancreas; AA5 ascorbicacid; MDA 5 malondialdehyde; 4-HNE5 4-hydroxynoneal; GSH5 glutathione; GSSG5 glutathione disulfide. Differences between groups:Controls (2–6 h). Saline (2–6 h), and Taurocholate (2–6 h) were tested by 2-way ANOVA with significance levels as indicated:† p , .05, ‡ p ,.001, ns5 not significant.n 5 5 in control 0 h and 6 h,n 5 6 in the rest of the groups.

Table 3. Cerulein Model: Pancreatitis Markers

CON group CER group

P0 h 4 h 6 h 12 h 4 h 6 h 12 h

P-Lipase (U/l3 100) 0.16 0.1 0.36 0.4 0.16 0.1 0.96 0.7 686 14 886 17 1176 7 §

P-amylase (U/l3 1000) 4.66 2.4 6.76 3.1 3.66 1.0 4.76 0.8 346 7 546 11 1306 21 §

Peritoneal exudate (g) 0.16 0.1 0.16 0.04 0.16 0.1 0.36 0.1 0.46 0.2 0.76 0.3 1.56 0.9 §

Pan-protein concentration (mg/g tissue) 1716 21 1706 25 1666 21 1586 27 1176 24 916 13 896 9 §

Results are given as means6 SD. CON5 Controls; CER5 Cerulein; P5 plasma; Pan5 pancreas. Differences between groups: Controls (4–12h) and Cerulein (4–12 h) were tested by 2-way ANOVA with significance levels as indicated:§ p , .0001.n 5 6 in all groups.

313Minor role of oxidative stress during intermediate phase of acute pancreatitis

over time in the control group of the cerulein model andwas significantly lower than in the cerulein-treated rats( p , .0001).Plasma-dehydroascorbic acid showed nosignificant difference between the groups (p 5 .86,datanot shown). Total-ascorbic acid in the pancreas, whenrelated to wet weight, was significantly lower in thecerulein group as compared with the control group (p ,.0001). At 6 h ascorbic acid in the pancreas was 0.6times lower in the cerulein group compared to the con-trols. Dehydroascorbic acid in the pancreas was notsignificantly different between the groups (p 5 .13,datanot shown).

In plasma, the levels of malondialdehyde and 4-hy-droxynoneal were not significantly different from thelevels in the control group (p 5 0.91 andp 5 0.34,respectively; data not shown). Malondialdehyde1 4-hy-droxynoneal in pancreatic tissue per mg protein, showedno difference when comparing the cerulein group to thecontrol group (p 5 0.91).

Correlation between antioxidants was done by pool-ing the time points of the cerulein groups with the controlgroup at t 5 0 (n 5 24). Correlations were foundbetween plasma-glutathione and ascorbic acid levels inpancreas per g tissue (r 5 0.62,p , .05) andbetweenpancreas glutathione per mg protein and pancreas ascor-bic acid in mg protein (r 5 0.57,p , .05). Glutathionedisulfide (as a percentage of total glutathione) in thepancreas correlated inversely with pancreas ascorbic acidlevels per g tissue (r 5 20.56, p , .05). No othersignificant correlations between any of the other antioxi-dants were found.

The 8-oxodG/deoxyguanosine ratio in pancreatic tis-sue was not significantly different in the cerulein groupcompared with the control group (p 5 .93). Theaver-age 8-oxodG/deoxyguanosine ratio was 0.856 0.368-oxodG/105 deoxyguanosine (Mean6 SD, Table 4).

DISCUSSION

Our current study in two models of acute pancreatitisshows modest depletion of pancreatic glutathione andascorbic acid with increases in glutathione oxidation.The reductions in antioxidant levels were concomitantwith overt signs of pancreatitis such as increases inlipase, amylase, edema of pancreas, and peritoneal exu-date. However, there was no increase in markers ofoxidation of lipids and DNA at 2 h after induction ofpancreatitis, suggesting that reactive oxygen species playa minor role in the intermediate phase of acute pancre-atitis.

The reduction in total glutathione was smaller thanpreviously reported, especially when calculated per mgprotein. Previous studies indicated a reduction of 70–80% glutathione per mg/g wet weight in either the tau-rocholate [7] or the cerulein model [30–32]. Consistentwith previous studies, our work shows that acute pan-creatitis produces dramatic pancreatic edema with time,leading to a concomitant decrease in protein concentra-tion (Tables 1 and 3). Thus, glutathione or other sub-stances not freely available from, for example, plasmamay show a decrease in concentration during acute pan-creatitis edema when expressed per g wet weight.Changes due to acute edema may consist of both in-creased water and unspecific proteins in the injured tis-sue. Thus, when pancreatic glutathione is reported permg protein, as in this study, it may only partially com-pensate for the changes due to edema. A better way ofreporting compounds such as glutathione is by relatingthem to DNA content in the tissue.

Previous studies using the taurocholate model of acutepancreatitis have shown that there is a very rapid (0.5 h)and large (52%) decrease in pancreatic glutathione levels(per mg protein) as well as an increase in the glutathionedisulfide/glutathione ratio from 2.7–13.8% after 3.5 h

Table 4. Cerulein Model: Markers of Oxidative Stress

CON group CER group

P0 h 4 h 6 h 12 h 4 h 6 h 12 h

P-total GSH (mM) 19.96 1.3 19.86 3.8 19.66 3.4 16.76 1.6 17.06 4.0 17.06 1.9 16.36 2.9 nsPan-GSSG (% of total GSH) 1.56 0.2 1.86 0.4 1.96 0.3 1.66 0.2 3.66 1.7 3.66 1.4 7.36 2.2 §

Pan-total GSH (mmol/g tissue) 1.96 0.4 2.36 0.6 2.06 0.3 2.16 0.6 0.86 0.4 0.86 0.3 1.06 0.2 §

Pan-total GSH (nmol/mg protein) 11.56 2.8 13.76 2.4 12.36 2.5 13.86 3.8 7.56 3.3 8.76 2.6 11.16 2.3 §

P-total AA (mM) 75 6 14 956 11 726 12 376 8 1016 25 966 11 836 30 §

Pan-total AA (nmol/g tissue) 4436 102 4296 31 4456 96 4086 68 2386 58 2706 62 2566 57 §

Pan-MDA 1 4HNE (pmol/mgprotein)

2196 137 1876 84 1736 34 1616 49 1696 69 1646 40 1796 37 ns

Pan-8-oxodG (/105) ' 1.36 0.5 0.86 0.2 1.16 0.4 0.86 0.7 0.86 0.2 0.86 0.4 0.96 0.4 ns

Results are given as means6 SD. CON5 Controls; SAL5 Saline; TAU5 Taurocholate; P5 plasma; Pan5 pancreas; AA5 ascorbic acid;MDA 5 malondialdehyde; 4-HNE5 4-hydroxynoneal; GSH5 glutathione; GSSG5 glutathione disulfide. Differences between groups: Controls(4–12 h) and Cerulein (4–12 h) were tested by 2-way ANOVA with significance levels as indicated:§ p , .0001, ns5 not significant.n 5 6 inall groups except in' wheren 5 9 in controls 0 h andn 5 3–6 in other groups.

314 P. KRUSE et al.

[7]. Another study reports that total sulfhydryl groupconcentrations measured both in plasma and in the pan-creas decreased after 1 h [8]. Our findings with thetaurocholate model confirm a decrease in pancreatic glu-tathione levels at 2 h, although the increase in the glu-tathione disulfide/glutathione ratio failed to reach statis-tical significance. The lack of an increase in glutathionedisulfide might be attributed to our use of a low tauro-cholate concentration; 3% taurocholate induces severebut nonlethal acute pancreatitis while the 5% tauro-cholate induces severe acute pancreatitis with 50–60%lethality. The current study also shows that plasma totalglutathione decreases modestly. These findings suggestthat while glutathione levels decrease somewhat, there isapparently limited oxidative damage as demonstrated byglutathione disulfide formation in the taurocholate modelat the times studied.

Previous studies using the cerulein model of acutepancreatitis have also shown that total glutathione levelsdecrease in the pancreas, [30–32] with a concomitantincrease in glutathione disulfide/glutathione-ratio. Inter-estingly, Luthen et al. noted no increase in pancreasglutathione disulfide [30]. By increasing the intracellularsupply of glutathione by pretreatment with glutathionemonoethyl ester in cerulein-induced pancreatitis in mice,the severity of pancreatitis was reduced, although pan-creatitis was not prevented [31]. Our study shows thattotal glutathione decreases by wet weight or by mg ofprotein and that the glutathione disulfide/glutathione ra-tio increases with time. These findings suggest that re-active oxygen species may play a role in cerulein in-duced acute pancreatitis. The decreases in pancreatictotal glutathione are greater for the cerulein model thanfor the taurocholate model, suggesting that larger de-creases in glutathione levels are needed for a change inthe glutathione disulfide/glutathione ratio. Despite thesedecreases, markers of oxidative stress are not signifi-cantly increased between 2 and 12 h. Previous studies inboth the cerulein and the taurocholate model found in-creased oxidative stress and oxidative protein modifica-tion to occur very early, within the first 1–2 h [33,34].Very early oxidative stress in acute pancreatitis mightexplain the lack of effect when treatment is initiated late,after onset of the disease [11].

Cellular glutathione levels may decrease due to anincreased use of glutathione, for example, to detoxifyreactive oxygen species enzymatically with formation ofglutathione disulfide. The glutathione disulfide/glutathi-one ratio, thiol redox, may participate in various impor-tant cellular functions, such as protein folding, cytoskel-eton integrity, and acinar stimulus-secretion coupling[32]. A high glutathione disulfide/glutathione ratio mayindicate low nicotineamide-adenine dinucleotide phos-phate (NADPH) levels because NADPH is used by glu-

tathione disulfide reductase to re-form glutathione fromglutathione disulfide. Glutathione levels may also de-crease due to the formation of glutathione S-conjuates,either enzymatically or nonenzymatically; therefore, thecellular glutathione disulfide/glutathione ratio need notnecessarily increase when glutathione is used for protec-tion.

Glutathione levels may also decrease due to insuffi-cient substrates for its synthesis. Glutathione is synthe-sized intracellularly from its constituent amino acidsusing adenosine triphosphate (ATP). Cysteine is nor-mally the limiting amino acid for the synthesis of gluta-thione, and under certain conditions ATP might also belimiting. ATP levels are depleted in cerulein (to 22%after 4 h) and taurocholate (to 10% after 0.5 h) models ofacute pancreatitis suggesting that ATP could becomelimiting for glutathione synthesis [7,30]. Neuschwander-Tetri et al. showed, using the cerulein model, that pre-treatment with an intracellular cysteine delivery drug didnot prevent pancreatic glutathione depletion, nor did itprevent histological damage of the pancreas [32]. Suchstudies, however, may be difficult to interpret because ofmultiple drug effects. In our study, in both the tauro-cholate and the cerulein model pancreatic glutathionelevels decreased, suggesting either decreased synthesisand/or increased conjugation. Such a decrease in gluta-thione levels might sensitize cells to reactive oxygenspecies damage.

This study is the first to measure ascorbic acid (vita-min C) and its oxidized form, dehydroascorbic acid, inexperimental acute pancreatitis. It was expected that ifoxidative stress and reactive oxygen species are impor-tant in experimental acute pancreatitis, then pancreaticascorbic acid would decrease and dehydroascorbic acidwould increase. We observed decreased pancreas ascor-bic acid levels in both the taurocholate and the ceruleinmodel when related to wet weight. While ascorbic acid,unlike GSH, is freely diffusible and is continuouslysupplied by the high levels in plasma, ascorbic acidlevels need not be corrected for edema in pancreatictissue. Interestingly, plasma ascorbic acid appeared toincrease after cerulein treatment, although this finding isbased on comparison with the control group that exhib-ited an unexplained large decrease in this marker withmaximum at 12 h. A cerulein-induced increase in plasmaascorbic acid may be compatible with rodent studies[35]; when glutathione levels were depleted by buthi-onine sulfoximine, liver ascorbic acid levels increased.When liver glutathione in mice was depleted by a com-bination of three different glutathione depleting agents,liver necrosis was accompanied by increased lipid per-oxidation, but only occurred when glutathione was se-verely depleted, that is, to 85–90% of control values [36].Severe depletion of glutathione in the pancreas of adult

315Minor role of oxidative stress during intermediate phase of acute pancreatitis

mice did not cause any morphological damage to thepancreas [37]. Depletion of total glutathione in rat lungtissue by 50% did not lead to structural changes of thelungs, even when exposed to lethal hyperoxia [38]. Invitro studies using human umbilical vein endothelialcells that were highly depleted (.90%) of glutathione,showed that addition of ascorbic acid either intra- orextracellularly, could protect against the toxicity of ni-trogen dioxide [39]. These results suggest that minorglutathione depletion per se does not lead to oxidativecellular damage.

Other markers of oxidative stress, malondialdehydeand 4-hydroxynoneal, were assessed in both models;8-oxodG/deoxyguanosine was assessed in the ceruleinmodel. In both the taurocholate and the cerulein modelwe found no relevant increases in malondialdehyde and4-hydroxynoneal levels. In the taurocholate model, pan-creas malondialdehyde1 4-hydroxynoneal levels werelower in the taurocholate group, suggesting that tauro-cholic acid might act as a scavenger locally in the pan-creas. As malondialdehyde and 4-hydroxynoneal wererelated to mg protein, a possible artifact due to proteinincreases during edema should be taken into consider-ation. Other cerulein model studies measuring markers oflipid peroxidation, such as malondialdehyde, conjugateddienes, and thiobarbituric acid reactive substances,showed significant increases after 3–6 h [6,40–42]. Themalondialdehyde content in rat pancreas and plasma hasbeen reported to increase from 0.4 to 1.1 nmol/mg pro-tein [7] and 0.34 to 4.0 nmol/ml, [43] respectively, wellwithin the detection limit of the malondialdehyde assay(0.1 nmol/ml) in our study. Nevertheless, neither thecerulein nor the taurocholate models in our study showany increases in either malondialdehyde or 4-hy-droxynoneal levels. These results suggest that at most,there are only mild effects of oxidative stress at the timestudied. The 8-oxodG/deoxyguanosine ratio has not pre-viously been assessed in experimental acute pancreatitis.Oxidative DNA damage may lead to an increased cancerrisk in man [44] and acute pancreatitis has been related toincreased incidence of pancreatic cancer in humans [45];therefore, oxidative DNA damage is of interest withseveral experimental studies reviewed recently [21]8-oxodG has been used as a marker of early oxidativestress after ischemia/reperfusion injury in liver and smallintestine transplantation in pigs. As early as 1–3 h afterreperfusion, the levels of 8-oxodG in urine were signif-icantly increased [46]. Chemically induced oxidativestress in rodents showed formation of 8-oxodG in kid-ney, and combined pre- and posttreatment of animalswith glutathione inhibited this DNA damage [47]. Lipidperoxidation products were shown to induce 8-oxodG inisolated DNA [48]. In our study, between 2 and 12 h afterinduction of acute pancreatitis with cerulein, there was

no elevation in the pancreatic 8-oxodG/deoxyguanosineratio. Our study with the cerulein model indicates thatthere was only oxidative DNA damage that did notexceed cellular repair capacity.

In conclusion, our study shows in two experimentalmodels of acute pancreatitis that both glutathione and ascor-bic acid levels decrease and that these changes occur 2 to12 h after initiation concomitantly with overt signs of pan-creatitis. However, during the time course of study (2–12 h)there was no increase in markers of oxidative damage toDNA or oxidation of lipids measured by 8-oxodG andmalondialdehyde1 4-hydroxynoneal, respectively. Ourfindings suggest that reactive oxygen species play a minorrole in the pathogenesis at an intermediate stage of exper-imental acute pancreatitis, which may explain the lack ofclinical effect of antioxidants in fulminate pancreatitis. It ispossible that oxidative stress markers may be increasedearlier, and precede glutathione and ascorbic acid decreases.Further studies on this complex pathology are needed andwill increase our understanding and guide the developmentof future therapies of this fatal condition.

Acknowledgements—This work is supported in part by a PHS/NIHgrant #AI31804, Novo Nordisk Foundation, King Christian X’s Foun-dation and Danish Foundation for the Advancement of Medical Sci-ence. The authors thank Anette Hauman, Anni Jensen, Anna Hansen,Sven Edelfors, and Anders Fuglsang for their technical assistance.

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ABBREVIATIONS

8-oxodG—7-hydro-8-oxo-29-deoxyguanosineATP—adenosine triphosphateNADPH—nicotineamide-adenine dinucleotide phos-

phateHPLC—High Performance Liquid Chromatography

317Minor role of oxidative stress during intermediate phase of acute pancreatitis