Gut 1993; 34: 732-737

Role of active oxygen, lipid peroxidation, andantioxidants in the pathogenesis of gastric mucosalinjury induced by indomethacin in rats

T Yoshikawa, Y Naito, A Kishi, T Tomii, T Kaneko, S linuma, H Ichikawa, M Yasuda,S Takahashi, M Kondo

AbstractThe roles of active oxygen, lipid peroxidation,and the antioxidative defence mechanism ingastric mucosal injury induced by treatmentwith indomethacin in rats were investigated.The total area of gastric erosions and concen-tration of lipid peroxides in the gastric mucosaincreased with time after administration ofindomethacin (20 mg/kg, orally). The a-tocopherol:total cholesterol ratio in serum wassignificantly decreased and the activity ofglutathione peroxidase, an important enzymeto scavenger of lipid peroxides, was inhibitedby the administration of indomethacin. Treat-ments with superoxide dismutase and catalaseinhibited the increases in gastric mucosalerosions and lipid peroxides in the gastricmucosa, and the reduction of seruma-tocopherol. Treatment with these scav-engers did not improve the decreasedglutathione peroxidase activity. These findingssuggest that active oxygen species and lipidperoxidation play an important part in thepathogenesis ofgastric mucosal injury inducedby indomethacin, and that the decreasedglutathione peroxidase activity aggravated theinjury due to accelerated accumulation ofhydrogen peroxide and lipid peroxides in thegastric mucosal cell.(Gut 1993; 34: 732-737)

role of reactive oxygen species in mediating themicrovascular disturbance that preceded gastricmucosal injury induced by several kinds of stressand ischaemia-reperfusion." 12 Furthermore,lipid peroxidation mediated by oxygen freeradicals is believed to be an important cause ofdestruction and damage to cell membranes,because polyunsaturated fatty acids of thecellular membranes are degraded by the lipidperoxidation with consequent disruption ofmembrane integrity.'3 Membrane peroxidationcan lead to changes in membrane fluidity andpermeability, enhanced rates of protein degrada-tion, and ultimately, cell lysis. We have alreadyreported that lipid peroxidation plays a signifi-cant part in the pathogenesis of gastric mucosallesions induced by water immersion restraintstress, burn shock, and ischemia-reperfusion.I'l6The present study was undertaken in rats tomeasure changes in lipid peroxides and anti-oxidants in serum samples and gastric mucosaafter administration of indomethacin. Also, weinvestigated the effects of a superoxide radicalscavenger (superoxide dismutase (SOD)), ahydrogen peroxide scavenger (catalase), and ahydroxyl radical scavenger (dimethylsulphoxide(DMSO)) on gastric mucosal injury and lipidperoxide formation induced by giving indo-methacin to rats, and the effects ofa combinationof SOD and catalase on indomethacin inducedchanges in antioxidants.

First Department ofMedicine, KyotoPrefectural University ofMedicine, Kamigyo-ku,Kyoto 602, JapanT YoshikawaY NaitoA KishiT TomiiT KanekoS IinumaH IchikawaM YasudaS TakahashiM KondoCorrespondence to:Dr T Yoshikawa, FirstDepartment of Medicine,Kyoto Prefectural Universityof Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto602, Japan.Accepted for publication28 September 1992

Indomethacin, a non-steroidal anti-inflamma-tory drug (NSAID), is known to induce erosionsand ulcers in the gastrointestinal tract.' 2Although it has been proposed that a deficiencyof endogenous prostaglandins due to inhibitionofcyclooxygenase by indomethacin is involved inthese effects, the exact pathogenic mechanismremains to be elucidated.-' Recent studiesshowed that a low dose of indomethacin oraspirin inhibited cyclooxygenase activity in thegastric mucosa of rats by 90% without causingany haemorrhagic erosions.6 Takeuchi et al7postulated that the enhanced gastric motilityinduced by indomethacin causes microcircula-tory disturbances that lead to increased micro-vascular permeability and cellular damage.Rainsford'0 also noted, with transmissionelectron microscopy, that microvascular injurywas present during gastric mucosal injury pro-duced by indomethacin. These reports sug-gested that inhibition of prostaglandin synthesiswas unlikely to be the sole mechanism respon-sible for the gastric damage induced byindomethacin.Much recent attention has been focused on the

Materials and methodsMale Sprague-Dawley rats, weight range 190-210 g from Keari Co Ltd, Osaka, were used. Theanimals were deprived of food but allowed freeaccess to water for 24 hours before the experi-ment. Gastric haemorrhagic damage wasinduced by oral administration of indomethacin(Sigma Chemical Co, St Louis, MO) at a dose of20 mg/kg, suspended in 0*5% carboxymethyl-cellulose solution with a few drops ofTween 80 ina volume of 0 5 ml/I00 g body weight. In thecontrol groups, the rats received an equivalentvolume of the vehicle.

EXPERIMENTAL PROCEDURE

Time course study ofindomethacin induced gastricmucosal injuryGroups of indomethacin treated rats were killedthree or six hours after administration of indo-methacin. Animals were killed by exsanguina-tion via the abdominal aorta under inhaled etheranaesthesia. The stomachs were removed,

732

on April 7, 2021 by guest. P

rotected by copyright.http://gut.bm

j.com/

Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. D

ownloaded from

http://gut.bmj.com/

Role ofactive oxygen, lipid peroxidation, and antioxidants in the pathogenesis ofgastric mucosal injuty induced by indomethacin in rats

opened along the greater curvature, and exam-ined under a dissecting microscope with a squaregrid for lesions developed in the glandularportion. The extent of the gastric damage wasexpressed as the total area (mm2) of haemor-rhagic erosion. The gastric mucosa was scrapedoff by means of two glass slides on ice, andhomogenised with 1-5 ml of 10 mM potassiumphosphate buffer (pH 7 8) containing 30 mMKCI in a Potter-Elvehjem homogeniser, tomeasure concentrations of lipid peroxides andtocopherols. To measure the activities of SODand glutathione peroxidase, the homogenateswere sonicated over ice for two minutes. Thesonicated samples were centrifuged at 20000 gfor 20 minutes and the supernatants were storedfrozen at - 80°C until assay.

Effects ofscavengers ofactive oxygen onindomethacin induced gastric mucosal injuryActivity of SOD in serum reached a maximum ofabout 100 U/ml after 150 minutes when SODwas subcutaneously injected at a dose of 50 000U/kg.'7 Therefore, the interval of injection isimportant to maintain effective plasma SODactivity. To assess the effect of SOD andcatalase, recombinant human Cu, Zn-SOD(Nippon Kayaku Co Ltd, Tokyo) at a dose of50 000 U/kg, or catalase from bovine liver (SigmaChemical Co, St Louis, MO) at a dose of 90 000U/kg dissolved in 1 ml of physiological saline,or both were injected subcutaneously one hourbefore and three hours after giving indo-methacin. The same amount of physiologicalsaline was injected in the same manner into thecontrol rats. Dimethylsulphoxide (Wake PureChemical Co, Osaka) at a dose of 550 mg/kgdiluted in 0 5 ml of physiological saline wasadministered by intraperitoneal injection 30minutes before and three hours after indo-methacin to keep DMSO at a sufficiently highconcentration to force efficient scavenging ofhydroxyl radicals. Control animals received 0-5ml of physiological saline in the same manner.Six hours after indomethacin treatment therats were killed by exsanguination via theabdominal aorta and the extent of gastric damagewas expressed as the total area of haemor-rhagic erosion. The concentration of lipidperoxides in the gastric mucosa was alsomeasured.

Effect ofSOD plus catalase on indomethacininduced changes in antioxidantsThe effects of treatment with SOD plus catalaseon the activities of SOD and glutathione peroxi-dase in gastric mucosa, on the concentration ofa-tocopherol in gastric mucosa and serumsamples and on the concentrations of reducedand oxidized glutathione after administration ofindomethacin were investigated. Rats receivedSOD (recombinant human Cu,Zn-SOD) andcatalase at the same doses as previously des-cribed, by subcutaneous injection one hourbefore and three hours after indomethacinadministration. Control animals received thesame amount of physiological saline. Gastricmucosal homogenates were prepared as des-

cribed earlier, serum samples were collected,and the activities or the concentrations of anti-oxidants were measured by the methods des-cribed next.

ASSAYSConcentrations of thiobarbituric acid (TBA)-reactive substances, an index of lipid peroxida-tion, were measured in serum samples by themethod of Yagi," and the concentrations intissue homogenates were measured according toOhkawa et al."9 The concentration of the TBA-reactive substances were expressed as nmolmalodialdehyde. Thiobarbituric acid (BDHChemicals, Poole, England) and 1,1,3,3-trimethoxypropane (Tokyo Kasei Co, Tokyo)were used for the TBA assay, and all otherchemicals were ofreagent grade. Protein concen-tration in the gastric mucosal homogenates wasmeasured by the method of Lowry et al.'0 Theconcentration of a-tocopherol in serum samplesand gastric mucosa was measured by the methodof Abe et al2' with a high speed LC-6A liquidchromatograph (Shimazu Co, Kyoto). Toeliminate the influence of lipids, the ratio ofa-tocopherol:total cholesterol in serum sampleswas determined. The serum cholesterol concen-tration was assayed according to the method ofRichmond.22 The activity of SOD was measuredby a recently developed chemiluminescenceassay,23 which involved inhibition of a Cypridinaluciferin analog with chemiluminescencedependent on superoxide generated by thehypoxanthine-xanthine oxidase system. Recom-binant human Cu,Zn-SOD (a gift from NipponKayaku Co, Tokyo) was used as a standard andits activity was determined by the cytochrome cmethod. Activity ofSOD in tissue was expressedas U/mg protein. The activity of glutathioneperoxidase in the gastric mucosa was assayedspectrophotometrically by the method ofGinzler et al24 with t-butyl hydroperoxidase asthe substrate. This assay is based on the oxida-tion of reduced glutathione by glutathioneperoxidase coupled to the oxidation ofNADPHby glutathione reductase. The rate of NADPHoxidation was monitored photometrically. Forthe glutathione assay, the stomach was perfusedintraluminally with 5% sulphosalicylic acid andthen homogenised in 10 vol/g of the samesolution. The tissue homogenate was centrifugedfor five minutes at 10 000 g, and then thesupernatant was stored on ice until use. Theamount of reduced glutathione (GSH) wasmeasured by the method of Griffith25 and theamount of oxidised glutathione (GSSG) wasmeasured by masking GSH with N-ethyl-malemide (NEM).

STATISTICAL ANALYSISResults are presented as means (SEM). Forstatistical analysis, the tests used were theBartlett test for homogenicity of variance,Kruskal-Wallis analysis of variance for effects intime and comparison of differences betweencontrol and groups treated with scavengers, andthe Mann-Whitney test for individual compari-son of the group means. Differences between the

733

on April 7, 2021 by guest. P

rotected by copyright.http://gut.bm

j.com/

Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. D

ownloaded from

http://gut.bmj.com/

Yoshikawa, Naito, Kishi, Tomii, Kaneko, Iinuma, Ichikawa, Yasuda, Takahashi, Kondo

Figure 1: Total area ofgastric erosions afterindomethacin. Values aremean (SEM) ofnineexperiments. ***p

Role ofactive oxygen, lipid peroxidation, and antioxidants in thepathogenesis ofgastric mucosal injury induced by indomethacin in rats

Effects ofsuperoxide dismutase (SOD), catalase, SOD pluscatalase, and dimethylsulphoxide (DMSO) on total area ofhaemorrhagic erosions and thiobarbituric acid (TBA) reactivesubstances in the gastric mucosa at six hours after givingindomethacin

Lesion TBA-reactiveNo of area substances (nmollmganimals (mm2) protein)

Control 1 6 26-8 (4 5) 0-663 (0-030)SOD 6 12-5 (1-6)* 0 589(0 085)Catalase 6 18-5 (4-7) 0 575 (0-097)SOD plus catalase 6 13-3 (3-8)* 0-559 (0.035)*Control 2 9 22-8 (3-5) 0-715 (0 069)DMSO 9 12-1 (2-4)* 0-524(0.048)*

Values are mean (SEM) for that group. *p

Yoshikawa, Naito, Kishi, Tomii, Kaneko, Iinuma, Ichikawa, Yasuda, Takahashi, Kondo

mucosal injury, '25I-albumin loss produced byischaemia. Cochran et al26 and Terano et al2' alsoreported that DMSO inhibited gastric mucosalinjury induced by stress or ethanol. Ratsreceived intraperitoneal DMSO at a dose of 550mg/kg, because previous work had shown thatpeak concentrations occur within 30 minutes andthat the average half life is about six hours at thatdose and route of administration.27 In the presentstudy, indomethacin produced gastric haemor-rhagic erosions and increased TBA-reactivesubstances, which are indicators of lipid peroxi-dation, in the gastric mucosa with time afteradministration. Also, lipid peroxide accumula-tion closely paralleled the development of gastricmucosal injury. This indicates that lipid peroxi-dation plays a significant part in the pathogenesisof the gastric mucosal lesions induced by indo-methacin, as well as gastric injuries produced byburn shock,'5 water immersion restraint stress,'4and ischaemia-reperfusion.'6 Also, the treatmentwith SOD, SOD plus catalase, and DMSOsignificantly inhibited the pathological changesinduced by indomethacin. As is well known, thesuperoxide radical can interact with hydrogenperoxide in the presence of iron to generate thehydroxyl radical, which is thought to be the mosttoxic reactant and to abstract methylene hydro-gen atoms from polyunsaturated fatty acids,which initiates lipid peroxidation. The protec-tion by radical scavengers, therefore, suggeststhat lipid peroxidation mediated by oxygenradicals, especially hydroxyl radicals, plays animportant part in the formation and develop-ment of the gastric mucosal lesions induced byindomethacin.The source of oxygen radicals in gastric

mucosal injury induced by indomethacin in ratsis not clear. Recent studies, however, suggest apossible role of neutrophils in the early patho-genic process. The severity of gastric damageinduced by indomethacin can be significantlyreduced by prior depletion of circulatory neutro-phils and by treatment with a monoclonal anti-body against the CD18 leucocyte adhesionmolecule.2829 Both SOD and DMSO have beenshown to diminish leucocyte adherence in micro-vessels.303' Therefore, the protective action ofthese scavengers in this model is consistent withthe proposal by Wallace et al.28 29

Vitamin E, a lipid soluable antioxidant, inter-acts with oxygen and lipid radicals and preventsthe propagation of free radical lipid peroxida-tion. Our previous work showed that the concen-trations of a-tocopherol decreased both in serumsamples and in gastric mucosa during ischaemia-reperfusion injury in rats,32 which suggests thata-tocopherol was consumed in the process oflipid peroxidation mediated by oxygen radicalsin ischemia-reperfusion to prevent the develop-ment of tissue damage. When the radicals areformed initially in the aqueous phase of wholeblood, the water soluble antioxidants in theplasma such as vitamin C and plasma vitamin Eparticipate in the primary defence and latervitamin E in the erythrocyte membranedecreases.33 In the present study, a-tocopherol inthe serum but not that in the gastric mucosasignificantly decreased with time after indo-methacin administration, and also the decrease

of a-tocopherol in the serum was returned to thenormal range by concomitant treatment withSOD and catalase. Our previous study showedthat subcutaneously injected SOD was pre-dominantly localised in extracellular compart-ments,'7 and our present study showed that theSOD activity in intracellular compartments ofthe rat stomach did not increase after the sub-cutaneous injection of human SOD. Thus wespeculated that a free radical reaction occurredfirst in the extracellular space after indomethacinadministration, and SOD and catalase after theconcentration of ct-tocopherol by their actions asscavengers of superoxides and inhibitors of theproduction of hydroxyl radicals.

Glutathione is an important constituent ofintracellular protective mechanisms against anumber of noxious stimuli including oxidativestress. Reduced glutathione is known as a majorlow molecular weight scavenger of free radicalsin cytoplasm. On the other hand, the antioxidantactivity of GSH peroxidase is coupled with theoxidation of GSH to GSSG, which can subse-quently be reduced by GSH reductase withNADPH as the reducing agent. Concentration ofglutathione is exceedingly high in the glandularstomach compared with concentrations in otherportions of the gastrointestinal tract or in mostother organs.34 Boyd et al35 reported that deple-tion of gastric glutathione by diethyl maleateproduced gastric ulceration. Mutoh et a136showed that intracellular glutathione was mainlyresponsible for protecting against gastric cellinjury induced by ethanol. These findingssuggest that in the gastric mucosa free radicals orlipid peroxides are injurious offensive factorsand GSH is a protecting defensive factor. Deple-tion of glutathione results in enhanced lipidperoxidation37 and excessive lipid peroxidationcan cause increased GSH consumption.38 Thedecrease in GSH in our study was accompaniedby an increase in lipid peroxides measured as aTBA-reactive substance in the gastric mucosa.The content of GSSG, however, showed nosignificant increases in response to the depletionof GSH and the activity of GSH peroxidase wassignificantly inhibited by indomethacin admin-istration, which indicates that GSH was con-sumed during the reaction with oxygen radicals,organic radicals, and peroxide radicals.

Glutathione peroxidase is important in theelimination of hydrogen peroxide and lipidhydroperoxides in the gastric mucosal cell. Thusinhibition of this enzyme may result in theaccumulation of hydrogen peroxide with subse-quent oxidation of lipids. The GSH peroxidaseactvity of this study was decreased six hours afterindomethacin administration and its activitycould not be recovered by the elimination ofoxygen radicals. These findings suggest thatindomethacin itself, or depletion of prosta-glandins, inhibits GSH peroxidase activity of thegastric mucosa. Our findings are in line withthose of Banerjee,39 who recently showed theinhibition of peroxidase activity in the mito-chondrial fraction of rat gastric mucosa byindomethacin.

In summary, the results indicate that excessivegeneration of oxygen radicals in the extracellularspace and the depletion of GSH in conjunction

736

on April 7, 2021 by guest. P

rotected by copyright.http://gut.bm

j.com/

Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. D

ownloaded from

http://gut.bmj.com/

Role ofactive oxygen, lipidperoxidation, and antioxidants in the pathogenesis ofgastric mucosal injury induced by indomethacin in rats 737

with inhibition of GSH peroxidase activityis responsible for the oxidative tissue damageof gastric mucosa after administration ofindomethacin.

1 Djahanguiri B. The production of acute gastric ulceration byindomethacin in the rat. Scand J Gastroenterol 1969; 4:265-7.

2 Fries JF, Miller SR, Spitz BW, Williams CA, Hubert HB,Block DA. Toward an epidemiology of gastropathy associ-ated with nonsteroidal antiinflammatory drug use. Gastro-enterology 1989; %: 647-55.

3 Whittle BJR. Temporal relationship between cyclooxygenaseinhibition, as measured by prostaglandin biosynthesis, andthe gastrointestinal damage induced by indomethacin in therat. Gastroenterology 1981; 80: 94-8.

4 Rainsford KD, Willis C. Relationship of gastric mucosaldamage induced in pigs by antiinflammatory drugs to theireffects on prostaglandin production. Dig Dis Sci 1982; 27:624-35.

5 Kobayashi K, Arakawa T, Satoh H, Fukuda T, Nakamura H.Effect of indomethacin, tiaprofenic acid and dicronfenac onrat gastric mucosal damage and content of prostacyclin andprostaglandin E2. Prostaglandins 1985; 30: 609-18.

6 Ligmusky M, Golanska EM, Hansen DG, Kauffman GL.Aspirin can inhibit gastric mucosal cyclooxygenase withoutcausing lesions in rats. Gastroenterology 1983; 84: 756-61.

7 Takeuchi K, Ueki S, Okabe S. Importance of gastric motilityin the pathogenesis of indomethacin-induced gastric lesionsinrats. DigDisSci 1986; 31: 1114-21.

8 Ueki S, Takeuchi K, Okabe S. Gastric motility is an importantfactor in the pathogenesis of indomethacin-induced gastricmucosal lesions in rats. Dig Dis Sci 1988; 33: 209-16.

9 Takeuchi K, Okada M, Ebara S, Osano H. Increased micro-vascular permeability and lesion formation during gastrichypermotility caused by indomethacin and 2-deoxy-D-glucose in the rat.J Clin Gastroenterol 1990; 12: S76-84.

10 Rainsford KD. Microvascular injury during gastric mucosaldamage by antiinflammatory drugs in pigs and rats. Agentsand Actions 1983; 13: 5-6.

11 Ito M, Guth PH. Role of oxygen-derived free radicals inhemorrhagic shock-induced gastric lesions in the rat. Gastro-enterology 1985; 88: 1162-7.

12 Perry MA, Wadhwa S, Parks DA, Pickard W, Granger DN.Role ofoxygen radicals in ischemia-induced lesions in the catstomach. Gastroenterology 1986; 90: 362-7.

13 Tappel AL. Lipid peroxidation damage to cell components.Fed Proc 1973; 32: 1870-4.

14 Yoshikawa T, Miyagawa H, Yoshida N, Sugino S, Kondo M.Increase in lipid peroxidation in rat gastric mucosal lesionsinduced by water-immersion restraint stress. J Clin BiochemNutr 1986; 1: 271-7.

15 Yoshikawa T, Yoshida N, Miyagawa H, Takemura T,Tanigawa T, Sugino S, Kondo M. Role of lipid peroxidationin gastric mucosal lesions induced by burn shock in rats.J Clin Biochem Nutr 1987; 2: 163-70.

16 Yoshikawa T, Ueda S, Naito Y, Takahashi S, Oyamada H,Morita Y, et al. Role of oxygen-derived free radicals ingastric mucosal injury induced by ischema or ischemia-reperfusion in rats. Free Radic Res Comms 1989; 7: 285-91.

17 Miyagawa H, Yoshikawa T, Tanigawa T, Yoshida N, SuginoS, Kondo M. Measurement of serum superoxide dismutaseactivity by electron spin resonance. J Clin Biochem Nutr1988; 5: 1-7.

18 Yagi K. A simple fluorometric assay for lipid peroxides inblood plasma. Biochemical Medicine 1976; 15: 212-6.

19 Ohkawa H, Ohnishi N, Yagi K. Assay for lipid peroxides foranimal tissues by thiobarbituric acid reaction. Anal Biochem1979; 95: 351-8.

20 Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the folin phenol reagent. J Biol Chem1951; 193: 265-75.

21 Abe KY, Yuguchi Y, Katsui G. Quantitative determination oftocopherols by high-speed liquid chromatography. J NutrSci Vitaminol (Tokyo) 1975; 21: 183-8.

22 Richmond W. Preparation and properties of a cholesteroloxidase from Nacardia sp and its application to the enzy-matic assay of total cholesterol in serum. Clin Chem 1973; 19:1350-6.

23 Nakano M, Kimura H, Hara M, Kuroiwa M, Kato M,Totsune K, Yoshikawa T. A highly sensitive method fordetermining both Mn- and Cu-Zn superoxide dismutaseactivities in tissue and blood cells. Anal Biochem 1990; 187:277-80.

24 Gunzler WA, Flohe L. Glutathione peroxidase. In: GreenwaldRA, ed. CRC handbook of methods for oxygen radicalresearch. Boca Raton: CRC Press 1985: 285-90.

25 Griffith OW. Determination of glutathione and glutathionedisulfide using glutathione reductase and 2-vinylpyridine.Anal Biochem 1980; 106: 207-12.

26 Cochran T, Stefanko J, Moore C. Dimethylsulfoxide protec-tion against gastric stress ulceration. Current Surg 1983;Nov-Dec: 435-7.

27 Terano A, Hiraishi H, Ota S, Shiga J, Sugimoto T. Role ofsuperoxide and hydroxyl radicals in rat gastric mucosalinjury induced by ethanol. GastroenterolJ3pn 1989; 24: 488-93.

28 Wallace JL, Kennan CM, Granger DN. Gastric ulcerationinduced by non-steroidal anti-inflammatory drugs is aneutrophil-dependent process. Am J Physiol 1990; 259;G462-7.

29 Wallace JL, Arfors K-E, McKnight W. A monoclonal anti-body against theCD18 leukocyte adhesion molecule preventsindomethacin-induced gastric damage in the rabbit. Gastro-enterology 1991; 100: 878-83.

30 Suzuki M, Inauen W, Kvietys PR, Grisham MB, Meininger C,Schelling ME, et al. Superoxide mediates reperfusion-induced leukocyte-endothelial cell interactions. Am JPhysiol 1989; 257: H1740-5.

31 Sekizuka E, Benoit JN, Grisham MB, Granger DN.Dimethylsulfoxide prevents chemoattractant-inducedleukocyte adherence. Am J Physiol 1989; 256:H594-7.

32 Yoshikawa T, Yasuda M, Ueda S, Naito Y, Tanigawa T,Oyamada H, et al. Vitamin E in gastric mucosal injuryinduced by ischemia-reperfusion. AmJ Clin Nutr 1991; 53:210S-4S.

33 Niki E, Komura E, Takahashi M, Urano S, Ito E, Terao K.Oxidative hemolysis of erythrocytes and its inhibitionby free radical scavengers. J Biol Chem 1988; 263: 19809-14.

34 Boyd SC, Sasame HA, Boyd MR. High concentration ofglutathione in glandular stomach: possible implications forcarcinogenesis. Science 1979; 205: 1010-2.

35 Boyd SC, Sasame HA, Boyd MR. Gastric glutathione deple-tion and acute ulcerogenesis by diethylmaleate given sub-cutaneously to rats. LifeSci 1981; 28: 2987-92.

36 Mutoh H, Hiraishi H, Ota S, Yoshida H, Ivey KJ, Terano A,et al. Protective role of intracellular glutathione againstethanol-induced damage in cultured rat gastric mucosalcells. Gastroenterology 1990; 98: 1452-9.

37 Younes M, Siegers CP. Mechanistic aspects of enhanced lipidperoxidation following glutathione depletion in vivo. ChemBiol Interact 1981; 34: 257-66.

38 Comporti M. Biology of disease: lipid peroxidation andcellular damage in toxic liver injury. Lab Invest 1985; 53:599-623.

39 Banerjee RK. Nonsteroidal anti-inflammatory drugs inhibitgastric peroxidase activity. Biochim Biophys Acta 1990; 1034:275-80.

on April 7, 2021 by guest. P

rotected by copyright.http://gut.bm

j.com/

Gut: first published as 10.1136/gut.34.6.732 on 1 June 1993. D

ownloaded from

http://gut.bmj.com/