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Digestive Diseases and Sciences, Vol. 48, No. 1 (January 2003), pp. 54–58 (C© 2003)

A Novel Vitamin E Derivative (TMG) ProtectsAgainst Gastric Mucosal Damage Induced

by Ischemia and Reperfusion in Rats

HIROSHI ICHIKAWA, MD, PhD,* NORIMASA YOSHIDA, MD, PhD,* HIROSHISA TAKANO, MD, PhD,*TAKESHI ISHIKAWA, MD, PhD,* OSAMU HANDA, MD, PhD,* TOMOHISA TAKAGI, MD, PhD,*YUJI NAITO, MD, PhD,* HIRONOBU MURASE, PhD,† and TOSHIKAZU YOSHIKAWA, MD, PhD*

The aim of the present study was to investigate the antioxidative effects of water-soluble vitaminE derivative, 2-(α-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), on ischemia–reperfusion (I/R) -induced gastric mucosal injury in rats. Gastric ischemia was induced by applyinga small clamp to the celiac artery and reoxygenation was produced by removal of the clamp. Thearea of gastric mucosal erosion, the concentration of thiobarbituric acid-reactive substances, and themyeloperoxidase activity in gastric mucosa significantly increased in I/R groups compared with thoseof sham-operated groups. These increases were significantly inhibited by pretreatment with TMG.The contents of both mucosal TNF-α and CINC-2β in I/R groups were also increased comparedwith the levels of those in sham-operated groups. These increases of the inflammatory cytokineswere significantly inhibited by the treatment with TMG. It is concluded that TMG inhibited lipidperoxidation and reduced development of the gastric mucosal inflammation induced by I/R in rats.

KEY WORDS: ischemia–reperfusion; vitamin E; free radical; cytokine; neutrophil.

Transient ischemia of tissues causes anoxia of cells andeventually results in death of cells. However, further dam-age can occur when oxygen is reintroduced to the tissue,causing ischemia–reperfusion (I/R) injury. At the onset ofreperfusion when ischemic tissue is being reoxygenated,there seems to be a burst in the generation of oxygen-derived free radicals, which may attack and damage impor-tant biological molecules. Granger et al (1) demonstratedthat superoxide dismutase (SOD) or allopurinol could at-tenuate the increase in intestinal mucosal injury producedby I/R. Yoshikawa et al also have been shown that active

Manuscript received February 28, 2002, revised manuscript receivedAugust 20, 2002; accepted October 2, 2002.

From the *First Department of Medicine, Kyoto Prefectural Univer-sity of Medicine, Kyoto 602-8566, Japan and†CCI Corporation, Gifu501-3923, Japan.

Address for reprint requests: Dr. Hiroshi Ichikawa, First Depart-ment of Medicine, Kyoto Prefectural University of Medicine 465Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.

oxygen species and lipid peroxidation may play a rolein the pathogenesis of gastric mucosal injury induced byI/R (2).

Vitamin E is a major lipophilic antioxidant in cellu-lar membranes and plasma lipoproteins and its excellentantioxidant activity is well recognized (3–6). It has beenreported that vitamin E scavenges not only chain-carryingperoxyl radicals but also singlet oxygen (7) and superox-ide anion radical (8, 9). Therefore, it has been expectedthat vitamin E or its derivatives can reduce the gastric orintestinal mucosal injury induced by I/R.

Murase et al have succeeded in the synthesis ofa novel water-soluble vitamin E derivative, 2-(α-D-glucopyranosy)methyl-2,5,7,8-tetramethylchroman-6-ol(TMG) (Figure 1), byα-glucosidase-catalyzed transg-lycosylation using as aqueous-organic solvent system,and elucidated its excellent water-solubility (>1×103 mg/ml) (18). It has been reported that TMG is some-what located within the membrane surface and acts as a

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Fig 1. Structures of 2-(α-D-glucopyranosyl)methyl-2,5,7,8-tetramethy-lchroman-6-ol (TMG). Both structures share a chromanol ring. TMGhas glucosyl group, which gives it water solubility, instead of phytylside chain ofα-tocopherol.

powerful antioxidant by scavenging radicals generatedeither in the aqueous or in the lipid phase. In the presentinvestigation, reperfusion-induced gastric mucosalinjury was produced in rats by applying a small clamp tothe celiac artery for 30 min followed by the removal ofthe vascular clamp to measure changes in lipid peroxidein the gastric mucosa during I/R and to evaluate theantioxidative effect of TMGin vivo.

MATERIALS AND METHODS

Experimental Animals. Male Sprague-Dawley (SD) ratsweighing 190–210 g were obtained from Keari Co. Ltd.(Osaka, Japan). They were housed in stainless steel cages withwire bottoms and maintained on a 12-hr light–12-hr dark cyclewith the temperature and relative humidity of the animal roomcontrolled at 21–23◦C and 55–65%, respectively. They were notfed for 18 hr prior to the experiments but were allowed free accessto water. All experimental procedures described below were ap-proved by the Animal Care Committee of the Kyoto PrefecturalUniversity of Medicine (Kyoto, Japan).

Preparation of Rats for Gastric Ischemia–Reperfusion.Gastric ischemia was induced for 30 min by applying a smallclamp to the celiac artery in rats given intraperitoneal urethaneanesthesia (1000 mg/kg). Reoxygenation was produced by re-moval of the clamp. Sixty minutes after reperfusion, the rats werekilled by exsanguination via the abdominal aorta under urethaneanethesia (1000 mg/kg). Stomachs were removed, opened alongthe greater curvature, and rinsed with physiological saline. TMG(a gift from CCI Pharmacy, Gifu, Japan) was dissolved in phys-iological saline at a dose of 0.4–4 mg/kg and was given to ratsintraveneously just before removal of the vascular clamping. Inthe I/R group, rats received an equivalent volume of the vehicle(physiological saline).

Assessment of Gastric Mucosal Injury Induced byIsohemia–Reperfusion.Macroscopic gastric damage was ex-amined under a dissecting microscope with a square grid and wasexpressed as the total area (square millimeters) of hemorrhagicerosions (erosion index) (2). The concentration of thiobarbituric

acid-reactive substances (TBARS) in the gastric mucosa, anindex of lipid peroxidation, was measured by the method ofOhkawa et al (10) and was expressed as nanomoles of malon-dialdehyde. The protein concentration in the gastric mucosalhomogenates was measured by the method of Lowry et al (11).Myeloperoxidase (MPO) activity in the gastric mucosa, an in-dex of polymorphonuclear leukocyte accumulation, was deter-mined by a modification of the method of Krawisz et al (12).The content of tumor necrosis factor-α(TNF-α) in the gastricmucosa was determined by ELISA using a rat tumor TNF-αELISA kit (Bio Source International, Inc., Camarilla, California,USA). Furthermore, the content of cytokine-induced neutrophilchemoattractants-2β (CINC-2β) in the gastric mucosa wasdetermined by ELISA using Rat GRO/CINC-2β ELISA kit(Immuno Biological Laboratories, Gunma, Japan) according tothe manufacturer’s instructions.

Statistical Analysis. All values were expressed as means± SEM. Data were compared using an analysis of variance(ANOVA) followed by Scheffer’s test. A value ofP < 0.05 wasconsidered significant.

RESULTS

Macroscopic Gastric Mucosal Findings. Multipleerosions and bleeding were revealed in the gastric mu-cosa after 60 min of reperfusion following 30 min ofischemia in the I/R with vehicle group. The total areaof erosions indeced by I/R was significantly decreasedby treatment with TMG at a dose of 0.4 and 4 mg/kg(Figure 2).

TBARS in Gastric Mucosa. TBA-RS in the gastricmucosa significantly increased 60 min after reperfusion inI/R with vehicle group. This increases was significantlyinhibited by treatment with TMG at a dose of 0.4 and4 mg/kg (Figure 3).

MPO Activity in Gastric Mucosa. The I/R with ve-hicle group showed a significant increase in mucosalMPO activity compared with the sham-operated group(Figure 4). However, this increase was significantlyreduced in all groups treated with TMG.

Inflammatory Cytokines in Gastric Mucosa.Figure 5demonstrated that the content of both mucosal TNF-α andCINC-2β in the I/R with vehicle group were significantlyincreased compared with the levels of those in the sham-operated group. These increases of the levels of TNF-α

and CINC-2β were significantly inhibited by the treatmentwith TMG at a dose of 4 mg/kg (Figure 5a, 5b).

DISCUSSION

Recently it has been recognized that oxidative stressappears to play a role in the pathogenesis of a numberof gastrointestinal disease states, including pancreatitis,peptic ulcers, inflammatory bowel diseases, cancer, hep-atitis, and ischemia–reperfusion injury. In particular it is

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Fig 2. Effect of TMG on gastric mucosal injury induced by I/R in rats.One milliliter of TMG solution (0.4–4 mg/kg) was given to rats intra-venously just before removal of the vascular clamping of celiac artery.Rats in the sham-operated group and the I/R group received an equiva-lent volume of physiological saline instead of TMG. Data are expressedas means± SEM. ∗P < 0.05 vs normal;#P < 0.05 vs control.

well known that a variety of bioactive compounds andeffector cells have been invoked as mediators of reper-fusion injury, including reactive oxygen species (ROS),platelet-activating factor, nitric oxide, platelets, mast cells,and granulocytes. A number ofin vivo models have been

Fig 3. Effect of TMG on the level of TBARS in gastric mucosa inI/R-treated rats. TMG was administered to rats in the same manner asdescribed in Figure 2. Data are expressed as means± SEM. ∗P < 0.05vs normal;#P < 0.05 vs control.

Fig 4. Effect of TMG on gastric mucosal MPO activities induced by I/Rin rats. TMG was administered to rats in the same manner as describedin Figure 2. Data are expressed are means± SEM. ∗P < 0.05 vs normal;#P < 0.05 vs control.

developed to clarify the mechanisms of I/R-induced tis-sue injury. We have already reported that oxygen-derivedfree radicals and lipid peroxidation may play a rolein the formation of gastric mucosal injury induced byI/R (2).

There are some major lines of evidence that implicateROS as a mediator of reperfusion-induced mucosal injury:(1) this type of injury was significantly attenuated by su-peroxide dismutase, catalase, and other free radical scav-engers (2); (2) allopurinol, a competitive inhibitor of xan-thine oxidase, significantly ameliorated gastric mucosalinjury (13); (3) the serumα-tocophero–choresterol ratioand gastric mucosalα-tocopherol, an internal antioxida-tive agent, were significantly decreased after reperfusion(14); and (4) the reduction of neutrophils, which are one ofthe major sources of ROS, prevented reperfusion-inducedmucosal injury (15).α-Tocopherol, a major constituent of lipid-soluble vita-

min E, is well known as the major antioxidant preventingoxidative attack of membrane lipids and other membranecompounds, and its excellent antioxidant activity is wellrecognized (3–6). It has been reported thatα-tocopherolscavenges not only chain-carrying peroxyl radicals (4, 6)but also singlet oxygen (7) and superoxide anion radi-cal (8, 9). In our previous study (14), we have reportedthat the gastric mucosal injury induced by I/R in vita-min E-deficient rats was more severe than that in vitaminE-nondeficient rats, indicating that vitamin E was con-sumed in the process of lipid peroxidation induced byoxygen radicals in I/R to prevent the development of tis-sue damage. We also have shown that the inhibition of lipid

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Fig 5. Effect of TMG on inflammatory cytokine levels: (a) TNF-α and(b) CINC-2β of gastric mucosa induced by I/R in rats. TMG was ad-ministered to rats in the same manner as described in Figure 2. Data areexpressed as means± SEM. ∗P < 0.05 vs normal;#P < 0.05 vs control.

peroxidation and interference with neutrophil infiltrationby vitamin E may be responsible for its cytoprotectiveeffects in I/R in nitric oxide-depleted rats (16).

In our previous study (14), oral administration ofα-tocopherol could not prevent reperfusion-inducedmucosal injury; however, we showed that TMG, a water-soluble vitamin E derivative, was effective in gastricmucosal damage induced by I/R in rats. Furthermore, ourpresent study showed that TMG inhibited lipid peroxida-tion of the gastric mucosa and prevented the accumulationof neutrophils into the gastric mucosa induced by I/R.Additionally, TMG reduced the content of inflammatory

cytokines (TNF-α, CINC-2β) in gastric treated withI/R.

It has been reported that the I/R-induced hyperadhe-siveness of neutrophils to endothelial cells was involvedin the expression of adhesion molecules stimulated byactive oxygen species (17). It is thought that TMG caninhibit the ovenexpression of neutrophoil or endothe-lial cell adhesion molecules by quenching I/R-inducedROS. Consequently the neutrophil infiltration into the mu-cosal tissue could be inhibited. It appeared that inflam-matory cytokines were produced both from neutrophilsitself and from endothelial cells stimulated by adheredneutrophils. We suggest that TMG has an antiinflamma-tory effect through scavenging ROS derived from thexanthine–anthine oxidase system, vascular endothelialcells, or neutrophils.

In this study we aimed to determine the origin of in-flammatory cytokines in this rat model. We measuredthe total value of cytokines in whole intestinal mucosain these experiments. It is assumed that the origin ofcytokines is neutrophils, macrophages and endothelialcells etc. Further examination is needed to resolve thisproblem.

In terms of MPO activity in this study, it should bepointed out that TMG did not decrease the concentrationof TNF-α and CINC-2β at the dose of 0.4 mg/kg andthat TMG inhibited MPO activity at the same dose. Itwas recognized that neutrophil and endothelial cell inter-action was inhibited by treatment with TMG at a doseof 0.4 mg/kg due to its strong antioxidative effects. Asmentioned above, proinflammatory cytokines may be pro-duced from neutrophils without adhering to endothelialcells. it was suggested that proinflammatory cytokineswere produced by both neutrophils and endothelial cellsstimulated with adhered neutrophils.

TMG, vitamin E glucoside, was synthesized from 2-hydroxymethyl-2,5,7,8-tetramethychroman-6-ol and mal-tose in a solution containing dimethyl sulfoxide by trans-glycosylation withα-glucosidase fromSaccharomycesspecies (18). The high water solubility of TMG enables itto be carried to organs and supplied to tissue rapidly. TMGalso has excellent antioxidative activity, equal to that ofα-tocopherol or ascorbic acid (18,19).

We presumed that TMG is located near or withinthe membrane surface and can scavenge both the chain-initiating radicals and chain-propagating radicals in thelipid or the aqueous phase (20). The next step was to de-termine the detailed ROS scavenging mechanisms of TMGin this model.

In summary, the results of the present study indicatethat TMG inhibited lipid peroxidation and reduced de-velopment of the gastric mucosal inflammation induced

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by I/R in rats. This investigation suggests that TMG mayuseful as a new therapeutic agent for reperfusion injury.

REFERENCES

1. Granger DN, Rutili G, McCord JM: Superoxide radicals in felineintestinal ischemia. Gastroenterology 81:22–29, 1981

2. Yoshikawa T, Ueda S, Naito Y, Takahashi S, Oyamada H, Morita Y,Yoneta T, Kondo M: Role of oxygen-derived free radical in gastricmucosal injury induced by ischemia or ischemia–reperfusion in rats.Free Radic Res Commun 7:285–291, 1989

3. Burton GW, Ingold KU: Vitamin E: Application of the principlesof physical organic chemistry to the exploration of its structure andfunction. Accounts of Chem Res 19:194–201, 1986

4. Niki B: Antioxidants in relation to lipid peroxidation. Chem PhysLipids 44:227–232, 1987

5. Burton GW, Traber MG: Vitamin E: Antioxidant activity, biokinet-ics, and bioavailability. Annu Rev Nutr 10:357–382, 1990

6. Burton GW, Joyce A: Is vitaminte E the only lipid-soluble chain-breaking antioxidant in human blood plasma and erythrocy mem-brane? Arch Biochem Biophys 221:281–290, 1983.

7. Grams GW: Oxidation ofα-tocopherol by singlet oxygen. Tetrahe-dron Lett 50:823–825, 1971

8. Nishikimi M, Yamada H, Yagi K: Oxidation by superoxide of to-copherols dispersed in aqueous media with deoxycholate. BiochimBiophys Acta 627:101–108, 1980

9. Fukuzawa K, Gebicki JM: Oxidation ofα-tocopherol in micelles andliposomes by the hydroxy, perhydroxy and superoxide free radicals.Arch Biochem Biophys 226:242–251, 1983

10. Ohkawa H, Ohnishi N, Yagi K: Assay for lipid peroxides for animaltissues by thiobarbituric acid reaction. Anal Biochem 95:351–358,1979

11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measure-ment with the Folin phenol reagent. J Biol Chem 193:265–275, 1951

12. Krawisz JE, Sharon P, Stenson WF: Quantitative assay for acute in-testinal inflammation based on myeloperoxidase activity. Gastroen-terology 87:1344–1350, 1984

13. Yoshikawa T, Ueda S, Takahashi S, Naito Y, Oyamada H, MoritaY, Tanigawa T, Takemura T, Sugino S, Kondo M: [Role of freeradicals and lipid peroxidation in gastric mucosal injury induced byischemia-reperfusion in rats]. Nippon Shokakibyo Gakkai Zasshi87:8–15, 1990

14. Yoshikawa T, Yasuda M, Ueda S, Naito Y, Tanigawa T, Oyamada H,Kondo M: Vitamin E in gastric mucosal injury induced by ischemia-reperfusion. Am J Clin Nutr 53:210S–214S, 1991

15. Naito Y, Yoshikawa T, Matsuyama K, Yagi N, Arai M, NakamuraY, Kaneko T, Yoshida N, Kondo M: Neutrophils, lipid peroxidation,and nitric oxide in gastric reperfusion injury in rats. Free Radic BiolMed 24:494–502, 1998

16. Naito Y, Yoshikawa T, Matsuyama K, Yagi N, Kasai K, SugimotoN, Masui Y, Yoshida N, Kondo M: Effect of vitamin E in gastricmucosal injury induced by ischaemia–reperfusion in nitric oxide-depleted rats. Aliment Pharmacol Ther 13:553–559, 1999

17. Ichikawa H, Flores S, Kvietys PR, Wolf RE, Yoshikawa T, GrangerDN, AW TY: Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells. Circ Res81:922–931, 1997

18. Murase H, Yamauchi R, Kato K, Kunieda T, Terao J: Synthesis ofa novel vitamin E derivative, 2-(alpha-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol, by alpha-glucosidase-catalyzedtransglycosylation. Lipids 32:73–78, 1997

19. Yoshida N, Yoshikawa T, Yamaguchi T, Naito Y, Tanigawa T, MuraseH, Kondo M: A novel water-soluble vitamin E derivative protectsagainst experimental colitis in rats. Antioxid Redox Signal 1:555–562, 1999

20. Murase H, Moon J, Yamauchi R, Kato K, Kunieda T, YoshikawaT, Terao J: Antioxidant activity of a novel vitamin E derivative, 2-(αD-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol. FreeRadic Biol Med 24:217–225, 1998

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