|Year : 2011 | Volume
| Issue : 5 | Page : 578-581
Effect of folic acid supplementation on oxidative gastric mucosa damage and acid secretory response in the rat
KO Ajeigbe1, SB Olaleye2, EO Oladejo3, AO Olayanju3
1 Department of Physiology, School of Basic Medical Sciences, Igbinedion University, Okada; Department of Physiology, Gastrointestinal Research Unit, College of Medicine, University of Ibadan, Ibadan, Nigeria
2 Department of Physiology, Gastrointestinal Research Unit, College of Medicine, University of Ibadan, Ibadan, Nigeria
3 Department of Physiology, School of Basic Medical Sciences, Igbinedion University, Okada, Nigeria
|Date of Submission||29-Dec-2010|
|Date of Decision||04-Feb-2011|
|Date of Acceptance||01-Jul-2011|
|Date of Web Publication||15-Sep-2011|
K O Ajeigbe
Department of Physiology, School of Basic Medical Sciences, Igbinedion University, Okada; Department of Physiology, Gastrointestinal Research Unit, College of Medicine, University of Ibadan, Ibadan
Source of Support: None, Conflict of Interest: None
Objective : This study investigated the antioxidative and antisecretory properties of folic acid in the rats' stomach. Materials and Methods: Male Wistar rats were treated with 2 mg/kg diet of folic acid for 21 days. Gastric ulceration was induced by indomethacin, scored, and assayed to determine the concentration of total protein, mucus, malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) in homogenized samples. Normal saline and Ranitidine treated group served as negative and positive control, respectively. Basal and stimulated acid secretion was measured by continuous perfusion method.
Result : Indomethacin caused severe damage to the rats' stomach with an ulcer index of 4.32 ± 0.13, increase in MDA concentration and reduction in the concentration of protein, mucus, catalase and superoxide dismutase (P < 0.001). Pre-treatment with folic acid prevented the formation of ulcers by 32%, and attenuated the inhibition of mucus by 14%, CAT, 51% and SOD, 150%. Ranitidine afforded 56% prevention in ulcer formation with 67%, 55% and 78% attenuation of the inhibition of mucus, CAT and SOD, respectively, by indomethacin. While indomethacin-induced lipid peroxidation was attenuated by 58% reduction in MDA concentration on pretreatment with folic acid, Ranitidine offered 65% reduction. Basal and stimulated acid secretions were significantly reduced in the treated when compared with control animals. Folic acid produced a 21% reduction in the basal acid output when compared with the control animals (P < 0.05), and 140% reduction in histamine-induced acid response. Conclusion : The results indicate the gastroprotective activity of folic acid due its antioxidative and anti-secretory properties.
Keywords: Acid secretion, folic acid, malondialdehyde, ulcer
|How to cite this article:|
Ajeigbe K O, Olaleye S B, Oladejo E O, Olayanju A O. Effect of folic acid supplementation on oxidative gastric mucosa damage and acid secretory response in the rat. Indian J Pharmacol 2011;43:578-81
|How to cite this URL:|
Ajeigbe K O, Olaleye S B, Oladejo E O, Olayanju A O. Effect of folic acid supplementation on oxidative gastric mucosa damage and acid secretory response in the rat. Indian J Pharmacol [serial online] 2011 [cited 2020 Sep 23];43:578-81. Available from: http://www.ijp-online.com/text.asp?2011/43/5/578/84976
| » Introduction|| |
Gastric ulcer is characterized by disruption of mucosal integrity of the stomach leading to local defect or excavation due to active inflammation.  Its molecular basis depicts increased formation of Reactive Oxygen Species (ROS) and/or decreased antioxidant reserve in the gastric mucosa, a condition termed as oxidative stress. Hence, its occurrence is associated with enhancement of oxidative stress by pro-ulcerative factors in the gut like Helicobacter pylori;  use of non-steroidal anti-inflammatory drugs (NSAIDs);  smoking;  psychological stress  and dietary intake of potential ulcerogens.  Lipid peroxidation, a result of the reaction of oxyradicals and polyunsaturated fatty acids, has been suggested as an attack factor in the gastric mucosa,  while glutathione and other anti-oxidative enzymes are important substances in the cellular defense system.  Moreover, gastric acid secretion still remains a force to reckon with in the pathogenesis of peptic ulceration.  When levels of acid and proteolytic enzymes overwhelm the mucosal defense mechanism, ulcers occur.
Folate modulates a number of disorders as a result of its anti-apoptotic and anti-oxidative properties.  This includes cardiovascular diseases.  neural tube and congenital defects,  subfertility,  and several malignancies like cancer of the colorectum, lungs, pancreas, esophagus, stomach, cervix, breast  and neuroblastoma and leukemia.  Though its prevalence is high in Africa and Asia,  peptic ulcer disease is common throughout the World and in the past one or two decades there has been a phenomenal increase in the knowledge on its treatment.  Consequently, efforts in the search of gastro-protective agents remain inexhaustible.
Currently, there is little or no attention drawn to the role folic acid may possibly play on the development of gastric ulcer, an important inflammatory disorder of the gastrointestinal system. This study, therefore, presents findings on the protective effect of folic acid on oxidative gastric mucosal damage induced by indomethacin, and acid secretion in the rat.
| » Materials and Methods|| |
Thirty-two male albino rats of the Wistar strain weighing between 180-250 g were used for this study. The animals were obtained from the Animal House of Igbinedion University, Okada, and then, separated randomly into four cages of eight rats each where they were kept for four weeks before the start of the experiment. The animals were housed under standard conditions of temperature (23 ± 2°C), humidity (55 ± 15%) and 12 hour light (7.00 am - 7.00 pm). The cages were constantly cleaned in order to prevent the animals from contracting disease. They were fed with standard commercial rat pellets (Ladokun Feeds Limited, Nigeria) and allowed water ad libitum. The Animal Care Committee of Igbinedion University, Okada approved the experimental protocols.
Folic acid tablets, indomethacin and ranitidine were obtained from a local pharmacy duly registered by the Pharmacists' Council of Nigeria (PCN). Protein estimation kit was procured from the Randox Laboratories, United Kingdom (UK). All other reagents were of analytical grade and obtained from British Drug Houses, Poole, UK.
The animals were divided into four groups of eight rats each.
- Group One: Animals were treated with normal saline. They were called the overall control group.
- Group Two: Animals were treated with indomethacin (25 mg/kg) after 24 hour fasting. This served as the treated control.
- Group Three: Animals were treated with 2 mg/kg of folic acid for three weeks before indomethacin administration.
- Group Four: Animals were treated with ranitidine (4 mg/kg) sixty minutes prior to indomethacin administration. This group served as the positive control group.
The route of administration for both folic acid and indomethacin is oral. Indomethacin was dissolved in sodium bicarbonate to form a clear solution; and folic acid was dissolved in normal saline to give a suspension. All the animals were sacrificed under sodium pentobarbitone anaesthesia.
Ulcer Induction and Index Determination
Four hours after the oral administration of indomethacin, the stomachs were opened along the greater curvature, washed in normal saline to remove debris and pinned on a cork mat for ulcer scoring. This was done by locating the wounds in the glandular region under a simple microscope. The length (mm) of all the elongated black-red lines parallel to the long axis of the stomach in the mucosa was measured. The ulcer index was calculated by adding the lengths of all the lesions in the glandular region of the stomach.
Determination of malondialdehyde (MDA)
The assay method of Hunter et al.0, modified by Gutteridge and Wilkins  was adopted. Malondialdehyde (MDA), a product of lipid peroxidation, when heated with 2-thiobarbituric acid (TBA) under acid conditions forms a pink colored product which has a maximum absorbance of 532 nm.
The stomach homogenate was supplemented with 1 g of TBA in 100 ml of 0.2% Sodium Hydroxide (NaOH) and 3 ml of glacial acetic acid, thoroughly mixed, and incubated in boiling water bath for 15 minutes It was then allowed to cool, and centrifuged. Absorbance was read at 532 nm and the results expressed as nanomoles MDA/mg wet tissue.
Determination of catalase activity
Activity of catalase in gastric mucosa was determined according to the procedure of Sinha.  This method is based on the reduction of dichromate in acetic acid to chromic acetate when heated in presence of hydrogen peroxide (H 2 O 2 ), with the formation of perchromic acid as an unstable intermediate. The chromic acetate so produced is measured. Absorbance was read at 480 nm within 30-60 seconds against distilled water.
Determination of superoxide dismutase (SOD) activity
A method originally described by Misra and Fridovich  as reported by Magwere et al., was employed. The homogenate was supplemented with 2.5 ml of carbonate buffer, followed by equilibration at room temperature; 0.3 ml of 0.3 nM adrenaline solution was then added to the reference and the test solution, followed by mixing and reading of absorbance at 420 nm.
Determination of gastric mucus
Adherent gastric glandular mucous was measured by the method of Corne et al. The excised stomach was soaked for 2 hours in 0.1% alcian blue dissolved in buffer solution containing 0.1 M sucrose and 0.05 M sodium acetate (pH adjusted to 5.8 with hydrochloric acid). After washing the stomach twice in 0.25 M sucrose (15 and 45 minutes), the dye complexed with mucous was eluted by immersion in 10 ml aliquots of 0.5 m MgCl 2 for 2 hours. The resulting blue solution was shaken with equal volumes of diethyl ether, and optical density of the aqueous phase was measured at 605 nm using a spectrophotometer. Using a standard curve, the absorbance of each solution was then used to calculate the concentration of the dye and the its weight (expressed in mg). The weight of the dye was then expressed over the weight of the stomach.
Determination of total protein concentration
Total protein concentration of the stomach homogenate was determined using the Randox Laboratories' protein estimation kit as described by Tietz. 
The data obtained was expressed as mean ± SEM (Standard Error of Means of seven observations) and analyzed statistically by application of the Statistical Package for Social Science (SPSS) version 11. The student's t-test was applied and P-values < 0.05 were considered to besignificant.
| » Results|| |
Development of Gastric Lesions
Indomethacin caused severe damage to the stomachs of the rats with an ulcer index of 4.32 ± 0.13 mm. Pre-treatment of rats with 2 mg/kg diet of folic acid prevented the formation of ulcers by 32%, and attenuated inhibition of mucus by 14%, while ranitidine afforded 56% prevention in ulcer formation and 67% attenuation of the inhibition of mucus [Table 1].
|Table 1: Effect of folic acid supplementation on indomethacin-induced gastric mucosal injury in rats, and its protein and mucus content, in the study|
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Lipid Peroxidation and Anti-oxidative Enzymes
Indomethacin produced lipid peroxidation with marked increase in MDA concentration, and significant reduction in the concentration of catalase and superoxide dismutase when compared with the normal saline treated group. The lipid peroxidation was attenuated by 58% reduction in malondialdehyde (MDA) concentration, following pretreatment with 2 mg/kg diet of folic acid, while ranitidine did it to the extent of 65%. Also, pre-treatment with 2 mg/kg diet of folic acid attenuated the indomethacin-induced inhibition of CAT by 51% and that of SOD by 150%. Ranitidine afforded a 55% and 78% attenuation of the inhibition of CAT and SOD respectively, by indomethacin [Table 2].
|Table 2: Effect of folic acid supplementation on rats' gastric mucosa malondialdehyde, catalase and superoxide dismutase following indomethacin administration|
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Acid Secretion Profile
The basal acid secretion in the control animals was 0.65 ± 0.01 mmol/10 minutes, while that of folic acid pretreated animals was 0.51 ± 0.02 mmol/10 minutes. Histamine stimulated secretion peaked at 2.60 ± 0.01 mmol/10 minutes in the control group, and 1.56 ± 0.02 mmol/10 minutes in the folic acid pretreated animals [Figure 1].
|Figure 1: Basal and histamine stimulated secretion in control and folic acid pretreated rats|
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| » Discussion|| |
Non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin induce an injury to gastrointestinal mucosa in experimental animals and humans, and their use is associated with a significant risk of hemorrhage, erosions, and perforation of both gastric and intestinal ulcers.  The molecular basis for the gastrointestinal toxicity of NSAIDs is widely believed to be their inhibitory activity against cyclooxygenase, which causes them to block the production of prostaglandins. This is associated with reduction of gastric mucosal blood flow, disturbance of microcirculation, decrease in mucus secretion, lipid peroxidation, and neutrophil activation, which are involved in the pathogenesis of gastrointestinal mucosal disorders. 
The findings of the present study show that folate, an important factor in the de novo synthesis of purines, and thymidine, Deoxyribonucleic acid (DNA) stability, and apoptosis,  was able to attenuate the development of gastric ulcer. The effects of folic acid dietary manipulation have been extensively studied in experimental models of cancer and cardiovascular disease,  however, there is a relative paucity of data regarding the effects of folic acid supplementation on peptic ulceration.
Gastrointestinal wall integrity is known to be controlled by two opposing forces: Aggressive and defensive.  The aggressive force encompasses the increase in acid output and subsequent lipid peroxidation, which is a result of the reaction between oxyradicals and the polyunsaturated fatty acids. The defensive actions are gastroprotective in nature and involve the anti-oxidative enzymes; superoxide dismutase (SOD) which catalyses the dismutation of superoxide radical anion (O2− ) into less noxious hydrogen peroxide (H 2 O 2 ), and catalase (CAT) or glutathione peroxidase that inactivate H 2 O 2 to water.  Indomethacin has been known to cause lipid peroxidation  with depletion of endogenous antioxidants. In the present study, this is confirmed by decrease activity of both catalase (CAT) and superoxide dismutase (SOD) with the concomitant increase in malondialdehyde (MDA) concentration in the homogenized gastric mucosal samples after indomethacin administration. Depletion of the antioxidant reserve and mucus in the gastric mucosa is an important factor in the pathogenesis of peptic ulceration. Hence, increase in the superoxide dismutase and mucus concentration observed in the folic acid pre-treated group suggests gastroprotective activity of folates, because they are scavengers which mop up and resist free radicals predisposing the stomach to inflammation. Moreover, this is underscored by a decrease in the MDA concentration observed in this group of animals which agrees with the mild severity of the wound in the glandular portion of the stomach when viewed macromorphologically. This could mean that folate inhibits the lipid peroxidative activity of indomethacin. Earlier reports of Cao et al., had even showed that both the epithelial apoptosis rate and the tumor suppressor p53 expression in gastric mucosa were significantly increased, while the expression of Bcl-2 oncogene protein decreased after folic acid treatment of patients with premalignant gastric lesions.
The presence of acid in the lumen of the stomach may not be a primary factor in the pathogenesis of indomethacin-induced gastropathy, it can make an important contribution to the severity of these lesions by impairing the restitution process, interfering with hemostasis and inactivating several growth factors that are important in mucosal defense and repair.  This necessitated the acid secretion study; which clearly showed that both basal and stimulated acid secretory rates are reduced by folic acid. Further studies to investigate the mechanistic events involved in the reduction of acid output by folic acid are on-going in our laboratory.
In conclusion, folic acid attenuates indomethacin induced gastric ulceration via anti-oxidative and anti-secretory mechanisms. It ameliorates the oxidative stress in the gastric mucosa by strengthening the defensive forces and weakening the attack factor. It inhibits acid output, an important factor in the pathogenesis of gastric ulceration.
| » References|| |
|1.||Valle DL. Peptic ulcer diseases and related disorders. In: Braunwald E, Fauci AS, Kasper DL, Hauser SL, Longo DL, Jameson JL, editors. Harrison's Principles of Internal Medicine. 15 th ed. New York: McGraw-Hill; 2002. p. 1649-65. |
|2.||Yamaguchi N, Kakizoe T. Synergistic interaction between Helicobacter pylori and gastric cancer. Lancet Oncol 2001;2:88-95. |
|3.||Rostom A, Wells G, Tugwell P, Welch V, Dube C, McGowan J. Prevention of chronic NSAID induced upper gastrointestinal toxicity. Cochrane Database Syst Rev 2000;15:37-41. |
|4.||Ma L, Wang WP, Chow JY, Yuen ST, Cho CH. Reduction of EDP is associated with delay of ulcer healing by cigarette smoking. Am J Physiol Gastrointest Liver Physiol 2000;278: G10-7. |
|5.||Mawdsley JE, Rampton S. The role of psychological stress in inflammatory bowel disease. Neuroimmunomodulation 2006;13:327-36. |
|6.||Ibironke GF, Olaleye SB, Balogun O, Aremu DA. Antiulcerogenic effect of diet containing seeds of Garcinia kola (Heckel). Phytother Res 1997;11:312-3. |
|7.||Guo JS, Chau JF, Cho CH, Koo MW. Partial sleep deprivation compromises gastric mucosal integrity. Life Sci 2005;77:220-9. |
|8.||Hung CR. Importance of histamine, glutathione and oxyradicals in modulating gastric haemorrhagic ulcer in septic rats. Clin Exp Pharmacol Physiol 2000;27:306-12. |
|9.||Al Mofleh IA. Spices, herbal xenobiotics and the stomach: Friends or foes? World J Gastroenterol 2010;16:2710-9. |
|10.||Kim YI. Role of folate in cancer development and progression. J Nutr 2003;133 Suppl 1:3731S-9. |
|11.||Boushey CJ, Beresford AA, Omen GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. JAMA 1995;274:1049-57. |
|12.||Berry RJ, Zhu L, Erickson DJ, Song L, Moore CA, Wang H, et al. Prevention of neural tube defects with folic acid in China. N Engl J Med 1999;341:1485-90. |
|13.||Ebisch IM, Thomas CM, Peters WH, Braat DD, Steegers-Theuniseen RP. The importance of folate, zinc, and antioxidants in the pathogenesis and prevention of subfertility. Hum Reprod Update 2007;13:163-74. |
|14.||Mason JB. Folate status: Effect on Carcinogenesis. In: Bailey LB, editor. Folate in Health and Disease. New York: Marcel Dekker, Inc.; 1995. p. 361-78. |
|15.||Sonnenberg A. Geographic and temporal variations in the occurrence of peptic ulcer disease. Scand J Gastroenterol Suppl 1985;110:11-24. |
|16.||Odaibo SK. Current concept in the management of peptic ulcer disease. Niger Med Pract 1998;15:37-41. |
|17.||Hunter GD, Millson GC, Chandler RL. Observations on the comparative infectivity of cellular fractions derived from homogenates of mouse-scrapie brain. Res Vet Sci 1963;4:543-9. |
|18.||Gutteridge JM, Wilkins S. Copper-dependent hydroxyl radical damage to ascorbic acid: Formation of a thiobarbituric acid reactive product. FEBS Lett 1982;137:327-30. |
|19.||Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972;47:389-94. |
|20.||Misra HP, Fridovich I. The role of superoxide anion in the auto-oxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972;247:3170-5. |
|21.||Magwere T, Naik YS, Hassler JA. Effect of chloroquine treatment on antioxidant enzymes in rat liver and kidney. Free Radic Biol Med 1997;22:321-7. |
|22.||Corne SJ, Morrissey SM, Woods RJ. A method for the quantitative estimation of gastric barrier mucous. J Physiol 1974;242:116P-7. |
|23.||Tietzs NW. Total protein determination. Clinical Guide to Laboratory Tests. 3 rd ed. Philadelphia: W. B. Saunders; 1998. p. 518-9. |
|24.||Naito Y, Kurroda M, Mizushima K, Takagi T, Handa O, Kokura S, et al. Transcriptome analysis for cytoprotective actions of Rebamipide against indomethacin-induced gastric mucosal injury in rats. J Clin Biochem Nutr 2007;41:202-10. |
|25.||Wallace JL. Pathogenesis of NSAID-induced gastro duodenal mucosal injury. Best Pract Res Clin Gastroenterol 2001;15:691-703. |
|26.||Huang RF, Ho YH, Lin HL, Wei JS, Liu TZ. Folate deficiency induces a cell cycle specific apoptosis in HepG2 cells. J Nutr 1999;129:25-31. |
|27.||Kim YI. Folate and DNA methylation: A mechanistic link between folate deficiency and colorectal cancer? Cancer Epidemiol Biomarkers Prev 2004;13:511-9. |
|28.||Masuda E, Kawano S, Nagano K, Tsuji S, Takei Y, Tsujii M, et al. Endogenous nitric oxide modulates ethanol induced gastric mucosal injury in rats. Gastroenterology 1995;108:58-64. |
|29.||Kapui Z, Boer K, Rozsa I, Blasko GY, Hermecz I. Investigations of indomethacin-induced gastric ulcer in rats. Arzneimittelforschung 1993;43:767-71. |
|30.||Cao DZ, Sun WH, Ou XL, Yu Q, Yu T, Zhang YZ, et al. Effects of folic acid on epithelial apoptosis and expression of Bcl-2 and p53 in premalignant gastric lesions. World J Gastroenterol 2005;11:1571-6. |
|31.||Naito Y, Iinuma S, Yagi N, Boku Y, Imamoto E, Takagi T, et al. Prevention of indomethacin-induced gastric mucosal injury in Helicobacter pylori-Negative Healthy volunteers: A comparison study Rebamipide vs Famotidine. J Clin Biochem Nutr 2008;43:34-40. |
[Table 1], [Table 2]
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