Journal of Gastroenterology and Hepatology (1992) 7 , 497-501

ALIMENTARY T R A C T A N D P A N C R E A S

Hydroxyl radical formation in human gastric juice S. N A L I N I , B. S. RAMAKRISHNA, A. M O H A N T Y A N D K. A. B A L A S U B R A M A N I A N The Wellcome Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College Hospital, Vellore, India

Abstract The hydroxyl radical is the most potent free radical derived from oxygen, and has been implicated in damage caused to the gastroduodenal mucosa. The ability of human gastric juice to generate hydroxyl radicals has been investigated in 54 adults with endoscopically normal gastroduodenal mucosa and in 39 patients with chronic duodenal ulcer. Hydroxyl radical production was measured by the formation of formaldehyde from dimethylsulfoxide. Unlike other body fluids, this reaction could proceed without the extraneous addition of catalysts such as hydrogen peroxide (H202), ascorbate and iron. Measurement of H202, iron and ascorbate showed that these catalysts are already present in the gastric juice. There was no significant difference in the concentration of these components in gastric juice between normal subjects and patients with duodenal ulcer, except that H 2 0 2 levels were slightly higher in duodenal ulcer patients. Although generation of free radicals has been investigated in other body fluids, this is the first reported case regarding the production of these active species in normal human gastric juice. Since hydroxyl production is not significantly enhanced in duodenal ulcer, we suggest that attention may be turned to mucosal antioxidant defences in this disease.

Key words: duodenal ulcer, gastric juice, hydroxyl radicals. INTRODUCTION Free radicals derived from oxygen have been implicated in the pathogenesis of several gastrointestinal diseases. 1-3 These free radicals include the superoxide anion and hydrogen peroxide (H202)which are produced in a wide range of biological reactions involving the metabolism of oxygen. Hydroxyl radicals are produced from H202 in the presence of a metal catalyst. This reaction is enhanced by superoxide or ascorbate which keeps the metal in reduced form.4 Hydroxyl radicals are extremely powerful oxidants and will react with almost all biologic substrates unlike superoxide and H202, suggesting a great capacity for cellular damage close to its site of production. The gastrointestinal epithelium is continuously exposed to reactive oxygen metabolites that are generated both in the lumen and in the tissue. Experimental evidence indicates that these active species may mediate or perpetuate cellular injury in the gastric mucosa brought about by stress as well as various structurally unrelated chemical^.^-'^ Similarly, a role has been postulated for oxygen-derived free radicals in the genesis of duodenal ~ l c e r a t i o n . ~ ~Although ” oxygen-derived free radicals have been implicated in damage to the gastric mucosa, we are not aware of any study on the possible source of free radicals in the gastric juice. T h e aim of the

present study was; to measure levels of various compounds such as H202, ascorbic acid and iron, which might facilitate the generation of oxygen-derived free radicals; to assess the capacity of gastric juice to generate the active hydroxyl radicals; and to assess soluble antioxidant protective thiols in the gastric juice. These parameters were studied in gastric juice obtained from normal subjects and patients with duodenal ulcer.

METHODS Chemicals Bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), 5,5’ dithiobis 2-nitrobenzoic acid (DTNB) 2,4 dinitrophenyl hydrazine and 2,6 dinitrophenol indophenol were all purchased from Sigma Chemical Co. All other chemicals used were of analytical grade.

Subjects Ninety-three subjects undergoing upper gastrointestinal

(GI) endoscopy for dyspepsia as part of their investigation were enrolled in this study. Fifty-four of these (36 male,

Correspondence: K. A. Balasubramanian, The Wellcome Research Laboratory, Department of GastrointestinalSciences, Christian Medical College Hospital, Vellore 632 004, Tamilnadu, India. Accepted for publication 19 March 1992.

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498 18 female) had a normal endoscopy, while 39 (32 male, seven female) were found to have a chronic duodenal ulcer. The normal controls ranged in age from 15 to 66 years (median 32 years), while the duodenal ulcer group ranged in age from 22 to 80 years (median 38 years). Patients with endoscopic gastritis were excluded from the study.

Gastric juice collection All the subjects underwent upper GI endoscopy after an oveynight fast. At endoscopy, a sterile teflon cannula was passed through the biopsy channel of the endoscope and the residual gastric juice in the stomach was aspirated into a syringe and discarded. Gastric juice was then aspirated for the next 15 min, taking care to avoid trauma to the mucosa. Gastric juice was centrifuged at 10 OOOx g for 20 min in a refrigerated centrifuge to remove mucus and other debris. The clear, colourless juice was used for the study. Juice contaminated with bile or blood was not used. The pH was measured and the juice was then frozen at - 20°C and analysed within 4 weeks.

Hydroxyl radical production Hydroxyl radicals or hydroxyl radical-like species were measured in gastric juice by quantitating the production of formaldehyde from DMSO. The reaction mixture consisted of 100 mmol/L potassium phosphate buffer pH7.4, 50 mmol/L DMSO (all final concentrations) and 0.5 mL gastric juice in a total volume of 1 mL.” After addition of DMSO the mixture was incubated at 37°C for 30 min and the reaction was terminated with the addition of 50 pL of 100% trichloroacetic acid (TCA). Production of formaldehyde in the supernatant was determined after centrifugation by the Nash r e a ~ t i 0 n . I ~Briefly, equal volumes of TCA supernatant and the Nash reagent (50 mmol/L acetic acid, 20 mmol/L acetyl acetone and 2 mmol/L ammonium acetate) were incubated at 37°C for 40 min in the dark and the absorption of the resulting diacetyldihydrolutidine was measured at 412 nm. A standard curve was prepared using formaldehyde and the product was expressed as nmol formed per mL of gastric juice. In preliminary studies with gastric juice from normal controls, exogenous ferrous iron (10 pmol/L) was added to the incubation mixture and compared with incubations without exogenous iron. N o significant difference in hydroxyl radical production was noted.

H,O, estimation H 2 0 2present in the gastric juice was measured using the ferric thiocyanate method.14 The standard peroxidase method could not be used owing to the acid pH of gastric juice. However the ferric thiocyanate method has been shown to be comparable with the peroxidase method in biological system^.'^ Briefly, to 1 m L of juice, 0.2 mL of 10 mmol/L ferrous ammonium sulfate and 0.1 mL of 2.5 mol/L potassium thiocyanate were added. The absorb-

ance of the red ferrithiocyanate complex formed in the presence of peroxide was measured at 480 nmol/L and compared with H,02 standard.

Ascorbic acid estimation Ascorbic acid and dehydroascorbic acid were measured spectrophotometrically using 2,4 dinitrophenyl hydrazine,15 and the values were expressed as pg/mL juice.

Iron The amount of iron present in the gastric juice was quantified using bathophenanthroline sulfonate.16 The colour that was formed was measured at 535 nm. A corresponding standard was run and the values were expressed as pg/mL.

Thiol compounds The thiol compounds present in gastric juice were measured using Ellmann’s reagent.” T h e intense yellow colour of the nitromercaptobenzoate anion formed was measured at 412 nm. Glutathione was used as a standard and the values were expressed as nmol/mL juice. Protein content of the gastric juice was quantified using BSA as is standard.”

Statistical analysis Statistical analysis was carried out using the SYSTAT package on a DCM Tandy computer and the results expressed as mean and standard error (s.e.m.). The statistical significance of the difference was evaluated by Student’s t-test.

RESULTS Gastric juice was obtained from 54 subjects who had endoscopically normal gastric mucosa, and from 39 patients who had a duodenal ulcer. T h e pH of the gastric juice was 3.06 k 0.10 (mean k s.e.m.) in controls and 2.9 +- 0.11 in duodenal ulcer. T h e protein content was 2.56 k 0.14 and 2.62 f 0.16 mg/mL juice in controls and duodenal ulcer patients, respectively. Comparison of H,O, levels in the gastric juice showed an increase in its level in duodenal ulcer patients when compared with normal subjects (Fig. 1). The mean f s.e.m. value in patients was 288 f 36 nmol/mL (range 27-960nmol/mL) and was higher than in controls 209 f 23 nmol/mL (range 16-591 nmol/mL) ( P < 0.05). Gastric juice from fasting patients contained small amounts of iron, both in controls (2.14 f 0.18 pg/mL, range 0.3 to 8.2pg/mL) and in patients with duodenal ulcer (2.12 f 0.13 pg/mL, range 0.95 to 4.6 pg/mL). The iron content of gastric juice was similar in patients and controls (Fig. 1).

Hydroxyl radical formation

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Figure 1 Concentration of iron, H,O, and amount of hydroxyl radicals formed in the gastric juice of normal subjects and duodenal ulcer patients. C: control; DU: duodenal ulcer. (-) Median.

The formation of hydroxyl radicals was measured in the gastric juice without the exogenous addition of H,O, and iron. As shown in Fig. 1 formation of hydroxyl radicals was observed in gastric juice both from normal and duodenal ulcer subjects. However no significant difference was observed between normal subjects 15 1.29 nmol/mL (range 4.7-51.3 nmol/mL), and duodenal ulcer patients 14.2 f 1.23 nmol/mL (range 4.638.3 nmol/mL). Total ascorbic acid and dehydroascorbic acid levels were measured in the gastric juice as parameters of the extent of oxidation. Figure 2 shows that gastric juice ascorbic acid levels were not significantly different between normal subjects and duodenal ulcer patients. Dehydroascorbic acid concentrations were 5.58 f 0.52 pg/mL (range 0.6-15.3 pg/mL) in normals and 5.14 rt 0.66 pg/mL (range 0.6-22.7 pg/mL) in patients. Ascorbic acid content was 3.3 rt 0.32 pg/mL (range 0.39-13.7 pg/mL) in controls and 2.82 ? 0.23 pg/mL (range 0.6-7.45 pg/mL) in patients. Thiol levels were measured in the gastric juice obtained from patients and controls to measure protective ability against free radical-mediated damage. Since the proteins in the juice were not precipitated by acid, total levels measured include protein thiol, although the contribution by protein thiol may be minimal in the juice. As shown in Fig. 3, the thiol concentration was 84 9 nmol/mL (range 8-377 nmol/mL) in controls and 93 f 15 nmol/mL in patients (range 8-466 nmol/mL).

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DISCUSSION The sequential univalent reduction of molecular oxygen in biological systems results in the production of reactive oxygen metabolites such as the superoxide anion, hydrogen peroxide (H202)and the hydroxyl r a d i ~ a l , ' ~which .~~ are all implicated in the genesis of cellular damage in various tissues. Of the three, the hydroxyl radical is the most powerful oxidant, reacting with almost all biological substrates unlike superoxide and H,O,. T h e cytotoxic effects of both superoxide and H,O, may be largely due to their role as precursors of the hydroxyl radical, through the iron-catalysed Fenton reaction, which is facilitated by the presence of ascorbic acid. This process has been investigated in certain body fluids such as plasma, syn-

Figure 2 Concentration of ascorbic acid (reduced and oxidized) in the gastric juice of control and duodenal ulcer patients. (-) Median.

ovial fluid and lymph.12 However this is the first report of the ability of the gastric juice in humans to spontaneously generate hydroxyl radicals. Experimental studies have shown that oxygen-derived free radicals may be implicated in the genesis of gastric ulceration induced by haemorrhagic shock, ethanol, aspirin and restraint-induced ~ t r e s s . ~Experiments -~ using cultured rat gastric mucosal cells have shown that H202 and hydroxyl radicals are important mediators of injury to these cells.2',22 Since gastric juice is in constant contact with the mucosal cells, it was appropriate to test this as a possible source of these active metabolites. The present study has shown that H,02 is present in the gastric juice. It is possible that this H,02 is derived both from gastric epithelial cells and from the phagocytes in the gastric mucosa. The study also shows that free iron, which is essential for hydroxyl radical production, is present in the gastric juice. Free iron is unusual in biological systems, but the acidity of gastric juice may explain these observations. 500

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Figure 3 Total thiol groups in the gastric juice of controls and duodenal ulcer patients. (-) Median.

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Department of Science and Technology, Government of The presence of H202, free iron and ascorbic acid in India, through a research grant is acknowledged. The gastric juice explains the ability of human gastric juice to authors thank Professor V. I. Mathan for his keen interest generate hydroxyl radicals as shown in this study. This in this work. suggests the possibility that there is a continuous production of this active species in wiwo. In contrast to this, the external addition of catalysts is necessary for the REFERENCES generation of hydroxyl radicals in other body fluids tested.12 In wivo formation of hydroxyl radicals following the intragastric administration of ferrous salts to rats has D. A. Oxygen free radicals: Mediators of gastrointes1. PARKS been ~bserved.'~In the present study iron concentrations tinal pathophysiology. Gut 1989;30: 293-8. in gastric juice were around 40 kmol/L, which is conJ. J. M., MARXJ. J. M. & VAN HUTTUM J. 2. KONINGSBERGER siderably higher than the concentration necessary for Free radicals in gastroenterology: A review. Scand. 3. hydroxyl radical generation." Gustroenterol. 1987;23 (Suppl. 154): 30-40. T h e presence of ascorbic acid in gastric juice has been T. & SJODAHL R. Oxygen radicals: their role in 3. OTAMIRI shown by others, and has been found to be altered in selected gastrointestinal disorders. Dig. Dis. 1991; 9: certain pathological condition^.^^,'^ T h e present study 133-41. confirms the presence of ascorbic acid in fasting gastric C. C. Comparison of superoxide with other 4. WINTERBOURN juice. The glandular stomach has a high content of reducing agents in the biological production of hydroxyl reduced glutathione (GSH),26 which protects the gastric radicals. Biochem. 3. 1979;182: 625-8. Wucosa against oxygen to xi city."^'* Efflux of cellular 5. ITOHM.& GUTHP. H. Role of oxygen-derived free radicals glutathione into the extracellular fluid is known and this in hemorrhagic shock induced gastric lesions in the rat. glutathione may contribute to the acid soluble thiol comGastroenterology 1985;88: 1162-7. pounds in gastric juice. This study documents the presence P. R., LIVINGSTONE E. H., JACOBSK. M., 6. TARNASKY of these compounds in normal gastric juice after fasting. ZIMMERMAN B. J., GUTHP. H. & GARRICK T. R. Role of In addition to considerable evidence suggesting a role oxyradicals in cold water immersion restraint-induced for free radicals in the genesis of gastric u l ~ e r a t i o n , ~ - ' ~ gastric mucosal injury in the rat. Dig. Dis. Sci. 1990;35: there is also experimental evidence that free radicals may 173-7. be responsible for the genesis or perpetuation of duodenal 7. PIHANG., REGILLO C. & SZABOS. Free radicals and lipid ~lceration.~~ The " present study shows that while the peroxidation in ethanol or aspirin induced gastric mucosal normal human gastric juice provides a conducive medium injury. Dig. Dis. Sci. 1987;32: 1395-401. for the generation of oxygen-derived free radicals, no T., YOSHIDAN., NAITOY.et al. Role of oxygen 8. YOSHIKAWA significant differences other than slightly higher H,O2 radicals in the pathogenesis of gastric mucosal lesions levels were noted between normal controls and patients induced by water immersion restraint stress and burn stress with duodenal ulcer disease. in rats. 3. Clin. Biochem. Nutr. 1990;8: 227-34. The mucosa of the gastrointestinal tract has antioxidant G., BALTASB. & NAGYS. Role of 9. ZOLLEII., KARACSONY defences to protect it from damage by free radicals. oxygen-derived free radicals in hemorrhagic shockGastric mucus forms a protective barrier against the induced gastric lesions of rats. Acta Physiol. 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Hydroxyl radical production in body both normal controls and patients with duodenal ulcer fluids: Role of metal ions, ascorbate and superoxide. can spontaneously generate hydroxyl radicals, and hyBiochem. 3. 1981;198: 125-31. droxyl radical scavengers can prevent duodenal ulcer 13. NASHT. The colorimetric estimation of formaldehyde by In conclusion, this report provides evidence of the ability of gastric juice to support the spontaneous formation of the highly reactive hydroxyl radicals, while at the same time it is suggested that this ability does not significantly change in duodenal ulcer disease. Further studies of mucosal production of free radicals and of mucosal antioxidant defences in duodenal ulcer are likely to be useful.

ACKNOWLEDGEMENTS The Wellcome Research Unit is supported by The Wellcome Trust, London. Financial assistance from the

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Hydroxyl radical formation in human gastric juice.

The hydroxyl radical is the most potent free radical derived from oxygen, and has been implicated in damage caused to the gastroduodenal mucosa. The a...
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