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Department of Pharmacology, College of Medicine, National Cheng-Kung University, Tainan 70101 Taiwan, Republic of China
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED
ABSTRACT 
The aggravation of acid-induced gastric damage and its prevention by glucose, ascorbate or glutathione precursors was studied in fed and food-deprived rats. The stomachs of fed rats and those starved for 1, 3 or 5 d were vagotomized just before irrigating for 3 h with solutions containing 0-150 mmol HCl/L. Mucosal glutathione, mucus, lipid peroxides and acid back-diffusion were measured. Stomach ulcers were evaluated by morphological and histological examination. The preventive effects of glucose, ascorbate and a mixture of L-glutamine, L-glycine and L-cysteine were evaluated in the stomachs of rats that were starved for 5 d, vagotomized, then perfused for 3 h with 100 mmol HCl/L. Greater acid back-diffusion and ulcer formation, and lower glutathione and mucus levels in starved rats were dependent on the duration of starvation and luminal acidity. Increased acid back-diffusion and decreased glutathione and mucus production were negatively correlated (r < 
0.80, P < 0.05) with ulcer formation. A significant enhancement in mucosal lipid peroxide concentration and serious damage of forestomach and corpus mucosal cells were observed in starved rats exposed to 100 mmol HCl/L. These ulcerogenic factors were effectively inhibited in acid-perfused stomachs of food-deprived rats by daily intraperitoneal injection of the amino acid mixture (150 mg/kg) or by an average daily consumption via drinking water of glucose (10 g) or ascorbate (1.2 g). Starvation aggravated acid-induced gastric damage and was associated with greater acid back-diffusion and oxygen radical generation, and lower mucosal glutathione and mucus production.
INTRODUCTION
Starvation is commonly found in undeveloped countries. It is also observed in patients with gastrointestinal obstruction or terminal cancer. Since nutrients such as glucose and amino acids are essential for maintaining homeostatic functions of gastric cells, it is possible that deprivation of food leads to pathological changes of the gastric mucosa. Furthermore, hypoglycemia caused by starvation may result in copious secretion of gastric acid. The influence of gastric acid on the stomach of long-term starved animals is unclear, however.
In general, the integrity of the gastric mucosa is greatly affected by both offensive and defensive factors. Gastric acid back-diffusion and free radicals are two offensive factors that are related to ulcer formation. For example, when gastric mucosal barriers are disrupted by aspirin, ethanol or bile salts, the luminal free H+ diffuses back to the mucosa. Meanwhile, the mucosal Na+, K+ and Ca2+ move to the lumen. The extent of this gastric acid back-diffusion is closely correlated with ulcer formation (Davenport and Chavre 1968
). Our previous work demonstrated that acid back-diffusion plays an important role in the formation of mucosal hemorrhagic ulcer in either diabetic (Hung and Huang 1995) or chemical-stimulated rats (Hung et al. 1994). Reports have indicated that oxygen radicals or oxygen-derived reactive species are related to various diseases (Marx 1987) and oxidative stresses (Fukumura et al. 1995). These radicals are also involved in acute mucosal ulceration induced by indomethacin (Vaananen et al 1991) and ethanol (Takuji et al. 1987). The product of these free radicals, the lipid peroxides, can elicit tissue inflammation (Link 1993). However, it is unknown whether ulcer formation induced by acid back-diffusion or oxygen free radicals can be aggravated by starvation.
Defensive factors, such as mucus and reduced glutathione, may protect gastric mucosa against a variety of noxious agent-induced damages. Glutathione, a 
-glutamyl cysteinyl glycine tripeptide, plays a pivotal role in the cellular defense system (Mutoh et al. 1991). It acts to prevent lipid peroxidation by free radicals and oxygen-derived reactive species that may damage gastric mucosal cells. In studies in vivo, either exogenous or endogenous glutathione is able to protect gastric mucosa from ethanol- (Takeuchi et al. 1988) or aspirin- (Strubelt and Hoppenkamp 1983) induced damage. Patients with peptic ulcer were found to have lower gastric glutathione concentrations, thus weakening the defensive mechanism (Hirokawa and Kawasaki 1995). The protective effect of gastric mucus on the gastric mucosa has been reviewed by Allen et al. (1988). For example, gastric mucus protects gastric mucosa from aspirin-induced damage (Ransford 1978). Whether changes in endogenous glutathione and in mucus production are involved in the food deprivation-induced gastric injury remains unknown.
The aims of this study were to evaluate several factors in the formation of mucosal ulceration in starved rats. These factors include 1) the role of gastric acid, 2) the involvement of free radicals, 3) changes in endogenous glutathione and mucus production, and 4) preventive effects of glucose, amino acids and ascorbate.
MATERIALS AND METHODS
). The pathological differences in gastric mucosa exposed to acid solution were compared with those treated with saline.
Experiment 4.
Because free radicals also may be involved in the formation of mucosal ulcerations in starved rats, lipid peroxides, the metabolites of free radicals, were assessed. To determine the role of acid back-diffusion in the generation of oxygen free radicals, the stomachs of fed and 5-d starved rats were vagotomized and irrigated with 4 mL of either saline (154 mmol NaCl/L) or acid solution (100 mmol HCl and 54 mmol NaCl/L). After 3 h irrigation, stomachs were immediately removed, weighed and homogenized. The concentrations of free radicals in supernatants were measured by the thiobarbituric acid test (Ohkawa et al. 1979).
Experiment 5.
This experiment was designed to study the preventive effects of several nutrients on mucosal damage provoked by an acid solution containing 100 mmol HCl and 54 mmol NaCl/L in 5-d starved rats. In this experiment, rats were deprived of food at 0900 h on the first day of starvation and were divided into 4 groups. Group 1 received daily a single intraperitoneal injection of 1.0 mL amino acid mixture (150 mg/kg), and was allowed free access to tap water. The components of the amino acid mixture are glutathione precursors: 50 mg/kg L-glutamine, 50 mg/kg L-cysteine and 50 mg/kg L-glycine. The amino acid mixture was prepared fresh before use every day. The amino acid mixture was used to determine whether chronic administration of this combination may elevate mucosal glutathione levels in starved rats. The composition, concentration, as well as the delivery of this amino acid mixture was determined by the response test in our pilot study in which we observed a potent inhibitory effect of this amount of amino acid mixture on gastric acid back-diffusion and mucosal ulceration in starved rats.
). Group 3 received daily free access to tap water containing 30 g ascorbate/L and a single intraperitoneal injection of 1.0 mL saline. The average consumption of ascorbate by each starved rat was 1.2 g/d. The solution of ascorbate in drinking bottles was renewed fresh each day. To prevent ascorbate from oxidation upon light exposure, drinking bottles were wrapped with aluminum foil. Earlier reports indicated that ulcer formation induced by starvation in rats can be attenuated by this concentration and delivery of ascorbate (Cheney and Rudrud, 1974). Group 4 served as the control and received daily free access to tap water and an intraperitoneal administration of 1.0 mL saline.
. A small incision was made in the forestomach. The stomach contents were gently expelled from the incision. A polypropylene tube (1.0 mm i.d. × 20 mm) was inserted through the same incision and secured with a ligature. Subsequently the stomach was rinsed meticulously with warm saline (37°C). Care was taken to avoid gastric distension. The residues were gently removed.
). Isotonic solutions (7 mL) containing 0, 50, 100 or 150 mmol HCl/L were instilled into the rat stomachs with a syringe. The luminal contents were mixed with the same syringe by three repeated aspirations and injections, and 3 mL of the fluid was taken as an initial sample. The incision of the forestomach was tightly closed. The abdominal wound was also sutured. After 3 h, the rats were killed with an overdose of ether. The gastric sample (final sample) was collected and centrifuged at 1000 × g for 20 min.
(7 Iv) × Ic, where Fv and Iv are the volume (mL) of final sample and initial sample, respectively, while Fc and Ic are the ionic concentration (mmol/L) in the final sample and initial sample, respectively. Negative values indicate that the net ion flux was from the lumen to the mucosa. Positive values mean that the net ion flux was from the mucosa to the lumen.
Morphological and histological studies of gastric mucosa (Experiment 3).
As soon as the the final sample was collected, the stomach was filled with 1.0% formalin for 10 min. The mucosa was exposed by opening the stomach along the greater curvature. The length (mm) and the width (mm) of ulcers on the gastric mucosa were measured with a planimeter (1 × 1 mm) under a dissecting microscope (×0.7-3.0; American Optical Scientific Instrument 569, Buffalo, NY). The ulcer areas were determined as previously described (Hung et al. 1994): ulcer area = length × width × 
/4. The total ulcer area (mm2) of each stomach was recorded. Gastric mucosal damage was determined by a person unaware of the experimental procedures. Histological studies of the stomach were conducted by methods previously described (Hung et al. 1994). Briefly, after gross examination, the specimens taken from corpus were blocked and immersed in 10% neutral formalin for 2 d. Blocks were then dehydrated in a series of alcohols, cleared in xylene and embeded in paraffin. Sections (7 mm thickness) were cut and stained with hematoxylen and eosin as routine histological procedures. Each section was examined under a microscope (Nikon HF, X-IIA, Tokyo, Japan), and tissue damage was quantitated using a method similar to that illustrated by Masuda et al. (1993). The section was scored with an index of 0-5 in which 0 indicates normal appearance; 1, mild injury in the epithelial cells; 2, mild injury in the upper mucosal cells; 3, hemorrhage or edema in the mid or lower part of mucosal cells; 4, degranulation or necrosis of the epithelial cells; 5, serious cell disruption of lower part of the mucosa. The index of each section was evaluated on a cumulative basis to give a maximal score of 15.
Assessment of gastric mucus (Experiments 1, 2 & 5).
Gastric mucus was assessed by the method described by Corne et al. (1974). Briefly, the rat stomach was excised and washed with tap water. The sample was immersed in 10 mL of a solution (pH 5.8) containing alcian blue (1.0 g/L ), sucrose (0.16 mol/L) and sodium acetate (0.05 mol/L) for 2 h. To remove the unbound dye, the sample was washed for 15 and then 45 min in a solution of 0.25 mol sucrose/L. The mucus-bound dye was eluted by immersing the mucosa in 10 mL of 0.5 mol MgCl2/L for 2 h. The eluate was mixed with 10 mL of diethylether, and the absorbance of the solution was measured on a spectrophotometer (Hitachi U-3210, Tokyo, Japan) at 605 nm. The amount of alcian blue extracted from the tissue was analyzed against a standard curve which was obtained from known graded concentrations (5-50 mg/L) of alcian blue solutions. The results were expressed as µg alcian blue/g wet tissue.
Assay of mucosal glutathione (Experiments 1, 2 & 5).
The method of measuring gastric mucosal glutathione was similar to that reported by Kaplowitz et al. (1980), but with slight modification. After the final sample was collected, the rat stomach was weighed and homogenized in 2 mL of phosphate buffer (0.1 mol NaH2PO4, 0.25 mol sucrose/L, pH 7.4). Acivicin (250 µmol/L), an irreversible inhibitor of -glutamyltransferase, was added to the homogenate to inhibit the catabolism of glutathione. The samples were then centrifuged at 1300 × g for 15 min at 4°C. To determine the recovery of reduced thiol, the supernatant was added with or without glutathione (200 µmol of reduced glutathione contained in phosphate buffer solution, pH 7.0). Subsequently, 0.5 mL of 250 g trichloroacetic acid/L was added to 1.0 mL of the supernatant of each sample and kept for 30 min at 4°C. After centrifugation at 1000 × g for 15 min, the supernatant was used to determine glutathione using 2,2
-dinitro-5,5-dithiodibenzoic acid. The optical density was measured at 412 nm on a Hitachi spectrophotometer (model U-3210, Tokyo, Japan). All samples were measured in duplicate. Recovery of added internal standard was greater than 90% in all experiments. Absorbances of the samples were measured against a standard curve constructed with freshly prepared glutathione solutions (0.05-0.5 mmol/L) which were treated in the same manner as the tissue samples. The results obtained from tissue samples were expressed as µmol/g wet tissue.
Determination of lipid peroxides (Experiments 4 & 5).
Lipid peroxides were determined by estimating malondialdehyde (MDA) using the thiobarbituric acid test as demonstrated by Ohkawa et al. (1979). Namely, the stomachs of rats were promptly excised and rinsed with cold saline. To minimize the possibility of interference of hemoglobin with radicals, any blood adhering to the mucosa was carefully removed. The stomach was weighed and homogenized in 10 mL of 100 g KCl/L. The homogenate (0.5 mL) was added to a solution containing 0.2 mL of 80 g SDS/L, 1.5 mL of 200 g acetic acid/L, 1.5 mL of 8 g 2-thiobarbiturate/L and 0.3 mL distilled water. The mixture was incubated at 98°C for 1 h. Upon cooling, 5 mL of n-butanol:pyridine (15:1) was added. The mixture was vortexed for 1 min and centrifuged at 1300 × g for 10 min. The absorbance of the supernatant was measured at 532 nm. A standard curve was obtained by using 1,1,3,3-tetramethoxypropane. The recovery was over 90%. All samples were measured in duplicate. The results were expressed as nmole malondialdehyde/g wet tissue.
Chemicals.
The following reagent grade chemicals were used: acivicin, alcian blue, ascorbic acid, cysteine, 2,2-dinitro-5,5-dithiodibenzoic acid, glutamine, glycine, sodium laurylsulfate, 1,1,3,3-tetramethoxypropane, 2-thiobarbituric acid, reduced glutathione, glucose, pyridine, n-butanol, sodium acetate, trichloroacetic acid and sucrose. The purity of chemicals was over 98%. They were obtained from Sigma, St. Louis, MO., Wako, Tokyo, Japan and Fluka, Buchs, Switzerland. All drugs were freshly prepared before use.
Statistical analysis.
Experimental data are expressed as means ± SEM. Significant differences in single measurement traits of Experiments 1, 2 and 5 were analyzed statistically using the Tukey honestly significant difference (HSD) test for pairwise comparison after ANOVA (Montgomery 1984). In Experiments 3 and 4, data were first analyzed by a two-way ANOVA, then post-hoc comparisons were made using the Tukey HSD procedure when the interaction in the full two-way ANOVA model was statistically significant. A P-value 
0.05 was considered statistically significant.
RESULTS
Effect of graded acid concentrations on lowering of glutathione and mucus levels and on aggravation of gastric ulcer in starved rats (Experiment 2). When the stomachs of 5-d starved rats with vagotomy were irrigated with saline or isotonic acid solutions (50, 100 or 150 mmol HCl/L), an acid concentration-dependent elevation of acid back-diffusion accompanied by the reduction of gastric glutathione and mucus levels was found. The mucosal ulceration was also aggravated in an acid concentration-related manner (Fig. 2). In this experiment, no visible ulcers were observed on the gastric mucosa of saline-treated starved rats. However, in the acid-irrigated (100 or 150 mmol HCl/L) stomachs of 5-day starved rats, a significant enhancement in ulceration was observed on the mucosa of both forestomach and corpus. The formation of mucosal ulcerations produced by acid solutions in 5-d starved rats was correlated with luminal Na+ output (r = 0.81), luminal H+ loss (r =
0.93), mucosal mucus production (r = 0.83) and gastric glutathione concentration (r = 0.86) (Fig. 3). The mucosal ulceration produced in the acid-perfused stomach of starved rats was mainly due to enhancement in acid back-diffusion and the attenuation of mucus and glutathione production.
Table 1.
Effects of daily supplements of various nutrients on gastric acid back-diffusion, glutathione, mucus, lipid peroxides
and ulcer formation in rats starved for 5 days1
DISCUSSION
Fig. 4.
Histological studies of 5-d starved rat gastric mucosa exposed to either saline (A) or acid solution containing 100 mmol HCl and 54 mmol NaCl/L (B) for 3 h. A mild injury of epigastric cells is found in saline-perfused rats. However, in the acid-irrigated rat, a complete disruption of the upper cell layer and lamina propria in the mucosa is obtained. The damaged cells are characterized by karyohexis and dense homogenous acidophylic cytoplasm.
[View Larger Versions of these Images (163 + 163K GIF file)]
Fig. 5.
Histological scores of acid-induced gastric mucosal damage in fed and 5-d starved rats. The stomachs of fed and starved rats were vagotomized and irrigated with either saline (154 mmol NaCl/L) or acid solution (100 mmol HCl and 54 mmol NaCl/L) for 3 h. Values are means ± SEM, n = 6. Bars labeled with different letters are significantly different at P < 0.05 based on Tukey method for all pairwise multiple comparison.
[View Larger Version of this Image (17K GIF file)]
Fig. 6.
Influence of acid on gastric lipid peroxide generation in fed and 5-d starved rats. The stomachs of both fed and starved rats were vagotomized and irrigated with either saline or acid solution containing 100 mmol HCl and 54 mmol NaCl/L for 3 h. MDA = malondialdehyde. Values are means ± SEM, n = 6. Bars labeled with different letters were significantly different at P < 0.05 based on Tukey method for all pairwise multiple comparison.
[View Larger Version of this Image (19K GIF file)]
). The lowering of gastric mucosal glutathione in acid-perfused starved rats might partly result from damage of mucosal cells induced by acid back-diffusion, from deficiency of glutathione precursors, or from lowering of glutathione biosynthetic ability. The mucosal lipid peroxide levels in starved rats were significantly higher than those in fed rats. Our unpublished data showed that starvation-produced augmentation of lipid peroxides could be abolished by antioxidants, such as sodium benzoate and superoxide dismutases. Therefore, these observations suggest that long term starvation might cause oxidative stress. Furthermore, intraluminal acid markedly elevated lipid peroxide production in starved rats. Because reduced glutathione can inactivate oxygen free radicals and reactive oxygen species, the consumption of this mucosal protective substance may be increased during starvation.
). The nonsteroid anti-inflammatory drug-produced mucosal hemorrhagic ulcer may be due to a decrease of gastric mucus production (Ransford 1978). This study also showed that gastric mucus was lowered in an intraluminal acid concentration-dependent and starvation time-related manner. This lowered mucus production was also found to be closely correlated with ulcer formation in starved rats. Both the precursors and energy supply for biosynthesis of mucus might be reduced during starvation.
). Glucose, an energy source, is important for the maintenance of cellular functions and biochemical reactions. Our study indicated that glucose [10 g/(rat·d)] diminished the greater acid back-diffusion and lipid peroxide levels produced by infusing acid solutions in starved rats, whereas lower glutathione and mucus generation produced by starvation were potently elevated.
). Although rats can synthesize ascorbate from glucose, long term deprivation of glucose may cause hypoglycemia and deficiency of ascorbate, and may lead to the pathological changes of gastric mucosal cells. Our results showed that ascorbate [1.2 g/(rat·d)] possessed potent cytoprotective effects on the gastric mucosa by increasing glutathione and mucus production, and by decreasing acid back-diffusion and lipid peroxide generation. The supplementation of ascorbate produced more potent inhibitory effects on acid-induced lipid peroxide generation and enhanced mucus production in starved rats more than the supplementation of glucose or amino acid mixture. Actually, ascorbate possesses potent reducing properties, and acts as an antioxidant during biological oxidative stress. It can also spare glutathione and serve as an essential antioxidant in severely glutathione deficienct newborn rats (Martensson and Meister, 1991). In addition, ascorbate was reported to be a very effective antioxidant in human plasma (Frei et al. 1990), and can protect various tissues of guinea pigs against lipid peroxidation (Chakraborty 1994). Therefore, oxygen free radicals and/or oxygen-derived reactive species may play an important role in the formation of mucosal ulcers induced by acid in starved rats. Starvation-produced acid back-diffusion and oxygen free radical generation can lower endogenous mucosal glutathione and mucus production, and results in aggravation of gastric mucosal ulceration. A daily administration of glucose, an amino acid mixture of glutathione precursors, or ascorbate can effectively protect gastric mucosa against acid-induced injury in starved rats.
FOOTNOTES
Manuscript received 7 June 1996. Initial reviews completed 8 July 1996. Revision accepted 26 November 1996.
LITERATURE CITED
II.
Modification of amino acids. J. Biol. Chem.
1987;
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