Effect of a zinc l-carnosine compound on acid-induced injury in canine gastric mucosa ex vivo

Tracy L. Hill Department of Clinical Science, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Anthony T. Blikslager Department of Clinical Science, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

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Abstract

Objective—To examine whether a zinc l-carnosine compound used for treatment of suspected gastric ulcers in dogs ameliorates acid-induced injury in canine gastric mucosa.

Sample—Gastric mucosa from 6 healthy dogs.

Procedures—Mucosa from the gastric antrum was harvested from 6 unadoptable shelter dogs immediately after euthanasia and mounted on Ussing chambers. The tissues were equilibrated for 30 minutes in neutral Ringer's solution prior to incubation with acidic Ringer's solution (HCl plus Ringer's solution [final pH, 1.5 to 2.5]), acidic Ringer's solution plus zinc l-carnosine compound, or zinc l-carnosine compound alone. Tissues were maintained for 180 minutes in Ussing chambers, during which permeability was assessed by measurement of transepithelial electrical resistance. After the 180-minute treatment period, tissues were removed from Ussing chambers and labeled with immunofluorescent anti–active caspase-3 antibody as an indicator of apoptosis.

Results—Permeability of the gastric mucosa was significantly increased in a time-dependent manner by addition of HCl, whereas control tissues maintained viability for the study period. Change in permeability was detected within the first 15 minutes after acid application and progressed over the subsequent 150 minutes. The zinc l-carnosine compound had no significant effect on this increase in permeability. Apoptosis was evident in acid-treated tissues but not in control tissues. The zinc l-carnosine compound did not protect against development of apoptosis.

Conclusions and Clinical Relevance—Addition of HCl caused a dose-dependent increase in gastric permeability over time and apparent induction of apoptosis as determined on the basis of immunofluorescence. However, there was no significant protective effect of a zinc l-carnosine compound. Nonetheless, results suggested the utility of this method for further studies of canine gastric injury.

Abstract

Objective—To examine whether a zinc l-carnosine compound used for treatment of suspected gastric ulcers in dogs ameliorates acid-induced injury in canine gastric mucosa.

Sample—Gastric mucosa from 6 healthy dogs.

Procedures—Mucosa from the gastric antrum was harvested from 6 unadoptable shelter dogs immediately after euthanasia and mounted on Ussing chambers. The tissues were equilibrated for 30 minutes in neutral Ringer's solution prior to incubation with acidic Ringer's solution (HCl plus Ringer's solution [final pH, 1.5 to 2.5]), acidic Ringer's solution plus zinc l-carnosine compound, or zinc l-carnosine compound alone. Tissues were maintained for 180 minutes in Ussing chambers, during which permeability was assessed by measurement of transepithelial electrical resistance. After the 180-minute treatment period, tissues were removed from Ussing chambers and labeled with immunofluorescent anti–active caspase-3 antibody as an indicator of apoptosis.

Results—Permeability of the gastric mucosa was significantly increased in a time-dependent manner by addition of HCl, whereas control tissues maintained viability for the study period. Change in permeability was detected within the first 15 minutes after acid application and progressed over the subsequent 150 minutes. The zinc l-carnosine compound had no significant effect on this increase in permeability. Apoptosis was evident in acid-treated tissues but not in control tissues. The zinc l-carnosine compound did not protect against development of apoptosis.

Conclusions and Clinical Relevance—Addition of HCl caused a dose-dependent increase in gastric permeability over time and apparent induction of apoptosis as determined on the basis of immunofluorescence. However, there was no significant protective effect of a zinc l-carnosine compound. Nonetheless, results suggested the utility of this method for further studies of canine gastric injury.

Gastric ulcer disease is an increasingly recognized cause of morbidity and death in dogs, although reports of prevalence of ulcer disease are lacking. The causes of gastric and pyloric ulcers are regarded as multi-factorial, including liver disease, NSAID treatment, and corticosteroid treatment. Gastritis, inflammatory bowel disease, renal failure, shock, recent surgery, intervertebral disk disease, stress, mast cell disease, gastric neoplasia, Helicobacter spp infection, and gastrinoma have also been associated with ulcers.1–4 Gastric perforation is a severe, potentially fatal sequela of gastric ulcers that may affect up to 27% of dogs with gastric ulcers.4 Of dogs that had perforation of gastric ulcers, between 37% and 69% were euthanized or died because of their disease.1,4,5

Several drugs have been investigated in dogs for treatment and prevention of gastric ulcers, including H2-receptor antagonists, proton pump inhibitors, and prostaglandin analogs. Although some of these have efficacy in both preventing and treating gastric ulceration and erosion, there remains the desire to optimize ulcer prevention. A zinc l-carnosine compound6 is a product that purportedly achieves optimal gastric health by adhering to the stomach lining, reinforcing natural defenses, and providing enhanced antioxidant activity, thereby providing protection from gastric ulcer disease.7 The zinc l-carnosine compound is composed of a zinc-carnosine complex plus vitamin E. There are multiple reports on the efficacy of the zinc l-carnosine compound zinc-carnosine complex in rats and humans. In rat epithelial cell culture, zinc l-carnosine compound prevented indomethacin-, H2O2-, or ethanol-induced cytolysis.8,9 A zinc l-carnosine compound prevented ulcer formation induced by indomethacin and acetylsalicylic acid in live rats.9–11 Administration of zinc l-carnosine compound was also associated with more rapid healing of ulcers induced in rats with induced arthritis, diabetes mellitus, and portal hypertension.12,13 Humans administered zinc l-carnosine compound had reduced signs of ulcer disease and improved endoscopic scores.14 In another study,10 zinc l-carnosine compound prevented a change in gastrointestinal permeability induced by indomethacin. A zinc l-carnosine compound has been marketed for ulcer prevention and gastric health in dogs.

To date, there is only 1 reporta of the clinical efficacy of this compound in dogs as a protective agent against acetylsalicylic acid–induced gastric injury in 18 healthy mixed-breed dogs; the zinc l-carnosine compound did not prevent gastric lesions in that study. The purpose of the study reported here was to investigate the efficacy of a commercially available zinc l-carnosine compound in an ex vivo study of acid-induced injury of canine gastric mucosa.

Materials and Methods

Tissue acquisition—Tissue samples were obtained from dogs that were scheduled to be euthanized at a local animal shelter. The age of the dogs was unknown but ranged from approximately 10 months to 7 years. The dogs were typically of mixed breed, ranging in size from approximately 10 to 30 kg. All dogs were euthanized with an overdose of sodium pentothal. Immediately following euthanasia, the entire antral section of the stomach was excised. This tissue was incised along the greater curvature and placed mucosal side down in oxygenated (95% O2 and 5% CO2) Ringer's solution (114.0mM NaCl, 5.0mM KCl, 1.25mM CaCl2, 1.10mM MgCl2, 25.0mM NaHCO3, 0.3mM NaH2PO4, and 1.65mM Na2HPO4) at room temperature (25°C). After transport to the laboratory, the tissue was transferred to oxygenated Ringer's solution at room temperature, and the seromuscular layer was removed via blunt dissection. The remaining antral mucosa tissue was mounted on Ussing chambers (area, 1.14 cm2), with 1 tissue sample/treatment group.

Ussing chambers—Mucosa was bathed on the mucosal and serosal sides of the chambers with 10 mL of oxygenated Ringer's solution maintained at 37°C via water-jacketed reservoirs. Then, 10 mmol of glucose/L was added to the serosal bathing solution, which was balanced with the addition of 10 mmol of mannitol/L in the mucosal bathing solution. The spontaneous PD was measured with Ringer-agar bridges connected to calomel electrodes, and the PD was short circuited through silver-silver chloride electrodes with a voltage clamp that corrected for fluid resistance. Resistance (Ω•cm2) was calculated from the spontaneous PD and Isc. If the spontaneous PD was between −1 and 1 mV, tissues were current clamped at ± 100 μA for 5 seconds and the PD was recorded. The Isc and PD were recorded every 15 minutes for 2 hours. Data were entered into spreadsheets that calculated TER from Isc and PD via Ohm's law. After 30 minutes of incubation, 1 of 3 treatments was added to the mucosal surface, signified as time 0. For each dog, all 3 treatments were applied to tissue mounted in separate chambers. For dogs in the first treatment group, neutral Ringer's solution was replaced with acidic Ringer's solution (pH, 1.45 to 1.55). In the second treatment group, tissues remained in Ringer's solution at a pH of 7.4, with the addition of 70 mg of pulverized zinc l-carnosine compound in a stock 100 mg/mL solution (7 mg/mL in chamber solution). This dose approximates the canine gastric concentration in vivo on the basis of gastric volume (using 10 mL/kg for gastric volume) for a 15-kg dog when given the recommended dose of half of a 70-mg tablet and extrapolated to the estimated dose for 10 mL of gastric juice. For the third treatment, tissues were subjected to both acidic Ringer's solution and the zinc l-carnosine compound treatment. A fourth chamber was kept as a control with neutral Ringer's solution. Tissues were maintained on the Ussing chambers with applied treatments for an additional 150 minutes. The pH was measured every 30 minutes, and HCl was added as needed to maintain pH between 1.45 and 1.55 for acid treatment groups. After 180 minutes (including the initial equilibration period and 150-minute treatment period), the tissues were removed.

Histologic examination—Gastric mucosal samples were obtained for each dog prior to mounting on Ussing chambers. After 180 minutes, the tissues were collected from each of the 4 treatment groups and placed in neutral-buffered 10% formalin. All 5 samples from each dog were sectioned at a thickness of 5 μm, stained with H&E, and viewed with a light microscope.

Immunofluorescence—Tissue sections were obtained for each treatment group (neutral Ringer's solution with and without zinc l-carnosine compound or acidic Ringer's solution with and without zinc l-carnosine compound) and embedded in optimal cutting temperature medium.b Sections were thawed, fixed in cold acetone, and blocked with 10% goat serum. Following blocking with goat serum, sections were incubated with rabbit anti–active caspase-3c antibody in 2% goat serum overnight at 4°C. After several washes with PBS solution and 0.1% Tween in PBS solution, sections were incubated with goat anti-rabbit antibody labeled with fluorescein isothiocyanated and diluted in 2% goat serum for 60 minutes. Slides were washed with PBS solution and 0.1% Tween in PBS solution and hard mounted with mounting medium with 4′,6-diamidino-2-phenylindole stain for DNA.e Sections were examined with a fluorescent microscope,f and softwareg was used to record images.

Data analysis—A 2-way repeated-measures ANOVA was used to compare TER data among the 4 treatment groups (control, acidic Ringer's solution, zinc l-carnosine compound, and acidic Ringer's solution with zinc l-carnosine compound) over the time period the tissues were in the Ussing chambers. The Holm-Sidak post hoc test was used to detect differences among treatments and time when significance was detected during the initial ANOVA. Values of P < 0.05 were considered significant.

Results

For the first 30 minutes of incubation time prior to application of treatments, there were no significant changes in TER for any of the treatment groups. There was no significant change in TER over the entire treatment period in either control group (neutral Ringer's solution or neutral Ringer's solution with zinc l-carnosine compound). Treatment with acidic Ringer's solution induced a significant decrease in the TER beginning 75 minutes after acid application in both acidic Ringer's treatment groups (Figure 1). There was no effect of zinc l-carnosine compound on TER with acid treatment. There was a significant effect of individual dog, reflecting the large variability in resistance among individual dogs.

Figure 1—
Figure 1—

Mean ± SE values of TER in ex vivo samples of canine gastric mucosa after various treatments.

Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.659

Histologic examination—Compared with baseline gastric mucosa, tissue that had been maintained in neutral Ringer's solution for 180 minutes (control) appeared normal with no remarkable crypt destruction in studied samples (Figure 2). Alternatively, tissues exposed to acidic Ringer's had moderate to marked crypt destruction. These lesions were also observed to the same degree in tissue that had been treated with acidic Ringer's solution and zinc l-carnosine compound. The tissue that was treated with zinc l-carnosine compound in neutral Ringer's solution did not appear affected and was similar to control tissue.

Figure 2—
Figure 2—

Photomicrographics of ex vivo samples of canine gastric mucosa after 180 minutes of various treatments. A—Control tissue. Notice there is no evidence of morphological changes. B—Zinc l-carnosine compound treatment with neutral Ringer's solution. Notice that treatment did not result in any change in morphology, compared with control tissues. C—Acid-injured tissue. Notice moderate to severe disruption of normal epithelium. D—Acidic injury with zinc l-carnosine compound treatment. Notice a similar degree of injury, compared with that of acid-injured tissue alone. H&E stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.659

Immunofluorescence—Neither control tissues nor control tissues treated with zinc l-carnosine compound had evidence of fluorescein isothiocyanate staining for active caspase-3, indicative of apoptosis. In acid-injured tissue, active caspase-3 staining was notably increased (Figure 3). Similarly, active caspase-3 was observed in the acid-injured tissue cotreated with zinc l-carnosine compound, indicating that zinc l-carnosine compound did not protect against the development of apoptosis induced by acid.

Figure 3—
Figure 3—

Photomicrographic views of ex vivo samples of canine gastric mucosa after various treatments and staining for immunofluorescence of active caspase-3 with a fluorescein isothiocyanate (FITC)–labeled anti–active caspase-3 antibody. Notice that use of a 4′,6-diamidino-2-phenylindole (DAPI) stain reveals nuclear material in epithelial cells for reference. The control and zinc l-carnosine compound–only groups have little to no staining for active caspase-3, indicating low apoptosis. Acid-injured tissue, both with and without zinc l-carnosine compound treatment, had moderate apoptosis of gastric epithelium. Merged = Merged images of staining with FITC and DAPI. Bar = 100 μm.

Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.659

Discussion

In this study of acid-induced gastric injury, zinc l-carnosine compound was not protective against mucosal damage or apoptosis. There are several mechanisms by which zinc-carnosine compound has been proposed to prevent gastric ulceration. For instance, it inhibits lipid peroxidation, decreases apoptosis, inhibits neutrophil activity, and suppresses nuclear factor κB and interleukin-8. In addition, zinc l-carnosine compound reduces tumor necrosis factor-α, induces insulin-like growth factor 1, and induces heat shock protein 72-kDa expression.9,11–13,15–19 However, these effects may be dependent on factors that are not applicable in Ussing chamber studies, such as mucosal blood flow. In support of the findings of the present study, Baan et al6 found that the same commercially available zinc l-carnosine compound was not protective against acetylsalicylic acid–induced ulcers in live dogs.

For all dogs, sections of gastric mucosa were collected exclusively from the antral and pyloric regions of the stomach. Although this region, along with the proximal portion of the duodenum, is the most likely site for ulcers in dogs,4 it may be that the effects of zinc l-carnosine compound depend on the region of the stomach examined. Therefore, future studies examining additional regions of gastric mucosa may be warranted.

In canine gastric mucosa, acidic Ringer's solution at a pH of 1.5 induced a reliable and repeatable decrease in TER. This pH was within the reference range for a dog from which food had been withheld.18,19 In live dogs, multiple mechanisms protect against epithelial injury in the presence of acidic pH in this range. The gastric mucosa is coated by an unstirred and a stirred layer of mucus and bicarbonate, both of which are secreted by surface epithelial cells. Mucin units form a gel that lines the apical surface of the gastric epithelium, trapping acid within the gel as well as providing a space for bicarbonate and other factors important for protection.20 Bicarbonate, secreted by surface epithelial cells, lies within the mucus layer and serves as a buffering mechanism for any penetrating acid. Phospholipids, both within the mucus layer and lining the surface epithelium, are secreted by epithelial cells and repel acid- and water-soluble agents that could cause cell damage or necrosis. Ussing chamber–mounted tissue likely loses the most superficial layer of mucus because of circulation of Ringer's solution within the chamber but maintains mucus and bicarbonate adjacent to the epithelium.h In addition, mounted tissue does not have changes in mucosal blood flow that allow for rapid clearance of penetrating acid as would be expected to occur in live dogs. In addition, cell mediators important for stimulation of restitution and repair, such as epidermal growth factor, transforming growth factor-α, insulin-like growth factor 1, and tumor necrosis factor-α, may be lacking.20 The loss of these mechanisms in mounted mucosal tissue may explain the significant decrease in resistance and histologically evident loss of normal mucosal epithelium after acid application.

Regardless of the limitations of the method, including significant variability of TER among dogs, acidic Ringer's solution induced a repeatable decrease in TER and evidence of mucosal epithelial damage. Therefore, this method may prove useful for further studies of acid-induced injury and repair. On the basis of the method used in the present study, zinc l-carnosine compound did not prevent acid-induced mucosal injury.

ABBREVIATIONS

Isc

Short circuit current

PD

Potential difference

TER

Transepithelial resistance

a.

Gastricalm, DVM Pharmaceuticals, Teva Animal Health Inc, Saint Joseph, Mo.

b.

Tissuetek, Sakura, Torrence, Calif.

c.

Promega, Madison, Wis.

d.

Invitrogen, Carlsbad, Calif.

e.

Vector Laboratories Inc, Burlingame, Calif.

f.

Meiji Techno America, Santa Clara, Calif.

g.

Infinity Analyze, Lumenera Corp, Ottawa, ON, Canada.

h.

Argenzio RA, College of Veterinary Medicine, North Carolina State University Raleigh, NC: Personal communication, 1994.

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