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Sucralfate interactions with gastric mucus
Sucralfate Interactions
with Gastric Mucus
CLIFFORDTASMAN-JONES, M.B.Ch.B.,F.R.C.P.,F.R.A.C.P., BSc., GAILMORRISON, Dip. Med. Tech., LINDYTHOMSEN,BSc., MARKVANDERWEE, BSc. Auckland, NOW Zealand
Sucralfate protects the stomach against a number of experimental damaging agents and is efficacious in the treatment of peptic ulcer disease. It binds with acidity to the base of an ulcer to form a protective barrier. Sucralfate also enhances prostaglandin synthesis and release in the mucosa. In this study, the rat stomach was examined to determine sucralfate’s interaction with gastric mucus. Mucus in the rat stomach forms a distinct and continuous blanket. In snap-frozen samples, pretreatment with phosphate-buffered saline as a control shows a layer of mucus of homogeneous structure thinner than the homogeneous layer after pretreatment with antibodies developed against rat gastric mucus. Pretreatment with the surface protective agent sucralfate shows some increase in the thickness of mucus with a thin dense sublayer adjacent to the epithehum and a less dense-appearing outer zone of variable thickness. Analysis of x-rays generated by the electron beam on windows of mucus and epithelium showed the expected gradients of sodium, potassium, chloride, and sulfur. The percentage of aluminum and sulfur in the mucus was higher in sucralfate-treated samples than in controls. Interaction between sucralfate and gastric mucus needs further investigation.
ytoprotection refers to gastroduodenal mucosal protection by mechanisms other than acid reducC tion. The specific mechanisms involved in cytoprotection include increased gastric mucus secretion, increased bicarbonate secretion, reduced gastric epithelial permeability, preservation of circulation, and increased endogenous prostaglandin production [ll. Sucralfate, a commercially available aluminum salt of sucrose octasulfate, polymerizes in an acid medium to a viscous substance binding to the gastric and duodenal mucosa [Z]. In both rats [3,41 and humans, [51 sucralfate protects the gastric mucosa from damage by intraluminal noxious agents such as aspirin, nonsteroidal anti-inflammatory drugs, and alcohol and has a proven efficacy in the treatment of peptic ulcer disease [6]. In rats, sucralfate increases prostaglandin release into the gastric lumen [41 suggesting that prostaglandin is a factor in sucralfate’s protective mechanisms. Increased endogenous prostaglandin production does not, however, completely explain the effects of sucralfate [5]. The effect of sucralfate on the normal gastric mucus layer includes reduction of hydrogen ion diffusion through mucus and modification of sodium/hydrogen ion exchange of gastric mucus [7]. Because of its high water content, the layer of mucus is distorted during dehydration [8]. Antibodies raised against colonic mucus incubated with colonic mucosa “stabilized” a layer that can be examined [8]. Snap-freezing provides another method for maintaining a layer of mucus during specimen preparation. In this study, we compared the scanning electronmicroscopical features of snap-frozen rat gastric epithelium after being treated with phosphate-buffered saline (PBS), specific antigastric mucus antisera, and sucralfate. In addition, we performed x-ray microanalysis of the retained mucus to determine the relative concentrations of selected elements present in the gastric mucus and epithelium.
MATERIALS AND METHODS
From the Departments of Medlclne and Pathology, University of Auckland, Auckland, New Zealand. Requests for reprints should be addressed to Dr. Clifford Tasman-Jones, Departments of Medlclne and Pathology, Umverslty of Auckland, Auckland, New Zealand.
Young male Wistar rats were used in this study. The animals were fed a polymeric liquid diet (Ensure osmolite) for the seven days prior to the experiment to ensure that the stomach was empty of food debris at the time of experimental sampling. The diet was prepared at quarter strength in water and fed to the animals ad lib&m. The animals were weighed before and after the seven days of feeding. After anesthetization with sodium pentobarbital, the abdominal cavity was opened, the pylorus was hgated, and one of the following solutions was injected (1 to 2.5 ml) into the stomach until it was mildly and approximately equally dilated: (1) PBS pH 7.4, 0.15 M (four rats); (2) rabbit serum (heat inactivated) continuing a high titer of antibodies to rat gastric mucus in PBS (four rats); and (3) 1 percent solution of sucralfate in PBS (four rats). June 9, 1989
The American Journal of Medicine
Volume 86 (suppl 6A)
5
SYMPOSIUM
ON SUCRALFATElTASMANJONES
Figure 1. Snap-frozen, freeze-fractured pattern.
ET AL
rat gastric mucosa pretreated
with PBS alone (0.15 M, pH 7.4). The muc
After injection into the stomach, the abdominal cavity was partially closed and maintained moist with saline-soaked swabs. The rats were kept warm and maintained under light anesthesia for 1 hour before the stomachs were removed. The animals were killed by an intracardiac injection of a rapidly acting barbiturate. Tissue samples (approximately 1 cm’) of the body of the glandular stomach were taken. One sample from each rat was prepared for scanning electron microscopy and x-ray microanalysis Scanning Electron Microscopy
Tissue samples were snap-frozen in melting Freon 12 (-156°C) and then quenched in liquid nitrogen (- 196°C). They were transferred to a brass block, also at liquid nitrogen temperature, where they were fractured with a razor blade pre-cooled in liquid nitrogen. The fractured specimens were freeze-dried at -60°C for 24 to 48 hours in an Edwards-Pearse tissue drier using phosphorus pentoxide as a vapor trap and then mounted on aluminum stubs with double-sided tape such that the fractured surface lay uppermost and parallel to the stub surface. Carbon paint was applied to the specimen sides and the stub surface to ensure conductivity and reduce extraneous x-ray signals. They were coated with carbon by vacuum evapo-
6
June 9, 1989
The American Journal of Medicine
Volume 86 (suppl 6A)
ration and then examined in an IS1 DS-130 scanning electron microscope fitted with a Robinson back-scattered electron detector and x-ray microanalytical facilities. Following the collection of x-ray spectra, the specimens were coated with palladium/gold and fractures through the mucosa and its associated mucus were photographed using an accelerating voltage of 20 kv. A section of mucus of each sample where the fracture was perpendicular through the gastric glands was measured to obtain some index of thickness. The two measurements of each section were made to indicate the thinnest and thickest mucus layer. Radiographic Analysis
Analyses were performed on every sample at the outer and inner aspects of mucus and the epithelium of the mucosa. X-rays were detected using an EG&G Ortec detector with a beryllium window. Analyses were performed for 200 live seconds using an accelerating voltage of 20 kv, spot size of 500 nm, and gun emission of 110 to 120 ,uA. The x-ray take-off angle was 36.5” and the count-rate varied between 800 and 3,200 counts/second. A minimum of three analyses were performed at different locations on the specimen, for each of the three areas of interest. The accumulated spectra were stored on floppy disk
SYMPOSIUM
Figure 2. Snap-frozen, freeze-fractured seen with a vertical fibrillar pattern.
rat gastric mucosa pretreated
ON SUCftALFATE /TASMAN.JONES
with antibodies against rat gastric mucus and suspended in PBS. A homogeneous
and used to determine the relative concentrations of elements present (normalized to 100 percent) using an EDAX semi-quantitative software routine that removed background, analyzed peak areas, and finally applied atomic number, absorption, and fluorescence correction to the results.
RESULTS All animals thrived on the liquid diet and increased their weights. At operation, stomachs were clear of food fibers and a significant layer of mucus above the glandular stomach could be defined. The appearance of the layer of mucus was consistent within each group. The scanning electron microscopical appearance of mucus in the pretreated PBS sample was a layer 40 to 125 pm thick with “fibrilles” that ran parallel and approximately perpendicular to the mucosal surface. While generally homogeneous, the more superficial layer was less dense than the remainder of the specimen (Figure 1). When antibodies raised against rat gastric mucus were used, the layer was similar in structure but with the fibrillar pattern more clearly defined and more parallel (Figure 2). This layer was 50 to 220 pm thick. Sucralfate-treated stomachs had two distinct layers: a very thin, deep layer that appeared dense and
ET AL
mucus layer is
homogeneous and a superficial layer that appeared to have a fibrillar pattern but less regular than that seen in Figure 2. The thickness (‘70 to 180 pm) was similar to that for the antibody-treated samples (Figure 3). Analysis of the x-rays induced by the electron bombardment of a specimen in the scanning electron microscope produced .a spectrum of peaks, the energies and heights of which represent specific elements and their relative concentrations, respectively (Table I). The average concentrations at each place of analysis (minimum, three analyses) are listed in Table I and are expressed as the percentage of that element present, relative to the other elements measured. All samples contained a small percentage of aluminum, whether or not sucralfate was used.
COMMENTS Scanning electron microscopical examination of unfixed, freeze-fractured, and freeze-dried material produced adequate morphologic preservation of both mucus and mucosa. The complete absence of chemical intervention in this methodology, although partially compromising the ultrastructural detail, was necessary to retain as accurately as possible the in viva elemental composition of the specimen for x-ray microanalytical purposes. Snap-frozen tissue specimens maintained a well-
June 9, 1989
The American Journal of Medicrne
Volume 86 (suppl 6A)
7
SYMPOSIUM
ON SIJCRALFATE /TASMAN-JONES
Figure 3. Snap-frozen, freeze-fractured a looser vertical frbrillar oattern.
ET AL
rat gastric mucosa pretreated with 1 percent sucralfate in PBS. Two layers appear visible with a dense but thinner basal layer and
formed layer of mucus in all specimens. Although variable in thickness, freeze-fractured sections were of consistent appearance within each treatment group. Although the mucosal samples were treated with great care, distortions of the mucus layer are likely to occur on opening the stomach and the manipulations during snap-freezing and mounting. The apparent differences in thickness need to be confirmed by other methods of preparation. Sucralfate does not appear to form a layer on top of mucus. The drug seems to penetrate the whole mucus layer. The apparent increase in thickness with both antibody and sucralfate preparation may be because of increased mucus release by increased prostaglandin secretion andlor by binding TABLE
I
Distribution
of Elements
Treatment Group PBS Mucus antibody Sucralfate
Mucus Outer Inner Outer Inner Outer Inner
in Gastric
Aluminum 1.1 i 1.8 t 0.7 f 0.4 2 4.2 * 3.4 k
0.6 2.0 0.3 0.1 2.1 1.8
Mucus*
Sodium 48.6 t 43.7 + 30.1 k 37.4 k 36.5 f 33.4 k
6.4 7.5 6.1 7.5 6.0 5.4
Sulfur
Others (K, Cl, P)
3.3 _c 1.6 3.5 i- 1.7 6.5 f 1.4 6.2 k 1.1 6.1 _c 1.4 7.2 A 1.6
41.2 51.2 50.3 51.7 50.8 53.9
= potassium; Cl = chlorine; P = phosphorus. Ixpressed as percent of elements measured; mean i SD. 8
June 9, 1989
The American Journal of Medicine
Volume 86 (suppl 6A)
superficial soluble mucus that would not normally be retained. Both the control and gastric mucus antibodytreated samples showed a “fibrillar” pattern to the mucus. Mucus is a highly hydrated substance with a normal water content that is very difficult to achieve with biologic samples. In particular, variations in texture and topography have a great influence on the x-rays detected. For this reason, we have taken multiple analyses at different sites, and used a scanning electron beam over a small selected area to overcome local variability. In all cases, the gradients of sodium and potassium are as expected, with the sodium being higher in the mucus than in the glandular epithelial layer, and potassium being the reverse. Aluminum was present in low relative concentrations (approximately 1 percent) at all sites in every treatment group except sucralfate, for which the levels were higher. It is uncertain whether this 1 percent concentration of aluminum is a “background” level due to extraneous x-rays arising from the aluminum stub supporting the specimens or a true indication of the levels in the tissue. The sucralfate treatment group, however, showed higher aluminum concentration with a decreasing gradient from the outer to the inner aspect of the mucus layer. The level of aluminum in the epithelium has not apparently been raised by this one-dose situation. Other workers reported that plasma aluminum concentration in patients with ulcers was not
SYMPOSIUM
raised after sucralfate treatment [91. However, there is evidence that sucralfate-treated patients have an increased urinary excretion of aluminum [lo]. Previous studies have shown that sucralfate protects the stomach against injury by necrotizing or irritating luminal substances. The mechanisms of this protection are complex with modification of proton diffusion, mucus viscosity, enzyme degradation of mucus, and the stimulation of endogenous prostaglandin secretion. It has been shown that protection by sucralfate is by stimulation of endogenous prostaglandin secretion [4,111. A recent primate study has demonstrated protection independent of prostaglandin [12]. Gastric mucus has a sodium/hydrogen ion exchange property [13]. Sucralfate bound to mucus maintains the moderate sodium/hydrogen ion exchange property of gastric mucus [7] and improves sodium/hydrogen ion exchange when this is impaired as in infection with Campylobacter pylori [71. This study shows that a layer of mucus is present at the gastric luminal surface. The layer appears to be thicker when treated with antibody or sucralfate, compared with PBS control. Although the thickness will add physical protection to the stomach, this is probably not the only protective mechanism induced by sucralfate. The effect of mucus complexed with sucralfate on hydrogen ion movement needs further clarification.
ON SlJCRALFATE/TASMANJONES
ET AL
REFERENCES 1. Roberts A, Nezamrs JE, Lancaster C, Hanchar Al: Cytoprotectron by prostaglandrns rn rats. Prevention of gastric necrosis by alcohol, HCI, NaOH, hypertonic NaCl and thermal injury. Gastroenterology 1979; 77: 433-443. 2. Guth PH: Mucosal coating agents and other nonantisecretory agents. Are they cytoprotective? Dig Dis Sci 1987; 32: 647-654. 3. Hollander D, Tarnawski A, Krause WJ, Gergely H: Protective effect of sucralfate against alcohol induced gastric mucosal injury in the rat. Macroscopic, histological, ultrastructural and functional time sequence analysis. Gastroenterology 1985; 88: 366-374. 4. Hollander D, Tarnawski A, Gergely H, Zipser RD: Sucralfate protection of gastric mucosa against alcohol-induced necrosis: a prostaglandin medrated process. Stand J Gastroenterol 1984; 19 (suppl 101): 97-102. 5. Wu WC, Semble EL, Caste11DO, et al: Sucralfate therapy of nonsteroidal antiinflammatory drug-induced gastritis (abstr). Gastroenterology 1985; 88: 1636. 6. Coste T, Rautureau J, Beaugrand M: Comparison of two sucralfate dosages presented in tablet form in duodenal ulcer healing. Am J Med 1987; 83 (suppl 36): 86-90. 7. Thomsen L, Tasman-Jones C, Morris A: Na+/H+ ion exchange property of postmortem human gastric mucus-the effect of Catnpylobacterpyloriinfection and sucralfate. Stand J Gastroenterol 1989; (in press). 8. Bollard JE, Vanderwee MA, Smith GW, Tasman-Jones C, Gavin JB, Lee SP: Preservation of mucus in situ in rat colon. Dig Dis Sci 1986; 31: 1338-1344. 9. Kinoshita H, Kumaki K, Nakano H, et at Plasma aluminum levels of patients on long term sucralfate therapy. Res Commun Chem Pathol Pharmacol 1982; 35: 515-518. 10. Haram EM, Webert R, Berstad A: Urinary excretion of alumirium after ingestion of sucralfate and an aluminium-containing antacid In man. Stand J Gastroenterol 1987; 22: 615-618. 11. Quodros E, Ramsamoo] E, Wilson DE: Relationshrp between sucralfate, gastrrc cytoprotection and prostaglandin and mucus synthesis and secretion (abstr). Gastroenterology 1985; 88: 1548. 12. Shea-Donohue T, Steel L, Montcalm E, Dubois A: Gastric protection by sucralfate. Role of mucus and prostaglandin. Gastroenterology 1986; 91: 660-666, 13. Thomsen L, Tasman-Jones C, Maher K, Wiggrns P, Lee S, Morris A: Na+/H+ ion exchange property of postmortem gastric mucus. Stand J Gastroenterol 1988; 23: 701704.
June 9, 1989
The American Journal of Medicine
Volume 86 (suppl 6A)
9
with Gastric Mucus
CLIFFORDTASMAN-JONES, M.B.Ch.B.,F.R.C.P.,F.R.A.C.P., BSc., GAILMORRISON, Dip. Med. Tech., LINDYTHOMSEN,BSc., MARKVANDERWEE, BSc. Auckland, NOW Zealand
Sucralfate protects the stomach against a number of experimental damaging agents and is efficacious in the treatment of peptic ulcer disease. It binds with acidity to the base of an ulcer to form a protective barrier. Sucralfate also enhances prostaglandin synthesis and release in the mucosa. In this study, the rat stomach was examined to determine sucralfate’s interaction with gastric mucus. Mucus in the rat stomach forms a distinct and continuous blanket. In snap-frozen samples, pretreatment with phosphate-buffered saline as a control shows a layer of mucus of homogeneous structure thinner than the homogeneous layer after pretreatment with antibodies developed against rat gastric mucus. Pretreatment with the surface protective agent sucralfate shows some increase in the thickness of mucus with a thin dense sublayer adjacent to the epithehum and a less dense-appearing outer zone of variable thickness. Analysis of x-rays generated by the electron beam on windows of mucus and epithelium showed the expected gradients of sodium, potassium, chloride, and sulfur. The percentage of aluminum and sulfur in the mucus was higher in sucralfate-treated samples than in controls. Interaction between sucralfate and gastric mucus needs further investigation.
ytoprotection refers to gastroduodenal mucosal protection by mechanisms other than acid reducC tion. The specific mechanisms involved in cytoprotection include increased gastric mucus secretion, increased bicarbonate secretion, reduced gastric epithelial permeability, preservation of circulation, and increased endogenous prostaglandin production [ll. Sucralfate, a commercially available aluminum salt of sucrose octasulfate, polymerizes in an acid medium to a viscous substance binding to the gastric and duodenal mucosa [Z]. In both rats [3,41 and humans, [51 sucralfate protects the gastric mucosa from damage by intraluminal noxious agents such as aspirin, nonsteroidal anti-inflammatory drugs, and alcohol and has a proven efficacy in the treatment of peptic ulcer disease [6]. In rats, sucralfate increases prostaglandin release into the gastric lumen [41 suggesting that prostaglandin is a factor in sucralfate’s protective mechanisms. Increased endogenous prostaglandin production does not, however, completely explain the effects of sucralfate [5]. The effect of sucralfate on the normal gastric mucus layer includes reduction of hydrogen ion diffusion through mucus and modification of sodium/hydrogen ion exchange of gastric mucus [7]. Because of its high water content, the layer of mucus is distorted during dehydration [8]. Antibodies raised against colonic mucus incubated with colonic mucosa “stabilized” a layer that can be examined [8]. Snap-freezing provides another method for maintaining a layer of mucus during specimen preparation. In this study, we compared the scanning electronmicroscopical features of snap-frozen rat gastric epithelium after being treated with phosphate-buffered saline (PBS), specific antigastric mucus antisera, and sucralfate. In addition, we performed x-ray microanalysis of the retained mucus to determine the relative concentrations of selected elements present in the gastric mucus and epithelium.
MATERIALS AND METHODS
From the Departments of Medlclne and Pathology, University of Auckland, Auckland, New Zealand. Requests for reprints should be addressed to Dr. Clifford Tasman-Jones, Departments of Medlclne and Pathology, Umverslty of Auckland, Auckland, New Zealand.
Young male Wistar rats were used in this study. The animals were fed a polymeric liquid diet (Ensure osmolite) for the seven days prior to the experiment to ensure that the stomach was empty of food debris at the time of experimental sampling. The diet was prepared at quarter strength in water and fed to the animals ad lib&m. The animals were weighed before and after the seven days of feeding. After anesthetization with sodium pentobarbital, the abdominal cavity was opened, the pylorus was hgated, and one of the following solutions was injected (1 to 2.5 ml) into the stomach until it was mildly and approximately equally dilated: (1) PBS pH 7.4, 0.15 M (four rats); (2) rabbit serum (heat inactivated) continuing a high titer of antibodies to rat gastric mucus in PBS (four rats); and (3) 1 percent solution of sucralfate in PBS (four rats). June 9, 1989
The American Journal of Medicine
Volume 86 (suppl 6A)
5
SYMPOSIUM
ON SUCRALFATElTASMANJONES
Figure 1. Snap-frozen, freeze-fractured pattern.
ET AL
rat gastric mucosa pretreated
with PBS alone (0.15 M, pH 7.4). The muc
After injection into the stomach, the abdominal cavity was partially closed and maintained moist with saline-soaked swabs. The rats were kept warm and maintained under light anesthesia for 1 hour before the stomachs were removed. The animals were killed by an intracardiac injection of a rapidly acting barbiturate. Tissue samples (approximately 1 cm’) of the body of the glandular stomach were taken. One sample from each rat was prepared for scanning electron microscopy and x-ray microanalysis Scanning Electron Microscopy
Tissue samples were snap-frozen in melting Freon 12 (-156°C) and then quenched in liquid nitrogen (- 196°C). They were transferred to a brass block, also at liquid nitrogen temperature, where they were fractured with a razor blade pre-cooled in liquid nitrogen. The fractured specimens were freeze-dried at -60°C for 24 to 48 hours in an Edwards-Pearse tissue drier using phosphorus pentoxide as a vapor trap and then mounted on aluminum stubs with double-sided tape such that the fractured surface lay uppermost and parallel to the stub surface. Carbon paint was applied to the specimen sides and the stub surface to ensure conductivity and reduce extraneous x-ray signals. They were coated with carbon by vacuum evapo-
6
June 9, 1989
The American Journal of Medicine
Volume 86 (suppl 6A)
ration and then examined in an IS1 DS-130 scanning electron microscope fitted with a Robinson back-scattered electron detector and x-ray microanalytical facilities. Following the collection of x-ray spectra, the specimens were coated with palladium/gold and fractures through the mucosa and its associated mucus were photographed using an accelerating voltage of 20 kv. A section of mucus of each sample where the fracture was perpendicular through the gastric glands was measured to obtain some index of thickness. The two measurements of each section were made to indicate the thinnest and thickest mucus layer. Radiographic Analysis
Analyses were performed on every sample at the outer and inner aspects of mucus and the epithelium of the mucosa. X-rays were detected using an EG&G Ortec detector with a beryllium window. Analyses were performed for 200 live seconds using an accelerating voltage of 20 kv, spot size of 500 nm, and gun emission of 110 to 120 ,uA. The x-ray take-off angle was 36.5” and the count-rate varied between 800 and 3,200 counts/second. A minimum of three analyses were performed at different locations on the specimen, for each of the three areas of interest. The accumulated spectra were stored on floppy disk
SYMPOSIUM
Figure 2. Snap-frozen, freeze-fractured seen with a vertical fibrillar pattern.
rat gastric mucosa pretreated
ON SUCftALFATE /TASMAN.JONES
with antibodies against rat gastric mucus and suspended in PBS. A homogeneous
and used to determine the relative concentrations of elements present (normalized to 100 percent) using an EDAX semi-quantitative software routine that removed background, analyzed peak areas, and finally applied atomic number, absorption, and fluorescence correction to the results.
RESULTS All animals thrived on the liquid diet and increased their weights. At operation, stomachs were clear of food fibers and a significant layer of mucus above the glandular stomach could be defined. The appearance of the layer of mucus was consistent within each group. The scanning electron microscopical appearance of mucus in the pretreated PBS sample was a layer 40 to 125 pm thick with “fibrilles” that ran parallel and approximately perpendicular to the mucosal surface. While generally homogeneous, the more superficial layer was less dense than the remainder of the specimen (Figure 1). When antibodies raised against rat gastric mucus were used, the layer was similar in structure but with the fibrillar pattern more clearly defined and more parallel (Figure 2). This layer was 50 to 220 pm thick. Sucralfate-treated stomachs had two distinct layers: a very thin, deep layer that appeared dense and
ET AL
mucus layer is
homogeneous and a superficial layer that appeared to have a fibrillar pattern but less regular than that seen in Figure 2. The thickness (‘70 to 180 pm) was similar to that for the antibody-treated samples (Figure 3). Analysis of the x-rays induced by the electron bombardment of a specimen in the scanning electron microscope produced .a spectrum of peaks, the energies and heights of which represent specific elements and their relative concentrations, respectively (Table I). The average concentrations at each place of analysis (minimum, three analyses) are listed in Table I and are expressed as the percentage of that element present, relative to the other elements measured. All samples contained a small percentage of aluminum, whether or not sucralfate was used.
COMMENTS Scanning electron microscopical examination of unfixed, freeze-fractured, and freeze-dried material produced adequate morphologic preservation of both mucus and mucosa. The complete absence of chemical intervention in this methodology, although partially compromising the ultrastructural detail, was necessary to retain as accurately as possible the in viva elemental composition of the specimen for x-ray microanalytical purposes. Snap-frozen tissue specimens maintained a well-
June 9, 1989
The American Journal of Medicrne
Volume 86 (suppl 6A)
7
SYMPOSIUM
ON SIJCRALFATE /TASMAN-JONES
Figure 3. Snap-frozen, freeze-fractured a looser vertical frbrillar oattern.
ET AL
rat gastric mucosa pretreated with 1 percent sucralfate in PBS. Two layers appear visible with a dense but thinner basal layer and
formed layer of mucus in all specimens. Although variable in thickness, freeze-fractured sections were of consistent appearance within each treatment group. Although the mucosal samples were treated with great care, distortions of the mucus layer are likely to occur on opening the stomach and the manipulations during snap-freezing and mounting. The apparent differences in thickness need to be confirmed by other methods of preparation. Sucralfate does not appear to form a layer on top of mucus. The drug seems to penetrate the whole mucus layer. The apparent increase in thickness with both antibody and sucralfate preparation may be because of increased mucus release by increased prostaglandin secretion andlor by binding TABLE
I
Distribution
of Elements
Treatment Group PBS Mucus antibody Sucralfate
Mucus Outer Inner Outer Inner Outer Inner
in Gastric
Aluminum 1.1 i 1.8 t 0.7 f 0.4 2 4.2 * 3.4 k
0.6 2.0 0.3 0.1 2.1 1.8
Mucus*
Sodium 48.6 t 43.7 + 30.1 k 37.4 k 36.5 f 33.4 k
6.4 7.5 6.1 7.5 6.0 5.4
Sulfur
Others (K, Cl, P)
3.3 _c 1.6 3.5 i- 1.7 6.5 f 1.4 6.2 k 1.1 6.1 _c 1.4 7.2 A 1.6
41.2 51.2 50.3 51.7 50.8 53.9
= potassium; Cl = chlorine; P = phosphorus. Ixpressed as percent of elements measured; mean i SD. 8
June 9, 1989
The American Journal of Medicine
Volume 86 (suppl 6A)
superficial soluble mucus that would not normally be retained. Both the control and gastric mucus antibodytreated samples showed a “fibrillar” pattern to the mucus. Mucus is a highly hydrated substance with a normal water content that is very difficult to achieve with biologic samples. In particular, variations in texture and topography have a great influence on the x-rays detected. For this reason, we have taken multiple analyses at different sites, and used a scanning electron beam over a small selected area to overcome local variability. In all cases, the gradients of sodium and potassium are as expected, with the sodium being higher in the mucus than in the glandular epithelial layer, and potassium being the reverse. Aluminum was present in low relative concentrations (approximately 1 percent) at all sites in every treatment group except sucralfate, for which the levels were higher. It is uncertain whether this 1 percent concentration of aluminum is a “background” level due to extraneous x-rays arising from the aluminum stub supporting the specimens or a true indication of the levels in the tissue. The sucralfate treatment group, however, showed higher aluminum concentration with a decreasing gradient from the outer to the inner aspect of the mucus layer. The level of aluminum in the epithelium has not apparently been raised by this one-dose situation. Other workers reported that plasma aluminum concentration in patients with ulcers was not
SYMPOSIUM
raised after sucralfate treatment [91. However, there is evidence that sucralfate-treated patients have an increased urinary excretion of aluminum [lo]. Previous studies have shown that sucralfate protects the stomach against injury by necrotizing or irritating luminal substances. The mechanisms of this protection are complex with modification of proton diffusion, mucus viscosity, enzyme degradation of mucus, and the stimulation of endogenous prostaglandin secretion. It has been shown that protection by sucralfate is by stimulation of endogenous prostaglandin secretion [4,111. A recent primate study has demonstrated protection independent of prostaglandin [12]. Gastric mucus has a sodium/hydrogen ion exchange property [13]. Sucralfate bound to mucus maintains the moderate sodium/hydrogen ion exchange property of gastric mucus [7] and improves sodium/hydrogen ion exchange when this is impaired as in infection with Campylobacter pylori [71. This study shows that a layer of mucus is present at the gastric luminal surface. The layer appears to be thicker when treated with antibody or sucralfate, compared with PBS control. Although the thickness will add physical protection to the stomach, this is probably not the only protective mechanism induced by sucralfate. The effect of mucus complexed with sucralfate on hydrogen ion movement needs further clarification.
ON SlJCRALFATE/TASMANJONES
ET AL
REFERENCES 1. Roberts A, Nezamrs JE, Lancaster C, Hanchar Al: Cytoprotectron by prostaglandrns rn rats. Prevention of gastric necrosis by alcohol, HCI, NaOH, hypertonic NaCl and thermal injury. Gastroenterology 1979; 77: 433-443. 2. Guth PH: Mucosal coating agents and other nonantisecretory agents. Are they cytoprotective? Dig Dis Sci 1987; 32: 647-654. 3. Hollander D, Tarnawski A, Krause WJ, Gergely H: Protective effect of sucralfate against alcohol induced gastric mucosal injury in the rat. Macroscopic, histological, ultrastructural and functional time sequence analysis. Gastroenterology 1985; 88: 366-374. 4. Hollander D, Tarnawski A, Gergely H, Zipser RD: Sucralfate protection of gastric mucosa against alcohol-induced necrosis: a prostaglandin medrated process. Stand J Gastroenterol 1984; 19 (suppl 101): 97-102. 5. Wu WC, Semble EL, Caste11DO, et al: Sucralfate therapy of nonsteroidal antiinflammatory drug-induced gastritis (abstr). Gastroenterology 1985; 88: 1636. 6. Coste T, Rautureau J, Beaugrand M: Comparison of two sucralfate dosages presented in tablet form in duodenal ulcer healing. Am J Med 1987; 83 (suppl 36): 86-90. 7. Thomsen L, Tasman-Jones C, Morris A: Na+/H+ ion exchange property of postmortem human gastric mucus-the effect of Catnpylobacterpyloriinfection and sucralfate. Stand J Gastroenterol 1989; (in press). 8. Bollard JE, Vanderwee MA, Smith GW, Tasman-Jones C, Gavin JB, Lee SP: Preservation of mucus in situ in rat colon. Dig Dis Sci 1986; 31: 1338-1344. 9. Kinoshita H, Kumaki K, Nakano H, et at Plasma aluminum levels of patients on long term sucralfate therapy. Res Commun Chem Pathol Pharmacol 1982; 35: 515-518. 10. Haram EM, Webert R, Berstad A: Urinary excretion of alumirium after ingestion of sucralfate and an aluminium-containing antacid In man. Stand J Gastroenterol 1987; 22: 615-618. 11. Quodros E, Ramsamoo] E, Wilson DE: Relationshrp between sucralfate, gastrrc cytoprotection and prostaglandin and mucus synthesis and secretion (abstr). Gastroenterology 1985; 88: 1548. 12. Shea-Donohue T, Steel L, Montcalm E, Dubois A: Gastric protection by sucralfate. Role of mucus and prostaglandin. Gastroenterology 1986; 91: 660-666, 13. Thomsen L, Tasman-Jones C, Maher K, Wiggrns P, Lee S, Morris A: Na+/H+ ion exchange property of postmortem gastric mucus. Stand J Gastroenterol 1988; 23: 701704.
June 9, 1989
The American Journal of Medicine
Volume 86 (suppl 6A)
9