Aust. N.Z. J. Med. (1975), 5, pp. 507-514
Gastric Glycoproteins in Chronic Peptic Ulcer 1. C. Roberts-Thornson",A. E. Clarke?, V. M. Maritzf and M . A. Denborough"' From the University of Melbourne Department of Medicine, Royal Melbourne Hospital
SUmmary: Gastric glycoproteins in chronic peptic ulcer. I. C. Roberts-Thomson, A. E. Clarke, V. M. Maritz and M. A. Denborough, Aust. N.Z. J. Med., 5, pp. 507-514.
The output and concentration of gastric glycoproteins in gastric juice from patients with chronic duodenal and gastric ulcer and from controls, have been determined in the basal state and following pentagastrin stimulation. Patients with gastric ulcer had a significantly higher basal glycoprotein output, basal glycoprotein concentration and stimulated glycoprotein concentration than patients with duodenal ulcer or controls. The basal and stimulated glycoprotein output in gastric juice from patients with duodenal ulcer and controls was independent of ABO blood group and secretor status. The carbohydrate composition of the gastric glycoproteins has also been determined in the basal state, and following stimulation of gastric juice by pentagastrin, which did not influence the carbohydrate composition of the molecules. The principal carbohydrate components were galactose, N acetylglucosamine, fucose, N-acetylgalactosamine and sialic acid. Small amounts of mannose and glucose were detected in some gastric glycoprotein samples. The carbohydrate composition of the glycoproteins varied according to the ABO blood group and secretor status of the individual. Glycoproteins from stimulated gastric juice from non-secretors of groups A and 0 had a higher. sialic acid content than glycoproteins from secretors of the same blood groups. There were no significant differences in the carbohydrate composition of glycoproteins from patients with chronic gastric 'Medical Registrar. ?Research Fellow. $Research Assistant. * Reader in Medicine. Correspondence: Professor M. A. Denborough, Department of Clinical Science, J o h n Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601 Accepted for publication: 15 July. 1975.
and duodenal ulcer compared with gastric glycoproteins from control subjects of the same blood group and secretor status. Gastric mucus contains a high concentration of blood group specific glycoproteins', which are synthesized and secreted by gastric epithelial cells.49 These high molecular weight glycoproteins consist of a polypeptide core to which multiple carbohydrate chains are attached in covalent linkage. The blood group activity is specified by particular sequences of the component monosaccharides at the end of a proportion of the carbohydrate chains. These carbohydrate chains form an effective barrier to the degradation of the polypeptide core of the glycoproteins by the proteolytic action of pepsin in the stomach. This resistance to proteolytic enzyme action and the presence of a high concentration of these glycoproteins in the mucous layer covering the gastric mucosa suggest a protective function for these macromolecules in the stomach. In this study gastric glycoproteins have been isolated by density gradient ultracentrifugation from patients with duodenal and gastric ulcers and from controls. The total glycoprotein output and concentration has been determined in all thret groups and correlated with acid output. The possibility that changes in the carbohydrate codposition of gastric glycoproteins might exist in patients with chronic duodenal and gastric ulcer has also been investigated. The neutral and amino sugars were determined by gas-liquid chromatography, while sialic acid was determined by the Warren method.6 The findings were correlated with ABO blood group and secretor status as associations have been demonstrated between duodenal ulcer and both group 0' and non-secretors,* '
9
Materials and Methods
Subjects Included in this study were 27 adults, 23 males and four females, aged 17 to 78 years. Nine patients had duodenal ulcers, eight patients had gastric ulcers and ten apparently
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ROBERTS-THOMSON ET AL.
healthy individuals who did not suffer from dyspepsia and did not have a family history of peptic ulcer acted as controls. The diagnosis of chronic duodenal and gastric ulcer was made by barium meal X-ray examination and was confirmed by endoscopy. In patients with duodenal ulcer anticholinergic drugs were ceased 24 hours prior to the collection of gastric juice, while in patients with gastric ulcer the gastric collection was performed prior to the introduction of carbenoxolone therapy. Collection of Gastric Juice Gastric juice was collected through a No. 16F nasogastric tube following an overnight or eight hour fast. The position of the tube was checked radiographically. The basal collection lasted for one hour. Following this, a subcutaneous injection of pentagastrin (6 & k g ) was given and the collection continued for a further hour. The gastric juice was removed by manual aspiration every two to three minutes and immediately neutralised with 1 M sodium hydroxide. The volume and acid output of the basal and stimulated gastric collection was determined. Throughout the test the patient expectorated all saliva. Cell debris and any food particles were removed by centrifugation at loo0 g for ten minutes. Determination of Glycoprotein Output and Concentration The neutralised gastric juice was concentrated by ultrafiltration through Visking 8/32 dialysis tubing, and was fractionated after equilibrium density gradient ultracentrifugation in caesium chloride as described by Denborough er a1.' Twelve fractions were collected. The glycoprotein was isolated in the higher density fractions seven to 12 and the glycoprotein output was determined as the sum of the total neutral sugarg in fractions seven to 12. The glycoprotein concentration was calculated knowing the glycoprotein output from a given volume of gastric juice. Carbohydrate Analysis Reagents All reagents used were from BDH Chemicals Ltd., Poole, England. Hydrolysis of Glycoprotein The glycoprotein was hydrolysed in 3 M HCl for three hours at 100°C at a total carbohydrate concentration not exceeding 0-5 mg/ml. Determination of Neutral and Amino Sugars Neutral and amino sugars were determined as their alditol acetates by gas-liquid chromatography under the conditions described by Clarke et al." except that the temperature was from 170-230°C with a program rate of 1.7"C/min. Determination of Sialic Acid Sialic acid was determined by the Warren method6 following hydrolysis in 0 . 2 M sulphuric acid for one hour at 80°C. The method was modified to the extent that 2 methoxyethanol was substituted for cyclohexanone, and the optical density at 549 mu was determined in the supernatant following centrifugation at lo00 g for five minutes. ABO Blood Group The ABO blood-group was determined by standard methods using anti-A and anti-B antisera. ABH Secretor Status The secretor status was determined in saliva and fraction
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11 of fractionated gastric juice by the doubling-dilution haemagglutination-inhibition test as described by Denborough et al." All subjects were readily categorised as secretors or non-secretors by this method and the results from salivary specimens were identical to those from gastric juice. Statistical Analysis The results were analysed by the t-test for unpaired data.
Results
Acid Output The basal acid output (BAO) and maximal acid output (MAO) for patients with duodenal and gastric ulcer and controls are shown in Table 1. In this small series the BAO was significantly higher in patients with duodenal ulcer (P < 0.05), and significantly lower in patients with gastric ulcer ( P < 0.05) when compared to controls. The M A 0 was significantly lower in patients with gastric ulcer (P < 0.005) when compared to controls. Glycoprotein Output and Concentration The glycoprotein output and concentration both in the basal state and following pentagastrin stimulation are also shown in Table 1. When patients with duodenal ulcer were compared to controls there were no significant differences in glycoprotein output or concentration in either the basal state or following pentagastrin stimulation. However, when patients with gastric ulcer were compared to controls the patients with gastric ulcer had a significantly higher basal glycoprotein output (P < 0.05), basal glycoprotein concentration (P < 0.02) and stimulated glycoprotein concentration ( P < 0.05). In controls and patients with duodenal ulcer the mean stimulated glycoprotein output was higher than the mean basal glycoprotein output but the differences were not statistically significant. In patients with gastric ulcer, the mean stimulated glycoprotein output was similar to the mean basal glycoprotein output. Relationship Between Acid Output and Glycoprotein Output The correlation coefficient ( I ) between acid output and glycoprotein output was determined for all three groups. There was no significant correlation between BAO and basal glyco-
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TABLE 1 The acid output, glycoprotein output and glycoprotein concentration in the basal state and following pentagastrin stimulation in patients with duodenal and gastric ulcer and in controls (Mean k SE)
Duodenal ulcer patients Gastric ulcer patients Controls
MA0 (mEq/hr)
Basal glycoprotein output (mg/hr)
Stimulated glycoprotein output (melhr)
Basal glycoprotein concentration (mgll)
Stimulated glycoprotein concentration (mgl4
5.8k1.0
33.3k3.7
13.4k1.3
19.1k2.5
142.8+10.0
65.9k7.5
1.0+0.4 3.1k0.7
13.7k2.7 27.1k2.3
19.7k2.7 13.1k1.6
19.1k2.5 19.8k2.9
302.1k57.9 165.4k17.2
113.8k12.8 79.0k10.2
Number of subjects
BAO (mEq/hr)
9
8 10
protein output in controls'(r = 0.06, P > O.l), patients with duodenal ulcer (r = 0.30, P > 0.1) or patients with gastric ulcer (r = 0.23, P > 0.1); and no significant correlation between M A 0 and stimulated glycoprotein output in controls (r = -0.29, P > O.l), patients with duodenal ulcer (r = 0.47, P > 0.1) or patients with gastric ulcer (r = 0.41, P > 0.1). The mean stimulated glycoprotein output was higher in four patients with duodenal ulcer who had a M A 0 greater than 35 mEq/hour (22.5 mg/hour) when compared to five patients with duodenal ulcer who had a M A 0 less than 35 mEq/hour (16.5 mg/hour). Glycoprotein Output, Blood-group and Secretor Status The relationship between glycoprotein output, blood-group and secretor status was evaluated in a group consisting of controls and patients with duodenal ulcer. Ten subjects of bloodgroup A had a mean basal and stimulated glycoprotein output of 13.5 and 19.5 mg/hour respectively, while seven subjects of bloodgroup 0 had a mean basal and stimulated glycoprotein output of 13.4 and 21.6 mg/hour respectively. There were no statistically significant differences (P > 0.1) between bloodgroups A and 0 for either basal or stimulated glycoprotein output. Thirteen secretors of bloodgroups A and 0 had a mean basal and stimulated glycoprotein output of 13.2 and 18.6 mg/hour respectively, while four non-secretors of bloodgroups A and 0 had a mean basal and stimulated glycoprotein output of 14.2 and 21.6 mg/hour
respectively. These differences between secretors and non-secretors were not statistically significant (P > 0.1). Age and Glycoprotein Output As patients with gastric ulcer had a higher mean age (58 years) than the combined group of patients with duodenal ulcer and controls (32 years), it was possible that the elevated basal glycoprotein output was due to age alone and not due to the presence of chronic gastric ulcer. However, there was no significant correlation between age and basal glycoprotein output in controls and patients with duodenal ulcer (r = -0.22, P > 0 . l), and age did not influence the basal glycoprotein output within the gastric ulcer group (r = -0.07, P > 0.1). Carbohydrate Analysis The carbohydrate composition of gastric glycoproteins was initially determined in the higher density fractions 7-12 in both the basal state and following stimulation of gastric juice by pentagastrin. However, in samples from two controls, two patients with duodenal ulcer and two patients with gastric ulcer there were no significant differences in the carbohydrate composition of fractions 7-12, a typical example of which is shown in Table 2. Because of this only one glycoprotein fraction from basal gastric juice and one from stimulated gastric juice was analysed to screen for major differences in carbohydrate composition. Fraction 11, which had a high glycoprotein concentration, was chosen.
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ROBERTS-THOMSON ET AL.
~
7 8 9 10 11 12
Fuc. ~
Gal.
Glc NAc. Gal NAc.
S.A.
~~~
22.6 20.4 23.1 22.7 20.1 24 2
30.2 30.3 27.7 30.4 31.4 30.6
27.9 29.8 30.3 26.8 29.8 26.2
10.9 12.6 11.7 13.9 11.1 10.4
5, NO.6
The carbohydrate composition of glycoproteins from all individuals in this study was then related to ABO blood group and secretor status. The following results were obtained : (i) Glycoproteins from non-secretors of blood groups A and 0 had a significantly lower percentage composition of fucose compared with glycoproteins from secretors of the same blood group (P < 0.01) (Fig. 1). This applied to glycoproteins from both basal gastric juice and from gastric juice stimulated by pentagastrin. (ii) Glycoproteins from secretors of blood group A had a significantly higher percentage composition of N-acetylgalactosamine compared with glycoproteins from secretors of blood group 0 ( P < 0.01) (Fig. 2). This again applied to glycoproteins from both basal gastric juice and from gastric juice stimulated by pentagastrin. (iii) Glycoproteins from one secretor of blood group B had a higher percentage composition of galactose 436.4 %) compared with glycoproteins from secretors of blood group 0 (mean 30.5 %). (iv) Glycoproteins from gastric juice stimulated by pentagastrin from non-secretors of blood groups A and 0 had a significantly higher percentage composition of sialic acid compared with glycoproteins from secretors
TABLE 2 The percentage carbohydrate composition of gastric glycoproteins in fractions 7-12 in stimulated gastric juice from a group A secretor. Mannose and glucose were not detected in any fractions Fraction -
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8.4 6.9 7.2 6.2 7.5 8.6
Fuc. = Fucose Gal. = Galactose S.A. = Sialic acid Glc NAc. = N-acetylglucosamine Gal NAc. = N-acetylgalactosamine
The carbohydrate composition of glycoproteins from patients with duodenal and gastric ulcer and controls, in the basal state and following pentagastrin stimulation, are shown in Tables 3 and 4. Pentagastrin stimulation does not appear to alter the carbohydrate composition of glycoproteins, and glycoproteins from patients with peptic ulcer have a similar carbohydrate composition to glycoproteins from controls matched for blood group and secretor status. Small amounts of mannose and glucose were detected in glycoproteins from many samples but the presence of these sugars was not related to blood group, secretor status or peptic ulcer.
TABLE 3 The mean percentage carbohydrate composition of gastric glycoproteins in basal gastric juice. The results have been divided according to ABO blood group and ABH secretor status in control subjects and patients with chronic duodenal and gastric ulcer No. of subjects Controls
Duodenal ulcer
Gastric ulcer
5 3 1 2 2 2 1 1 1 2 1 1
1
s = Secretor
Blood Secretor group status
A 0 0 A A 0 0 AB
S S NS S NS S NS
B
NS S NS S S
A A 0 B
S
Fuc.
Man.
Gal.
Glu.
20.5 21.9 13.3 17-4 11.0 22.3 7.7 21.8 10.6 21.1 13.6 17.1 16.9
0.4
28.7 32.7 32.2 27-2 28.1 29-1 36.6 32.1 32.1 27.4 31.0 25.0 37.1
4.2 1.4 1.6 0.4 0.6 1.0 1.6
1.1
1.2 0.8 1.5
1.0 1.5 1.5 1.7 1.6 0.8 2.0 -
-
2.2 -
Glc NAc. Gal NAc. 25.6 26.3 30.3 34.2 37.6 29.6 32.8 21.8 30.9 28.3 33.3 27.2 27.7
NS = Non-secretor Fuc. = Fucose Man. = Mannose Gal. = Galactose S.A. = Sialic acid Glc NAc. = N-acetylglucosamine Gal NAc. = N-acetylgalactosamine
13.5 1.2 10.7 12.6 13.3 11.7 12.2 14.8 14.5 12.6 12.8 9.7 8.2
S.A
5.7 9.3 10.7 7.6 8 .0 6.5 8.2 6.5 11.1 7.4 8.6 19.0 10.1
Glu. = Glucose
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GASTRIC GLY COPROTEINS IN CHRONIC PEPTIC ULCER
TABLE 4 The mean percentage carbohydrate composition of gastric glycoproteins in gastric juice stimulated by pentagastrin. Results have been divided according to ABO blood group and ABH secretor status in control subjects and patients with chronic duodenal and gastric ulcer No. of subjects Controls
Duodenal ulcer
Gastric ulcer
6 3 1 2 2 2 1 1 1 2 1 2 1 1
Blood Secretor group status A 0 0 A A 0 0 AB B A A 0 0 B
S S NS S
NS S NS S NS S NS S NS S
Fuc.
M?n.
Gal.
Glu.
20.1 21.2 13.1 18.1 10.9 21.8 8.1 20.7 14.7 16.8 11.6 19.9 14.6 19.2
1.0 0.9 1.3 1.3 1.6 1.4 2.5 2.2 1.6 1.3 1.4 2.1
27.8 33.4 30.6 27.9 30.2 26.1 34.4 23.1 31.5 31.8 26.3 30.7 31.1 36.4
0.6 1.6 8.9 7.0 0.7 3.6 1.9 2.3 2.6
-
-
GlcNAc. Gal NAc. 29.6 26.9 26.8 24.6 35-4 31.6 30.9 23.0 28.3 28.8 33.2 27.7 22.4 26.5
S = Secretor NS = Non-secretor Fuc. = Fucose Man. = Mannose Gal. = Galactose Glc NAc. = N-acetylglucosamine S.A. = Sialic acid Gal NAc. = N-acetylgalactosamine
of the same blood group ( P < 0.01) (Fig. 3). In basal gastric juice, however, no significant difference in sialic acid levels was found in glycoproteins from non-secretors when compared with glycoproteins from secretor s. Discussion
The results from this study show no significant difference in either gastric glycoprotein output or concentration in patients with duodenal ulcer when compared with controls. However, patients with chronic gastric ulcer have a significantly higher basal glycoprotein output than both controls and patients with duodenal ulcer. Theoretically, the concentration of glycoprotein in gastric juice might be impcrtant in protecting against peptic ulceration, and it is of interest that glycoprotein concentration in both fasting and stimulated gastric juice is also significantly greater in patients with gastric ulcer when compared with both controls and patients with duodenal ulcer. The reason for the increased output and concentration of gastric glycoprotein in patients with gastric ulcer is uncertain, but there are two possible explanations. First, chronic gastric ulceration is almost always associated with
13.4 9.6 7.6 12.8 12.8 11.4 11.0 11.9 12.1 13.2 13.0 9.1 12.0 9.0
S.A. 7.6 6.4 11.6 8.3 8.5 7.9 9.5 8.3 11.8 5.8 14.5 8.0 14.1 8.9
Glu. = Glucose
gastritis and goblet cell metaplasia in the region of the ulcer and throughout the antrum of the stomach.lZ This abnormal accumulation of goblet cells may increase glycoprotein secretion and account for the elevated basal glycoprotein output in gastric juice. It is unlikely that glycoprotein secretion is increased from other surface epithelial cells as histochemical studies of these cells suggest that the amount of glycoprotein is dirnini~hed.~ On the other hand, in the present study, as in other larger series13, the amount of gastric juice and the hydrogen ion concentration in gastric juice following pentagastrin stimulation was higher in controls and in patients with duodenal ulcer, than in patients with gastric ulcer. The elevated glycoprotein concentrations in patients with gastric ulcer might be explained by the lower volumes of gastric juice both in the basal state and following pentagastrin. In this study, as in another14, there was no significant correlation between acid output and glycoprotein output. Thus the relationship between these two indices does not appear to be relevant to the aetiology of peptic ulcer. The relationship between glycoprotein output, the ABO blood-group and secretor status has not been previously investigated. However,
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ROBERTS-THOMSON ET AL.
results from the present study indicate that glycoprotein output is independent of ABO blood-group and secretor status, and therefore, quantitative changes in gastric glycoproteins do not appear to explain the increased susceptibility of non-secretors and people of bloodgroup 0 to duodenal ulcer. In this study, as in other reports’. ’, 15-19, the principal carbohydrate components of human gastric glycoproteins were found to be galactose, N-acetylglucosamine, fucose, Nacetylgalactosamine and sialic acid. Schrager’ using gas-liquid chromatography to identify sugar residues found that the quantitative relationship of galactose : N-acetylglucosamine : N-acetylgalactosamine was 4 : 3 : 1. Analysis of our results in secretors of blood groups A and 0 has shown a slightly different relationship with a galactose : N-acetylglucosamine : Nacetylgalactosamine ratio of 5 : 5 : 2. The presence of small amounts of glucose and mannose in some glycoprotein samples was apparently unrelated to blood group, secretor status or peptic ulcer. The possibility that these sugars arose during the hydrolytic procedure by a mechanism involving acid reversion was considered. However, a mixture of fucose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid in the proportions found in gastric juice samples at a final con-
30
20
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centration of 0.5 % contained only the component monosaccharides after treatment with 3 M HCl for three hours at 100°C. Thus mannose and glucose were considered to be components of glycoprotejns in gastric juice. The source of these sugars may be glycoproteins synthesised and secreted by gastric epithelial cells or, perhaps, glycoproteins from saliva.l9
T I
I
+
BLOOD GROUP A SECRETORS
BLOOD GROUP 0 SECRETORS
FIGURE 2. The percentage composition of N-acetylgalactosamine in glycoproteins from stimulated gastric juice from secretors of blood groups A and 0. Blood group A secretors had a significantly higher percentage composition of N-acetylgalactosamine (P < 0.01 ).
9
.
I
i
-
I
10
. -
i 4.
~~
NON-SECRETORS BLOOD GROUPS A 0
~
~~~
SECRETORS BLOOD.GROUPS A . 0
FIGURE 1 The percentage composition of fucose in glycoproteins from stimulated gastric juice from secretors and non-secretors of blood groups A and 0 Nonsecretors had a significantly lower percentage composition of fucose (P < 0 01)
HON-SECRETORS BLOOD-GROUPS A D .
SECRETORS 01000-GROUPS A.O.
FIGURE 3. The percentage composition of sialic acid in glycoproteins from stimulated gastric juice from secretors and non-secretors of blood groups A and 0. Non-secretors had a significantly higher percentage composition of sialic acid (P < 0.01).
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GASTRIC GLYCOPROTEINS IN CHRONIC PEPTIC ULCER
The preparation of volatile derivatives for gas-liquid chromatography involved reduction of the free neutral sugars to the corresponding alditol. As the alditols could be derived not only from the free sugars but from the corresponding acids by borohydride reduction, the possibility that the carbohydrate components were derived wholly or in part from the corresponding uronic acids was considered. Paper chromatography of neutralised hydrolysates showed only the neutral sugars, and no uronic acids could be detected in the glycoprotein sample by the method of Bitter and Muir.20 However, the sensitivity of gas-liquid chromatography as compared with colorimetric methods leaves open the possibility that small amounts of connective tissue glycosaminoglycans may be present in the sample. Results from this study indicate that the carbohydrate composition of gastric glycoproteins is similar in patients with chronic gastric and duodenal ulcer when compared with controls of the same blood group and secretor status. However, our findings conflict with a recent report by Domschke et aL21 These authors measured free and bound sialic acid by a thiobarbituric acid method similar to that described by Warren6 and found that the quantity of sialic acid in basal and stimulated gastric juice was low in patients with gastric ulcer when compared with controls. The different conclusions reached may have been due to differences in methodology, Domschke et d2' did not separate glycoproteins from proteins and did not relate the sialic acid content to the glycoprotein content. In all samples analysed in this study the carbohydrate composition of glycoproteins varied according to the ABO blood group and secretor status. The finding that glycoproteins from non-secretors have a low proportion of fucose, and that glycoproteins from secretors of blood group A and B have a high proportion of N-acetylgalactosamine and galactose respectively, indicates that gastric glycoproteins are similar to glycoproteins from ovarian cystszz and support the view2 that the majority of gastric glycoproteins are blood group specific substances. The finding of an increased proportion of sialic acid in glycoproteins from
513
stimulated gastric juice from non-secretors is interesting and has not been reported previously. This observation needs to be confirmed by study of a larger series. The low proportion of fucose in glycoproteins from non-secretors has been noted p r e v i o ~ s l y ~ ~ , and is vnlikely to have a significant influence on the physical properties of mucus as fucose is a neutral sugar. Although the proportion of N-acetylgalactosamine and galactose is higher in secretors of blood groups A and B respectively there is no evidence to suggest that these sugars influence the physical properties of mucus and enhance its protective function; so that the higher incidence of duodenal ulcer in group 0 secretors compared with group A and B secretors cannot be explained on the basis of the carbohydrate composition of the gastric glycoproteins. Acknowledgements
We are grateful to Mrs. Ruth Walker for skilled technical assistance and to Dr. Ross Ullman for help with statistical analyses. References 1. SCHRAGUI,I. (1970): The chemical composition and function of gastrointestinal mucus, Guf 11, 450. 1. DENBOROUGH, M. A., PRESSER,1. C. and UNGAR,B. (1971): Isolation of
glycoproteins from human gastric juice and saliva by density gradient ultracentrifugation, Clin. Chem. 17, 335. 3. ALLEN,A. (1972): The structure and function of gastric mucus, Gut 13, 666. 4. KENT.P. W. and ALLEN,A. (1968): The biosynthesis of intestinal mucins. The effect of salicylates on glycoprotcin biosynthesis by sheep colonic and human mucosal tissues in virro, Biochem. J . 106, 645. 5. LEV,R.(1970): The histochemistry of mucus-producing cells in the normal and diseased gastrointestinal mucosn, Progr. Gasrroenterol. 1, 13. 6 . WARREN,L. (1959): The thioharbituric acid assay of sialic acids, J . biol. Chem. 234, 1971. H. H., MEHIGAN, I. A. and ROBERTS, I. A. F. (1954): The 7. AIRD,I., BENTALL, blood groups in relation to peptic ulceration and carcinoma of colon, rectum, breast and bronchus. Brit. med. J . 2, 315. 8 . CLARKE, C. A,, EDWARDS, J. W., HADDOCK, D. R. W., HOWEL-EVANS, A. W., R. B. and SHEPPARD, P.M. (1956): ABO blood groups and MCCONNELL. secretor character in duodenal ulcer, Brit. med. J . 2, 725. 9. Dusors, M., GILL^, K. A,, HAMILTON, 1. K., REBERS, P. A. and SMITH,F. (1957): Colorimetric method for the determination of sugars and related substances, Anal. Chem. 28, 350. 10. CLARKE,A. E.. MARITZ,V. M..PRESSER, 1. and DENBOROUGH, M. A. (1974): Quantitative estimation of neutral and amino sugars of glycoproteins from human secretions by gas-liquid chromatography, Biochem. Med. 9, 342. 11. DENBOROUGH, M. A., DOWNING, H. 1. and DOE, A. G. (1969): Serum blood group substances and ABO haemolytic disease, Brit. J . Haemat. 16, 103. 12. Cox, A. I. (1963): Gastric mucosal changes in peptic ulcer, Gastroenterology 45, 558. 13. WORMSLEY. K. G. and GROSSMAN. M. L. (1965): Maximal histolog test in control subjects and patients with peptic ulcer, Gut 6, 427. 14. GLASS,G. B. J., PUOH,B. L. and WOLF,S. (1950): Correlation of acid, pepsin and mucoprotein secretion by human gastric glands, J. appl. Physiol. 2, 571. IS. HOSKINS.L. C. and ZAMCHECK, N. (1963): Studies on gastric mucins in health and disease, Ann. N.Y. Acad. Sci. 106, 767. 16. RICHMOND, V.. CAPUTTO,R. and WOLF,S. (1955): Biochemical study of the large molecular constituents of gastric juice, Gastroenterology 29, 1017. 17. GLASS,G. B. I., RICH,M.and STfPmNsoN, L. (1958): Comparative study of mucoproteins of human gastricjuice and serum. Gasrroenrerology 34,598. 18. WALDRON-EDWARD, D. ,and SKORYNA,S. C , (1970): Studies on human gastric gel mucin. Isolation and characterisation of a major glycoprotein component, Gastroenterology 59. 671.
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19 MARSHALL, R. D. (1972): Glycoproteins, Ann. Rev. Biochem 41, 673. 20. BITTER, T. and MUIR,H. M. (1962): A modified uronic acid carbazole reaction, A n d . Biocliern 4. 110 21. DOMSCHE,W., DOMSCHKE, S., CLASSEN, M. and DEMLING, I,. (1972). Some properties of mucus in patients with gastric ulcer. Effects of treatment with carbenoxolone sodium, Scnnd. J. Gostroent. 7, 641
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22. MORGAN,W. T. J. (1963): Some observations on the carbohydratecontaining components of human ovarian cyst mucm, Ann. N.Y. Acad. Sci. 106, 177. 23. EVANS,D. A. P., MCDONNELL. R. B , DONOHUE, W. T A., SlRCUs. W. and CREAN, G. P. (1963): Fucose and agglutinogen contents of gastric p i c e in subjects with peptic ulcers, J . Lab. d i n . Med. 61, 660.
CORRIGENDUM
The following segment of the Case Report TROPHIC HORMONE PRODUCTION C. M. Dunn, which appeared in the June Journal of Medicine, pp. 270-273, has been be inserted on page 272, second column.
entitled “ECTOPIC ADRENOCORTICOAND NEOPLASM” by R. F. Pinerua and 1975 issue of Australian and New Zealand omitted from the original article and should
Obesity, osteoporosis and striae are most unusual. The rapid development of pigmentation is common. The capacity of tumours to produce more than one peptide hormone has been emphasised.’ Alpha-melanocyte stimulating hormone (a-MSH) and beta-melanocyte stimulating hormone (P-MSH) have been demonstrated in turnours’, with P-MSH being the major melanocyte stimulating factor. However, a heterogeneous form of MSH structurally different from both a-MSH and P-MSH has been described.” Although plasma ACTH and MSH were not measured, there was no autopsy evidence of an autonomous adrenal lesion to explain the undoubted hypercorticism. The pigmentation observed was in all probability due to MSH secretion. Severe electrolyte disturbances are typical of ectopic ACTH production in contra distinction to the hypercorticism of pituitary origin. Hypokalaemic hypochloraemic alkalosis was prominent in all patients. Muscle weakness was severe in two patients and was no doubt produced by hypokalaemia which contributed to the production of ventricular ectopic beats in one patient. The presence of such a metabolic alkalosis in the absence of more usual causes is a clue to an ACTH producing or adrenal tumour which may initially be unsuspected. Electrolyte abnormalities may occasionally precede by months or years recognition of an ACTH producing tumour.” The widespread rash which in Patient 2 developed pari passu with his malignant disease may have been a dermatological paramalignant manifestation and bore some similarities to the sign of Leser-Trelat.” The neuropathy found in Patient 3 could have been a neurological manifestation of his neoplasm although there was a far more common alternative explanation. The response to synacthen stimulation in the patients tested showed that despite high plasma cortisol levels and florid evidence of acute adrenal overactivity, the suprarenals had still significant reserve capacity and were capable of response to further trophic hormone stimulation. Patient 3 had considerable adrenal responsiveness when tested 23 days before autopsy which showed extensive metastatic replacement of the suprarenal glands. It is noteworthy that in the same patient three days before death the plasma cortisol wus 100 pg/lOO mi.
Gastric Glycoproteins in Chronic Peptic Ulcer 1. C. Roberts-Thornson",A. E. Clarke?, V. M. Maritzf and M . A. Denborough"' From the University of Melbourne Department of Medicine, Royal Melbourne Hospital
SUmmary: Gastric glycoproteins in chronic peptic ulcer. I. C. Roberts-Thomson, A. E. Clarke, V. M. Maritz and M. A. Denborough, Aust. N.Z. J. Med., 5, pp. 507-514.
The output and concentration of gastric glycoproteins in gastric juice from patients with chronic duodenal and gastric ulcer and from controls, have been determined in the basal state and following pentagastrin stimulation. Patients with gastric ulcer had a significantly higher basal glycoprotein output, basal glycoprotein concentration and stimulated glycoprotein concentration than patients with duodenal ulcer or controls. The basal and stimulated glycoprotein output in gastric juice from patients with duodenal ulcer and controls was independent of ABO blood group and secretor status. The carbohydrate composition of the gastric glycoproteins has also been determined in the basal state, and following stimulation of gastric juice by pentagastrin, which did not influence the carbohydrate composition of the molecules. The principal carbohydrate components were galactose, N acetylglucosamine, fucose, N-acetylgalactosamine and sialic acid. Small amounts of mannose and glucose were detected in some gastric glycoprotein samples. The carbohydrate composition of the glycoproteins varied according to the ABO blood group and secretor status of the individual. Glycoproteins from stimulated gastric juice from non-secretors of groups A and 0 had a higher. sialic acid content than glycoproteins from secretors of the same blood groups. There were no significant differences in the carbohydrate composition of glycoproteins from patients with chronic gastric 'Medical Registrar. ?Research Fellow. $Research Assistant. * Reader in Medicine. Correspondence: Professor M. A. Denborough, Department of Clinical Science, J o h n Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601 Accepted for publication: 15 July. 1975.
and duodenal ulcer compared with gastric glycoproteins from control subjects of the same blood group and secretor status. Gastric mucus contains a high concentration of blood group specific glycoproteins', which are synthesized and secreted by gastric epithelial cells.49 These high molecular weight glycoproteins consist of a polypeptide core to which multiple carbohydrate chains are attached in covalent linkage. The blood group activity is specified by particular sequences of the component monosaccharides at the end of a proportion of the carbohydrate chains. These carbohydrate chains form an effective barrier to the degradation of the polypeptide core of the glycoproteins by the proteolytic action of pepsin in the stomach. This resistance to proteolytic enzyme action and the presence of a high concentration of these glycoproteins in the mucous layer covering the gastric mucosa suggest a protective function for these macromolecules in the stomach. In this study gastric glycoproteins have been isolated by density gradient ultracentrifugation from patients with duodenal and gastric ulcers and from controls. The total glycoprotein output and concentration has been determined in all thret groups and correlated with acid output. The possibility that changes in the carbohydrate codposition of gastric glycoproteins might exist in patients with chronic duodenal and gastric ulcer has also been investigated. The neutral and amino sugars were determined by gas-liquid chromatography, while sialic acid was determined by the Warren method.6 The findings were correlated with ABO blood group and secretor status as associations have been demonstrated between duodenal ulcer and both group 0' and non-secretors,* '
9
Materials and Methods
Subjects Included in this study were 27 adults, 23 males and four females, aged 17 to 78 years. Nine patients had duodenal ulcers, eight patients had gastric ulcers and ten apparently
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healthy individuals who did not suffer from dyspepsia and did not have a family history of peptic ulcer acted as controls. The diagnosis of chronic duodenal and gastric ulcer was made by barium meal X-ray examination and was confirmed by endoscopy. In patients with duodenal ulcer anticholinergic drugs were ceased 24 hours prior to the collection of gastric juice, while in patients with gastric ulcer the gastric collection was performed prior to the introduction of carbenoxolone therapy. Collection of Gastric Juice Gastric juice was collected through a No. 16F nasogastric tube following an overnight or eight hour fast. The position of the tube was checked radiographically. The basal collection lasted for one hour. Following this, a subcutaneous injection of pentagastrin (6 & k g ) was given and the collection continued for a further hour. The gastric juice was removed by manual aspiration every two to three minutes and immediately neutralised with 1 M sodium hydroxide. The volume and acid output of the basal and stimulated gastric collection was determined. Throughout the test the patient expectorated all saliva. Cell debris and any food particles were removed by centrifugation at loo0 g for ten minutes. Determination of Glycoprotein Output and Concentration The neutralised gastric juice was concentrated by ultrafiltration through Visking 8/32 dialysis tubing, and was fractionated after equilibrium density gradient ultracentrifugation in caesium chloride as described by Denborough er a1.' Twelve fractions were collected. The glycoprotein was isolated in the higher density fractions seven to 12 and the glycoprotein output was determined as the sum of the total neutral sugarg in fractions seven to 12. The glycoprotein concentration was calculated knowing the glycoprotein output from a given volume of gastric juice. Carbohydrate Analysis Reagents All reagents used were from BDH Chemicals Ltd., Poole, England. Hydrolysis of Glycoprotein The glycoprotein was hydrolysed in 3 M HCl for three hours at 100°C at a total carbohydrate concentration not exceeding 0-5 mg/ml. Determination of Neutral and Amino Sugars Neutral and amino sugars were determined as their alditol acetates by gas-liquid chromatography under the conditions described by Clarke et al." except that the temperature was from 170-230°C with a program rate of 1.7"C/min. Determination of Sialic Acid Sialic acid was determined by the Warren method6 following hydrolysis in 0 . 2 M sulphuric acid for one hour at 80°C. The method was modified to the extent that 2 methoxyethanol was substituted for cyclohexanone, and the optical density at 549 mu was determined in the supernatant following centrifugation at lo00 g for five minutes. ABO Blood Group The ABO blood-group was determined by standard methods using anti-A and anti-B antisera. ABH Secretor Status The secretor status was determined in saliva and fraction
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11 of fractionated gastric juice by the doubling-dilution haemagglutination-inhibition test as described by Denborough et al." All subjects were readily categorised as secretors or non-secretors by this method and the results from salivary specimens were identical to those from gastric juice. Statistical Analysis The results were analysed by the t-test for unpaired data.
Results
Acid Output The basal acid output (BAO) and maximal acid output (MAO) for patients with duodenal and gastric ulcer and controls are shown in Table 1. In this small series the BAO was significantly higher in patients with duodenal ulcer (P < 0.05), and significantly lower in patients with gastric ulcer ( P < 0.05) when compared to controls. The M A 0 was significantly lower in patients with gastric ulcer (P < 0.005) when compared to controls. Glycoprotein Output and Concentration The glycoprotein output and concentration both in the basal state and following pentagastrin stimulation are also shown in Table 1. When patients with duodenal ulcer were compared to controls there were no significant differences in glycoprotein output or concentration in either the basal state or following pentagastrin stimulation. However, when patients with gastric ulcer were compared to controls the patients with gastric ulcer had a significantly higher basal glycoprotein output (P < 0.05), basal glycoprotein concentration (P < 0.02) and stimulated glycoprotein concentration ( P < 0.05). In controls and patients with duodenal ulcer the mean stimulated glycoprotein output was higher than the mean basal glycoprotein output but the differences were not statistically significant. In patients with gastric ulcer, the mean stimulated glycoprotein output was similar to the mean basal glycoprotein output. Relationship Between Acid Output and Glycoprotein Output The correlation coefficient ( I ) between acid output and glycoprotein output was determined for all three groups. There was no significant correlation between BAO and basal glyco-
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TABLE 1 The acid output, glycoprotein output and glycoprotein concentration in the basal state and following pentagastrin stimulation in patients with duodenal and gastric ulcer and in controls (Mean k SE)
Duodenal ulcer patients Gastric ulcer patients Controls
MA0 (mEq/hr)
Basal glycoprotein output (mg/hr)
Stimulated glycoprotein output (melhr)
Basal glycoprotein concentration (mgll)
Stimulated glycoprotein concentration (mgl4
5.8k1.0
33.3k3.7
13.4k1.3
19.1k2.5
142.8+10.0
65.9k7.5
1.0+0.4 3.1k0.7
13.7k2.7 27.1k2.3
19.7k2.7 13.1k1.6
19.1k2.5 19.8k2.9
302.1k57.9 165.4k17.2
113.8k12.8 79.0k10.2
Number of subjects
BAO (mEq/hr)
9
8 10
protein output in controls'(r = 0.06, P > O.l), patients with duodenal ulcer (r = 0.30, P > 0.1) or patients with gastric ulcer (r = 0.23, P > 0.1); and no significant correlation between M A 0 and stimulated glycoprotein output in controls (r = -0.29, P > O.l), patients with duodenal ulcer (r = 0.47, P > 0.1) or patients with gastric ulcer (r = 0.41, P > 0.1). The mean stimulated glycoprotein output was higher in four patients with duodenal ulcer who had a M A 0 greater than 35 mEq/hour (22.5 mg/hour) when compared to five patients with duodenal ulcer who had a M A 0 less than 35 mEq/hour (16.5 mg/hour). Glycoprotein Output, Blood-group and Secretor Status The relationship between glycoprotein output, blood-group and secretor status was evaluated in a group consisting of controls and patients with duodenal ulcer. Ten subjects of bloodgroup A had a mean basal and stimulated glycoprotein output of 13.5 and 19.5 mg/hour respectively, while seven subjects of bloodgroup 0 had a mean basal and stimulated glycoprotein output of 13.4 and 21.6 mg/hour respectively. There were no statistically significant differences (P > 0.1) between bloodgroups A and 0 for either basal or stimulated glycoprotein output. Thirteen secretors of bloodgroups A and 0 had a mean basal and stimulated glycoprotein output of 13.2 and 18.6 mg/hour respectively, while four non-secretors of bloodgroups A and 0 had a mean basal and stimulated glycoprotein output of 14.2 and 21.6 mg/hour
respectively. These differences between secretors and non-secretors were not statistically significant (P > 0.1). Age and Glycoprotein Output As patients with gastric ulcer had a higher mean age (58 years) than the combined group of patients with duodenal ulcer and controls (32 years), it was possible that the elevated basal glycoprotein output was due to age alone and not due to the presence of chronic gastric ulcer. However, there was no significant correlation between age and basal glycoprotein output in controls and patients with duodenal ulcer (r = -0.22, P > 0 . l), and age did not influence the basal glycoprotein output within the gastric ulcer group (r = -0.07, P > 0.1). Carbohydrate Analysis The carbohydrate composition of gastric glycoproteins was initially determined in the higher density fractions 7-12 in both the basal state and following stimulation of gastric juice by pentagastrin. However, in samples from two controls, two patients with duodenal ulcer and two patients with gastric ulcer there were no significant differences in the carbohydrate composition of fractions 7-12, a typical example of which is shown in Table 2. Because of this only one glycoprotein fraction from basal gastric juice and one from stimulated gastric juice was analysed to screen for major differences in carbohydrate composition. Fraction 11, which had a high glycoprotein concentration, was chosen.
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~
7 8 9 10 11 12
Fuc. ~
Gal.
Glc NAc. Gal NAc.
S.A.
~~~
22.6 20.4 23.1 22.7 20.1 24 2
30.2 30.3 27.7 30.4 31.4 30.6
27.9 29.8 30.3 26.8 29.8 26.2
10.9 12.6 11.7 13.9 11.1 10.4
5, NO.6
The carbohydrate composition of glycoproteins from all individuals in this study was then related to ABO blood group and secretor status. The following results were obtained : (i) Glycoproteins from non-secretors of blood groups A and 0 had a significantly lower percentage composition of fucose compared with glycoproteins from secretors of the same blood group (P < 0.01) (Fig. 1). This applied to glycoproteins from both basal gastric juice and from gastric juice stimulated by pentagastrin. (ii) Glycoproteins from secretors of blood group A had a significantly higher percentage composition of N-acetylgalactosamine compared with glycoproteins from secretors of blood group 0 ( P < 0.01) (Fig. 2). This again applied to glycoproteins from both basal gastric juice and from gastric juice stimulated by pentagastrin. (iii) Glycoproteins from one secretor of blood group B had a higher percentage composition of galactose 436.4 %) compared with glycoproteins from secretors of blood group 0 (mean 30.5 %). (iv) Glycoproteins from gastric juice stimulated by pentagastrin from non-secretors of blood groups A and 0 had a significantly higher percentage composition of sialic acid compared with glycoproteins from secretors
TABLE 2 The percentage carbohydrate composition of gastric glycoproteins in fractions 7-12 in stimulated gastric juice from a group A secretor. Mannose and glucose were not detected in any fractions Fraction -
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8.4 6.9 7.2 6.2 7.5 8.6
Fuc. = Fucose Gal. = Galactose S.A. = Sialic acid Glc NAc. = N-acetylglucosamine Gal NAc. = N-acetylgalactosamine
The carbohydrate composition of glycoproteins from patients with duodenal and gastric ulcer and controls, in the basal state and following pentagastrin stimulation, are shown in Tables 3 and 4. Pentagastrin stimulation does not appear to alter the carbohydrate composition of glycoproteins, and glycoproteins from patients with peptic ulcer have a similar carbohydrate composition to glycoproteins from controls matched for blood group and secretor status. Small amounts of mannose and glucose were detected in glycoproteins from many samples but the presence of these sugars was not related to blood group, secretor status or peptic ulcer.
TABLE 3 The mean percentage carbohydrate composition of gastric glycoproteins in basal gastric juice. The results have been divided according to ABO blood group and ABH secretor status in control subjects and patients with chronic duodenal and gastric ulcer No. of subjects Controls
Duodenal ulcer
Gastric ulcer
5 3 1 2 2 2 1 1 1 2 1 1
1
s = Secretor
Blood Secretor group status
A 0 0 A A 0 0 AB
S S NS S NS S NS
B
NS S NS S S
A A 0 B
S
Fuc.
Man.
Gal.
Glu.
20.5 21.9 13.3 17-4 11.0 22.3 7.7 21.8 10.6 21.1 13.6 17.1 16.9
0.4
28.7 32.7 32.2 27-2 28.1 29-1 36.6 32.1 32.1 27.4 31.0 25.0 37.1
4.2 1.4 1.6 0.4 0.6 1.0 1.6
1.1
1.2 0.8 1.5
1.0 1.5 1.5 1.7 1.6 0.8 2.0 -
-
2.2 -
Glc NAc. Gal NAc. 25.6 26.3 30.3 34.2 37.6 29.6 32.8 21.8 30.9 28.3 33.3 27.2 27.7
NS = Non-secretor Fuc. = Fucose Man. = Mannose Gal. = Galactose S.A. = Sialic acid Glc NAc. = N-acetylglucosamine Gal NAc. = N-acetylgalactosamine
13.5 1.2 10.7 12.6 13.3 11.7 12.2 14.8 14.5 12.6 12.8 9.7 8.2
S.A
5.7 9.3 10.7 7.6 8 .0 6.5 8.2 6.5 11.1 7.4 8.6 19.0 10.1
Glu. = Glucose
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GASTRIC GLY COPROTEINS IN CHRONIC PEPTIC ULCER
TABLE 4 The mean percentage carbohydrate composition of gastric glycoproteins in gastric juice stimulated by pentagastrin. Results have been divided according to ABO blood group and ABH secretor status in control subjects and patients with chronic duodenal and gastric ulcer No. of subjects Controls
Duodenal ulcer
Gastric ulcer
6 3 1 2 2 2 1 1 1 2 1 2 1 1
Blood Secretor group status A 0 0 A A 0 0 AB B A A 0 0 B
S S NS S
NS S NS S NS S NS S NS S
Fuc.
M?n.
Gal.
Glu.
20.1 21.2 13.1 18.1 10.9 21.8 8.1 20.7 14.7 16.8 11.6 19.9 14.6 19.2
1.0 0.9 1.3 1.3 1.6 1.4 2.5 2.2 1.6 1.3 1.4 2.1
27.8 33.4 30.6 27.9 30.2 26.1 34.4 23.1 31.5 31.8 26.3 30.7 31.1 36.4
0.6 1.6 8.9 7.0 0.7 3.6 1.9 2.3 2.6
-
-
GlcNAc. Gal NAc. 29.6 26.9 26.8 24.6 35-4 31.6 30.9 23.0 28.3 28.8 33.2 27.7 22.4 26.5
S = Secretor NS = Non-secretor Fuc. = Fucose Man. = Mannose Gal. = Galactose Glc NAc. = N-acetylglucosamine S.A. = Sialic acid Gal NAc. = N-acetylgalactosamine
of the same blood group ( P < 0.01) (Fig. 3). In basal gastric juice, however, no significant difference in sialic acid levels was found in glycoproteins from non-secretors when compared with glycoproteins from secretor s. Discussion
The results from this study show no significant difference in either gastric glycoprotein output or concentration in patients with duodenal ulcer when compared with controls. However, patients with chronic gastric ulcer have a significantly higher basal glycoprotein output than both controls and patients with duodenal ulcer. Theoretically, the concentration of glycoprotein in gastric juice might be impcrtant in protecting against peptic ulceration, and it is of interest that glycoprotein concentration in both fasting and stimulated gastric juice is also significantly greater in patients with gastric ulcer when compared with both controls and patients with duodenal ulcer. The reason for the increased output and concentration of gastric glycoprotein in patients with gastric ulcer is uncertain, but there are two possible explanations. First, chronic gastric ulceration is almost always associated with
13.4 9.6 7.6 12.8 12.8 11.4 11.0 11.9 12.1 13.2 13.0 9.1 12.0 9.0
S.A. 7.6 6.4 11.6 8.3 8.5 7.9 9.5 8.3 11.8 5.8 14.5 8.0 14.1 8.9
Glu. = Glucose
gastritis and goblet cell metaplasia in the region of the ulcer and throughout the antrum of the stomach.lZ This abnormal accumulation of goblet cells may increase glycoprotein secretion and account for the elevated basal glycoprotein output in gastric juice. It is unlikely that glycoprotein secretion is increased from other surface epithelial cells as histochemical studies of these cells suggest that the amount of glycoprotein is dirnini~hed.~ On the other hand, in the present study, as in other larger series13, the amount of gastric juice and the hydrogen ion concentration in gastric juice following pentagastrin stimulation was higher in controls and in patients with duodenal ulcer, than in patients with gastric ulcer. The elevated glycoprotein concentrations in patients with gastric ulcer might be explained by the lower volumes of gastric juice both in the basal state and following pentagastrin. In this study, as in another14, there was no significant correlation between acid output and glycoprotein output. Thus the relationship between these two indices does not appear to be relevant to the aetiology of peptic ulcer. The relationship between glycoprotein output, the ABO blood-group and secretor status has not been previously investigated. However,
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ROBERTS-THOMSON ET AL.
results from the present study indicate that glycoprotein output is independent of ABO blood-group and secretor status, and therefore, quantitative changes in gastric glycoproteins do not appear to explain the increased susceptibility of non-secretors and people of bloodgroup 0 to duodenal ulcer. In this study, as in other reports’. ’, 15-19, the principal carbohydrate components of human gastric glycoproteins were found to be galactose, N-acetylglucosamine, fucose, Nacetylgalactosamine and sialic acid. Schrager’ using gas-liquid chromatography to identify sugar residues found that the quantitative relationship of galactose : N-acetylglucosamine : N-acetylgalactosamine was 4 : 3 : 1. Analysis of our results in secretors of blood groups A and 0 has shown a slightly different relationship with a galactose : N-acetylglucosamine : Nacetylgalactosamine ratio of 5 : 5 : 2. The presence of small amounts of glucose and mannose in some glycoprotein samples was apparently unrelated to blood group, secretor status or peptic ulcer. The possibility that these sugars arose during the hydrolytic procedure by a mechanism involving acid reversion was considered. However, a mixture of fucose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid in the proportions found in gastric juice samples at a final con-
30
20
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centration of 0.5 % contained only the component monosaccharides after treatment with 3 M HCl for three hours at 100°C. Thus mannose and glucose were considered to be components of glycoprotejns in gastric juice. The source of these sugars may be glycoproteins synthesised and secreted by gastric epithelial cells or, perhaps, glycoproteins from saliva.l9
T I
I
+
BLOOD GROUP A SECRETORS
BLOOD GROUP 0 SECRETORS
FIGURE 2. The percentage composition of N-acetylgalactosamine in glycoproteins from stimulated gastric juice from secretors of blood groups A and 0. Blood group A secretors had a significantly higher percentage composition of N-acetylgalactosamine (P < 0.01 ).
9
.
I
i
-
I
10
. -
i 4.
~~
NON-SECRETORS BLOOD GROUPS A 0
~
~~~
SECRETORS BLOOD.GROUPS A . 0
FIGURE 1 The percentage composition of fucose in glycoproteins from stimulated gastric juice from secretors and non-secretors of blood groups A and 0 Nonsecretors had a significantly lower percentage composition of fucose (P < 0 01)
HON-SECRETORS BLOOD-GROUPS A D .
SECRETORS 01000-GROUPS A.O.
FIGURE 3. The percentage composition of sialic acid in glycoproteins from stimulated gastric juice from secretors and non-secretors of blood groups A and 0. Non-secretors had a significantly higher percentage composition of sialic acid (P < 0.01).
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The preparation of volatile derivatives for gas-liquid chromatography involved reduction of the free neutral sugars to the corresponding alditol. As the alditols could be derived not only from the free sugars but from the corresponding acids by borohydride reduction, the possibility that the carbohydrate components were derived wholly or in part from the corresponding uronic acids was considered. Paper chromatography of neutralised hydrolysates showed only the neutral sugars, and no uronic acids could be detected in the glycoprotein sample by the method of Bitter and Muir.20 However, the sensitivity of gas-liquid chromatography as compared with colorimetric methods leaves open the possibility that small amounts of connective tissue glycosaminoglycans may be present in the sample. Results from this study indicate that the carbohydrate composition of gastric glycoproteins is similar in patients with chronic gastric and duodenal ulcer when compared with controls of the same blood group and secretor status. However, our findings conflict with a recent report by Domschke et aL21 These authors measured free and bound sialic acid by a thiobarbituric acid method similar to that described by Warren6 and found that the quantity of sialic acid in basal and stimulated gastric juice was low in patients with gastric ulcer when compared with controls. The different conclusions reached may have been due to differences in methodology, Domschke et d2' did not separate glycoproteins from proteins and did not relate the sialic acid content to the glycoprotein content. In all samples analysed in this study the carbohydrate composition of glycoproteins varied according to the ABO blood group and secretor status. The finding that glycoproteins from non-secretors have a low proportion of fucose, and that glycoproteins from secretors of blood group A and B have a high proportion of N-acetylgalactosamine and galactose respectively, indicates that gastric glycoproteins are similar to glycoproteins from ovarian cystszz and support the view2 that the majority of gastric glycoproteins are blood group specific substances. The finding of an increased proportion of sialic acid in glycoproteins from
513
stimulated gastric juice from non-secretors is interesting and has not been reported previously. This observation needs to be confirmed by study of a larger series. The low proportion of fucose in glycoproteins from non-secretors has been noted p r e v i o ~ s l y ~ ~ , and is vnlikely to have a significant influence on the physical properties of mucus as fucose is a neutral sugar. Although the proportion of N-acetylgalactosamine and galactose is higher in secretors of blood groups A and B respectively there is no evidence to suggest that these sugars influence the physical properties of mucus and enhance its protective function; so that the higher incidence of duodenal ulcer in group 0 secretors compared with group A and B secretors cannot be explained on the basis of the carbohydrate composition of the gastric glycoproteins. Acknowledgements
We are grateful to Mrs. Ruth Walker for skilled technical assistance and to Dr. Ross Ullman for help with statistical analyses. References 1. SCHRAGUI,I. (1970): The chemical composition and function of gastrointestinal mucus, Guf 11, 450. 1. DENBOROUGH, M. A., PRESSER,1. C. and UNGAR,B. (1971): Isolation of
glycoproteins from human gastric juice and saliva by density gradient ultracentrifugation, Clin. Chem. 17, 335. 3. ALLEN,A. (1972): The structure and function of gastric mucus, Gut 13, 666. 4. KENT.P. W. and ALLEN,A. (1968): The biosynthesis of intestinal mucins. The effect of salicylates on glycoprotcin biosynthesis by sheep colonic and human mucosal tissues in virro, Biochem. J . 106, 645. 5. LEV,R.(1970): The histochemistry of mucus-producing cells in the normal and diseased gastrointestinal mucosn, Progr. Gasrroenterol. 1, 13. 6 . WARREN,L. (1959): The thioharbituric acid assay of sialic acids, J . biol. Chem. 234, 1971. H. H., MEHIGAN, I. A. and ROBERTS, I. A. F. (1954): The 7. AIRD,I., BENTALL, blood groups in relation to peptic ulceration and carcinoma of colon, rectum, breast and bronchus. Brit. med. J . 2, 315. 8 . CLARKE, C. A,, EDWARDS, J. W., HADDOCK, D. R. W., HOWEL-EVANS, A. W., R. B. and SHEPPARD, P.M. (1956): ABO blood groups and MCCONNELL. secretor character in duodenal ulcer, Brit. med. J . 2, 725. 9. Dusors, M., GILL^, K. A,, HAMILTON, 1. K., REBERS, P. A. and SMITH,F. (1957): Colorimetric method for the determination of sugars and related substances, Anal. Chem. 28, 350. 10. CLARKE,A. E.. MARITZ,V. M..PRESSER, 1. and DENBOROUGH, M. A. (1974): Quantitative estimation of neutral and amino sugars of glycoproteins from human secretions by gas-liquid chromatography, Biochem. Med. 9, 342. 11. DENBOROUGH, M. A., DOWNING, H. 1. and DOE, A. G. (1969): Serum blood group substances and ABO haemolytic disease, Brit. J . Haemat. 16, 103. 12. Cox, A. I. (1963): Gastric mucosal changes in peptic ulcer, Gastroenterology 45, 558. 13. WORMSLEY. K. G. and GROSSMAN. M. L. (1965): Maximal histolog test in control subjects and patients with peptic ulcer, Gut 6, 427. 14. GLASS,G. B. J., PUOH,B. L. and WOLF,S. (1950): Correlation of acid, pepsin and mucoprotein secretion by human gastric glands, J. appl. Physiol. 2, 571. IS. HOSKINS.L. C. and ZAMCHECK, N. (1963): Studies on gastric mucins in health and disease, Ann. N.Y. Acad. Sci. 106, 767. 16. RICHMOND, V.. CAPUTTO,R. and WOLF,S. (1955): Biochemical study of the large molecular constituents of gastric juice, Gastroenterology 29, 1017. 17. GLASS,G. B. I., RICH,M.and STfPmNsoN, L. (1958): Comparative study of mucoproteins of human gastricjuice and serum. Gasrroenrerology 34,598. 18. WALDRON-EDWARD, D. ,and SKORYNA,S. C , (1970): Studies on human gastric gel mucin. Isolation and characterisation of a major glycoprotein component, Gastroenterology 59. 671.
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19 MARSHALL, R. D. (1972): Glycoproteins, Ann. Rev. Biochem 41, 673. 20. BITTER, T. and MUIR,H. M. (1962): A modified uronic acid carbazole reaction, A n d . Biocliern 4. 110 21. DOMSCHE,W., DOMSCHKE, S., CLASSEN, M. and DEMLING, I,. (1972). Some properties of mucus in patients with gastric ulcer. Effects of treatment with carbenoxolone sodium, Scnnd. J. Gostroent. 7, 641
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22. MORGAN,W. T. J. (1963): Some observations on the carbohydratecontaining components of human ovarian cyst mucm, Ann. N.Y. Acad. Sci. 106, 177. 23. EVANS,D. A. P., MCDONNELL. R. B , DONOHUE, W. T A., SlRCUs. W. and CREAN, G. P. (1963): Fucose and agglutinogen contents of gastric p i c e in subjects with peptic ulcers, J . Lab. d i n . Med. 61, 660.
CORRIGENDUM
The following segment of the Case Report TROPHIC HORMONE PRODUCTION C. M. Dunn, which appeared in the June Journal of Medicine, pp. 270-273, has been be inserted on page 272, second column.
entitled “ECTOPIC ADRENOCORTICOAND NEOPLASM” by R. F. Pinerua and 1975 issue of Australian and New Zealand omitted from the original article and should
Obesity, osteoporosis and striae are most unusual. The rapid development of pigmentation is common. The capacity of tumours to produce more than one peptide hormone has been emphasised.’ Alpha-melanocyte stimulating hormone (a-MSH) and beta-melanocyte stimulating hormone (P-MSH) have been demonstrated in turnours’, with P-MSH being the major melanocyte stimulating factor. However, a heterogeneous form of MSH structurally different from both a-MSH and P-MSH has been described.” Although plasma ACTH and MSH were not measured, there was no autopsy evidence of an autonomous adrenal lesion to explain the undoubted hypercorticism. The pigmentation observed was in all probability due to MSH secretion. Severe electrolyte disturbances are typical of ectopic ACTH production in contra distinction to the hypercorticism of pituitary origin. Hypokalaemic hypochloraemic alkalosis was prominent in all patients. Muscle weakness was severe in two patients and was no doubt produced by hypokalaemia which contributed to the production of ventricular ectopic beats in one patient. The presence of such a metabolic alkalosis in the absence of more usual causes is a clue to an ACTH producing or adrenal tumour which may initially be unsuspected. Electrolyte abnormalities may occasionally precede by months or years recognition of an ACTH producing tumour.” The widespread rash which in Patient 2 developed pari passu with his malignant disease may have been a dermatological paramalignant manifestation and bore some similarities to the sign of Leser-Trelat.” The neuropathy found in Patient 3 could have been a neurological manifestation of his neoplasm although there was a far more common alternative explanation. The response to synacthen stimulation in the patients tested showed that despite high plasma cortisol levels and florid evidence of acute adrenal overactivity, the suprarenals had still significant reserve capacity and were capable of response to further trophic hormone stimulation. Patient 3 had considerable adrenal responsiveness when tested 23 days before autopsy which showed extensive metastatic replacement of the suprarenal glands. It is noteworthy that in the same patient three days before death the plasma cortisol wus 100 pg/lOO mi.
