Clinical Science (1979) 56,533-538
Carbohydrate moieties of glycoproteins in human hepatic and gall-bladder bile, gall-bladder mucosa and gall stones
S . P. L E E , T. H . L I M A N D A. J . SCOTT Department of Medicine, University of Auckland, Grafton, Auckland, New Zealand
(Received 20 December 1977; accepted 17 January 1979) Summary 1. The soluble glycoproteins of human bile, gallbladder mucosa and gall stones have been extracted ‘and hydrolysed, and the monosaccharides analysed by gas-liquid chromatography. 2. Human biliary glycoproteins contained 55-75% of carbohydrate, the major monosaccharide components being galactose, fucose and N-acetylglucosamine,accounting for 70-85% of all the monosaccharides. Mannose, glucose, N-acetylgalactosamine and N-acetylneuraminic acid (sialic acid) were also present. N-Acetylneuraminic acid was present in large amounts in the gall-bladder mucosa and bile of one ulcerated and markedly inflamed gall bladder. 3. The proportion of monosaccharides in soluble glycoproteins of mucosa and bile were not different in samples from subjects with or without gall stones. 4. Gall stones were analysed for cholesterol, calcium and bilirubin and classified as ‘cholesterol stones’ (7/10) and ‘pigment stones’ (3/10). Both cholesterol and pigment stones contain a variable amount of glycoprotein. The pattern of carbohydrate constituents was similar to that present in the gall-bladder mucosa and bile in the same subject. There was also no major differencebetween the pattern found in ‘cholesterol’ and ‘pigment’ stones. 5. Evidence and argument are presented suggesting that some glycoprotein is secreted by the Correspondence: Dr S. P. Lee, Department of Medicine (Gastroenterology), The Peter Bent Brigham Hospital, Harvard Medical School, 72 1 Huntington Avenue, Boston, Massachusetts 021 15, U.S.A. 37
gall bladder and incorporated into gall stones. This calls for further work upon the influence of these carbohydrate-rich macromolecules on cholesterol solubilization in mixed micelles. Key words: bile, cholelithiasis, gall bFdder, glycoprotein, monosaccharides. Introduction
Mucous membranes respond to challenge or injury by secreting mucus and shedding epithelial cells (Florey, 1970). Mucus (mucopolysaccharide or glycoprotein) refers to particular macromolecules which can modify the physicochemical properties of various body fluids, as in the gastrointestinal tract, where its secretion may have a protective and lubricating function. Structurally, the constituents of mucus comprise a backbone polypeptide chain to which oligosaccharide units are attached. The carbohydrate side chains surround and protect the protein core from proteolysis. The oligosaccharide units are galactose, fucose, N-acetylglucosamine, mannose, N-acetylneuraminic acid (sialic acid), Nacetylgalactosamine and glucose. This monosaccharide composition may be changed in disease (Teague, Fraser & Clamp, 1973). Interest in the role of mucus in the genesis and growth of gall stones arose from histochemical demonstration that it forms a matrix for the gall stones (Womack, Zeppa & Irvin, 1963), as solution of the lipid components in cholesterol stones revealed a network of mucopolysaccharides (Sutor & Wooley, 1974). Serial observations of mucus secretion in models of experimental cholelithiasis have shown an increase in gall-bladder mucus 533
534
S . P . Lee, T. H . Lim and A . J. Scott
before stone formation (Freston, Bouchier & Newman, 1969; Womack, 1971), suggesting that the production of gall-bladder mucus plays an important role in the precipitation of cholesterol (Womack, 1971). A casting technique has shown that the gall-bladder mucosa contained multiple invaginations, where pockets of mucus are present (Hulten, 1968a, b) with precipitation of cholesterol crystals possibly occurring inside these spherules of mucus. Kleeberg (1953, 1956) has also demonstrated that precipitation of mineral salts within a colloid matrix in vitro produced remarkable resemblance to the laminated structure of human gall stones. Few attempts have been made to characterize biliary glycoprotein; the origin of these glycoproteins, and whether calculous biliary glycoprotein has a normal or abnormal chemical composition is unclear. As the carbohydrate components of gastrointestinal glycoproteins determine the physical, chemical and hence the biological behaviour of the whole molecule, we have therefore studied the extraction and partial characterization of the carbohydrate moiety of biliary gly coproteins. Material and methods Sample collection and preparation Gall-bladder bile was obtained by needle aspiration from 10 gall bladders during cholecystectomy (group A) and stored at -2OOC until used. After cholecystectomy the gall bladder was then prepared as follows. A longitudinal incision through the fundus, body, neck and cystic duct was made and the gall stones were removed. A 6 mm longitudinal strip was then cut by a second parallel incision. This was made into a ‘Swiss roll’, stabilized with a pin and fixed in 10% formalin for histological examination. The rest of the mucosa was washed with NaCl solution (155 mmol/l) three times and blotted dry. The mucosa was then scraped with a sharp scalpel held at right angles to the mucosa; a second 6 mm strip was fixed for histological examination to confirm the disappearance of the epithelial layer. The gall stones were washed three times with distilled water, airdried and then ground to a powder. All steps except gall-stone powdering were fully aseptic. Four samples of normal gall-bladder bile (group B) and gall bladder were obtained from renal transplant donors in vitro, with full consent of the coroner and/or relatives. Hepatic bile (group C)
was collected from T-tube drainage in four patients after exploration of the common bile duct for cholesterol gall stones. Gall-bladder mucosal scrapings were freezedried. Weighed samples of scrapings and gall-stone powder were homogenized (Vitra 35 000 rev./min for 15 min) in phosphate buffer (0-05 mmol/l), pH 7.4, immersed in ice. The disintegrated specimen was then centrifuged at 5000 g for 20 min and the supernatant collected. The residue was rehomogenized and centrifuged three times. Further extraction, purification and analysis of soluble glycoproteins from the supernatants were as described for bile. Separation of soluble glycoprotein fractions Bile samples were diluted (gall-bladder bile ten times, hepatic bile five times) with distilled water. Glycoproteins were extracted by a method similar to that of Bouchier & Clamp (1971). Hexadecyltrimethylammonium bromide (Cetavalon, ICI Ltd) was added to a final concentration of 0.05 mmol/l and left for 24-36 h at 4OC, and then centrifuged at 16 000 g for 30 min. The precipitate was stirred with NaCl solution (0.25 mmol/l) at 4OC for 16 h. The solution was again centrifuged at 16 000 g for 30 min; the supernatant was treated with absolute ethanol and the precipitate collected by centrifuging at 500 g for 10 min. This final precipitate was dissolved in distilled water and dialysed against running water for 48 h, freeze-dried and weighed. Carbohydrate determination The monosaccharides of the soluble glycoproteins of each sample were determined after methanolysis by gas-liquid chromatography as described by Bhatti, Chambers & Clamp (1970). The sample was heated in methanolic HCl (1-5 mmol/l) at 90°C for 24 h, re-N-acetylated, and the trimethylsilyl derivate analysed by gas-liquid chromatography (Hewlett-Packard 7620A, with a 2 m x 6 mm glass column, 3% SE 30 Gaschrome Q and a mesh size 80-100; a flame ionization detector was used). Chemical analysis of gall stones Cholesterol was measured with the LiebermannBurchard reaction (Abell, Levy, Brodie & Kendall, 1952), bilirubin was determined by the oxidation method (Ferro & Ham, 1967) and calcium by
Human biliary glycoproteins
atomic absorption spectrometry as modified by Carlisle & Tasman-Jones (1973). Gall stones were classified as ‘cholesterol stones’ if their cholesterol content exceeded 85% of the dry weight, and ‘pigment stones’ if the calcium plus bilirubin contents were more than 25% of the dry weight. Comparisons were made by Mann-Whitney U modification of the Wilcoxon rank test (Feinstein,
1977). Results
Glycoproteins were present in all samples. Biliary glycoproteins contained galactose, fucose, Nacetylglucosamine, mannose, glucose, N-acetylgalactosamine and N-acetylneuraminic acid in descending order of abundance. Galactose, fucose and N-acetylglucosamine together made up 70-85% of all the monosaccharides. Ribose in traces (0.32.7%) was present in three samples of gall stones (two ‘cholesterol’, one ‘pigment’) and was not detected in other specimens. There were seven ‘cholesterol stones’ and three ‘pigment stones’ in group A. Quantification and relative proportion of each monosaccharide moiety is shown in scattergrams (Figs. 1-3) and in Table 1. No significant differ-
535
ence (P > 0.05) was found between bile samples from groups A, B and C, and mucosal samples from groups A and B, when compared for their monosaccharide contents and proportions. No significant difference in either monosaccharide content or proportion was found when the ‘cholesterol’ and ‘pigment’ stone subgroup bile and mucosa samples within group A were compared, nor when bile and mucosa samples of each subgroup were compared with corresponding samples in group B (P > 0.05). The N-acetylneuraminic acid content was below 10% in all groups. In one sample in group A (6),in a grossly inflamed, ulcerated gall bladder containing pigment stones and purulent gall-bladder bile, the proportion of N-acetylneuraminic acid was very high (see Fig. 3). Histologically, dense polymorphonucleated leucocyte infiltration and mucosal ulceration were present. In total the carbohydrate (sum of all monosaccharides) made up 55-75% of the glycoprotein molecules extracted (Fig. 4). The amount of glycoproteins extractable by this method was higher in calculous gall-bladder bile than in normal gallbladder bile (P < 0.05), and higher in the mucosal scrapings from gall bladders containing stones than those from normal gall bladders (P< 0.05).
I
A
A
600
0
-
g 500
0
A o
100
L
t
II
.
40
. . . . . 0
0
0
I
.
c
4 -m
30
0
,-
L
cw O
8
U
20
I
a
.
l o t Stone
Bile
Mucosa
Bile
Group A
FIG. 1. Scatter diagram of monosaccharide (fucose) in the extracted and purified glycoproteins. (a) The quantity of the monosaccharide expressed as n m o l h g of glycoprotein; (b) the quantity of monosaccharide expressed as a percentage of the total carbohydrate content of the glycoprotein molecule. 0, ‘Cholesterol stone’; A, ‘pigment stone’.
S . P. Lee, T. H . Lim and A . J. Scott
536 700
-.-
-
(a)
600 -
.-e % 500 - 400 aJ
0 A
00
E! .
3 300 E v 0
g
200
-
C
I
100-
UDO rn
Bile ‘Mucosa Stone
Bile Mucosa
Bile
FIG. 2. Scatter diagram of monosaccharide (mannose) in the extracted and purified glycoproteins. (0) The quantity of the monosaccharide, expressed as nmol/mg of glycoprotein; (b) the quantity of monosaccharide expressed as a percentage of the total carbohydrate content of the glycoprotein molecule. 0, ‘Cholesterol stone’; 4 ‘pigment stone’.
20
t A ‘Z’
’OI-
:
D
I D
0
0 (-Group
A -1-Group
BiGroupC
I
FGroupA*-Group
B A G r o u p Cl
FIG. 3. Scatter diagram of monosaccharide (N-acetylneuraminic acid) in the extracted and purified glycoproteins. (a) The quantity of the monosaccharide expressed as nmol/mg of glycoprotein; (b) the quantity of monosaccharide expressed as a percentage of the total carbohydrate content of the glycoprotein molecule. 0, ‘Cholesterol stone’; A,‘pigment stone’. Sample no. 6 is indicated by (6).
Discussion
Carbohydrates in human biliary glycoprotein Our findings confirm that the major sugar components were galactose, fucose and N-acetylglucosamine, which together made up 70-85% of the carbohydrates. The carbohydrate fraction represented the major (55-75%) component of the biliary glycoprotein molecule. The carbohydrate moieties are similar to those of other known gastrointestinal mucins (Horowitz, 1963). Apart from the
detection of glucose, our data are comparable with those reported by Bouchier & Clamp (1971). Schrager, Oates & Rosbottom (1972) concluded that there is no mannose in biliary glycoproteins. Our findings are in variance with this. Mannose is present in all samples analysed. The presence of mannose and glucose suggests that with our method of extraction there may have been some small contamination with either glycolipids or cellmembrane glycoproteins. Others have also found a low N-acetylneuraminic acid content in human
Human biliary glycoproteins
537
(Bouchier & Clamp, 1971; Schrager et al., 1972) and pig (Neiderhiser, Pantner & Carlson, 1971) biliary glycoproteins. In samples from an ulcerated inflamed gall bladder containing pigment stones and purulent bile, the N-acetylneuraminic acid concentrations in mucosa, bile and stones were unusually high. This elevation of N-acetylneuraminic acid concentrations may be due to the metabolic products present after leucocytic infiltration. Individuals with different blood groups (ABO or Lewis system) and secretor status often have some differences in the proportions of monosaccharides in their secretory glycoproteins. Unfortunately, the blood groups and secretor status have not been determined in the present study. However, in human biliary mucus, little difference in either the proportion of monosaccharides or the total percentage of sugars has been found in relation to secretor and non-secretor status (Schrager et al., 1972).
Within the limits of this method of analysis, the monosaccharide proportions of glycoprotein molecules do not differ significantly when normal samples of bile and gall-bladder mucosa are compared with calculous ones. The difference seems therefore to be quantitative rather than qualitative.
Origins of glycoproteins Bouchier & Clamp (197 1) found no difference in the glycoprotein of the hepatic bile of patients who had gall stones and those who did not. The total hexosamine content, however, was found to be higher in calculous gall-bladder bile than in normal bile (Bouchier, Copperband & El Kidsi, 1965), suggesting a difference in the mucus content. Direct quantification of glycoproteins in the present study has confirmed this. It follows that the hepatic bile must have undergone some modification in the gall bladder which contains stones. This increased glycoprotein content of the gall-bladder bile can be a result of excessive concentrating function of the diseased gall bladder, for which there is no evidence, or, alternatively, an increase in production by the gall-bladder mucosa. The latter assumption is further supported by our finding of a higher glycoprotein content in the pathological gallbladder mucosa when compared with the normal mucosa. Histological studies have also shown that the calculous gall-bladder mucosa contains more mucus-secreting cells than the normal mucosa (Elfving, 1960).
S . P. Lee, T. H . Lim and A . J. Scott
538
(b)
I
. .
.
-
.
&
-
. -.. .
...
I
,
4-,
Bile 'Mucosa' Stone Bile 'Mucosa Bile -Group A . GroupB GroupC
Bile
Mucosa Stone
Bile
Mucosa
Bile
FIG.4. Scatter diagram of t h e quantities of glycoproteins extracted in test samples (a) expressed as mg/g of mucosa a n d mg/l of bile. ( b ) T h e sum of all monosaccharides (total carbohydrate) expressed as a percentage of the glycoprotein molecule.
The increased production of glycoproteins by the gall-bladder mucosa could be a secondary effect of the contained stones. The evidence from experimental lithogenesis in animals, however, has indicated that glycoproteins or mucus production are increased before stones are formed (Freston et al., 1969; Womack, 1971). Our findings of a higher glycoprotein content in the pathological mucosa and bile, and the similarity of glycoprotein structure found in mucosa, bile and gall stones, suggest that these macromolecules are secreted by the gall bladder into bile and are incorporated into stones.
CARLISLE,V.F. & TASMAN-JONES, C. (1973) The composition of New Zealand gallstones. American Journal of Surgery,
Acknowledgments
HULTEN,0. (1968a) Formation of gallstones I. Acta Chirurgica Scandinavica, 134, 125. HULTEN, 0. (1968b) Formation of gallstones 11. Acta Chirurgica Scandinavica, 134,557. KLEEBERG, J. (1953) Experimental studies on colloid-chemical mechanism of gall stone formation. Gastroenterologia, 80,
126,403-410.
ELFVING,G. (1960) Crypts and ducts in the gall-bladder wall. Acts Pathologica Microbiologica Scandinavica, Suppl. 135. FENSTEN,A.R. (1977) Clinical Biostatistics. C.V. Mosby Co., St Louis. FERRO, P.V. & HAM.A.B. (1967) A new method for total free and conjugated serum bilirubin. American Journal of Clinical Pathology, 47,472-480. FLOREY, H.W. (1970) General Pathology, 4th edn. Lloyd-Luke, London. FRESTON,J.W., BOUCHIER,I.A.D. & NEWMAN,J. (1969) Biliary mucous substances in dihydrocholesterol induced cholelithiasis. Gastroenterology, 57,670-678. HOROWITZ,M.I. (1963) Macromolecules of the gastrointestinal tract. Annals of the New York Academy of Sciences, 106, 278-287.
This study was supported by a grant from the Medical Research Council of New Zealand. We thank Mrs C. Millar for her excellent secretarial help. References ABELL, L.L., LEVY, B.B., BRODIE, B.B. & KENDALL,F.E. (1952) A simplified method for the estimation of total cholesterol in serum and the demonstration of its specificity. Journal of Biological Chemistry, 195,357-366. BHAITI, T., CHAMBERS, R.E. & CLAMP,J.R. (1970) The gas chromatographic properties of biologically important Nacetylglucosamine derivatives, monosaccharides, disaccharides, trisaccharides, tetrasaccharides and pentasaccharides. Biochimica et Biophysica Acta, 222,339-347. BOUCHIER,I.A.D. & CLAMP, J.R. (1971) Glycoproteins in human bile. Clinica Chimica Acta, 35.2 19-224. BOUCHIER, I.A.D., COPPERBAND,S.R. & EL KIDSI, B.M. (1965) Mucous substances and viscosity of normal and pathological human bile. Gastroenterology, 49,343-352.
313-339.
KLEEBERG, J. (1956) Further physico-chemical studies in model gallstone formation; role of dehydration. Gastroenterologia, 85,271-280.
NEIDERHISER, D.H., PANTNER,J.J. & CARLSON,D.M. (1971) The purification and properties of the glycoproteins of pig gall-bladder bile. Archives of Biochemistry and Biophysics, 145,155-163.
SCHRAGER, J., OATES,M.D.G. & ROSBOTTOM,A. (1972) The isolation and partial characterisation of the principal biliary glycoprotein. Digestion, 6,338-355. SUTOR, D.J. & WOOLEY,S.E. (1974) The organic matrix of gallstones. Gut, 15,487-491. TEAGUE,R.H., FRASER,D. & CLAMP,J.R. (1973) Changes in monosaccharide content of mucous glycoproteins in ulcerative colitis. British Medical Journal, ii. 645-646. WOMACK,N.A. (1971) The development of gallstones. Surgery, Gynecology and Obstetrics, 133,937-945. WOMACK,N.A., ZEPPA, R. & IRVIN,G.L., 111 (1963) The anatomy of gallstones. Annals of Surgety, 157,670-686.
Carbohydrate moieties of glycoproteins in human hepatic and gall-bladder bile, gall-bladder mucosa and gall stones
S . P. L E E , T. H . L I M A N D A. J . SCOTT Department of Medicine, University of Auckland, Grafton, Auckland, New Zealand
(Received 20 December 1977; accepted 17 January 1979) Summary 1. The soluble glycoproteins of human bile, gallbladder mucosa and gall stones have been extracted ‘and hydrolysed, and the monosaccharides analysed by gas-liquid chromatography. 2. Human biliary glycoproteins contained 55-75% of carbohydrate, the major monosaccharide components being galactose, fucose and N-acetylglucosamine,accounting for 70-85% of all the monosaccharides. Mannose, glucose, N-acetylgalactosamine and N-acetylneuraminic acid (sialic acid) were also present. N-Acetylneuraminic acid was present in large amounts in the gall-bladder mucosa and bile of one ulcerated and markedly inflamed gall bladder. 3. The proportion of monosaccharides in soluble glycoproteins of mucosa and bile were not different in samples from subjects with or without gall stones. 4. Gall stones were analysed for cholesterol, calcium and bilirubin and classified as ‘cholesterol stones’ (7/10) and ‘pigment stones’ (3/10). Both cholesterol and pigment stones contain a variable amount of glycoprotein. The pattern of carbohydrate constituents was similar to that present in the gall-bladder mucosa and bile in the same subject. There was also no major differencebetween the pattern found in ‘cholesterol’ and ‘pigment’ stones. 5. Evidence and argument are presented suggesting that some glycoprotein is secreted by the Correspondence: Dr S. P. Lee, Department of Medicine (Gastroenterology), The Peter Bent Brigham Hospital, Harvard Medical School, 72 1 Huntington Avenue, Boston, Massachusetts 021 15, U.S.A. 37
gall bladder and incorporated into gall stones. This calls for further work upon the influence of these carbohydrate-rich macromolecules on cholesterol solubilization in mixed micelles. Key words: bile, cholelithiasis, gall bFdder, glycoprotein, monosaccharides. Introduction
Mucous membranes respond to challenge or injury by secreting mucus and shedding epithelial cells (Florey, 1970). Mucus (mucopolysaccharide or glycoprotein) refers to particular macromolecules which can modify the physicochemical properties of various body fluids, as in the gastrointestinal tract, where its secretion may have a protective and lubricating function. Structurally, the constituents of mucus comprise a backbone polypeptide chain to which oligosaccharide units are attached. The carbohydrate side chains surround and protect the protein core from proteolysis. The oligosaccharide units are galactose, fucose, N-acetylglucosamine, mannose, N-acetylneuraminic acid (sialic acid), Nacetylgalactosamine and glucose. This monosaccharide composition may be changed in disease (Teague, Fraser & Clamp, 1973). Interest in the role of mucus in the genesis and growth of gall stones arose from histochemical demonstration that it forms a matrix for the gall stones (Womack, Zeppa & Irvin, 1963), as solution of the lipid components in cholesterol stones revealed a network of mucopolysaccharides (Sutor & Wooley, 1974). Serial observations of mucus secretion in models of experimental cholelithiasis have shown an increase in gall-bladder mucus 533
534
S . P . Lee, T. H . Lim and A . J. Scott
before stone formation (Freston, Bouchier & Newman, 1969; Womack, 1971), suggesting that the production of gall-bladder mucus plays an important role in the precipitation of cholesterol (Womack, 1971). A casting technique has shown that the gall-bladder mucosa contained multiple invaginations, where pockets of mucus are present (Hulten, 1968a, b) with precipitation of cholesterol crystals possibly occurring inside these spherules of mucus. Kleeberg (1953, 1956) has also demonstrated that precipitation of mineral salts within a colloid matrix in vitro produced remarkable resemblance to the laminated structure of human gall stones. Few attempts have been made to characterize biliary glycoprotein; the origin of these glycoproteins, and whether calculous biliary glycoprotein has a normal or abnormal chemical composition is unclear. As the carbohydrate components of gastrointestinal glycoproteins determine the physical, chemical and hence the biological behaviour of the whole molecule, we have therefore studied the extraction and partial characterization of the carbohydrate moiety of biliary gly coproteins. Material and methods Sample collection and preparation Gall-bladder bile was obtained by needle aspiration from 10 gall bladders during cholecystectomy (group A) and stored at -2OOC until used. After cholecystectomy the gall bladder was then prepared as follows. A longitudinal incision through the fundus, body, neck and cystic duct was made and the gall stones were removed. A 6 mm longitudinal strip was then cut by a second parallel incision. This was made into a ‘Swiss roll’, stabilized with a pin and fixed in 10% formalin for histological examination. The rest of the mucosa was washed with NaCl solution (155 mmol/l) three times and blotted dry. The mucosa was then scraped with a sharp scalpel held at right angles to the mucosa; a second 6 mm strip was fixed for histological examination to confirm the disappearance of the epithelial layer. The gall stones were washed three times with distilled water, airdried and then ground to a powder. All steps except gall-stone powdering were fully aseptic. Four samples of normal gall-bladder bile (group B) and gall bladder were obtained from renal transplant donors in vitro, with full consent of the coroner and/or relatives. Hepatic bile (group C)
was collected from T-tube drainage in four patients after exploration of the common bile duct for cholesterol gall stones. Gall-bladder mucosal scrapings were freezedried. Weighed samples of scrapings and gall-stone powder were homogenized (Vitra 35 000 rev./min for 15 min) in phosphate buffer (0-05 mmol/l), pH 7.4, immersed in ice. The disintegrated specimen was then centrifuged at 5000 g for 20 min and the supernatant collected. The residue was rehomogenized and centrifuged three times. Further extraction, purification and analysis of soluble glycoproteins from the supernatants were as described for bile. Separation of soluble glycoprotein fractions Bile samples were diluted (gall-bladder bile ten times, hepatic bile five times) with distilled water. Glycoproteins were extracted by a method similar to that of Bouchier & Clamp (1971). Hexadecyltrimethylammonium bromide (Cetavalon, ICI Ltd) was added to a final concentration of 0.05 mmol/l and left for 24-36 h at 4OC, and then centrifuged at 16 000 g for 30 min. The precipitate was stirred with NaCl solution (0.25 mmol/l) at 4OC for 16 h. The solution was again centrifuged at 16 000 g for 30 min; the supernatant was treated with absolute ethanol and the precipitate collected by centrifuging at 500 g for 10 min. This final precipitate was dissolved in distilled water and dialysed against running water for 48 h, freeze-dried and weighed. Carbohydrate determination The monosaccharides of the soluble glycoproteins of each sample were determined after methanolysis by gas-liquid chromatography as described by Bhatti, Chambers & Clamp (1970). The sample was heated in methanolic HCl (1-5 mmol/l) at 90°C for 24 h, re-N-acetylated, and the trimethylsilyl derivate analysed by gas-liquid chromatography (Hewlett-Packard 7620A, with a 2 m x 6 mm glass column, 3% SE 30 Gaschrome Q and a mesh size 80-100; a flame ionization detector was used). Chemical analysis of gall stones Cholesterol was measured with the LiebermannBurchard reaction (Abell, Levy, Brodie & Kendall, 1952), bilirubin was determined by the oxidation method (Ferro & Ham, 1967) and calcium by
Human biliary glycoproteins
atomic absorption spectrometry as modified by Carlisle & Tasman-Jones (1973). Gall stones were classified as ‘cholesterol stones’ if their cholesterol content exceeded 85% of the dry weight, and ‘pigment stones’ if the calcium plus bilirubin contents were more than 25% of the dry weight. Comparisons were made by Mann-Whitney U modification of the Wilcoxon rank test (Feinstein,
1977). Results
Glycoproteins were present in all samples. Biliary glycoproteins contained galactose, fucose, Nacetylglucosamine, mannose, glucose, N-acetylgalactosamine and N-acetylneuraminic acid in descending order of abundance. Galactose, fucose and N-acetylglucosamine together made up 70-85% of all the monosaccharides. Ribose in traces (0.32.7%) was present in three samples of gall stones (two ‘cholesterol’, one ‘pigment’) and was not detected in other specimens. There were seven ‘cholesterol stones’ and three ‘pigment stones’ in group A. Quantification and relative proportion of each monosaccharide moiety is shown in scattergrams (Figs. 1-3) and in Table 1. No significant differ-
535
ence (P > 0.05) was found between bile samples from groups A, B and C, and mucosal samples from groups A and B, when compared for their monosaccharide contents and proportions. No significant difference in either monosaccharide content or proportion was found when the ‘cholesterol’ and ‘pigment’ stone subgroup bile and mucosa samples within group A were compared, nor when bile and mucosa samples of each subgroup were compared with corresponding samples in group B (P > 0.05). The N-acetylneuraminic acid content was below 10% in all groups. In one sample in group A (6),in a grossly inflamed, ulcerated gall bladder containing pigment stones and purulent gall-bladder bile, the proportion of N-acetylneuraminic acid was very high (see Fig. 3). Histologically, dense polymorphonucleated leucocyte infiltration and mucosal ulceration were present. In total the carbohydrate (sum of all monosaccharides) made up 55-75% of the glycoprotein molecules extracted (Fig. 4). The amount of glycoproteins extractable by this method was higher in calculous gall-bladder bile than in normal gallbladder bile (P < 0.05), and higher in the mucosal scrapings from gall bladders containing stones than those from normal gall bladders (P< 0.05).
I
A
A
600
0
-
g 500
0
A o
100
L
t
II
.
40
. . . . . 0
0
0
I
.
c
4 -m
30
0
,-
L
cw O
8
U
20
I
a
.
l o t Stone
Bile
Mucosa
Bile
Group A
FIG. 1. Scatter diagram of monosaccharide (fucose) in the extracted and purified glycoproteins. (a) The quantity of the monosaccharide expressed as n m o l h g of glycoprotein; (b) the quantity of monosaccharide expressed as a percentage of the total carbohydrate content of the glycoprotein molecule. 0, ‘Cholesterol stone’; A, ‘pigment stone’.
S . P. Lee, T. H . Lim and A . J. Scott
536 700
-.-
-
(a)
600 -
.-e % 500 - 400 aJ
0 A
00
E! .
3 300 E v 0
g
200
-
C
I
100-
UDO rn
Bile ‘Mucosa Stone
Bile Mucosa
Bile
FIG. 2. Scatter diagram of monosaccharide (mannose) in the extracted and purified glycoproteins. (0) The quantity of the monosaccharide, expressed as nmol/mg of glycoprotein; (b) the quantity of monosaccharide expressed as a percentage of the total carbohydrate content of the glycoprotein molecule. 0, ‘Cholesterol stone’; 4 ‘pigment stone’.
20
t A ‘Z’
’OI-
:
D
I D
0
0 (-Group
A -1-Group
BiGroupC
I
FGroupA*-Group
B A G r o u p Cl
FIG. 3. Scatter diagram of monosaccharide (N-acetylneuraminic acid) in the extracted and purified glycoproteins. (a) The quantity of the monosaccharide expressed as nmol/mg of glycoprotein; (b) the quantity of monosaccharide expressed as a percentage of the total carbohydrate content of the glycoprotein molecule. 0, ‘Cholesterol stone’; A,‘pigment stone’. Sample no. 6 is indicated by (6).
Discussion
Carbohydrates in human biliary glycoprotein Our findings confirm that the major sugar components were galactose, fucose and N-acetylglucosamine, which together made up 70-85% of the carbohydrates. The carbohydrate fraction represented the major (55-75%) component of the biliary glycoprotein molecule. The carbohydrate moieties are similar to those of other known gastrointestinal mucins (Horowitz, 1963). Apart from the
detection of glucose, our data are comparable with those reported by Bouchier & Clamp (1971). Schrager, Oates & Rosbottom (1972) concluded that there is no mannose in biliary glycoproteins. Our findings are in variance with this. Mannose is present in all samples analysed. The presence of mannose and glucose suggests that with our method of extraction there may have been some small contamination with either glycolipids or cellmembrane glycoproteins. Others have also found a low N-acetylneuraminic acid content in human
Human biliary glycoproteins
537
(Bouchier & Clamp, 1971; Schrager et al., 1972) and pig (Neiderhiser, Pantner & Carlson, 1971) biliary glycoproteins. In samples from an ulcerated inflamed gall bladder containing pigment stones and purulent bile, the N-acetylneuraminic acid concentrations in mucosa, bile and stones were unusually high. This elevation of N-acetylneuraminic acid concentrations may be due to the metabolic products present after leucocytic infiltration. Individuals with different blood groups (ABO or Lewis system) and secretor status often have some differences in the proportions of monosaccharides in their secretory glycoproteins. Unfortunately, the blood groups and secretor status have not been determined in the present study. However, in human biliary mucus, little difference in either the proportion of monosaccharides or the total percentage of sugars has been found in relation to secretor and non-secretor status (Schrager et al., 1972).
Within the limits of this method of analysis, the monosaccharide proportions of glycoprotein molecules do not differ significantly when normal samples of bile and gall-bladder mucosa are compared with calculous ones. The difference seems therefore to be quantitative rather than qualitative.
Origins of glycoproteins Bouchier & Clamp (197 1) found no difference in the glycoprotein of the hepatic bile of patients who had gall stones and those who did not. The total hexosamine content, however, was found to be higher in calculous gall-bladder bile than in normal bile (Bouchier, Copperband & El Kidsi, 1965), suggesting a difference in the mucus content. Direct quantification of glycoproteins in the present study has confirmed this. It follows that the hepatic bile must have undergone some modification in the gall bladder which contains stones. This increased glycoprotein content of the gall-bladder bile can be a result of excessive concentrating function of the diseased gall bladder, for which there is no evidence, or, alternatively, an increase in production by the gall-bladder mucosa. The latter assumption is further supported by our finding of a higher glycoprotein content in the pathological gallbladder mucosa when compared with the normal mucosa. Histological studies have also shown that the calculous gall-bladder mucosa contains more mucus-secreting cells than the normal mucosa (Elfving, 1960).
S . P. Lee, T. H . Lim and A . J. Scott
538
(b)
I
. .
.
-
.
&
-
. -.. .
...
I
,
4-,
Bile 'Mucosa' Stone Bile 'Mucosa Bile -Group A . GroupB GroupC
Bile
Mucosa Stone
Bile
Mucosa
Bile
FIG.4. Scatter diagram of t h e quantities of glycoproteins extracted in test samples (a) expressed as mg/g of mucosa a n d mg/l of bile. ( b ) T h e sum of all monosaccharides (total carbohydrate) expressed as a percentage of the glycoprotein molecule.
The increased production of glycoproteins by the gall-bladder mucosa could be a secondary effect of the contained stones. The evidence from experimental lithogenesis in animals, however, has indicated that glycoproteins or mucus production are increased before stones are formed (Freston et al., 1969; Womack, 1971). Our findings of a higher glycoprotein content in the pathological mucosa and bile, and the similarity of glycoprotein structure found in mucosa, bile and gall stones, suggest that these macromolecules are secreted by the gall bladder into bile and are incorporated into stones.
CARLISLE,V.F. & TASMAN-JONES, C. (1973) The composition of New Zealand gallstones. American Journal of Surgery,
Acknowledgments
HULTEN,0. (1968a) Formation of gallstones I. Acta Chirurgica Scandinavica, 134, 125. HULTEN, 0. (1968b) Formation of gallstones 11. Acta Chirurgica Scandinavica, 134,557. KLEEBERG, J. (1953) Experimental studies on colloid-chemical mechanism of gall stone formation. Gastroenterologia, 80,
126,403-410.
ELFVING,G. (1960) Crypts and ducts in the gall-bladder wall. Acts Pathologica Microbiologica Scandinavica, Suppl. 135. FENSTEN,A.R. (1977) Clinical Biostatistics. C.V. Mosby Co., St Louis. FERRO, P.V. & HAM.A.B. (1967) A new method for total free and conjugated serum bilirubin. American Journal of Clinical Pathology, 47,472-480. FLOREY, H.W. (1970) General Pathology, 4th edn. Lloyd-Luke, London. FRESTON,J.W., BOUCHIER,I.A.D. & NEWMAN,J. (1969) Biliary mucous substances in dihydrocholesterol induced cholelithiasis. Gastroenterology, 57,670-678. HOROWITZ,M.I. (1963) Macromolecules of the gastrointestinal tract. Annals of the New York Academy of Sciences, 106, 278-287.
This study was supported by a grant from the Medical Research Council of New Zealand. We thank Mrs C. Millar for her excellent secretarial help. References ABELL, L.L., LEVY, B.B., BRODIE, B.B. & KENDALL,F.E. (1952) A simplified method for the estimation of total cholesterol in serum and the demonstration of its specificity. Journal of Biological Chemistry, 195,357-366. BHAITI, T., CHAMBERS, R.E. & CLAMP,J.R. (1970) The gas chromatographic properties of biologically important Nacetylglucosamine derivatives, monosaccharides, disaccharides, trisaccharides, tetrasaccharides and pentasaccharides. Biochimica et Biophysica Acta, 222,339-347. BOUCHIER,I.A.D. & CLAMP, J.R. (1971) Glycoproteins in human bile. Clinica Chimica Acta, 35.2 19-224. BOUCHIER, I.A.D., COPPERBAND,S.R. & EL KIDSI, B.M. (1965) Mucous substances and viscosity of normal and pathological human bile. Gastroenterology, 49,343-352.
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KLEEBERG, J. (1956) Further physico-chemical studies in model gallstone formation; role of dehydration. Gastroenterologia, 85,271-280.
NEIDERHISER, D.H., PANTNER,J.J. & CARLSON,D.M. (1971) The purification and properties of the glycoproteins of pig gall-bladder bile. Archives of Biochemistry and Biophysics, 145,155-163.
SCHRAGER, J., OATES,M.D.G. & ROSBOTTOM,A. (1972) The isolation and partial characterisation of the principal biliary glycoprotein. Digestion, 6,338-355. SUTOR, D.J. & WOOLEY,S.E. (1974) The organic matrix of gallstones. Gut, 15,487-491. TEAGUE,R.H., FRASER,D. & CLAMP,J.R. (1973) Changes in monosaccharide content of mucous glycoproteins in ulcerative colitis. British Medical Journal, ii. 645-646. WOMACK,N.A. (1971) The development of gallstones. Surgery, Gynecology and Obstetrics, 133,937-945. WOMACK,N.A., ZEPPA, R. & IRVIN,G.L., 111 (1963) The anatomy of gallstones. Annals of Surgety, 157,670-686.
