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Diet, butyric acid and differentiation of gastrointestinal tract tumours
Medical
Hypotheses
18:
113-118.
1985
DIET, BUTYRIC ACID AND DIFFERENTIATION TRACT TUMOURS J. R. Jass, Imperial Hospital, City Road,
OF GASTROINTESTINAL
Cancer Research Fund Colorectal London EClV 2PS, UK
Unit,
St Mark’s
ABSTRACT Butyric acid has two contrasting functional roles. As a product of fermentation within the human colon, it serves as the most important energy source for It also promotes the differentiation of cultured normal colorectal epithelium. malignant cells. A switch from aerobic to anaerobic metabolism accompanies neoplastic transformation in the colorectum. The separate functional roles for n-butyrate may reflect the different metabolic activities of normal and neoplastic tissues. Relatively low intracolonic levels of n-butyrate are associated with a low fibre diet. Deficiency of n -butyrate, coupled to the increased energy requirements of neoplastic tissues, may promote the switch to anaerobic metabolism. The presence of naturally occurring differentiating agents, such as n-butyrate, may modify the patterns of growth and differentiation of gastrointestinal turnours, INTRODUCTION Butyric acid, a short chain fatty acid, has been the subject of two unrelated lines of enquiry. First, its properties as a differentiating agent have been investigated in detail. Second, its normal physiological role within the gut has been explored. Workers concerned with tumour differentiation have considered the positive effects of butyrate on cultured malignant cells. On the other hand, the realisation that n-butyrate is normally the most important source of energy for colorectal epithelium, has invited investigation of the possible pathological consequences of butyrate deficiency. It is therefore necessary to explain why normal colorectal epithelial cells employ butyrate as an energy source whereas malignant colorectal cell lines are sensitive to the differentiating properties of this substance. 113
The role of butyric acid first as a tumour differentiating agent and second as a nutrient within the colonic microenvironment, will be briefly reviewed. An attempt will be made to construct a metabolic link between these two lines of inquiry and thereafter to construct a hypothesis which explains the varied patterns of differentiation characterising tumours of the gastrointestinal tract. BUTYRIC
ACID AS A DIFFERENTIATING
AGENT
Sodium butyrate is one of several agents that has been shown to promote the in vitro differentiation of malignant colorectal cell lines. The expression of a mature phenotype depends upon the availability of chromosomal DNA for RNA transcription. Histones normally render DNA inactive. The expression of genes is achieved by the interruption of histone-DNA interactions. Histones are suitably modified for this purpose by a process of hyperacetylation. Sodium butyrate causes histone hyperacetylation by inhibiting histone deacetylase (1, 2). HeLa cells grown in the presence of n-butyrate show nuclear changes, including a reduction in the amount of condensed hetero chromatin and conversely an increase in the proportion of metabolically active euchromatin. This signal of cellular differentiation probably represents the morphological counterpart of the histone modifications (3). Differentiation induced by n -butyrate is usually reversible, but exceptions have been recorded (4). Multiple n -butyrate induced changes have been documented in various cultured cell lines. The changes are of two principal types. The first is the relative loss of tissue culture characteristics associated with malignant transformation. This includes reduction in growth rate (5, 6), suppression of anchorage independent growth (5) and decreased efficiency of cloning (6). The second is the expression of new phenotypes (5-12). These may not necessarily reflect the normal, adult counterpart of the malignant cell. Indeed, some alterations may include the synthesis of products associated with heterologous fetal tissues (7 -12). Thus n butyrate will promote cellular differentiation in vitro, but at the same time lead to the expression of inappropriate genes. The biological significance of such inappropriate gene products is unclear. BUTYRIC
ACID AS A PRODUCT
OF FERMENTATION
Short chain fatty acids (SCFA), including butyric acid, are normally produced in the colon through the breakdown of carbohydrate by anaerobic bacteria. This process, termed fermentation, takes place mainly in the caecum and right colon in man. Most of the substrate is derived from undigested plant cell walls (tflbre’). Other less important sources are the small amounts of starch which have not been digested and absorbed in the small bowel and mucus secreted by colonic epithelium. The amount of carbohydrate fermented in the human colon each day is probably not less than 30 g and may reach several times that amount in populations where the
114
diet consists largely of starchy foods, with little meat and dairy produce (13). SCFA have at least two important physiological roles, first as anions in fluid and electrolyte balance (14) and secondly as the most important source of energy for colorectal epithelial cells (15). HYPOTHESIS It is necessary to explain how n-butyrate may serve as both a differentiating agent and a respiratory fuel. These contrasting roles are evidenced under Normal colonocytes will rapidly metabolise quite different circumstances. n-butyrate to ketones and carbon dioxide in vitro (15). This presumably will prevent the intracytoplasmic accumulation of n butyrate. Under pathological conditions, the metabolism of n-butyrate may be blocked. In colonocytes from patients with ulcerative colitis, butyrate oxidation is impaired, whereas glucose oxidation is increased (16). Similarly, ln carcinoma of the colon and rectum, the activities of glycolytic enzymes, pentose shunt enzymes and diaphorases are increased, whereas mitochondrial enzymes show reduced activity (17). This shift from aerobic to anaerobic metabolism might interfere with the oxidation of n -butyrate, which would therefore accumulate within epithelial cell cytoplasm. This might explain the sensitivity of malignant cells to the differentiating effects of n -butyrate. Both ulcerative colitis and carcinoma are diseases with a predilection for the left colon. Interestingly, this is the region of the colon most dependent upon n-butyrate as a respiratory fuel (15). Ulcerative colitis and carcinoma are more common in populations who take diets in which meat and dairy produce predominate over starch and ‘fllre’. There will be relatively low intracolonic levels of n-butyrate within such populations. These observations invite two suggestions, which may or may not be related. First, both ulcerative colitis and colorectal carcinoma may be exacerbated by n -butyrate deficiency. Secondly, the switch from aerobic to anaerobic metabolism may reflect cellular adaptation to n-butyrate depletion. The majority of colorectal cancers arise in pre -existing adenomas. Adenomas will be especially dependent upon a supply of n-butyrate as judged by their greatly increased surface area and the elevated mitochondrial enzyme activity of their lining epithelium (17). With increasing size, epithelial dysplasia and villosity, adenomas show a switch from aerobic to anaerobic metabolism (17). Thus, the acquired sensitivity to n-butyrate as a differentiating agent may be brought about by a metabolic switch within a precancerous lesion. This switch could reflect both an increased requirement for energy and the reduced availability of n -butyrate. However, even in Western colons, the levels of n-butyrate are likely to be comparable to the concentrations used in in vitro differentiation experiments (5). It is suggested that the presence of butyric acid and the acquired sensitivity to its differentiating effects account for the fact that colorectal carcinoma is relatively well differentiated.
115
Hyperplastic (metaplastic) polyps may represent non -neoplastic markers of n -butyrate depletion. They are usually found in n -butyrate dependent areas such as the left colon and rectum (18). They are uncommon in Japanese who take a diet which is high in starch and low in meat and dairy produce (19). However, they are common in Japanese who adopt a Western diet (19). Finally they show evidence of metabolic exhaustion including the incomplete synthesis of secretory glycoproteins (20, 21), diminished activity of mitochondrial enzymes (22) and a failure of the IgA secretory mechanism (23). It is interesting to speculate on the possibility of the stomach as a site of fermentation in man. Bacteria do not colonise the normal stomach. Hypochlorhydria occurs in stomachs showing atrophic gastritis and intestinal metaplasia. Such stomachs may be colonised by anaerobic bacteria including bacteroides and clostridia (24). It is in stomachs showing intestinal metaplasia that well differentiated (intestinal -type) adenocarcinomas arise (25). Populations with a high incidence of intestinal -type carcinoma consume large amounts of fermentable food e.g. rice in Japan. CONCLUSION It is suggested that a metabolic switch within carcinoma of the colorectum accounts for the reduced dependence upon n-butyrate as a respiratory fuel and the increased susceptibility to its differentiating effects. The role of butyric acid on the differentiation of gastrointestinal tumours would be most easily tested by looking for evidence of fermentation and butyric acid production in the stomachs of patients with hypochlorhydria. This hypothesis could be extended to a wider area of tumour pathology by suggesting that a particular pattern of differentiation is determined by an acquired sensitivity to naturally occurring differentiating agents. ACKNOWLEDGEMENTS I am grateful to Dr M J Hill, Dr V Murday and Mr J M A Northover helpful discussion, and to Jill Grimsey for typing the manuscript.
for their
REFERENCES 1.
Sealy L, Chalkley R. The effect of sodium modification. Cell 14: 115, 1978.
butyrate
2.
Buffa LC, Vidali G, Mann RS, Allfrey VG. deacetylation in vivo and in vitro by sodium 253: 3364, 1978.
Suppression of histone butyrate. J Biol Chem
3.
Tralka TS, Rabson AS, Thorgeirsson HP, Tsent JS. Sodium n -butyrate causes reversible decrease in condensed chromatin clumps in HeLa cells (40593). Proc Sot Exp Biol Med 161: 534,
116
on histone
1979.
4.
Emergence of permanently differentiated Augeron C, Laboisse CL. cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res 44: 3961, 1984
5.
Kim YS, Tsao D, Siddiqui B, Whitehead JS, Arnstein P, Bennett J, Hicks J. Effects of sodium butyrate and dimethylsulphoxide on biochemical properties of human colon cancer cells. Cancer 45: 1185, 1980
6.
Dexter DL, Lev R, McKendall GR, Mitchell P, Calabres P. Sodium butyrate -induced alteration of growth properties and glycogen levels in cultured human colon carcinoma cells. Histochem J 16: 137, 1984.
7.
Altenburg BC, Via DP, Steiner SH. Modification of the phenotype of murine sarcoma virus -transformed cells by sodium butyrate. Exptl Cell Res 102: 233, 1976.
8.
Tsao D, Morita A, Bella A, Luu P, Kim Y. Differential effects of sodium butyrate, dimethyl sulphoxide and retinoic acid on membrane associated antigen, enzymes and glycoproteins of human rectal adenocarcinoma cells. Cancer Res 42: 1052, 1982.
-
9.
Nozawa S, Engvall E, Kano S, Kurihara S, Fishman WH. Sodium butyrate produces concordant expression of ‘early placental’ alkaline phosphatase, pregnancy -specific beta -glycoprotein and human chorionic gonadotrophin beta -subunit in a newly established uterine cervical cancer cell line (SKG-III a). Int J Cancer 32: 267, 1983.
10.
Morita A, Tsao D, Kim YS. Effect of sodium phosphatase in HRT-18, a human cancer line. 4540, 1983.
11.
Dexter DL, Konieczky SF, Lawrence JB, Shaffer M, Mitchell P, Coleman JR, Induction by butyrate of differentiated properties in cloned murine rhabdomyosarcoma cells. Differentiation 18: 115, 1981.
12.
Testa U, Henri A, Bettaieb A, Tlteux M, Vainchenker W, Tonthat Docklear MC, Rochant H. Regulation of i and I-antigen expression in the K 562 cell line. Cancer Res 42: 4694, 1982.
13.
Cummings JH. Colonic absorption: the importance of short chain fatty acids in man. p 371 in Basic Science in Gastroenterology. (JM Poiak, SR Bloom, NA Wright, AG Butler, eds) Royal Postgraduate Medical School: Glaxon Group Research Ltd, 1984.
117
butyrate Cancer
on alkaline Res 42:
H,
14.
Roediger WEW, Moore A. Effect of short -chain fatty acid on sodium absorption in isolated human colon perfused through the vascular bed. Dig Dls Sci 26: 100, 1981.
15.
Roediger WEW. Anaerobic bacteria of the colonic mucosa in man. Gut
16.
Roediger WEW. energy deficiency
17.
Jass JR, Strudley I, Faludy metaplasia and dysplasia. 110, 1984.
support the metabolic 21: 793, 1980.
The colonic epithelium in ulcerative disease? Lancet 2: 712, 1984.
welfare
colitis:
J. Histochemistry of epithelial Stand J Gastroenterol 19 (suppl
an
104):
18.
Arthur JF. Structure and significance of metaplastic rectal mucosa. J Clin Path01 21: 735, 1968.
nodules
in
19.
Stemmermann GN, Yatani R. Dlverticulosls and polyps of the large intestine, A necropsy study of Hawaii Japanese. Cancer 31: 1260, 1972.
20.
Jass JR, Filipe A morphological the colorectum.
21.
Boland CR, Montgomery CK, Klm YS. A cancer -associated alteration in benign colonic polyps. Gastroenterology 82: 1982.
22.
Czernobilsky of the colon.
23.
Jass JR, Faludy J. Immunohistochemical demonstration of IgA and secretory component in relation to epithelial cell differentiation in normal colorectal mucosa and metaplastic polyp. A semiquantitative study. Histochem J 17: 1985 (ln press)
24.
Borriello SP. Bacteria and gastrointestinal secretion and motility. p 397 in Basic Science in Castroenterology. (JM Polak, SR Bloom, NA Wright, AG Butler, eds) Royal Postgraduate Medical School: Glaxo Group Research Ltd, 1984.
25.
Sipponen intestinal 1983.
MI, Abbas S, Falcon CAJ, Wilson Y, Love11 D. and histochemical study of metaplastic polyps of Cancer 53: 510, 1984
B, Tsou KC. Cancer 21:
Adenocarcinoma, 165, 1968.
P, Kekki M, Siurala M. Atrophic metaplasia in gastric carcinoma.
118
adenomas
mucin 664,
and polyps
chronic gastritis and C ancer 52: 1062
Hypotheses
18:
113-118.
1985
DIET, BUTYRIC ACID AND DIFFERENTIATION TRACT TUMOURS J. R. Jass, Imperial Hospital, City Road,
OF GASTROINTESTINAL
Cancer Research Fund Colorectal London EClV 2PS, UK
Unit,
St Mark’s
ABSTRACT Butyric acid has two contrasting functional roles. As a product of fermentation within the human colon, it serves as the most important energy source for It also promotes the differentiation of cultured normal colorectal epithelium. malignant cells. A switch from aerobic to anaerobic metabolism accompanies neoplastic transformation in the colorectum. The separate functional roles for n-butyrate may reflect the different metabolic activities of normal and neoplastic tissues. Relatively low intracolonic levels of n-butyrate are associated with a low fibre diet. Deficiency of n -butyrate, coupled to the increased energy requirements of neoplastic tissues, may promote the switch to anaerobic metabolism. The presence of naturally occurring differentiating agents, such as n-butyrate, may modify the patterns of growth and differentiation of gastrointestinal turnours, INTRODUCTION Butyric acid, a short chain fatty acid, has been the subject of two unrelated lines of enquiry. First, its properties as a differentiating agent have been investigated in detail. Second, its normal physiological role within the gut has been explored. Workers concerned with tumour differentiation have considered the positive effects of butyrate on cultured malignant cells. On the other hand, the realisation that n-butyrate is normally the most important source of energy for colorectal epithelium, has invited investigation of the possible pathological consequences of butyrate deficiency. It is therefore necessary to explain why normal colorectal epithelial cells employ butyrate as an energy source whereas malignant colorectal cell lines are sensitive to the differentiating properties of this substance. 113
The role of butyric acid first as a tumour differentiating agent and second as a nutrient within the colonic microenvironment, will be briefly reviewed. An attempt will be made to construct a metabolic link between these two lines of inquiry and thereafter to construct a hypothesis which explains the varied patterns of differentiation characterising tumours of the gastrointestinal tract. BUTYRIC
ACID AS A DIFFERENTIATING
AGENT
Sodium butyrate is one of several agents that has been shown to promote the in vitro differentiation of malignant colorectal cell lines. The expression of a mature phenotype depends upon the availability of chromosomal DNA for RNA transcription. Histones normally render DNA inactive. The expression of genes is achieved by the interruption of histone-DNA interactions. Histones are suitably modified for this purpose by a process of hyperacetylation. Sodium butyrate causes histone hyperacetylation by inhibiting histone deacetylase (1, 2). HeLa cells grown in the presence of n-butyrate show nuclear changes, including a reduction in the amount of condensed hetero chromatin and conversely an increase in the proportion of metabolically active euchromatin. This signal of cellular differentiation probably represents the morphological counterpart of the histone modifications (3). Differentiation induced by n -butyrate is usually reversible, but exceptions have been recorded (4). Multiple n -butyrate induced changes have been documented in various cultured cell lines. The changes are of two principal types. The first is the relative loss of tissue culture characteristics associated with malignant transformation. This includes reduction in growth rate (5, 6), suppression of anchorage independent growth (5) and decreased efficiency of cloning (6). The second is the expression of new phenotypes (5-12). These may not necessarily reflect the normal, adult counterpart of the malignant cell. Indeed, some alterations may include the synthesis of products associated with heterologous fetal tissues (7 -12). Thus n butyrate will promote cellular differentiation in vitro, but at the same time lead to the expression of inappropriate genes. The biological significance of such inappropriate gene products is unclear. BUTYRIC
ACID AS A PRODUCT
OF FERMENTATION
Short chain fatty acids (SCFA), including butyric acid, are normally produced in the colon through the breakdown of carbohydrate by anaerobic bacteria. This process, termed fermentation, takes place mainly in the caecum and right colon in man. Most of the substrate is derived from undigested plant cell walls (tflbre’). Other less important sources are the small amounts of starch which have not been digested and absorbed in the small bowel and mucus secreted by colonic epithelium. The amount of carbohydrate fermented in the human colon each day is probably not less than 30 g and may reach several times that amount in populations where the
114
diet consists largely of starchy foods, with little meat and dairy produce (13). SCFA have at least two important physiological roles, first as anions in fluid and electrolyte balance (14) and secondly as the most important source of energy for colorectal epithelial cells (15). HYPOTHESIS It is necessary to explain how n-butyrate may serve as both a differentiating agent and a respiratory fuel. These contrasting roles are evidenced under Normal colonocytes will rapidly metabolise quite different circumstances. n-butyrate to ketones and carbon dioxide in vitro (15). This presumably will prevent the intracytoplasmic accumulation of n butyrate. Under pathological conditions, the metabolism of n-butyrate may be blocked. In colonocytes from patients with ulcerative colitis, butyrate oxidation is impaired, whereas glucose oxidation is increased (16). Similarly, ln carcinoma of the colon and rectum, the activities of glycolytic enzymes, pentose shunt enzymes and diaphorases are increased, whereas mitochondrial enzymes show reduced activity (17). This shift from aerobic to anaerobic metabolism might interfere with the oxidation of n -butyrate, which would therefore accumulate within epithelial cell cytoplasm. This might explain the sensitivity of malignant cells to the differentiating effects of n -butyrate. Both ulcerative colitis and carcinoma are diseases with a predilection for the left colon. Interestingly, this is the region of the colon most dependent upon n-butyrate as a respiratory fuel (15). Ulcerative colitis and carcinoma are more common in populations who take diets in which meat and dairy produce predominate over starch and ‘fllre’. There will be relatively low intracolonic levels of n-butyrate within such populations. These observations invite two suggestions, which may or may not be related. First, both ulcerative colitis and colorectal carcinoma may be exacerbated by n -butyrate deficiency. Secondly, the switch from aerobic to anaerobic metabolism may reflect cellular adaptation to n-butyrate depletion. The majority of colorectal cancers arise in pre -existing adenomas. Adenomas will be especially dependent upon a supply of n-butyrate as judged by their greatly increased surface area and the elevated mitochondrial enzyme activity of their lining epithelium (17). With increasing size, epithelial dysplasia and villosity, adenomas show a switch from aerobic to anaerobic metabolism (17). Thus, the acquired sensitivity to n-butyrate as a differentiating agent may be brought about by a metabolic switch within a precancerous lesion. This switch could reflect both an increased requirement for energy and the reduced availability of n -butyrate. However, even in Western colons, the levels of n-butyrate are likely to be comparable to the concentrations used in in vitro differentiation experiments (5). It is suggested that the presence of butyric acid and the acquired sensitivity to its differentiating effects account for the fact that colorectal carcinoma is relatively well differentiated.
115
Hyperplastic (metaplastic) polyps may represent non -neoplastic markers of n -butyrate depletion. They are usually found in n -butyrate dependent areas such as the left colon and rectum (18). They are uncommon in Japanese who take a diet which is high in starch and low in meat and dairy produce (19). However, they are common in Japanese who adopt a Western diet (19). Finally they show evidence of metabolic exhaustion including the incomplete synthesis of secretory glycoproteins (20, 21), diminished activity of mitochondrial enzymes (22) and a failure of the IgA secretory mechanism (23). It is interesting to speculate on the possibility of the stomach as a site of fermentation in man. Bacteria do not colonise the normal stomach. Hypochlorhydria occurs in stomachs showing atrophic gastritis and intestinal metaplasia. Such stomachs may be colonised by anaerobic bacteria including bacteroides and clostridia (24). It is in stomachs showing intestinal metaplasia that well differentiated (intestinal -type) adenocarcinomas arise (25). Populations with a high incidence of intestinal -type carcinoma consume large amounts of fermentable food e.g. rice in Japan. CONCLUSION It is suggested that a metabolic switch within carcinoma of the colorectum accounts for the reduced dependence upon n-butyrate as a respiratory fuel and the increased susceptibility to its differentiating effects. The role of butyric acid on the differentiation of gastrointestinal tumours would be most easily tested by looking for evidence of fermentation and butyric acid production in the stomachs of patients with hypochlorhydria. This hypothesis could be extended to a wider area of tumour pathology by suggesting that a particular pattern of differentiation is determined by an acquired sensitivity to naturally occurring differentiating agents. ACKNOWLEDGEMENTS I am grateful to Dr M J Hill, Dr V Murday and Mr J M A Northover helpful discussion, and to Jill Grimsey for typing the manuscript.
for their
REFERENCES 1.
Sealy L, Chalkley R. The effect of sodium modification. Cell 14: 115, 1978.
butyrate
2.
Buffa LC, Vidali G, Mann RS, Allfrey VG. deacetylation in vivo and in vitro by sodium 253: 3364, 1978.
Suppression of histone butyrate. J Biol Chem
3.
Tralka TS, Rabson AS, Thorgeirsson HP, Tsent JS. Sodium n -butyrate causes reversible decrease in condensed chromatin clumps in HeLa cells (40593). Proc Sot Exp Biol Med 161: 534,
116
on histone
1979.
4.
Emergence of permanently differentiated Augeron C, Laboisse CL. cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res 44: 3961, 1984
5.
Kim YS, Tsao D, Siddiqui B, Whitehead JS, Arnstein P, Bennett J, Hicks J. Effects of sodium butyrate and dimethylsulphoxide on biochemical properties of human colon cancer cells. Cancer 45: 1185, 1980
6.
Dexter DL, Lev R, McKendall GR, Mitchell P, Calabres P. Sodium butyrate -induced alteration of growth properties and glycogen levels in cultured human colon carcinoma cells. Histochem J 16: 137, 1984.
7.
Altenburg BC, Via DP, Steiner SH. Modification of the phenotype of murine sarcoma virus -transformed cells by sodium butyrate. Exptl Cell Res 102: 233, 1976.
8.
Tsao D, Morita A, Bella A, Luu P, Kim Y. Differential effects of sodium butyrate, dimethyl sulphoxide and retinoic acid on membrane associated antigen, enzymes and glycoproteins of human rectal adenocarcinoma cells. Cancer Res 42: 1052, 1982.
-
9.
Nozawa S, Engvall E, Kano S, Kurihara S, Fishman WH. Sodium butyrate produces concordant expression of ‘early placental’ alkaline phosphatase, pregnancy -specific beta -glycoprotein and human chorionic gonadotrophin beta -subunit in a newly established uterine cervical cancer cell line (SKG-III a). Int J Cancer 32: 267, 1983.
10.
Morita A, Tsao D, Kim YS. Effect of sodium phosphatase in HRT-18, a human cancer line. 4540, 1983.
11.
Dexter DL, Konieczky SF, Lawrence JB, Shaffer M, Mitchell P, Coleman JR, Induction by butyrate of differentiated properties in cloned murine rhabdomyosarcoma cells. Differentiation 18: 115, 1981.
12.
Testa U, Henri A, Bettaieb A, Tlteux M, Vainchenker W, Tonthat Docklear MC, Rochant H. Regulation of i and I-antigen expression in the K 562 cell line. Cancer Res 42: 4694, 1982.
13.
Cummings JH. Colonic absorption: the importance of short chain fatty acids in man. p 371 in Basic Science in Gastroenterology. (JM Poiak, SR Bloom, NA Wright, AG Butler, eds) Royal Postgraduate Medical School: Glaxon Group Research Ltd, 1984.
117
butyrate Cancer
on alkaline Res 42:
H,
14.
Roediger WEW, Moore A. Effect of short -chain fatty acid on sodium absorption in isolated human colon perfused through the vascular bed. Dig Dls Sci 26: 100, 1981.
15.
Roediger WEW. Anaerobic bacteria of the colonic mucosa in man. Gut
16.
Roediger WEW. energy deficiency
17.
Jass JR, Strudley I, Faludy metaplasia and dysplasia. 110, 1984.
support the metabolic 21: 793, 1980.
The colonic epithelium in ulcerative disease? Lancet 2: 712, 1984.
welfare
colitis:
J. Histochemistry of epithelial Stand J Gastroenterol 19 (suppl
an
104):
18.
Arthur JF. Structure and significance of metaplastic rectal mucosa. J Clin Path01 21: 735, 1968.
nodules
in
19.
Stemmermann GN, Yatani R. Dlverticulosls and polyps of the large intestine, A necropsy study of Hawaii Japanese. Cancer 31: 1260, 1972.
20.
Jass JR, Filipe A morphological the colorectum.
21.
Boland CR, Montgomery CK, Klm YS. A cancer -associated alteration in benign colonic polyps. Gastroenterology 82: 1982.
22.
Czernobilsky of the colon.
23.
Jass JR, Faludy J. Immunohistochemical demonstration of IgA and secretory component in relation to epithelial cell differentiation in normal colorectal mucosa and metaplastic polyp. A semiquantitative study. Histochem J 17: 1985 (ln press)
24.
Borriello SP. Bacteria and gastrointestinal secretion and motility. p 397 in Basic Science in Castroenterology. (JM Polak, SR Bloom, NA Wright, AG Butler, eds) Royal Postgraduate Medical School: Glaxo Group Research Ltd, 1984.
25.
Sipponen intestinal 1983.
MI, Abbas S, Falcon CAJ, Wilson Y, Love11 D. and histochemical study of metaplastic polyps of Cancer 53: 510, 1984
B, Tsou KC. Cancer 21:
Adenocarcinoma, 165, 1968.
P, Kekki M, Siurala M. Atrophic metaplasia in gastric carcinoma.
118
adenomas
mucin 664,
and polyps
chronic gastritis and C ancer 52: 1062