- Email: [email protected]
BILE ACIDS IN A HUMAN MODEL OF COLORECTAL NEOPLASIA
BILE ACIDS IN A HUMAN MODEL OF COLORECTAL NEOPLASIA DM Bradburn' IR Rowland2, JC Mathers], A Gunn4, J Burn5, IDA Johnston' Departments of Surgery' , Biological and Nutritional Sciences' , and Human Genetics' Newcastle University, Newcastle upon Tyne. NE2 4HH Ashington Hospital', West View, Ashington, Northumberland and BIBRA' , Woodmansterne Rd, Carshalton, Surrey SM5 4DS
INTRODUCTION Familial Adenomatous Polyposis (FAP) is an autosomal dominant genetic condition characterised by the formation of multiple premalignant adenomatous colorectal and duodenal polyps. Affected individuals usually develop colonic polyps in their late teens and one or more of these will undergo malignant transformation within 10-15 years'. FAP is a useful model of sporadic colonic cancer (CC). The histological appearance and natural history of the colonic adenomas in FAP are identical to the premalignant lesions in sporadic CC2. The genetic defect in FAP (on chromosome 5q22) is the earliest changes in the genetic events of the adenoma carcinoma sequence and is thought to cause a hyperproliferative epithelium'. It therefore appears that the propensity to colonic cancer in FAP occurs because they are already "one step along the way" to malignant transformation. It is not unreasonable to suggest that the factors determining the subsequent genetic changes in the adenoma carcinoma sequence are the same in both FAP and CC. Epidemiological evidence has stressed the importance of diet as a risk factor for colonic neoplasia. It has been proposed that dietary complex carbohydrates are protective whilst a high fat intake has an adverse effect4. Fat increases faecal bile acid output, and bile acids are known promoters of CC in animal experiments5. In this context, it is interesting to note that abnormal duodenup and faecal7 bile acids have been noted in FAP. Changes in bile acid metabolism in FAP may therefore be additional to the genetic defect in stimulating the change from normal mucosa to carcinoma. The pattern of bile acid entering the colon in FAP is not known and a fuller understanding of the steroids entering and leaving the colon in this subject group may allow dietary strategies to be developed to delay the progression of polyposis.
1. To determine the amounts and patterns of bile acids arriving at and leaving the
Food and Cancer Prevention: Chemical and Biological Aspects
354
colon in FAP. 2. To examine the effect of colonic polyps on bacterial bile acid metabolism independent of any genetic influence. MATERIALS AND METHODS Jleostomv studies. Twelve ileostomists were studied, 6 whose surgery was for FAP, and 6 whose surgery was for ulcerative colitis (UC, controls). No subject had taken antibiotics for 6 weeks prior to the study. Haematological liver function tests were normal, no patient had gallstones or a cholecystectomy and ileal resection was less than 7cm in all cases. Subject details are presented in Table I.
Table I Ileostomy study, subject details.
FAP
uc
P3
aee bears)
2 male 4 female
3 male 3 female
wv mass index Otelmz)
duration of ileostomv beat$
mean 43.6 range 27-67
mean 26.7 range 18.0-43.9
mean 8.3 range 5-13
mean 50 range 25-80
mean 24.3 range 21.0-30.5
mean 9 range 1-28
Volunteers were admitted to a metabolic unit after an overnight fast (fluids allowed). A known, controlled diet was consumed for 24 hours and ileal effluent was collected 2 hourly and immediately snap frozen. Samples were thawed, pooled, homogenised, freeze dried, and sterols were extracted by established methods*. Individual bile acids were measured by HPLC using dexamethasone as an internal standard (25cm x 4.6mm Phase Sep [UK] ODs5 column, eluent [650ml ethanol, 30ml tetrahydrofuran, 320ml water, 3g NaH2P04, 0.29g tetrabutyl ammonium chloride per litre], pH 5.4, flow rate ImUminute, UV detector [Gilsonl 205nm). Faecal Studies. Using a combination of DNA markers, ophthalmoscopy and pedigree analysis, individuals undergoing endoscopic screening for FAP were divided into 3 groups as outlined in Table I1 '. No dietary restrictions were imposed and twenty four hour faecal collections were made from all subjects. After homogenising, pH was measured and samples were freeze dried. Bile acids were measured by GLCR.
355
Bile Acidr in a Human Model of Colorectal Neoplasia
Table I1 Faecal studies, subject details.
number
risk of
caniaee
a s -
control
7
< 1%
30.1
9 - 53
FAP gene carriers with no polyps (GCNP)
9
> 98%
25.2
7 - 53
FAP gene carriers with polyps (GCWP)
8
100%
19.9
11-32
statistics, Ileostomv data, Differences between groups were examined by ANOVA. Age sex and body mass index were examined as covariate factors. Faecal data. Data was not normally distributed so were examined by the Mann Whitney test. The null hypotheses tested were:1. There is no difference between gene camers with and without polyps. 2. There is no difference between gene camers and normal controls. RESULTS Abbreviations:- CA = cholic acid, CDCA = chenodeoxycholic acid, prefix T = taurine conjugate, prefix G = glycine conjugate. SED = standard error of the difference of the mean. There were no differences in ileal wet and dry weights between groups. There were significantly lower 24 hour outputs of TCDCA (p=O.O47), total taurine amidates (p=0.092) and total CDCA amidates (p=O.O88) in subjects with FAP. Complete results are presented in Table 111. There were no differences in the 24 hour faecal outputs of neutral sterols but the 24 hour output of deoxcholate (p=O.O2). lithocholate (p=O.O5) and total bile acids (p=O.O2) were significantly lower in FAP gene carriers compared to controls. There were no differences comparing FAP gene carriers with and without polyps. Complete results are presented in Table IV. DISCUSSION The current data agree with the range of ileal and faecal sterol output which may be calculated from the results of other workers’ lo. In addition the 24 hour ileal outputs are slightly higher than the faecal outputs consistent with the small amount of bile acid absorption from the colon. Increased molar ratios of duodenal chenodeoxycholate amidates have been noted in duodenal and gallbladder bile in FAP6. If this is reflected in ileal fluid, colonic polyp formation may be influenced. In contrast there were decreased ileal
Food and Cancer Prevention: Chemical and Biological Aspects
356
Table Ill Ileostorny 24 hour wet and dry weights and sterol outputs.
u c
FAP
La?w
mean
wet weight (g,
409
298-600
452
300-686
78.8
CS,
46
39 - 48
50
28-64
7.0
total bile acids
0.730
0.308-1.527
0.381
0.119-0.849
0.2483
TCA
0.068
0.004-0.137
0.056
0.017-0.117
0.0299
dry weight
SED
ileal oumut (mmoVdavl
TCDCA
0.157
0.052-0.288
0.065
0.0294.172
0.0402
total taurine amidates
0.225
0.065-0.306
0.122
0.035-0.271
0.0552
GCA
0.190
0.025-0.449
0.115
0.034-0.235
0.0884
GCDCA
0.310
0.012-0.772
0.140
0.053-0.412
0.1307
total glycine amidates
0.500
0.017-1.221
0.261
0.084-0.638
0.2131
total CA amidates
0.259
0.0080.583
0.172
0.052-0.334
0.1172
total CDCA amidates
0.467
0.140-0.944
0.206
0.071-0.516
0.1363
Table IV 24 hour faecal wet and dry weights and sterol outputs. no Dolvos
control
with oolvos
~~~~~~
wet
wt (g)
dry wt (g,
PH
117
6 5 - 341
89
53 - 260
79
5 8 - 210
50
1 7 - 111
26
16-51
20
1 4 - 38
6.37
6.11 - 6.66
6.51
6.07 - 7.10
6.24
5.9 - 7.04
171
701 - 1014
62
18-466
112
9 - 508
bile acia
0 lithocholate deoxycholate
198
138 - 633
37
15 - 366
136
65 - 371
total bile acids
406
219- 1647
147
3 2 - 747
288
112- 879
Bile Acids in a Human Model of ColorectafNeoplasia
357
outputs of taurochenodeoxycholate and total taurine and chenodeoxycholate amidates by FAP subjects in the present studies. This suggests that there is increased recirculation, rather than increased formation of bile acids. Duodenal polyposis in FAP clusters around the ampulla of Vater, implying that bile may be an aetiological factor". Increased recirculation of bile acids in FAP may therefore promote duodenal polyposis. Increased faecal concentrations of primary bile acids have been noted in FAP7,but we are unaware of other published data for total outputs. We could not detect any primary bile acids in faeces, but the decreased total and individual secondary bile acid outputs in FAP agrees with the results from the ileostomy studies. It is interesting to note that there was not a specific decrease in the output of lithwholate, the chenodeoxycholate metabolite.
In summary, the data d o not suggest a primary role for bile acids in colonic polyposis in FAP, but suggests that increased recirculation may augment duodenal polyposis. Duodenal cancer developing from polyps is the commonest extracolonic malignant cause of death in FAP'. Dietary bile acid ligands, such as complex carbohydrates decrease the small intestinal absorption of sterols" and decrease bile acid recirculation. Further study of the potential value of dietary intervention with such materials in attempts to decrease duodenal cancer in FAP is warranted.
1. 2. 3. 4. 5. 6.
M. Rhodes, D.M. Bradburn,
m,
78,
7. 8. 9. 10. 11.
a
321
A.L. Watne, H.L. Lai, T. Mance, S. Core, J S u a , 1976, 131, 42 E. Bailey, A.F.G. Brooks, J C h r o a , 1987, 421. 21 J. Bum, P. Chapman, J. Delhanty. C. Wood.F. Lalloo. M.B. Gonzales, K. Tsioupara, W. Church, M. Rhodes, A. GUM, , -J 1991. 28,
a
I. Bosaeus, B. Sandstrom, H. Andersson,
891
J G-
1 1986, 21,
A.D. Spigelman, C. Crofton-Sleigh, S. Venitt, R.K.S. Phillips, Br J S w . 1990, 77,
12.
W.1992, 33, U
H.J.R. Bussey, Path Ann& , 1979, 14, E.R. Fearon. B. Vogelstein, 1990, 61, S.A. Bingham, J Rov S w Me& 1990, 83, 4 2 B.S.Reddy, C a n er Res, 1981, 41, UlQ A.D. Spigelman, R. W. Owen, M.J. Hill, R.K.S. Phillips, 3t J Sure, 1991,
a
I. Bosaeus, N.G.Carlsson, A.S. Sandberg, H. Andersson, Bum Nutr: Clin
m. 1986, 40,
INTRODUCTION Familial Adenomatous Polyposis (FAP) is an autosomal dominant genetic condition characterised by the formation of multiple premalignant adenomatous colorectal and duodenal polyps. Affected individuals usually develop colonic polyps in their late teens and one or more of these will undergo malignant transformation within 10-15 years'. FAP is a useful model of sporadic colonic cancer (CC). The histological appearance and natural history of the colonic adenomas in FAP are identical to the premalignant lesions in sporadic CC2. The genetic defect in FAP (on chromosome 5q22) is the earliest changes in the genetic events of the adenoma carcinoma sequence and is thought to cause a hyperproliferative epithelium'. It therefore appears that the propensity to colonic cancer in FAP occurs because they are already "one step along the way" to malignant transformation. It is not unreasonable to suggest that the factors determining the subsequent genetic changes in the adenoma carcinoma sequence are the same in both FAP and CC. Epidemiological evidence has stressed the importance of diet as a risk factor for colonic neoplasia. It has been proposed that dietary complex carbohydrates are protective whilst a high fat intake has an adverse effect4. Fat increases faecal bile acid output, and bile acids are known promoters of CC in animal experiments5. In this context, it is interesting to note that abnormal duodenup and faecal7 bile acids have been noted in FAP. Changes in bile acid metabolism in FAP may therefore be additional to the genetic defect in stimulating the change from normal mucosa to carcinoma. The pattern of bile acid entering the colon in FAP is not known and a fuller understanding of the steroids entering and leaving the colon in this subject group may allow dietary strategies to be developed to delay the progression of polyposis.
1. To determine the amounts and patterns of bile acids arriving at and leaving the
Food and Cancer Prevention: Chemical and Biological Aspects
354
colon in FAP. 2. To examine the effect of colonic polyps on bacterial bile acid metabolism independent of any genetic influence. MATERIALS AND METHODS Jleostomv studies. Twelve ileostomists were studied, 6 whose surgery was for FAP, and 6 whose surgery was for ulcerative colitis (UC, controls). No subject had taken antibiotics for 6 weeks prior to the study. Haematological liver function tests were normal, no patient had gallstones or a cholecystectomy and ileal resection was less than 7cm in all cases. Subject details are presented in Table I.
Table I Ileostomy study, subject details.
FAP
uc
P3
aee bears)
2 male 4 female
3 male 3 female
wv mass index Otelmz)
duration of ileostomv beat$
mean 43.6 range 27-67
mean 26.7 range 18.0-43.9
mean 8.3 range 5-13
mean 50 range 25-80
mean 24.3 range 21.0-30.5
mean 9 range 1-28
Volunteers were admitted to a metabolic unit after an overnight fast (fluids allowed). A known, controlled diet was consumed for 24 hours and ileal effluent was collected 2 hourly and immediately snap frozen. Samples were thawed, pooled, homogenised, freeze dried, and sterols were extracted by established methods*. Individual bile acids were measured by HPLC using dexamethasone as an internal standard (25cm x 4.6mm Phase Sep [UK] ODs5 column, eluent [650ml ethanol, 30ml tetrahydrofuran, 320ml water, 3g NaH2P04, 0.29g tetrabutyl ammonium chloride per litre], pH 5.4, flow rate ImUminute, UV detector [Gilsonl 205nm). Faecal Studies. Using a combination of DNA markers, ophthalmoscopy and pedigree analysis, individuals undergoing endoscopic screening for FAP were divided into 3 groups as outlined in Table I1 '. No dietary restrictions were imposed and twenty four hour faecal collections were made from all subjects. After homogenising, pH was measured and samples were freeze dried. Bile acids were measured by GLCR.
355
Bile Acidr in a Human Model of Colorectal Neoplasia
Table I1 Faecal studies, subject details.
number
risk of
caniaee
a s -
control
7
< 1%
30.1
9 - 53
FAP gene carriers with no polyps (GCNP)
9
> 98%
25.2
7 - 53
FAP gene carriers with polyps (GCWP)
8
100%
19.9
11-32
statistics, Ileostomv data, Differences between groups were examined by ANOVA. Age sex and body mass index were examined as covariate factors. Faecal data. Data was not normally distributed so were examined by the Mann Whitney test. The null hypotheses tested were:1. There is no difference between gene camers with and without polyps. 2. There is no difference between gene camers and normal controls. RESULTS Abbreviations:- CA = cholic acid, CDCA = chenodeoxycholic acid, prefix T = taurine conjugate, prefix G = glycine conjugate. SED = standard error of the difference of the mean. There were no differences in ileal wet and dry weights between groups. There were significantly lower 24 hour outputs of TCDCA (p=O.O47), total taurine amidates (p=0.092) and total CDCA amidates (p=O.O88) in subjects with FAP. Complete results are presented in Table 111. There were no differences in the 24 hour faecal outputs of neutral sterols but the 24 hour output of deoxcholate (p=O.O2). lithocholate (p=O.O5) and total bile acids (p=O.O2) were significantly lower in FAP gene carriers compared to controls. There were no differences comparing FAP gene carriers with and without polyps. Complete results are presented in Table IV. DISCUSSION The current data agree with the range of ileal and faecal sterol output which may be calculated from the results of other workers’ lo. In addition the 24 hour ileal outputs are slightly higher than the faecal outputs consistent with the small amount of bile acid absorption from the colon. Increased molar ratios of duodenal chenodeoxycholate amidates have been noted in duodenal and gallbladder bile in FAP6. If this is reflected in ileal fluid, colonic polyp formation may be influenced. In contrast there were decreased ileal
Food and Cancer Prevention: Chemical and Biological Aspects
356
Table Ill Ileostorny 24 hour wet and dry weights and sterol outputs.
u c
FAP
La?w
mean
wet weight (g,
409
298-600
452
300-686
78.8
CS,
46
39 - 48
50
28-64
7.0
total bile acids
0.730
0.308-1.527
0.381
0.119-0.849
0.2483
TCA
0.068
0.004-0.137
0.056
0.017-0.117
0.0299
dry weight
SED
ileal oumut (mmoVdavl
TCDCA
0.157
0.052-0.288
0.065
0.0294.172
0.0402
total taurine amidates
0.225
0.065-0.306
0.122
0.035-0.271
0.0552
GCA
0.190
0.025-0.449
0.115
0.034-0.235
0.0884
GCDCA
0.310
0.012-0.772
0.140
0.053-0.412
0.1307
total glycine amidates
0.500
0.017-1.221
0.261
0.084-0.638
0.2131
total CA amidates
0.259
0.0080.583
0.172
0.052-0.334
0.1172
total CDCA amidates
0.467
0.140-0.944
0.206
0.071-0.516
0.1363
Table IV 24 hour faecal wet and dry weights and sterol outputs. no Dolvos
control
with oolvos
~~~~~~
wet
wt (g)
dry wt (g,
PH
117
6 5 - 341
89
53 - 260
79
5 8 - 210
50
1 7 - 111
26
16-51
20
1 4 - 38
6.37
6.11 - 6.66
6.51
6.07 - 7.10
6.24
5.9 - 7.04
171
701 - 1014
62
18-466
112
9 - 508
bile acia
0 lithocholate deoxycholate
198
138 - 633
37
15 - 366
136
65 - 371
total bile acids
406
219- 1647
147
3 2 - 747
288
112- 879
Bile Acids in a Human Model of ColorectafNeoplasia
357
outputs of taurochenodeoxycholate and total taurine and chenodeoxycholate amidates by FAP subjects in the present studies. This suggests that there is increased recirculation, rather than increased formation of bile acids. Duodenal polyposis in FAP clusters around the ampulla of Vater, implying that bile may be an aetiological factor". Increased recirculation of bile acids in FAP may therefore promote duodenal polyposis. Increased faecal concentrations of primary bile acids have been noted in FAP7,but we are unaware of other published data for total outputs. We could not detect any primary bile acids in faeces, but the decreased total and individual secondary bile acid outputs in FAP agrees with the results from the ileostomy studies. It is interesting to note that there was not a specific decrease in the output of lithwholate, the chenodeoxycholate metabolite.
In summary, the data d o not suggest a primary role for bile acids in colonic polyposis in FAP, but suggests that increased recirculation may augment duodenal polyposis. Duodenal cancer developing from polyps is the commonest extracolonic malignant cause of death in FAP'. Dietary bile acid ligands, such as complex carbohydrates decrease the small intestinal absorption of sterols" and decrease bile acid recirculation. Further study of the potential value of dietary intervention with such materials in attempts to decrease duodenal cancer in FAP is warranted.
1. 2. 3. 4. 5. 6.
M. Rhodes, D.M. Bradburn,
m,
78,
7. 8. 9. 10. 11.
a
321
A.L. Watne, H.L. Lai, T. Mance, S. Core, J S u a , 1976, 131, 42 E. Bailey, A.F.G. Brooks, J C h r o a , 1987, 421. 21 J. Bum, P. Chapman, J. Delhanty. C. Wood.F. Lalloo. M.B. Gonzales, K. Tsioupara, W. Church, M. Rhodes, A. GUM, , -J 1991. 28,
a
I. Bosaeus, B. Sandstrom, H. Andersson,
891
J G-
1 1986, 21,
A.D. Spigelman, C. Crofton-Sleigh, S. Venitt, R.K.S. Phillips, Br J S w . 1990, 77,
12.
W.1992, 33, U
H.J.R. Bussey, Path Ann& , 1979, 14, E.R. Fearon. B. Vogelstein, 1990, 61, S.A. Bingham, J Rov S w Me& 1990, 83, 4 2 B.S.Reddy, C a n er Res, 1981, 41, UlQ A.D. Spigelman, R. W. Owen, M.J. Hill, R.K.S. Phillips, 3t J Sure, 1991,
a
I. Bosaeus, N.G.Carlsson, A.S. Sandberg, H. Andersson, Bum Nutr: Clin
m. 1986, 40,