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Fecal steroids and bacterial flora in patients with polyposis coli
Fecal Steroids and Bacterial Flora in Patients with Polyposis Coli Alvin L. Watne, MD, Morgantown, West Virginia Hsiao-Ya L. Lai, PhD, Morgantown, West Virginia Thomas Mance, MS, Morgantown, West Virginia Sue Core, PhD, Morgantown, West Virginia
Polyposis coli was recognized by Lockhart-MumMendelian mery and Dukes [1] as a premalignant dominant characteristic giving rise to colorectal carcinoma. The inherited defect is unknown, but the fact that the patients are not born with polyps and that these develop after ten to fifteen years of life, the surface activation of the polyps [2], the disappearance of rectal polyps after ileorectostomy, and in the Gardner’s syndrome variant the occurrence of tumors from the ampulla of Vater to the rectum and the association of distant tumors of soft tissue and bone including sarcoma opens the question of some type of a systemic carcinogen possibly activated by the inherited trait. The recent interest in the role of cholesterol [3], bile acids, and variations of the fecal flora [4] led to this study to evaluate these parameters in patients with polyposis coli. Material and Methods Subjects. Three groups of volunteer subjects were studied. The first consisted of fourteen normal adults, the second was nine patients with untreated polyposis coli, and the third group comprised six patients with polyposis coli who had undergone ileorectostomy at least one month prior to the study. Four patients were studied for fecal flora before ileorectostomy and three after surgery. Two patients were studied both before and after surgery. Materials for Fecal Steroid Analysis. Cholic, chenodeoxycholic, lithocholic, deoxycholic, and hyodeoxycholic acids and their corresponding methyl esters were ob-
From the Charleston Foundation Laboratory for Cancer Research. West Virginia University Medical Center, Morgantown, West Virginia. This work was supported by Charleston Foundation for Cancer Research, West Virginia State Aerie Fraternal Order of Eagles, PHS Grant 5T12CA06071-10 from the National Cancer Institute, and PHS Grant 5TOlCA05170-11 from the National Cancer Institute. Reprint requests should be addressed to Alvin L. Watne. MD. Professor and Chairman, Department of Surgery, West Virginia University Medical Center, Morgantown, West Virginia 26506 Presented at the Sixteenth Annual Meeting of the Society for Surgery of the Alimentary Tract, San Antonio, Texas. May 20-21, 1975.
tained from Applied Science Laboratory and Supelco Inc. Cholesterol, coprostanol, coprostanone, and 5 (Ycholestane were obtained from the same companies. Collection
and Lyophilization
of Feces.
Stools were
collected into plastic bags, transferred to lyophilization flasks, and frozen to stop bacterial action. The entire sample was then lyophilized and mixed with a Virtis homogenizer to a fine powder to insure homogeneity. Saponification and Extraction. A modified procedure of Grundy, Ahrens, and Miettinen [5] was used for isolation and gas-liquid chromatographic (GLC) analysis of fecal bile acids and neutral steroids; 5 cY-cholestane and hyodeoxycholic acid were added as internal standards before saponification. The extracted neutral steroids were converted to Tri Fluoro Acetate (‘WA)-derivatives directly for gas-liquid chromatographic analysis. The extracted bile acids were converted to methyl esters [6], which were converted to TFA-derivatives for gasliquid chromatographic analysis. Gas-Liquid Chromatography. Final purification and quantification of bile acids and neutral steroids were carried out on an instrument equipped with a hydrogen flame ionization detector (Packard Model 421, Downers Grove, Illinois) and a computing integrator (Autolab System I, Spectra-Physics, Santa Clara, California). Six foot glass U-tubes of 0.25 inch internal diameter were packed with 3 per cent QF-1 on loo/l20 mesh GasChrom Q (Applied Science Laboratories Inc., State College, Pennsylvania). Nitrogen was used as the carrier gas. Column temperature was usually 23O*C and temperature of injection ports and the detector was 250°C.
The relative retention times of unknown were compared with those of the reference standards. Fecal Flora Studies. Fresh stools were collected at the laboratory and immediately placed under an atmosphere of CO:! for anaerobic fecal flora. No samples were exposed to oxygen for longer than 5 minutes. Approximately 1 gm of stool from the center of a fecal mass was diluted into a weighed tube of VP1 salt solution (9 ml) and shaken until the clump disintegrated. The sample was then weighed, 1 ml portions were serially diluted to 10mQ, and 1.0 ml aliquots from the 10m6 to 10eg tubes were added to four duplicate anaerobic roll tubes for each dilution. Molten anaerobic Rumen fluid-GlucoseCellobiose Agar (46%) was spun until the agar harden-
The American Journal of Surgery
PolyposisColi
ed, then incubated at 37V for five days. The colonies were counted and at least fifty-five colonies were picked to chopped meat or Sweet E media. These were grown for one day and an aliquot was lyophilized. The remaining culture was frozen at -7OV. The isolates were then subcultured and speciated by the technics described by Holdeman and Moore [7]. Quadruplicate 0.1 ml samples from dilutions of 10 to 106 for quantitative aerobic bacterial analysis were plated to petri dishes of Brain Heart Infusion Agar. These were incubated at 37’C for one day and counted.
COPROSTANOL CONTROL 114) _ POLYPOsls (9) 9..
CnOLESTEmL
TOTAL STEROL
16.4
6.0
22.4
3.99
14.69
20.66
p
p < 0.01
P>O.l r-?
Results The mean concentrations of neutral and acid fecal steroids of normal controls and patients with polyposis coli are given in Figures 1 and 2. The mean concentration of total cholesterol and coprostanol excretion is not significantly different in the controls and patients with polyposis coli; however, the polyposis coli group excreted a significantly higher concentration of cholesterol than the controls. The total bile acid excretion is not significantly different in the two groups, but the excretion of cholic and chenodeoxycholic acid is signficantly higher in the polyposis coli group. The mean concentrations of neutral and acid steroid excretion of patients with polyposis coli before and after ileorectostomy are given in Figures 3 and 4. Before ileorectostomy the six patients with polyposis coli excreted an average value of 6.5 mg coprostanol and 12.1 mg cholesterol per gram dry weight; after ileorectostomy they excreted no coprostanol and 17 mg cholesterol per gram dry weight. Before ileorectostomy the patients with polyposis coli excreted more secondary bile acids than primary bile acids; after ileorectostomy they excreted more primary bile acids than secondary bile acids. The reduction of cholesterol to coprostanol and the 7 a-dehydroxylation of bile acids are significantly diminished after ileorectostomy. The anaerobic/aerobic bacteria ratio samples from the preoperative polyposis coli group was unchanged from the normal ratio (2.7/l), whereas four postileorectostomy patients showed an increased ratio (3.5/l). The predominant anaerobic bacteria in the patients with polyposis coli before and after ileorectostomy are compared in Figure 5A with the flora found by Moore, Cato, and Holdeman [S] using identical technics in two normal adult subjects. Compared with this normal determination, the patients with polyposis coli before operation show an increase in the percentage of clostridia, bifidobacteria, and to a lesser extent lactobacillus, rumino-
Voluna 131, January 1976
0
CONTROL POLYPOSIS
Figure I. Fecal neutral steroid excretion of controfs and pattents with poryposrs coii.
mLmLE1M CONTROLS 04) POLYPOSIS (91
6.0 3.6
IO f .r t 6 ? :
6
6
4 2
q CONTROL q POLYPOSIS Figure 2. Fecal bife acids excretion of confrok and patients with polyp&s call
Coprostonol
Total
Sterel
Pre-op
6.5
18.6
Post-op
0
17.0
pc 0.01
pso.1
Pm Post
Pre Post
Z .p
15
2 0 p
IO
F 5
Pm post
F&we 3. Fecal neutral sterohl excretion of palhmts with polyposis coti before anti after lleorectostomy.
43
Watne et al
Lithochdic
DSOXyCtWlk
Chsnochdlc
Chdic
Total &Is Ac!d
PWOP
2.4
2.4
08
0.6
62
Post-op
0.5
0.6
2.1
2.4
5.2
PC 0.1
pqo.1
pao.1
p< 0.01
PC005
_
s
6
P
$
4
a P 2
Pre Port
Prs
Post
Pm Port
Pm Part
PR
post
Fmre 4. Fecal bile acids excretion of pafienfs wifh poiyposh3 toll before and affer ifeorecfosfomy. coccus, peptostreptococcus, and fusobacteria, with a decrease in the eubacteria and bacteroides, Four months or more after ileorectostomy the clostridia, ruminoccoccus, peptostreptococcus, and fusobacteria disappear, and eubacteria and lactobacilli decrease. There is a marked increase in bifidobacteria and bacteroides. This very same pattern is shown in Figure 5B comparing the anaerobic fecal flora of two patients with polyposis coli before and after ileorectostomy. Statistical evaluation of the small number of patients studied failed to provide any significant alteration.
60 Y.
FLORA M
NorInd
12)
Fwop
(31
PO*1 op
(4)
Pre op Pod op 12) (2)
Figure 5. Leff, the fecal flora of paflenfs wifh poiyposfs coil before and afler ifeorecfosfomy. Number In parentheses represent0 the number of samples sfudied. Rlghf, the fecal flora of the two paffenfs wffh poiyposis co/i before and affer Ileorecfosfomy.
44
Comments The fact that the patients with polyposis coli and the normal control group excreted the same amount of acid and neutral steroids daily was no surprise, since both groups were on a general American diet and had no known metabolic disorders, that is, normal serum cholesterol concentrations [9]. However, the fact that the patients with polyposis coli excrete a significantly increased amount of cholesterol as compared with coprostano1 suggests some alteration in the fecal flora and may be significant since Weil-Malherie and Dickens [3] as well as Altman, Pugachiov, and BalliniKerr [IO] have demonstrated that cholesterol promotes tumor formation by 3,4 benzpyrene or 3methylcholanthrene. This effect, however, is not supported by the observation that the cholesterol excretion by the patients with polyposis coli is further increased after ileorectostomy, whereas many of these patients show a decrease in the size and frequency of rectal polyps. Again, the total bile acid excretion is not significantly different in the controls and patients with polyposis coli. However, the patients with polyposis coli show a significant increase in the excretion of primary bile acids, cholic and chenodeoxycholic acid. The bacterial degradation of these bile acids is further decreased after ileorectostomy. Diarrhea is a common complaint among the patients with polyposis coli, and we have shown that their stool transit time is 19 hours compared with 55.8 hours for normal control subjects [ll]. This may be due to the primary bile acids such as chenodeoxycholic acid, which is known to cause diarrhea [12]. After ileorectostomy the transit time in two patients decreased from a preoperative average of 19.4 hours to 14.2 hours. Crowther et al [13] have shown that people who excreted mostly bacterial metabolites of cholesterol when placed on a low fat, low fiber diet had much slower transit times and excreted less bacterial metabolites of cholesterol. It appears that the time the substrate is exposed to the fecal bacteria is of minor importance in the degradation of fecal steroids. Eyssen, Piessens-Denef, and Parmentier [14] showed that the presence of anaerobic bacteria in the cecum not only allowed hydrogenation of cholesterol to coprostanol but after cecectomy in conventional or gnotobiotic rats, Eubacterium 21, 408 (an anaerobic bacteria) responsible for this conversion, could not inhabit the rat intestine. Lai and Watne [15] demonstrated the site of conversion in the human to be in the descending colon.
TheAmerican
Journal of Surgery
Polyposis Coli
With the loss of the conversion locale in the ileorectostomy patients, it is not surprising that they excrete only cholesterol and primary bile acids. Associated with such bile acid alterations is the disappearance of clostridia, ruminococcus, and peptostreptococcus and a slight reduction of eubacteria and lactobacilli. It should be noted, however, that the preoperative polyposis coli group excreted more cholesterol and primary bile acids than the normal controls, and had an increased number of clostridia, bifidobacteria, lactobacillus, ruminococcus, and peptostreptococcus and showed only slightly less numbers of eubacteria and bacteroides than the subjects reported by Moore, Cato, and Holdeman [a]. The only bacteria to show a reduction from normal in the patient with polyposis coli both before and after ileorectostomy were eubacteria and may be associated with the decreasing reduction of cholesterol to coprostanol. The many factors influencing the growth of the fecal bacteria and the role of these bacteria on the fecal steroid patterns are under study. Summary Fecal neutral and acid steroids and bacterial flora have been studied in a series of patients with polyposis coli and normal controls. The patients with polyposis coli showed a higher concentration of cholesterol and primary bile acids, which increased still more after ileorectostomy. The bacterial flora of the patients with polyposis coli showed an anaerobeiaerobe ratio of 2.111 with a relative increase in clostridia and bifidobacteria and decrease in eubacteria and bacteroides. After ileorectostomy clostridia disappear, as do rumenococcus, peptostreptococcus, and fusobacteria, whereas eubacteria and lactobacilli decrease and bifidobacteria and bacteroides increase. Eubacteria showed a decrease from normal in the pre- and postoperative patients with polyposis coli and paralleled the reduction of cholesterol to coprostanol. References 1. Lochhart-Mummery JP, Dukes CE: Familial adenomatosis of the colon and rectum: relationship to cancer. Lancet 2: 586.1939. 2. Cole JW, McKalen A: Studies on the morphogenesis of adenomatous polyps in the human colon. Cancer 16: 998, 1963. 3. Weil-Malherie H, Dickens F: Factors affecting carcinogenesis. IV. The effect of tricaprylin solutions of cholesterol and phospholipids. Cancer Res 6: 17 1, 1946. 4. Hill MJ: Steroid nuclear dehydrogenation and colon cancer. Am J Ciin Nufr 27: 1475, 1974. 5. Grundy SM, Ahrens Jr EH, Miettinen TA: Quantitative isolation and gas-liquid chromatographic analysis of total fecal
Volume 131, January 1976
bile acids. J Lipid Res 6: 397, 1965. 6. Mason ME, Wailer GR: Dimethoxypropane induced transesterification of fats and oils in preparation of methyl esters for gas chromatographic analysis. Anal Chem 36: 583, 1964. 7. Holdernan LV, Moore WEC: Anaerobe Laboratory M&ual. Virginia Polytechnic Institute and State University, Blacksburg, Va, 1973. 8. Moore WEC, Cato EP, HoMeman LV: Anaerobic bacteria of the gastrointestinal flora and their occurrence in clinical infection. Jlnfect Dis 119: 641, 1969. 9. Watne AL, Core SK, Carrier JM: Gardner’s Syndrome. Sung Gynecol Obsfef, 141: 53, 1975. 10. Altman RFA, Pugachiov 0, Balli&Kerr I: Phospholipids (pL) retard and cholesterol (chol) promotes the formation of tumors induced by carcinogenic hydrocarbons. Z Naturforsch [B] 23: 1277. 1968. 11. Watne AL, Johnson R: Bowel transit time in patients with Gardner’s syndrome. Dis Colon Rectum 17: 52, 1974. 12. Mitchell WD. Findlay JM. Prescott RJ, Eastwood MA. Horn DB: Bile acids in the diarrhoea of ileal resection. Gut 14: 382, 1973. 13. Crowther JS, Drasar BS, Hill MI, Johnson K: The effect of a chemically defined diet on the fecal flora and fecal steroid concentration. Gut 14: 790, 1973. 14. Eyssen H, Piessens-Denef M, Parmentier G: Role of cecum in maintaining A5-steroid and-fatty acibreducing activity of rat intestinal microflora. JNufr 102: 1501, 1972. 15. Lai HYL, Watne AL: The site of the reduction of cholesterol to coprostanol and the iradehydroxylatiin of bile acids. FedF’roc 34: 661, 1975. 16. Hill MJ, Drasar ES, Aries VC, Crowther JD, Hawksworth GM Williams REO: Bacteria and aetiology of cancer of large bowel. Lancet 1: 95, 1971.
Discussion Robert E. Condon (Milwaukee, WI): I thought you might be interested in some preliminary experience at the Vince Lombardi Colon Clinic in patients with multiple polyposis. These patients have recurrent polyps in the rectal segment after ileoproctostomy in recurrent crons and waves and then go through intervals when
new polyp formation is not occurring. A group of five patients who had been in the phase of an ongoing wave of new polyp formation have been treated with massive oral doses of vitamin C, which alters the redox potential in the colon. Among these five patients, two have had regression and two have had complete disappearance of rectal polyps. It is probable this treatment also results in an alteration of the bacterial flora. Malcolm M. Stanley (Hines, IL): How are the stools handled? It is very difficult to get the stools promptly and it requires a lot of specialized equipment. I would like to ask about the dietary control during the study. Were these normal subject& and patients taking different diets before or during the study? Lawrence DenBesten (Iowa City, IA): We may interpret the data presented as cause or effect. From the controls to the patients with polyposis on to the patients after ileorectostomy, there is decreasing evidence of bacterial action on the neutral and the acid steroids. There is less conversion to coprostanol, greater relative secretion of primary bile acids, and lesser secretion of secon-
45
Watne et al
dary bile acids, all indicating that there is less action of bacteria on the sterols, and therefore less conversion. This could be related, of course, to differing bacterial composition of the fecal flora. However, the phenomenon may also result from the increased time the sterols were in contact with the bacteria, as suggested by the decreased transit time. I would like to ask and thereby reiterate some of the questions of Doctor Stanley. What was the dietary intake? Diet, of course, will greatly influence fecal sterol excretion. What was the control diet; the length of time after operation these patients were studied; had the fecal flora been reestablished and stabilized; were these patients on any antibiotics shortly prior to the study? These and many other factors will greatly influence the action of bacteria on neutral and acid steroids. S. Swenson, Jr (Omaha, NE): This presentation points out the fact that the rapid transit of bile into and through the colon results in lack of conjugation and change. It is particularly important in the initial jejunocolic bypass that Paine did for obesity. This then really bypassed the entire area of the terminal ileum, and the bile acids themselves are conjugated in the terminal ileum. Some of these patients still survive and it might be extremely interesting to study these people. One of the problems with small bowel bypass for obesity is dietary and the rapid transit of bile into the colon with severe bile or chemical colitis; perhaps the answer to this is in diet, reduction of fat intake, and the utilization of antibiotics. Sherwood L. Gerbach (Sepulveda, CA): The studies of Hale and Drosser of people from Uganda, India, and Japan show these same patterns, that is, low conversion of neutral steroids from cholesterol to coprostanol and high excretion of primary bile acids, whereas westerners, mostly patients studied from England and Scotland, had high incidence of colon cancer risk, in fact, very avidly convert cholesterol to coprostanol and primary to secondary bile acids. Thus, we believe it was diet and these important metabolic conversions that relate to cancer risk, and now we are being told that one patient with perhaps the greatest colon cancer risk of all failed to show these changes. I wonder if the authors would speculate on what is the reason that these conversions are taking place. They are obviously environmental and metabolic, as well as gen-
46
eric, because the bypass experiment shows you can cause regression of the polyps. That was accomplished in animals as well. Alvin L. Watne (Morgantown, WV): There seems to be some alteration in the internal milieu of the colon, which will allow or prohibit the growth of certain species of bacteria. This may be related to the electropotential of the cells, to the pH, or to other factors. The stool specimens were collected at the laboratory with a plastic holder that fits to the local commode. Within five minutes a specimen is taken from the center of the stool and placed under carbon dioxide in a nutrient media to preserve anaerobic microflora. We elected to leave patients on their natural diet, to evaluate them in their present environment. We have not attempted to alter the flora by altering their diet and, indeed, the patients with polyposis cob ingest hamburgers, colas, and pizza just like you and me. Age groups were similar for the controls and the patients with polyposis coli. Hill [4] has pointed out that the Clostridium paraputrifrium may well convert bile acids to carcinogens or cocarcinogens. These specimens did not contain that particular species. These patients had both polyposis coli and Gardner’s syndrome. At this time we believe that Gardner’s syndrome represents a manifestation of the general problem of polyposis coli. We do not believe that the bacteria themselves are in any way responsible for the polyposis coli, and indeed primary bile acids can stimulate growth of bacteria such as bacteroides, and deoxycholic acid can inhibit others, so that it may be the alteration of the bile content alters in some way the bacterial flora, and not vice versa. All of the patients were studied at least four months or more after operation. Some were studied immediately after the operation and the changes occur within one month. None of the patients have been placed on any antibiotic at this time to alter the flora. Our observations on the relationship to cancer are contrary to Hill and Draser’s work. We cannot say at this time that the cholesterol or the primary bile acids are responsible for the high incidence of cancer. Doctor Burkitt visited us and we cannot explain why these people have rapid transit and yet have a high incidence of colonic cancer.
The American Journal of Surgery
Polyposis coli was recognized by Lockhart-MumMendelian mery and Dukes [1] as a premalignant dominant characteristic giving rise to colorectal carcinoma. The inherited defect is unknown, but the fact that the patients are not born with polyps and that these develop after ten to fifteen years of life, the surface activation of the polyps [2], the disappearance of rectal polyps after ileorectostomy, and in the Gardner’s syndrome variant the occurrence of tumors from the ampulla of Vater to the rectum and the association of distant tumors of soft tissue and bone including sarcoma opens the question of some type of a systemic carcinogen possibly activated by the inherited trait. The recent interest in the role of cholesterol [3], bile acids, and variations of the fecal flora [4] led to this study to evaluate these parameters in patients with polyposis coli. Material and Methods Subjects. Three groups of volunteer subjects were studied. The first consisted of fourteen normal adults, the second was nine patients with untreated polyposis coli, and the third group comprised six patients with polyposis coli who had undergone ileorectostomy at least one month prior to the study. Four patients were studied for fecal flora before ileorectostomy and three after surgery. Two patients were studied both before and after surgery. Materials for Fecal Steroid Analysis. Cholic, chenodeoxycholic, lithocholic, deoxycholic, and hyodeoxycholic acids and their corresponding methyl esters were ob-
From the Charleston Foundation Laboratory for Cancer Research. West Virginia University Medical Center, Morgantown, West Virginia. This work was supported by Charleston Foundation for Cancer Research, West Virginia State Aerie Fraternal Order of Eagles, PHS Grant 5T12CA06071-10 from the National Cancer Institute, and PHS Grant 5TOlCA05170-11 from the National Cancer Institute. Reprint requests should be addressed to Alvin L. Watne. MD. Professor and Chairman, Department of Surgery, West Virginia University Medical Center, Morgantown, West Virginia 26506 Presented at the Sixteenth Annual Meeting of the Society for Surgery of the Alimentary Tract, San Antonio, Texas. May 20-21, 1975.
tained from Applied Science Laboratory and Supelco Inc. Cholesterol, coprostanol, coprostanone, and 5 (Ycholestane were obtained from the same companies. Collection
and Lyophilization
of Feces.
Stools were
collected into plastic bags, transferred to lyophilization flasks, and frozen to stop bacterial action. The entire sample was then lyophilized and mixed with a Virtis homogenizer to a fine powder to insure homogeneity. Saponification and Extraction. A modified procedure of Grundy, Ahrens, and Miettinen [5] was used for isolation and gas-liquid chromatographic (GLC) analysis of fecal bile acids and neutral steroids; 5 cY-cholestane and hyodeoxycholic acid were added as internal standards before saponification. The extracted neutral steroids were converted to Tri Fluoro Acetate (‘WA)-derivatives directly for gas-liquid chromatographic analysis. The extracted bile acids were converted to methyl esters [6], which were converted to TFA-derivatives for gasliquid chromatographic analysis. Gas-Liquid Chromatography. Final purification and quantification of bile acids and neutral steroids were carried out on an instrument equipped with a hydrogen flame ionization detector (Packard Model 421, Downers Grove, Illinois) and a computing integrator (Autolab System I, Spectra-Physics, Santa Clara, California). Six foot glass U-tubes of 0.25 inch internal diameter were packed with 3 per cent QF-1 on loo/l20 mesh GasChrom Q (Applied Science Laboratories Inc., State College, Pennsylvania). Nitrogen was used as the carrier gas. Column temperature was usually 23O*C and temperature of injection ports and the detector was 250°C.
The relative retention times of unknown were compared with those of the reference standards. Fecal Flora Studies. Fresh stools were collected at the laboratory and immediately placed under an atmosphere of CO:! for anaerobic fecal flora. No samples were exposed to oxygen for longer than 5 minutes. Approximately 1 gm of stool from the center of a fecal mass was diluted into a weighed tube of VP1 salt solution (9 ml) and shaken until the clump disintegrated. The sample was then weighed, 1 ml portions were serially diluted to 10mQ, and 1.0 ml aliquots from the 10m6 to 10eg tubes were added to four duplicate anaerobic roll tubes for each dilution. Molten anaerobic Rumen fluid-GlucoseCellobiose Agar (46%) was spun until the agar harden-
The American Journal of Surgery
PolyposisColi
ed, then incubated at 37V for five days. The colonies were counted and at least fifty-five colonies were picked to chopped meat or Sweet E media. These were grown for one day and an aliquot was lyophilized. The remaining culture was frozen at -7OV. The isolates were then subcultured and speciated by the technics described by Holdeman and Moore [7]. Quadruplicate 0.1 ml samples from dilutions of 10 to 106 for quantitative aerobic bacterial analysis were plated to petri dishes of Brain Heart Infusion Agar. These were incubated at 37’C for one day and counted.
COPROSTANOL CONTROL 114) _ POLYPOsls (9) 9..
CnOLESTEmL
TOTAL STEROL
16.4
6.0
22.4
3.99
14.69
20.66
p
p < 0.01
P>O.l r-?
Results The mean concentrations of neutral and acid fecal steroids of normal controls and patients with polyposis coli are given in Figures 1 and 2. The mean concentration of total cholesterol and coprostanol excretion is not significantly different in the controls and patients with polyposis coli; however, the polyposis coli group excreted a significantly higher concentration of cholesterol than the controls. The total bile acid excretion is not significantly different in the two groups, but the excretion of cholic and chenodeoxycholic acid is signficantly higher in the polyposis coli group. The mean concentrations of neutral and acid steroid excretion of patients with polyposis coli before and after ileorectostomy are given in Figures 3 and 4. Before ileorectostomy the six patients with polyposis coli excreted an average value of 6.5 mg coprostanol and 12.1 mg cholesterol per gram dry weight; after ileorectostomy they excreted no coprostanol and 17 mg cholesterol per gram dry weight. Before ileorectostomy the patients with polyposis coli excreted more secondary bile acids than primary bile acids; after ileorectostomy they excreted more primary bile acids than secondary bile acids. The reduction of cholesterol to coprostanol and the 7 a-dehydroxylation of bile acids are significantly diminished after ileorectostomy. The anaerobic/aerobic bacteria ratio samples from the preoperative polyposis coli group was unchanged from the normal ratio (2.7/l), whereas four postileorectostomy patients showed an increased ratio (3.5/l). The predominant anaerobic bacteria in the patients with polyposis coli before and after ileorectostomy are compared in Figure 5A with the flora found by Moore, Cato, and Holdeman [S] using identical technics in two normal adult subjects. Compared with this normal determination, the patients with polyposis coli before operation show an increase in the percentage of clostridia, bifidobacteria, and to a lesser extent lactobacillus, rumino-
Voluna 131, January 1976
0
CONTROL POLYPOSIS
Figure I. Fecal neutral steroid excretion of controfs and pattents with poryposrs coii.
mLmLE1M CONTROLS 04) POLYPOSIS (91
6.0 3.6
IO f .r t 6 ? :
6
6
4 2
q CONTROL q POLYPOSIS Figure 2. Fecal bife acids excretion of confrok and patients with polyp&s call
Coprostonol
Total
Sterel
Pre-op
6.5
18.6
Post-op
0
17.0
pc 0.01
pso.1
Pm Post
Pre Post
Z .p
15
2 0 p
IO
F 5
Pm post
F&we 3. Fecal neutral sterohl excretion of palhmts with polyposis coti before anti after lleorectostomy.
43
Watne et al
Lithochdic
DSOXyCtWlk
Chsnochdlc
Chdic
Total &Is Ac!d
PWOP
2.4
2.4
08
0.6
62
Post-op
0.5
0.6
2.1
2.4
5.2
PC 0.1
pqo.1
pao.1
p< 0.01
PC005
_
s
6
P
$
4
a P 2
Pre Port
Prs
Post
Pm Port
Pm Part
PR
post
Fmre 4. Fecal bile acids excretion of pafienfs wifh poiyposh3 toll before and affer ifeorecfosfomy. coccus, peptostreptococcus, and fusobacteria, with a decrease in the eubacteria and bacteroides, Four months or more after ileorectostomy the clostridia, ruminoccoccus, peptostreptococcus, and fusobacteria disappear, and eubacteria and lactobacilli decrease. There is a marked increase in bifidobacteria and bacteroides. This very same pattern is shown in Figure 5B comparing the anaerobic fecal flora of two patients with polyposis coli before and after ileorectostomy. Statistical evaluation of the small number of patients studied failed to provide any significant alteration.
60 Y.
FLORA M
NorInd
12)
Fwop
(31
PO*1 op
(4)
Pre op Pod op 12) (2)
Figure 5. Leff, the fecal flora of paflenfs wifh poiyposfs coil before and afler ifeorecfosfomy. Number In parentheses represent0 the number of samples sfudied. Rlghf, the fecal flora of the two paffenfs wffh poiyposis co/i before and affer Ileorecfosfomy.
44
Comments The fact that the patients with polyposis coli and the normal control group excreted the same amount of acid and neutral steroids daily was no surprise, since both groups were on a general American diet and had no known metabolic disorders, that is, normal serum cholesterol concentrations [9]. However, the fact that the patients with polyposis coli excrete a significantly increased amount of cholesterol as compared with coprostano1 suggests some alteration in the fecal flora and may be significant since Weil-Malherie and Dickens [3] as well as Altman, Pugachiov, and BalliniKerr [IO] have demonstrated that cholesterol promotes tumor formation by 3,4 benzpyrene or 3methylcholanthrene. This effect, however, is not supported by the observation that the cholesterol excretion by the patients with polyposis coli is further increased after ileorectostomy, whereas many of these patients show a decrease in the size and frequency of rectal polyps. Again, the total bile acid excretion is not significantly different in the controls and patients with polyposis coli. However, the patients with polyposis coli show a significant increase in the excretion of primary bile acids, cholic and chenodeoxycholic acid. The bacterial degradation of these bile acids is further decreased after ileorectostomy. Diarrhea is a common complaint among the patients with polyposis coli, and we have shown that their stool transit time is 19 hours compared with 55.8 hours for normal control subjects [ll]. This may be due to the primary bile acids such as chenodeoxycholic acid, which is known to cause diarrhea [12]. After ileorectostomy the transit time in two patients decreased from a preoperative average of 19.4 hours to 14.2 hours. Crowther et al [13] have shown that people who excreted mostly bacterial metabolites of cholesterol when placed on a low fat, low fiber diet had much slower transit times and excreted less bacterial metabolites of cholesterol. It appears that the time the substrate is exposed to the fecal bacteria is of minor importance in the degradation of fecal steroids. Eyssen, Piessens-Denef, and Parmentier [14] showed that the presence of anaerobic bacteria in the cecum not only allowed hydrogenation of cholesterol to coprostanol but after cecectomy in conventional or gnotobiotic rats, Eubacterium 21, 408 (an anaerobic bacteria) responsible for this conversion, could not inhabit the rat intestine. Lai and Watne [15] demonstrated the site of conversion in the human to be in the descending colon.
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Journal of Surgery
Polyposis Coli
With the loss of the conversion locale in the ileorectostomy patients, it is not surprising that they excrete only cholesterol and primary bile acids. Associated with such bile acid alterations is the disappearance of clostridia, ruminococcus, and peptostreptococcus and a slight reduction of eubacteria and lactobacilli. It should be noted, however, that the preoperative polyposis coli group excreted more cholesterol and primary bile acids than the normal controls, and had an increased number of clostridia, bifidobacteria, lactobacillus, ruminococcus, and peptostreptococcus and showed only slightly less numbers of eubacteria and bacteroides than the subjects reported by Moore, Cato, and Holdeman [a]. The only bacteria to show a reduction from normal in the patient with polyposis coli both before and after ileorectostomy were eubacteria and may be associated with the decreasing reduction of cholesterol to coprostanol. The many factors influencing the growth of the fecal bacteria and the role of these bacteria on the fecal steroid patterns are under study. Summary Fecal neutral and acid steroids and bacterial flora have been studied in a series of patients with polyposis coli and normal controls. The patients with polyposis coli showed a higher concentration of cholesterol and primary bile acids, which increased still more after ileorectostomy. The bacterial flora of the patients with polyposis coli showed an anaerobeiaerobe ratio of 2.111 with a relative increase in clostridia and bifidobacteria and decrease in eubacteria and bacteroides. After ileorectostomy clostridia disappear, as do rumenococcus, peptostreptococcus, and fusobacteria, whereas eubacteria and lactobacilli decrease and bifidobacteria and bacteroides increase. Eubacteria showed a decrease from normal in the pre- and postoperative patients with polyposis coli and paralleled the reduction of cholesterol to coprostanol. References 1. Lochhart-Mummery JP, Dukes CE: Familial adenomatosis of the colon and rectum: relationship to cancer. Lancet 2: 586.1939. 2. Cole JW, McKalen A: Studies on the morphogenesis of adenomatous polyps in the human colon. Cancer 16: 998, 1963. 3. Weil-Malherie H, Dickens F: Factors affecting carcinogenesis. IV. The effect of tricaprylin solutions of cholesterol and phospholipids. Cancer Res 6: 17 1, 1946. 4. Hill MJ: Steroid nuclear dehydrogenation and colon cancer. Am J Ciin Nufr 27: 1475, 1974. 5. Grundy SM, Ahrens Jr EH, Miettinen TA: Quantitative isolation and gas-liquid chromatographic analysis of total fecal
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bile acids. J Lipid Res 6: 397, 1965. 6. Mason ME, Wailer GR: Dimethoxypropane induced transesterification of fats and oils in preparation of methyl esters for gas chromatographic analysis. Anal Chem 36: 583, 1964. 7. Holdernan LV, Moore WEC: Anaerobe Laboratory M&ual. Virginia Polytechnic Institute and State University, Blacksburg, Va, 1973. 8. Moore WEC, Cato EP, HoMeman LV: Anaerobic bacteria of the gastrointestinal flora and their occurrence in clinical infection. Jlnfect Dis 119: 641, 1969. 9. Watne AL, Core SK, Carrier JM: Gardner’s Syndrome. Sung Gynecol Obsfef, 141: 53, 1975. 10. Altman RFA, Pugachiov 0, Balli&Kerr I: Phospholipids (pL) retard and cholesterol (chol) promotes the formation of tumors induced by carcinogenic hydrocarbons. Z Naturforsch [B] 23: 1277. 1968. 11. Watne AL, Johnson R: Bowel transit time in patients with Gardner’s syndrome. Dis Colon Rectum 17: 52, 1974. 12. Mitchell WD. Findlay JM. Prescott RJ, Eastwood MA. Horn DB: Bile acids in the diarrhoea of ileal resection. Gut 14: 382, 1973. 13. Crowther JS, Drasar BS, Hill MI, Johnson K: The effect of a chemically defined diet on the fecal flora and fecal steroid concentration. Gut 14: 790, 1973. 14. Eyssen H, Piessens-Denef M, Parmentier G: Role of cecum in maintaining A5-steroid and-fatty acibreducing activity of rat intestinal microflora. JNufr 102: 1501, 1972. 15. Lai HYL, Watne AL: The site of the reduction of cholesterol to coprostanol and the iradehydroxylatiin of bile acids. FedF’roc 34: 661, 1975. 16. Hill MJ, Drasar ES, Aries VC, Crowther JD, Hawksworth GM Williams REO: Bacteria and aetiology of cancer of large bowel. Lancet 1: 95, 1971.
Discussion Robert E. Condon (Milwaukee, WI): I thought you might be interested in some preliminary experience at the Vince Lombardi Colon Clinic in patients with multiple polyposis. These patients have recurrent polyps in the rectal segment after ileoproctostomy in recurrent crons and waves and then go through intervals when
new polyp formation is not occurring. A group of five patients who had been in the phase of an ongoing wave of new polyp formation have been treated with massive oral doses of vitamin C, which alters the redox potential in the colon. Among these five patients, two have had regression and two have had complete disappearance of rectal polyps. It is probable this treatment also results in an alteration of the bacterial flora. Malcolm M. Stanley (Hines, IL): How are the stools handled? It is very difficult to get the stools promptly and it requires a lot of specialized equipment. I would like to ask about the dietary control during the study. Were these normal subject& and patients taking different diets before or during the study? Lawrence DenBesten (Iowa City, IA): We may interpret the data presented as cause or effect. From the controls to the patients with polyposis on to the patients after ileorectostomy, there is decreasing evidence of bacterial action on the neutral and the acid steroids. There is less conversion to coprostanol, greater relative secretion of primary bile acids, and lesser secretion of secon-
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Watne et al
dary bile acids, all indicating that there is less action of bacteria on the sterols, and therefore less conversion. This could be related, of course, to differing bacterial composition of the fecal flora. However, the phenomenon may also result from the increased time the sterols were in contact with the bacteria, as suggested by the decreased transit time. I would like to ask and thereby reiterate some of the questions of Doctor Stanley. What was the dietary intake? Diet, of course, will greatly influence fecal sterol excretion. What was the control diet; the length of time after operation these patients were studied; had the fecal flora been reestablished and stabilized; were these patients on any antibiotics shortly prior to the study? These and many other factors will greatly influence the action of bacteria on neutral and acid steroids. S. Swenson, Jr (Omaha, NE): This presentation points out the fact that the rapid transit of bile into and through the colon results in lack of conjugation and change. It is particularly important in the initial jejunocolic bypass that Paine did for obesity. This then really bypassed the entire area of the terminal ileum, and the bile acids themselves are conjugated in the terminal ileum. Some of these patients still survive and it might be extremely interesting to study these people. One of the problems with small bowel bypass for obesity is dietary and the rapid transit of bile into the colon with severe bile or chemical colitis; perhaps the answer to this is in diet, reduction of fat intake, and the utilization of antibiotics. Sherwood L. Gerbach (Sepulveda, CA): The studies of Hale and Drosser of people from Uganda, India, and Japan show these same patterns, that is, low conversion of neutral steroids from cholesterol to coprostanol and high excretion of primary bile acids, whereas westerners, mostly patients studied from England and Scotland, had high incidence of colon cancer risk, in fact, very avidly convert cholesterol to coprostanol and primary to secondary bile acids. Thus, we believe it was diet and these important metabolic conversions that relate to cancer risk, and now we are being told that one patient with perhaps the greatest colon cancer risk of all failed to show these changes. I wonder if the authors would speculate on what is the reason that these conversions are taking place. They are obviously environmental and metabolic, as well as gen-
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eric, because the bypass experiment shows you can cause regression of the polyps. That was accomplished in animals as well. Alvin L. Watne (Morgantown, WV): There seems to be some alteration in the internal milieu of the colon, which will allow or prohibit the growth of certain species of bacteria. This may be related to the electropotential of the cells, to the pH, or to other factors. The stool specimens were collected at the laboratory with a plastic holder that fits to the local commode. Within five minutes a specimen is taken from the center of the stool and placed under carbon dioxide in a nutrient media to preserve anaerobic microflora. We elected to leave patients on their natural diet, to evaluate them in their present environment. We have not attempted to alter the flora by altering their diet and, indeed, the patients with polyposis cob ingest hamburgers, colas, and pizza just like you and me. Age groups were similar for the controls and the patients with polyposis coli. Hill [4] has pointed out that the Clostridium paraputrifrium may well convert bile acids to carcinogens or cocarcinogens. These specimens did not contain that particular species. These patients had both polyposis coli and Gardner’s syndrome. At this time we believe that Gardner’s syndrome represents a manifestation of the general problem of polyposis coli. We do not believe that the bacteria themselves are in any way responsible for the polyposis coli, and indeed primary bile acids can stimulate growth of bacteria such as bacteroides, and deoxycholic acid can inhibit others, so that it may be the alteration of the bile content alters in some way the bacterial flora, and not vice versa. All of the patients were studied at least four months or more after operation. Some were studied immediately after the operation and the changes occur within one month. None of the patients have been placed on any antibiotic at this time to alter the flora. Our observations on the relationship to cancer are contrary to Hill and Draser’s work. We cannot say at this time that the cholesterol or the primary bile acids are responsible for the high incidence of cancer. Doctor Burkitt visited us and we cannot explain why these people have rapid transit and yet have a high incidence of colonic cancer.
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