Gastroenterology Unit, Guy's Hospital Campus, Guy's, King's and St. Thomas' School of Medicine, Kings College, London, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Prolonged large bowel transit time
and an associated increase in the proportion of deoxycholic acid (DCA)
in serum and bile have been implicated in the development of
cholesterol-rich gallstones and colon cancer. Prolongation of
intestinal transit also increases intracolonic pH that, we
hypothesized, should favor the solubilization and absorption of newly
formed DCA within the colon. To test this hypothesis, we performed in
vitro studies on homogenized cecal aspirates (obtained at colonoscopy)
that were incubated anaerobically with [14C]cholic acid
for 16 h after which the pH was adjusted to between 4.0 and 7.0 in
0.5-pH unit steps. The resultant reaction mixtures were centrifuged to
separate the supernatant from the precipitate, and the specific
activity of [14C]DCA was quantitated in both phases. As
the pH in the aspirates was manipulated from 4.0 to 7.0, the proportion
of newly formed, labeled DCA increased in the supernatant and fell in
the precipitate, particularly at a hydrogen ion concentration
of <100 × 107 (equivalent to pH 5.0-7.0).
These results show that the solubility of DCA in colonic contents
increases with increasing pH. If solubility is rate limiting, this
should lead to increased absorption that, in turn, would explain why
the proportion of DCA in serum and bile increases with the prolongation
of large bowel transit time.
cholesterol gallstones; colorectal cancer; deoxycholic acid metabolism; bile acid solubility
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DEOXYCHOLIC ACID
(DCA) has been implicated in the pathogenesis of both
cholesterol gallstones (16) and colon cancer (1, 22,
23). It is a secondary bile acid that is formed in the cecum and
ascending colon (9, 18) by the action of two intestinal bacterial enzymes, cholylglycine hydrolase (20) and
7-dehydroxylase (29).
We (10, 34, 36) and others (14, 16, 27, 38) have shown that the percentage of DCA in serum (34-36) and bile (10, 14, 16) and the formation rate and pool size (35) of DCA are related to large bowel transit time (LBTT). Moreover, pharmacological prolongation of LBTT increases, and shortening of transit through the intestine decreases, the percentage of DCA in serum and bile (14). These observations support the concept that a sluggish intestine might contribute to the pathogenesis of cholesterol gallstones. Although this concept is not new, it is not widely recognized.
Precisely how changes in intestinal transit affect DCA metabolism is not clear. In theory, they could affect DCA formation in, and/or absorption from, the colon. Certainly DCA and other bile acids can be absorbed from the colon, mainly by passive non-ionic diffusion (19, 24); but, given the fact that the pKa of DCA is ~5.0-5.3 (4, 5, 12, 18a, 23a, 28), the solubility and, therefore, at least some of the bioavailability (i.e., biological availability for absorption) of newly formed DCA is likely to be critically dependent on colonic luminal pH. Prolongation of LBTT is associated with an increase, and acceleration of LBTT with a decrease, in intracolonic and fecal pH (7, 13). Nonetheless, the effect of changes in pH on the solubilization of DCA in colonic contents has never been fully defined.
This report is one of a series of studies from our unit on the role of intestinal transit in the pathogenesis of cholesterol gallstones (10, 11, 21, 30, 31, 34-36). In a companion study (31), we measured the LBTT, the profile of intestinal luminal pH, and the percentage of DCA in serum and bile. We also studied the quantitative bacteriology and activities of the bile acid-metabolizing enzymes in aspirates obtained from the cecum and ascending colon during clinically indicated, unprepared (washout of the left colon by instant enema) colonoscopy (31). However, the aim of the present study was to measure the influence of varying pH values on the distribution of newly formed DCA in the supernatant and precipitate phases of fresh cecal aspirates in vitro.
![]() |
STUDY DESIGN, METHODS, AND MATERIALS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Study Design
The companion study (31) was carried out in 40 subjects; however, the present report was limited to cecal samples from six gallstone-free individuals. The techniques for collecting, homogenizing, and preparing the cecal samples for analysis are described in detail elsewhere (31).The homogenized cecal aspirates (1 sample from each of 6 individuals)
were divided into seven 1-ml aliquots, to each of which we
added 200,000 dpm of [14C]cholic acid (CA) plus 500 µl
of 2 mM "cold" or nonisotopic CA. The nonisotopic CA was added for
two reasons: 1) to enhance the 7-dehydroxylase activity
during the incubation (by substrate enzyme induction; Ref.
37) and 2) to increase the total amount of DCA
formed, thus facilitating its identification by TLC (8).
The CA-enriched aliquots were incubated under anaerobic conditions for 16 h at 37°C. The pH of the individual reaction mixtures was then adjusted with phosphate buffers to yield final pH values ranging from 4.0 to 7.0 in 0.5-pH unit steps. The mixtures were allowed to equilibrate for ~15 min. The samples were mixed with a vortex mixer for 10 min and centrifuged at 5,000 g for 15 min to separate the supernatant and precipitate phases, and the resultant fractions were acidified with 1 ml of 0.5 M HCl. (Strictly speaking, the term "precipitate phase" is a mixture of insoluble material of adequate density and size to the sediment under the centrifugation conditions.) The bile acids were then extracted twice with ethyl acetate, taken to dryness on a heating block, and redissolved in 100 µl of 100% methanol. The individual bile acids were separated by TLC as previously described (8). The areas corresponding to CA and DCA were scraped off the plates, and the 14C radioactivity was counted in an LKB 81,000 liquid scintillation counter with external standard quench correction (26). The distribution of the labeled DCA between the supernatant and precipitate phases was then expressed as a percentage of the total [14C]DCA radioactivity. The mean (± SE) conversion rate of CA to DCA was 48.4 ± 11.7%.
Materials
All reagents, including the bile acid standards, were obtained from Sigma-Aldrich Chemicals (Poole, UK). The [14C]CA was obtained from DuPont-NEN (Stevenage, UK).Ethical Considerations
The protocol for the colonoscopic sampling of cecal contents conformed to the Ethical Guidelines of the 1975 Declaration of Helsinki and was approved by the Research Ethics Committee of Guy's and St. Thomas' Hospitals. All patients gave their written informed consent.Statistical Analyses
Unless otherwise stated, the results are expressed as means ± SE. The significance of differences in means was tested with the nonparametric heteroscedastic t-test using Excel software 5.0 (Microsoft, Redmond, WA). P values <0.05 were considered statistically significant. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of pH on Distribution of Nascent [14C]DCA in Cecal Aspirates
The percentage distribution of [14C]DCA between the supernatant and precipitate phases of the fresh human cecal aspirates, as a function of induced changes in pH, is shown in Fig. 1. The same data plotted on linear/linear [% [14C]DCA vs. hydrogen ion concentration ([H+])] rather than on linear/log (% [14C]DCA vs. pH) axes are shown in Fig. 2.
|
|
As the pH of the cecal aspirates was manipulated to between 4.0 and 7.0, there was a reciprocal relationship between the percentage of DCA in the supernatant and the precipitate phases such that at pH 6.68 (the mean value in the proximal colon of 20 gallstone patients; Ref. 31), ~80% of the newly formed DCA was in the supernatant phase compared with only 55% at pH 5.24, (the mean value in the cecum of the 20 gallstone patients; Ref. 31) (Fig. 1).
When the distribution of [14C]DCA between supernatant and
precipitate was plotted against [H+] (Fig. 2), there was
a very different visual impression from that illustrated in Fig. 1.
Thus, over a wide range of hydrogen ion concentrations (100 to
1,000 × 107), the phase distribution of labeled DCA
between supernatant and precipitate was relatively constant. However,
when the [H+] dropped below 100 × 10
7
M (range 1 to 100 × 10
7 M, corresponding to pH
values of 5.0 to 7.0, i.e., the narrow range of pH values seen between
the right and left halves of the colon), there was a dramatic increase
in the percentage of DCA in the supernatant and, therefore, a
corresponding decrease in the percentage of DCA in the precipitate phase.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have recently shown (31) that compared with stone-free control individuals, patients with cholesterol-rich gallstones have longer LBTTs and higher colonic luminal pH values, probably because prolongation of LBTT allows more time for the absorption of short-chain fatty acids by the colonic mucosa. The aim of the present study was to test the hypothesis that these changes in intestinal transit and colonic luminal pH lead to increased solubilization, and therefore potentially to an increased bioavailability of newly formed unconjugated DCA, in the colon. Indeed, we have proposed that there are at least three stages in this process: 1) increased formation, 2) greater solubilization, and 3) enhanced absorption of DCA (31).
It could be argued that the systematic stepwise manipulation of pH with the use of phosphate buffers yields results analogous to those obtained in classic potentiometric titration studies (4, 5, 12, 28) of the sort used in the past to determine the solubility of the bile acid, its pH of precipitation, and its pKa in aqueous solutions. For two reasons, however, we believe that the present results contribute more than could have been obtained by simple titration. First, we were not studying the solubility characteristics of "exogenous" DCA but rather those of newly formed 14C-labeled DCA that was generated acutely during a 16-h incubation of fresh human cecal aspirates. Second, we were examining the effect of pH on the solubility of DCA in the presence of normal colonic contents including bacteria and unabsorbed dietary fiber. The amount of DCA available for absorption from the colonic lumen is almost certainly influenced by the nature of these contents and does not behave strictly according to solubility rules. Thus bile acids can adsorb to many substances found in the colonic lumen such as wood fiber, food residues, bacteria, and lignin (6), and this is likely to reduce the availability of DCA (and other bile acids) for absorption.
In designing these studies, we chose to modify the pH at the end of the 16-h incubation period. In other words, we were examining the effect of changing pH on the distribution of the newly formed DCA rather than the influence of pH on the activity of the dehydroxylating enzyme. However, we have previously shown that although this enzyme is pH dependent, its activity changes remarkably little over the pH range found in the colon (30).
In the present study, the speed and duration of centrifugation were chosen arbitrarily, with the hope/belief that this might yield a supernatant similar in composition to that of "fecal water" present in vivo. The problem of whether it does or not has been addressed previously (2), but there is no way of knowing that the arbitrary centrifugation settings will necessarily replicate physiologically relevant conditions.
Bile acid absorption from the colon is predominantly via the non-ionic route (15, 19, 24, 25). Therefore, to extrapolate from the results of these in vitro studies to normal physiology (or even to the pathophysiology of gallstone disease), we also need to make assumptions about the distribution of the solubilized DCA between the ionized and non-ionized fractions. This distribution depends on the apparent pKa values of the bile acids and how the pKa values are determined. Thus if the pKa of DCA were 5.0 (or even 5.3), by definition, at this pH, 50% of the DCA would be ionized and 50% protonated. However, the solubility of a bile acid in a "pure" solution is not confined exclusively to the ionized fractions above the critical micellar concentration, a small percentage of the otherwise insoluble protonated bile acid can be solubilized by the bile acid anion. However, as discussed above, predictions about bile acid solubility in vitro ignore phenomena such as adsorption and binding (6), in the distinctly "impure" milieu of human cecal contents. In any event, our results show that at pH 5.3, ~40% of the newly formed [14C]DCA was in the precipitate phase. This confirms the mismatch between predictions based on pKa and solubility and what we found in practice, although in this particular case, there was less rather than more [14C]DCA in the precipitate phase than would have been predicted from the pKa of DCA alone.
Whatever the distribution of the solubilized DCA between protonated and ionized species, the fact remains that prolongation of LBTT is associated with increases in the percentage of DCA in serum (34-36) and bile (10, 14, 16), including unconjugated (newly formed both from conjugated CA and from recycled conjugated DCA) DCA (36), and in the DCA input rate and pool size (35).
Implications of Present Observations for Future Research
Colorectal cancer. Although the present studies were stimulated by our wish to understand more about the pathogenesis of cholesterol gallstones, the results may also be relevant to the role of DCA in the development of colorectal cancer (1, 22, 23). Thus, if prolonged intestinal transit and the resultant changes in colonic luminal pH and DCA metabolism are important in gallstone formation, they may be equally important in patients developing colorectal neoplasia.
Gallstone prevention. If prolonged large bowel transit and the associated increase in colonic luminal pH are important in the development of cholelithiasis, it should be possible to prevent stone formation in high-risk groups either by using regimes that accelerate colonic transit or by lowering pH in the intestinal lumen. Indeed, as indicated above, pharmacological induction of rapid transit through the colon has already been shown to reduce the percentage of DCA and the cholesterol saturation index in bile (14, 32, 33). Moreover, Thornton and Heaton (32) showed that the administration of lactulose, which is known to lower colonic luminal pH (3, 33) and also to accelerate intestinal transit (17), reduces both the percentage of DCA and the cholesterol saturation index in gallbladder bile (33).
Controlled trials are now needed to prove or disprove whether intestinal prokinetic drugs, or agents that lower colonic luminal pH, not only change bile composition but also prevent stone formation and/or reduce the incidence of colorectal neoplasia in high-risk groups. ![]() |
ACKNOWLEDGEMENTS |
---|
We thank Professor Phil Hylemon (Virginia Commonwealth University, Richmond, VA) for his valued assistance in establishing our enzyme assay techniques.
![]() |
FOOTNOTES |
---|
This work was supported, in part, by grants from the John Ellerman Foundation and Novartis Pharma, Basel, Switzerland.
This work was presented in part, in abstract form, at the 6th United European Gastroenterology Meeting, Birmingham, UK, in 1997 [Thomas LA, Bathgate T, Veysey MJ, King A, French GR, Murphy GM, and Dowling RH. Do changes in colonic luminal pH explain the increased proportions of serum and biliary deoxycholic acid seen in patients with cholesterol gallbladder stones (GBS)? (Abstract) Gut 41: A32, 1997.]
Address for reprint requests and other correspondence: R. H. Dowling, Gastroenterology Lab., 4th Floor North Wing, St. Thomas Hospital, London SE1 7EH, UK (E-mail: h.dowling{at}talk21.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 January 2000; accepted in final form 27 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bayerdorffer, E,
Mannes GA,
Richter WO,
Ochsenkuhn T,
Wiebecke B,
Kopcke W,
and
Paumgartner G.
Increased serum deoxycholic acid levels in men with colorectal adenomas.
Gastroenterology
104:
145-151,
1993[ISI][Medline].
2.
Breuer, N,
Dommes P,
Tandon R,
and
Goebell H.
Isolation and quantification of non-sulfated bile acids in the faeces.
J Clin Chem Clin Biochem
22:
623-631,
1984[ISI][Medline].
3.
Bown, RL,
Gibson JA,
Sladen GE,
Hicks B,
and
Dawson AM.
Effects of lactulose and other laxatives on ileal and colonic pH as measured by a radiotelemetry device.
Gut
15:
999-1004,
1974[ISI][Medline].
4.
Carey, MC.
Bile Acids in Gastroenterology, edited by Barbara L,
Dowling RH,
Hofmann AF,
and Roda E.. Boston, MA: MTP, 1983, p. 19-56.
5.
Dowling, RH,
and
Small DM.
The effect of pH on the solubility of varying mixtures of free and conjugated bile salts in solution (Abstract).
Gastroenterology
54:
1291,
1968[ISI].
6.
Eastwood, MA,
and
Hamilton D.
Studies on the adsorption of bile salts to non-absorbed components of diet.
Biochim Biophys Acta
152:
165-173,
1968[ISI][Medline].
7.
El Oufir, L,
Flourie B,
Bruley des Varannes S,
Barry JL,
Cloarec D,
Bornet F,
and
Galmiche JP.
Relations between transit time, fermentation products, and hydrogen consuming flora in healthy humans.
Gut
38:
870-877,
1996[Abstract].
8.
Eneroth, P.
Thin layer chromatography of bile acids.
J Lipid Res
4:
11-16,
1963
9.
Hill, MJ,
and
Drasar BS.
Degradation of bile salts by human intestinal bacteria.
Gut
9:
22-27,
1968[ISI][Medline].
10.
Hussaini, SH,
Murphy GM,
Kennedy C,
Besser GM,
Wass JAH,
and
Dowling RH.
The role of bile composition and physical chemistry in the pathogenesis of octreotide-associated gallbladder stones.
Gastroenterology
107:
1503-1513,
1994[ISI][Medline].
11.
Hussaini, SH,
Pereira SP,
Veysey MJ,
Kennedy C,
Jenkins P,
Murphy GM,
Wass JAH,
and
Dowling RH.
The roles of gallbladder emptying and intestinal transit in the pathogenesis of octreotide-induced gallbladder stones.
Gut
38:
775-783,
1996[Abstract].
12.
Igimi, H,
and
Carey MC.
pH-Solubility relations of chenodeoxycholic and ursodeoxycholic acids: physical-chemical basis for dissimilar solution and membrane phenomena.
J Lipid Res
21:
72-90,
1980[Abstract].
13.
Lewis, SJ,
and
Heaton KW.
Increasing butyrate concentration in the distal colon by accelerating intestinal transit.
Gut
41:
245-251,
1997
14.
Marcus, SN,
and
Heaton KW.
Intestinal transit, deoxycholic acid and the cholesterol saturation of bile: three interrelated factors.
Gut
27:
550-558,
1986[Abstract].
15.
Marcus, SN,
and
Heaton KW.
Effects of a new, concentrated wheat fibre preparation on intestinal transit, deoxycholic acid metabolism and the composition of bile.
Gut
27:
893-900,
1986[Abstract].
16.
Marcus, SN,
and
Heaton KW.
Deoxycholic acid and the pathogenesis of gallstones.
Gut
29:
522-533,
1988[ISI][Medline].
17.
McJunkin, B,
Fromm H,
Sarva RP,
and
Amin P.
Factors in the mechanism of diarrhea in bile acid malabsorption: fecal pHa key determinant.
Gastroenterology
80:
1454-1464,
1981[ISI][Medline].
18.
Midtvedt, T.
Microbial bile acid transformation.
Am J Clin Nutr
27:
1341-1347,
1974[ISI][Medline].
18a.
Moroi, Y,
Kitagawa M,
and
Itoh H.
Aqueous solubility and acidity constants of cholic, deoxycholic, chenodeoxycholic and ursodeoxycholic acids.
J Lipid Res
33:
49-53,
1992[Abstract].
19.
Morris, JS,
Heaton KW,
and
Read AE.
Absorption of bile acids by the colon.
Gut
11:
1063,
1970[ISI][Medline].
20.
Nair, PP,
Gordon M,
and
Reback J.
The enzymatic cleavage of the carbon-nitrogen bond in 3,7
,12
-trihydroxy-5
-cholan-24-oylglycine.
J Biol Chem
243:
7-11,
1967.
21.
Pereira SP, Hussaini SH, Cassell TB, Murphy GM, Wass JAH, and Dowling
RH. Octreotide increases the proportions of arachadonic acid-rich
phospholipids in gallbladder bile. Aliment Pharm Ther. In
press.
22.
Reddy, BS,
Narisawa T,
Weisburger JH,
and
Wynder EL.
Promoting effect of deoxycholic acid on colonic adenocarcinomas in germ-free rats.
J Natl Cancer Inst
56:
441-442,
1976[ISI][Medline].
23.
Reddy, BS,
and
Wynder EL.
Large bowel carcinogenesis: fecal constituents of populations with diverse incidence rates of colon cancer.
J Natl Cancer Inst
50:
1437-1442,
1973[ISI][Medline].
23a.
Roda, A,
and
Fini A.
Effect of nuclear hydroxy substituents on aqueous solubility and acidic strength of bile acids.
Hepatology
4, Suppl5:
72S-76S,
1984[Medline].
24.
Samuel, P,
Saypol GM,
Meilman E,
and
Mosbach E.
Absorption of bile acids from the large bowel in man.
J Clin Invest
47:
2070-2078,
1968[ISI][Medline].
25.
Schiff, ER,
Small NC,
and
Dietschy JM.
Characterization of the kinetics of the passive and active transport mechanisms for bile acid absorption in the small intestine and colon of the rat.
J Clin Invest
51:
1351-1362,
1972[ISI][Medline].
26.
Schrodt, AG,
Gibbs JA,
and
Cavanagh RE.
Quench correction by automatic external standardisation.
Adv Tracer Meth
2:
155-162,
1965.
27.
Shoda, J,
He BF,
Tanaka N,
Matsuzaki Y,
Osuga T,
Yamamori S,
Miyazaki H,
and
Sjovall J.
Increase of deoxycholic acid in supersaturated bile of patients with cholesterol gallstone disease and its correlation with de novo syntheses of cholesterol and bile acids in liver, gallbladder emptying, and small intestinal transit.
Hepatology
21:
1291-1302,
1995[ISI][Medline].
28.
Small, DM.
The physical chemistry of cholanic acids.
In: The Bile Acids: Chemistry, Physiology and Metabolism, edited by Nair PP,
and Kritchevsky D.. New York: Plenum, 1971, p. 247-354.
29.
Stellwag, EJ,
and
Hylemon PB.
7-Dehydroxylation of cholic acid and chenodeoxycholic acid by Clostridium leptum.
J Lipid Res
20:
325-333,
1979[Abstract].
30.
Thomas, LA,
King A,
French GL,
Murphy GM,
and
Dowling RH.
Cholylglycine hydrolase and 7-dehydroxylase optimum assay conditions in vitro and caecal enzyme activities ex vivo.
Clin Chim Acta
268:
61-72,
1997[ISI][Medline].
31.
Thomas, LA,
Veysey MJ,
Bathgate T,
King A,
French GR,
Smeeton NC,
Murphy GM,
and
Dowling RH.
Mechanism for the transit-induced increase in colonic deoxycholic acid formation in cholesterol cholelithiasis.
Gastroenterology
119:
806-815,
2000[ISI][Medline].
32.
Thornton, JR,
and
Heaton KW.
Do colonic bacteria contribute to cholesterol gallstone formation? Effects of lactulose on bile.
Br Med J
282:
1018-1020,
1981[ISI][Medline].
33.
Van Berge Henegouwen, GP,
van der Werf SD,
and
Ruben AT.
Effect of long term lactulose ingestion on secondary bile salt metabolism in man: potential protective effect of lactulose in colonic carcinogenesis.
Gut
28:
675-680,
1987[Abstract].
34.
Veysey MJ, Malcolm P, Mallet AI, Jenkins PJ, Besser GM, Murphy GM, and
Dowling RH. The effects of cisapride on gallbladder emptying,
intestinal transit and serum deoxycholate: a prospective, randomised,
double-blind, placebo-controlled trial. Gut. In
press.
35.
Veysey MJ, Thomas LA, Mallet AI, Jenkins PJ, Besser GM, Murphy GM, and
Dowling RH. Prolonged colonic transit increases deoxycholic acid
input and pool size, in humans. Gastroenterology. In press.
36.
Veysey, MJ,
Thomas LA,
Mallet AI,
Jenkins PJ,
Besser GM,
Wass JA,
Murphy GM,
and
Dowling RH.
Prolonged large bowel transit increases serum deoxycholic acida risk factor for octreotide-induced gallbladder stones.
Gut
44:
675-681,
1999
37.
White, BA,
Lipsky RL,
Fricke RJ,
and
Hylemon PB.
Bile acid induction specificity of 7-dehydroxylase activity in an intestinal Eubacterium species.
Steroids
35:
103-109,
1980[ISI][Medline] .
38.
Xu, Q,
Scott RB,
Tan DTM,
and
Shaffer EA.
Slow intestinal transit: a motor disorder contributing to cholesterol gallstone formation in the ground squirrel.
Hepatology
23:
1664-1672,
1996[ISI][Medline].