Influence of pH on the phase distribution of nascent deoxycholic acid in fresh human cecal aspirates

Linzi A. Thomas, Martin J. Veysey, Gerard M. Murphy, and R. Hermon Dowling

Gastroenterology Unit, Guy's Hospital Campus, Guy's, King's and St. Thomas' School of Medicine, Kings College, London, United Kingdom


    ABSTRACT
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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 × 10-7 (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
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INTRODUCTION
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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 7alpha -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
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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 7alpha -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
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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.


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Fig. 1.   Percentage of 14C-labeled deoxycholic acid (DCA) in the supernatant and precipitate phases as a function of pH.



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Fig. 2.   Percentage of 14C-labeled DCA in the supernatant and precipitate phases as a function of hydrogen ion concentration ([H+]).

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 × 10-7), 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
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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.


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RESULTS
DISCUSSION
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