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Am J Physiol Gastrointest Liver Physiol 293: G256-G263, 2007. First published April 5, 2007; doi:10.1152/ajpgi.00027.2007
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LIVER AND BILIARY TRACT

Human cecal bile acids: concentration and spectrum

James P. Hamilton,1 Guofeng Xie,1 Jean-Pierre Raufman,1 Susan Hogan,2 Terrance L. Griffin,3 Christine A. Packard,3 Dale A. Chatfield,3 Lee R. Hagey,4 Joseph H. Steinbach,4 and Alan F. Hofmann4

1Division of Gastroenterology and Hepatology, Department of Medicine, Veterans Affairs Maryland Health Care System and University of Maryland School of Medicine, Baltimore; 2Office of the Chief Medical Examiner, State of Maryland, Baltimore, Maryland; 3Department of Chemistry, San Diego State University, San Diego; and 4Division of Gastroenterology, Department of Medicine, University of California, San Diego, California

Submitted 12 January 2007 ; accepted in final form 26 March 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3{alpha}-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3{alpha}-hydroxy bile acid concentration (µmol bile acid/ml cecal content) was 0.4 ± 0.2 mM (mean ± SD); the total 3-hydroxy bile acid concentration was 0.6 ± 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 ± 9%, mean ± SD); sulfated, nonamidated bile acids were 7 ± 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 ± 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 ± 16%), lithocholic (26 ± 10%), and ursodeoxycholic (6 ± 9), as well as their primary bile acid precursors cholic (6 ± 9%) and chenodeoxycholic acid (7 ± 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 ± 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.

bile acid deconjugation; bile acid dehydroxylation; intestinal bile acids; mass spectrometry of bile acids

BILE ACIDS ARE SECRETED FROM the hepatocyte into bile in predominantly conjugated form (28) and undergo efficient enterohepatic circulation (18). A small fraction of bile acids is not absorbed by the small intestine and passes into the large intestine. These bile acids are absorbed in part from the colon; the remaining bile acids are eliminated in feces. In the large intestine, before excretion from the body, bile acids undergo complex bacterial biotransformations. The first major pathway is deconjugation resulting in the formation of unconjugated bile acids, a process that begins in the distal small intestine (33). The second major pathway in the large intestine is dehydroxylation at C-7 resulting in the formation of 7-deoxy acids. In addition, hydroxy groups may undergo dehydrogenation to form hydroxy, oxo bile acids. The oxo group may in turn be reduced to either an {alpha}- or a beta-hydroxy group (9, 38, 43). Thus, cecal bile acids should include a variety of primary bile acids (synthesized in the hepatocyte) and secondary bile acids (derived from primary bile acids by the action of bacterial enzymes on the hydroxy groups of the steroid nucleus), and bile acids should be present in both unconjugated and conjugated form.

The apparent concentration of bile acids in the cecum, i.e., the mass of bile acids per millilter of water, is determined by the input of bile acids into the cecum, loss from absorption of bile acids by the cecal epithelium, and water movements. The aqueous concentration of bile acids in the cecal lumen, i.e., the true monomeric concentration of bile acids, might be less than the apparent concentration for two reasons. First, bile acids may adsorb to food residue. Second, cecal pH is considerably more acidic than ileal pH (10), and the aqueous solubility of bile acids decreases logarithmically as pH decreases (19).

On the basis of several lines of evidence, the aqueous concentration of bile acids in the large intestine may well have physiological significance. Perfusion of the colon of human subjects (30) or that of experimental animals (2, 4, 13) with deoxycholic acid (DCA) or chenodeoxycholic acid (CDCA) in both unconjugated and conjugated forms induces colonic secretion. Secretion is also induced by these dihydroxy bile acids in vitro by using mucosal preparations of colon (11, 29, 32, 37), colonic crypts (29), or polarized colonic epithelial cells (8, 21–23). Bile acid malabsorption may induce diarrhea that is responsive to bile acid sequestrants (20). In subjects without bile acid malabsorption, ingestion of cholestyramine, a bile acid sequestrant, induces constipation (23).

Little information is available on the concentration and spectrum of cecal bile acids in the human cecum, presumably because of the difficulty of obtaining samples from subjects that have not had colonic cleansing procedures for colonoscopy. In the present work, we determined the apparent and aqueous concentration as well as the spectrum of cecal bile acids in persons dying an unnatural death.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cecal samples. Cecal samples (n = 30) were obtained during autopsy from persons who had died an unnatural death. Such persons, who had expired fewer than 24 h before autopsy, were identified by a forensic pathologist (S. Hogan) in the Office of the Chief Medical Examiner for the State of Maryland in Baltimore, MD. The causes of death were trauma (vehicular accident, fall, or crush injury), n = 10; homicide or suicide, or drug overdose, n = 15; stroke/myocardial infarction, n = 2; and other, n = 3.

Total apparent bile acid concentration. Cecal content, 1 ml, was aspirated from the cecum of 19 cadavers and mixed with 4 ml of reagent-grade isopropanol. Samples were stored at 4°C. They were thawed, shaken thoroughly, and centrifuged. One aliquot of the supernatant was used for enzymatic determination. A second aliquot was used for determination of bile acid classes by electrospray ionization mass spectrometry (ESI-MS) and gas chromatography mass spectrometry (GC-MS) in the Department of Chemistry, San Diego State University (SDSU). Cecal content, 1 ml, from an additional 11 newly deceased persons was collected and stored at –80°C. These 11 samples were used to determine the concentration of bile acids in aqueous solution.

Bile acid standards. Unconjugated bile acids were high-grade commercial samples that were further purified by adsorption column chromatography on silica gel (7). N-acylamidated (glycine- or taurine-conjugated) bile acids were synthesized in the laboratory of A. F. Hofmann as described by Tserng et al. (48) and purified by column chromatography as described (7). Sulfated bile acid standards were either synthesized in this laboratory or generously donated by T. Iida, Department of Chemistry, Nihon University, Tokyo, Japan (14). Internal standards for liquid chromatography mass spectrometry (LC-MS) 2,2',4,4'-2H-cholic acid (CA) and 2,2',4.4'-2H-deoxycholic acid (DCA) were kindly provided by Dr. Ulrich Beuers, Ludwig-Maximilian University, Munich, Germany, and Dr. Franz Stellaard, University of Groningen, Groningen, The Netherlands.

Apparent and aqueous concentration of 3{alpha}-hydroxy bile acids. The apparent concentration of 3{alpha}-hydroxy bile acids was determined enzymatically using the method described by Porter et al. (36) for fecal bile acids. Briefly, lyophilized stool (100 mg) was refluxed for 120 min in ethylene glycol-0.7 M potassium hydroxide for bile acid desorption as well as deconjugation. After cooling, the solution was acidified, and bile acids were extracted with diethyl ether. The ether extract was evaporated, the residue redissolved in methanol, and its bile acid content determined by the enzymatic procedure. On the basis of recovery of 14C radioactivity by combustion in freeze dried feces of subjects ingesting 14C-CA, the alkaline hydrolysis-ether extraction procedure used in this method gives complete recovery of fecal bile acids (36). The same is likely to be true for bile acids in cecal content.

In the second group of 11 samples, 1 ml of cecal contents was freeze-dried and reconstituted with an equal volume of distilled water. After homogenization, aqueous bile acid concentration was determined by the enzymatic procedure in the supernatant solution generated by centrifuging samples at 15,000 g for 10 min, as described by Lapré et al. (24).

Determination of bile acid classes by ESI-MS and ESI-MS/MS. ESI-MS tandem mass spectrometry (ESI-MS/MS) was performed at SDSU on a Thermo TSQ Quantum quadrapole mass spectrometer operating in the negative mode. An aliquot of the isopropanol extract of cecal samples was diluted with 0.1 M sodium bicarbonate. Bile acids were then adsorbed to a C-8 reversed phase column (Isolute C8 SPE; International Sorbent Technology, Mid Glamorgan, UK), rinsed with water, and desorbed with methanol.

Spectra were quantified by using the detector response from samples injected into a flow of methanol/water, 95:5 vol/vol, and calibration factors obtained from model mixtures, after background subtraction. The sheath gas flow was 70 and the surface induced dissociation was 30 V. The following mass-to-charge ratio (m/z) values were measured: 373, 3-oxo-cholanoic acid; 375, monohydroxy bile acids [presumably lithocholic acid (LCA) or isolithocholic acid (isoLCA)]; 391, dihydroxy bile acids (DCA, CDCA, and epimers); 407, trihydroxy bile acids (CA and epimers); 448, glycine conjugates of dihydroxy bile acids; 455, monosulfate of monohydroxy bile acids; 464, glycine conjugates of trihydroxy bile acids; 471, monosulfates of dihydroxy bile acids; 482, taurine conjugates of monohydroxy bile acids; 498, taurine conjugates of dihydroxy bile acids; and 514, taurine conjugates of trihydroxy bile acids.

ESI-MS/MS was performed at the University of California, San Diego to confirm the identity of assigned m/z values using a Perkin Elmer Sciex API-III instrument (Perkin-Elmer, Alberta, Canada) modified with a nanoelectrospray source (Protana, Odense, Denmark). Palladium-coated borosilicate glass capillaries (Protana) were used for sample injection. The instrument was operated in the negative mode with Q1 ion spray voltage set to 600 V. The interface voltage was set to 100 V and the orifice voltage to 90 V. A curtain gas of ultrapure nitrogen was pumped into the interface at 0.6 l/min to aid evaporation of solvent droplets and prevent particulate matter from entering the analyzer. Chemical identity of peaks was confirmed by the fragmentation pattern of selected ions (Q3 mode) by using argon gas, as well as by comparison with known standards. In particular, conjugates giving rise to sulfate (m/z 97), taurine (m/z 124), or glycine (m/z 74) were identified. Results by the two mass spectrometry procedures for the 14 samples having satisfactory spectra agreed quite well for unconjugated bile acids, the major constituents identified (r = 0.97).

Identification of individual bile acids by GC-MS and LC-MS. Bile acids were determined by GC-MS of the methyl ester peracetates (40). Bile acids in the unconjugated fraction were determined by derivatizing an aliquot of the isopropanol extract without a solvolysis or deconjugation step (n = 6). The acetylation procedure was shown to hydrolyze the ester bond of sulfolithocholate, suggesting that any bile acids present as 3-sulfated bile acids would likely be measured by the GC-MS procedure. Therefore only amidated bile acids would not be determined in this subset of samples. Total bile acids (n = 13), i.e., bile acids in both conjugated and unconjugated forms, were determined by performing GC-MS on the diethyl ether extract of unconjugated bile acids that are generated by the deconjugation step in the enzymatic procedure of Porter et al. (36). Because most bile acids were present in unconjugated form (see RESULTS), GC-MS results of the sets of analyses did not differ statistically and were pooled, giving GC-MS results for 19 samples.

Methyl esterification was performed using ethereal diazomethane and peracetates were prepared as described by Roovers et al. (40). Chromatography was performed on a capillary column 30 m x 0.25 internal diameter with a stationery phase of methyl polysiloxane (65%)-phenylpolysiloxane (35%) coated at 0.25-µm thickness (J & W Scientific, Folsom, CA). The gas chromatogram was a Hewlett-Packard HP-5890 coupled to a mass-selective detector Hewlett-Packard HP-5970. Compounds were identified by their retention times and fragmentation pattern based on a mass spectrometry library of some 40 bile acids.

LC-MS/MS analysis of the samples was also performed on the TSQ Quantum at SDSU. Samples prepared as described above were evaporated to dryness, then redissolved in methanol containing five internal standards [tauroursocholic acid (3{alpha}, 7beta,12{alpha}-trihydroxy-5beta-cholan-24-oyltaurine), glycolagodeoxycholic acid (3{alpha},12beta-dihydroxy-5beta-cholan-24-oylglycine), 2,2',4,4'-2H-CA, 2,2',4.4'-2H-DCA, and 7{alpha}-hydroxy-5beta-cholan-24-oic acid]; these compounds acted as retention time standards. The samples were separated on a Hypersil-Keystone BetaBasic C18 column and eluted with a gradient of neat methanol into the aqueous phase consisting of 0.1 M ammonium acetate (adjusted to pH 5.7)-methanol (8:2, vol/vol). Detection was by selected reaction monitoring, and quantification was performed against a five-point standard curve prepared for each compound; the detector response for the reference compounds varied widely. Samples were analyzed for unconjugated bile acids ursodeoxycholic acid (UDCA), CA, CDCA, DCA, and LCA as well as their corresponding taurine N-acyl amidates, glycine N-acylamidates, and sulfate conjugates. For CDCA, the 3- and 7-sulfates were measured individually. In addition, the 3beta-hydroxy epimers of the unconjugated bile acids were measured. The taurine, glycine, and sulfate conjugates of these 3beta-hydroxy epimers not measured as standards were not available. In addition, 3beta,7{alpha}-dihydroxy and 3beta,12{alpha}-dihydroxy (isoCDCA and isoDCA) had identical retention times on the C18 columns and, because the detector response for the two compounds differed, it was not possible to include these two bile acids in the analyses. Proportions of 23 analytes were determined.

Statistical analysis. Results are expressed as means ± SD. Differences between groups were tested by Wilcoxon rank sum test. Correlation coefficients were determined by using SigmaPlot Software, Systat Software, San Jose CA. Bile acids conjugated with glycine or taurine are termed amidates although a more proper name would be N-acyl amidates.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Concentration of total bile acids. The apparent bile acid concentration of 3{alpha}-hydroxy bile acids in 19 subjects as determined by the enzymatic procedure was 0.43 ± 0.19 mM. The median concentration was 0.39 mM and the range was from 0.14 to 0.93 mM. The mean aqueous concentration in 11 different samples was 0.40 ± 0.16 mM. The median value was 0.37 mM, and the range was from 0.12 mM to 0.69 mM. The difference in apparent concentration and aqueous concentration was not significant, indicating that most bile acids were in solution in cecal content.

Measurement of 3{alpha}-hydroxy bile acids by using 3{alpha}-hydroxysteroid dehydrogenase gives an underestimate of total bile acids for three reasons. The first is that 3beta-hydroxy bile acids were present, and such bile acids are not dehydrogenated by the enzyme, which acts only on 3{alpha}-hydroxy bile acids. The second is that a small fraction of bile acids is present in the form of C-3 sulfates, precluding their measurement by the enzyme. Sulfated, nonamidated bile acids averaged 7% of total bile acids determined using LC-MS, but 4 of 15 subjects had values exceeding 10% of bile acids. The third is that the enzymatic method does not measure 3-oxo bile acids. However, compounds having an m/z value of the 3-oxo derivatives of mono-, di-, and trihydroxy bile acids comprised <5% of bile acids by ESI-MS.

Using the value obtained by GC-MS for 3beta-hydroxy bile acids, we calculated the total 3-hydroxy bile acid concentration ([BA]3-hydroxy) by using the equation [BA]3-hydroxy = [BA]enzymatic/(1 – fraction of 3beta-hydroxy bile acids in total bile acids), where [BA]enzymatic is the concentration of bile acids determined by the enzymatic procedure.

The total 3-hydroxy bile acid concentration was 0.6 ± 3 mM. Figure 1 shows 3{alpha}-hydroxy (enzymatic method) and total 3-hydroxy (enzymatic plus 3beta-hydroxy bile acids) apparent bile acid concentrations. The true total bile acid concentration is still slightly higher because of the small fraction of 3-sulfated bile acids that was present.


Figure 1
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  		Fig. 1. Comparison of 3{alpha}-hydroxy bile acid concentration as determined by the enzymatic method (left) and total (unsulfated) bile acid concentration (3{alpha}-hydroxy bile acids plus 3beta-hydroxy bile acids) as determined by gas chromatography mass spectrometry (right).

 
Bile acid classes. By ESI-MS, ESI-MS, and LC-MS, most bile acids were in unconjugated form. Data are summarized in Tables 1 and 2. The median value by ESI-MS was 92% for unconjugated bile acids, 8% for sulfated nonamidated bile acids, and 1% for amidated, nonsulfated bile acids. Individual values for the three major bile acid classes are illustrated in Fig. 2 by using triangular coordinates. Similar values were obtained by LC-MS/MS (Table 2).


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  		Table 1. Bile acid class and group composition (percent total) of human cecal contents by ESI-MS and ESI-MS-MS

 

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  		Table 2. Bile acid composition of human cecal contents by LC-MS

 

Figure 2
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  		Fig. 2. Relative proportions of bile acids groups for individual samples of cecal content as determined by electrospray ionization mass spectrometry and electrospray ionization mass spectrometry tandem mass spectrometry. Most bile acids are in unconjugated form.

 
The distribution of individual bile acid classes is also presented in Table 1. The most abundant class was dihydroxy bile acids (60%). Monohydroxy bile acids (15%) and trihydroxy bile acids (14%) were the second most prevalent classes. Sulfated, nonamidated bile acids were present in all samples, averaging 9 ± 8%. However, there was much between-subject variation with use of LC-MS; some samples had little sulfation, whereas 3 of 15 subjects had 13% of bile acids in sulfated form, and in one subject 40% of bile acids were sulfated. As shown in Fig. 3, the proportion of CDCA that was sulfated exceeded that of DCA (P = 0.02). By LC-MS, sulfation of CDCA was exclusively at carbon 3, i.e., no 7-sulfates of CDCA were present. The proportion of bile acids in sulfated form determined by ESI-MS correlated highly with that obtained by LC-MS (r = 0.90). The mass spectrum of the subject with 13% dihydroxy sulfates is shown in Fig. 4, which also shows a spectrum from a sample lacking sulfated bile acids. Monosulfates of trihydroxy bile acids were not present. C27 bile acids also were not present in appreciable proportions. On the basis of m/z values for the parent compounds as well as the MS/MS chromatograms for the precursors of sulfate, sulfated amidates of LC were not present. However, the m/z value corresponding to the doubly charged anion of sulfolithocholylglycine (m/z = 256) and m/z value of sulfolithocholyltaurine (m/z = 256) could not be quantified because of interference by fatty acid anions. The m/z value for the monosulfate of dihydroxy taurine amidates (m/z = 289) was present in only one sample and was 7% of total bile acids.


Figure 3
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  		Fig. 3. Relative proportions of bile acids as determined by liquid chromatography mass spectrometry. Left: percentage of deoxycholic acid (DCA, unconjugated) and 3-sulfo-DCA. Right: percentage of chenodeoxycholic acid (CDCA, unconjugated) and 3-sulfo-CDCA.

 

Figure 4
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  		Fig. 4. Two representative mass spectra. Mass-to-charge ratio (m/z) values for peaks corresponding to bile acids are shown. Top: peak A, m/z 375, monohydroxy bile acids; peak B, m/z 391, dihydroxy bile acids; peak C, m/z 407, trihydroxy bile acids, and peak D, m/z 471, monosulfated dihydroxy bile acids. Bottom: peak A, m/z 391, dihydroxy bile acids; peak B, m/z 407, trihydroxy bile acids, and peak C, m/z 471, monosulfated dihydroxy bile acids. The peak at 453 has an m/z value corresponding to the sulfate of an unsaturated monohydroxy bile acid, but by mass spectrometry tandem mass spectrometry at least 2 nonbile acids are present in the peak, one of which is a sulfate. In most samples, a peak at m/z 453 was present in only trace proportions. Peaks at m/z 471, 521, and 583 are not bile acids.

 
Individual bile acids by GC-MS. Results of GC-MS of the unconjugated bile acid fraction of cecal content are summarized in Table 3, and typical gas chromatograms are shown in Fig. 5. The dominant bile acids were DCA, the 7-deoxy derivative of CA and LCA, the 7-deoxy derivative of CDCA. CDCA was present in 11 of 19 samples and CA was present in 9 of 19 samples. UDCA, the 7beta-hydroxy epimer of CDCA, was present in 7 of 19 samples. In addition, the 3beta-hydroxy derivatives ("iso" bile acids) of LCA, CDCA, UDCA, and CA were present in 18 of 19 samples. Such 3beta-hydroxy ("iso") bile acids constituted 26 ± 32% of bile acids (median, 18%; range 0–58%). The 3beta-hydroxy epimers of LCA, DCA, and CDCA were greater in proportion than those of UDCA and CA. Thus isoLCA was present in 13 of 19 samples, isoDCA was present in 14 of 19 samples, and in 10 of 19 samples in which CDCA was present, 9 contained isoCDCA. In contrast, only 2 of 7 UDCA samples contained isoUDCA, and only 1 of 9 samples containing CA had isoCA.


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  		Table 3. Bile acid composition (percent total) of human cecal contents by GC-MS

 

Figure 5
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  		Fig. 5. Representative gas chromatograms of cecal bile acids, chromatographed as their methyl ester peracetate derivatives. Labeled peaks indicate bile acids identified by their mass spectra. Unlabeled peaks are not bile acids. Left: peak A, isolithocholic acid (isoLCA); peak B, lithocholic acid (LCA); peak C, isodeoxycholic acid (isoDCA) plus nonbile acid contaminant; peak D, DCA; peak E, isochenodeoxycholic acid (isoCDCA); peak F, CDCA; peak G, isocholic acid (isoCA) (second half of peak); peak H, CA. Right: peak A, isoLCA; peak B, LCA; peak C, isoDCA; peak D, DCA; peak E, isoCDCA; peak F, CDCA; peak G, isoCA; peak H, CA; peak I, ursodeoxycholic acid; and peak J, hyocholic acid (internal standard).

 
No other bile acids were identified except for one sample that contained <5% of the 12 oxo (keto) derivative of isodeoxycholic acid.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To the best of our knowledge, the data presented in this paper are the first describing the concentration and spectrum of bile acids in the human cecum. In the present study, GC-MS analyses of cecal samples indicated that 3beta-hydroxy bile acids were usually present. These averaged 26% of bile acids and varied in proportion from 0 to 56%. In addition, in a few subjects, a considerable proportion of bile acids was present in the sulfated form. Thus the enzymatic measurement of 3{alpha}-hydroxy bile acids gives a variable underestimate of the total apparent bile acid concentration in cecal contents.

Postprandial bile acid concentration in the human distal ileum averages 2 mM (33), whereas postprandial bile acid concentration in the proximal small intestine is considerably higher, averaging 10 mM (35). Most bile acids secreted into the small intestine during digestion are absorbed in the distal ileum. Therefore, the decrease in the luminal concentration of bile acids in the distal ileum by a factor of five indicates that here bile acid absorption is more efficient than water absorption. The same appears to be true in the cecum, as bile acid concentration here decreases by a factor of three.

The analyses showed that most cecal bile acids are present in unconjugated form. Deconjugation of bile acids begins in the distal small intestine (32), and the state of conjugation of bile acids crossing the ileocecal valve is not known. LCA, which is secreted into bile in part in sulfated, amidated form (41), was present in cecal samples largely in unconjugated form.

Although 3beta-hydroxy bile acids have been identified previously in fecal bile acids (9, 43), they are not generally considered in discussions of the enterohepatic circulation of bile acids. From their physicochemical properties, i.e., number of hydrogen bonding sites, isoLCA, isoDCA, isoCDCA, and isoUDCA are likely to be rapidly absorbed from the colon (1, 16, 39, 42). In agreement with this view, studies by Shefer et al. (44) showed that isoCDCA and isoUDCA are rapidly and efficiently absorbed from the intestine. Animal studies (6, 25, 44) and recent human studies (26, 27) indicate that 3beta-hydroxy bile acids are efficiently reepimerized to the physiologically occurring 3{alpha}-hydroxy bile acids during hepatocyte transport. Therefore, our results suggest strongly that 3beta-hydroxy bile acids are continuously absorbed from the colon and reepimerized during hepatocyte transport. This hidden pathway in the enterohepatic circulation of bile acids is analogous to the deconjugation-reconjugation pathway for conjugated bile acids, another hidden pathway known to occur in animals and humans (18). The 3-sulfate of isoDCA has very recently been shown to be a major urinary bile acid in humans (15), indicating that 3beta-hydroxy bile acids can also be sulfated and eliminated via the kidney. We did not quantify this bile acid sulfate, because a standard was not available to us when this work was performed.

Four subjects had a relatively high proportion of sulfated bile acids by LC-MS. As noted above, the sulfate of CDCA was predominantly 3-sulfo-CDCA. Because the 3-sulfo conjugates of DCA and CDCA are not present in bile in appreciable proportions (45, 46), it seems likely that these sulfated bile acids were formed in the intestine. We speculate that DCA and CDCA are formed by bacterial deconjugation in the distal small intestine. They are then absorbed by enterocytes and sulfated. The sulfated bile acid was then effluxed into the intestinal lumen. The efflux pump MRP2 is known to transport sulfated bile acids (31) and has been shown to be present in the human intestinal epithelium (12, 49). Both the small intestine and large intestine possess sulfotransferase activity, although there is considerable variability in the site of expression in the intestine as well as between individual subjects (5). Most studies of sulfated bile acids in feces have given values <10% (3, 47).

As noted in the introduction, the concentration and spectrum of cecal bile acids can be influenced by multiple factors and the analyses reported here represent the momentary composition of one sample of cecal content. A major limitation of our sample source is that postmortem changes may have occurred and that some subjects may have been ingesting antibiotics or other drugs that could have influenced the cecal bile acid profile. We elected not to use brain-dead tissue donors, because such patients commonly receive multiple antibiotics and antibiotic exposure could alter the cecal flora. Presumably the bile acid spectrum and concentration is altered by increased entry of bile acids across the ileocecal valve during digestion. Whether subjects were eating or fasting in our study is not known. Nor is it known whether cecal content is well mixed.

In this study, we used three complementary analytical methods. ESI-MS measures the apparent amount of compounds having m/z values corresponding to those of expected bile acids and their corresponding conjugates. It is a highly sensitive method, but is not specific because a given m/z value could contain compounds other than bile acids. GC-MS provides information on 3beta-hydroxy bile acids that cannot be determined by ESI-MS or LC-MS, because the 3beta-hydroxy epimers of DCA and CDCA have the same m/z values as their 3{alpha}-hydroxy epimers and the same retention time by LC. LC-MS permits individual compounds such as sulfated bile acids to be identified by both their retention time and their m/z values.

Despite these limitations, we believe that some meaningful conclusions can be drawn from these data. The first is that cecal bile acids are largely in unconjugated and 7-dehydroxylated form. Deconjugation and dehydroxylation render bile acids membrane-permeable, and their concentration falls as a result of absorption from the cecum. The second is that most cecal bile acids are in solution despite the acidic pH of the cecum (10) and the low aqueous solubility of protonated mono- and dihydroxy bile acids (19). The third is that cecal bile acids, in contrast to small intestinal bile acids, are present at a concentration below their critical micellization concentration. The critical micellization concentration of bile acids during lipid digestion has been estimated to be 1–2 mM (17). If micelles are not present, absorption of insoluble lipids from cecal content, for example toxic bacterial lipids, cannot occur. The fourth is that CDCA and DCA are present at a concentration well below that inducing secretion in the perfused human colon (30). In health, the colon absorbs ~90% of the fluid that enters it from the terminal ileum (34). Therefore, it is quite reasonable that bile acids are present at concentrations that do not induce secretion. Fifth, in some patients, an appreciable fraction of dihydroxy bile acids (DCA and CDCA) is present as the nonamidated 3-sulfate and is likely to be formed in distal intestinal enterocytes. Lastly, 3beta-hydroxy bile acids are present in cecal contents and are likely to be absorbed, returned to the liver, and reepimerized during hepatocyte transport.

The present data also provide values for previously healthy subjects and may be useful for comparison with the bile acid spectrum present in disease states such as bile acid malabsorption, inflammatory bowel disease, or colon cancer.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Work at the University of Maryland was supported by National Institutes of Health (NIH) Grants T32 DK-067872 (J. P. Hamilton and G. Xie) and CA-107345 (J.-P. Raufman), and by the Office of Research and Development, Department of Veterans Affairs. Work at the University of California, San Diego and SDSU was supported in part by NIH Grant DK-64891 (A. F. Hofmann).


   ACKNOWLEDGMENTS
 
Present address for J. P. Hamilton: Division of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, MD.


   FOOTNOTES
 

Address for reprint requests and correspondence regarding general aspects of the study: J.-P. Raufman, Univ. of Maryland School of Medicine, 22 S. Greene St., N3W62 Baltimore, MD 21201 (e-mail: jraufman{at}medicine.umaryland.edu). Address for correspondence regarding bile acid metabolism: A. F. Hofmann, Univ. of California, San Diego, San Diego, La Jolla, CA 92093-0813 (e-mail: ahofmann{at}ucsd.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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