The site-specific delivery of ursodeoxycholic acid to the rat colon by sulfate conjugation

GASTROENTEROLOGY 1995;109:1835-1844

The Site-Specific Delivery of Ursodeoxycholic Acid to the Rat Colon by Sulfate Conjugation CECILIA M. P. RODRIGUES,* BETSY T. KREN,* CLIFFORD J. STEER, t and KENNETH D. R. SETCHELL* *Department of Pediatrics, Clinical Mass Spectrometry Center, Children's Hospital Medical Center, Cincinnati, Ohio; and ~Division of Gastroenterology, Department of Medicine, University of Minnesota, Minneapolis, Minnesota

See editorial on page 2036.

Background & Aims: Because ursodeoxycholate has been shown to act as a tumor-suppressive agent in the colon, the absorption and metabolism of its sulfate conjugates were examined in rats to show that sulfation would facilitate the site-specific delivery of ursodeoxycholate to the colon. Methods: Bile acids were measured in intestinal contents, feces, urine, plasma, and liver tissue after oral administration of ursodeoxycholate and its 0-3, C-7, and 0-3,7 sulfate derivatives. Results: Ursodeoxycholate was found in the jejunum after administration of all bile acids, but the mass was greatest for ursodeoxycholic acid administration. In the colon, lithocholic acid, normally found in negligible amounts, became the major bile acid after ursodeoxycholate administration. In contrast, reductions in mass and proportions of lithocholate and deoxycholate occurred after administering the C-7 sulfates. The fecal lithocholate/deoxycholate ratio, a risk marker for colon cancer, increased markedly after administration of ursodeoxycholate and its 0-3 sulfate, but did not change after administering the C-7 sulfates. Unlike ursodeoxycholate or its 0-3 sulfate, which increased liver concentrations of lithocholate and ursodeoxycholate, the C-7 sulfates had the opposite effect, which was consistent with poor absorption. Conclusions: Sulfation of ursodeoxycholate, specifically at the 0-7 position, protects the molecule from bacterial degradation and inhibits its intestinal absorption, thereby facilitating delivery to the colon.

'rsodeoxycholic acid (UDCA) has been used clinically for more than 2 decades, initially proving effective for the treatment of patients with cholelithiasis 1 and more recently showing promise in the treatment of patients with cholestatic liver diseases. 2-~3 It is well established that oral administration of UDCA leads to a significant improvement in serum liver enzymes, and based on results from long-term clinical trials, the consensus opinion is that UDCA is beneficial for the treat-

U

ment of early-stage primary biliary cirrhosis. ~4 Despite the wealth of published data relating to the effects of UDCA, the mechanism of action of this drug remains unclear. However, it is apparent that UDCA has a wide range of effects that, consequently, have led to investigations of its potential use in extrahepatic diseases. In this respect, data are emerging from animal models of colonic carcinogenesis that suggests a protective role for UDCA. 15-1v In the course of examining the metabolic fate of UDCA, we have shown that, at the usual therapeutic doses administered orally (10-15 mg" kg body wt -~" day-i), this hydrophilic bile acid is absorbed from the intestine and efficiently biotransformed in the liver mainly by conjugation. As a consequence, negligible quantities of unconjugated UDCA are secreted in bile 18a9 or found in the liver tissue of rats administered UDCA. 2° These facts led us to question whether the beneficial effects of UDCA are perhaps mediated via its conversion to more hydrophilic conjugated bile acids. In healthy subjects and in patients with cholestatic liver disease, UDCA sulfate is a specific urinary metabolite of orally administered UDCA. 21 W e have shown recently in rats that UDCA sulfates reduce lipid secretion and have greater choleretic activity than UDCA (Setchell and Yamashita, unpublished data), suggesting that these conjugates may be more useful for treating liver disease. However, because bile acid sulfates are generally poorly absorbed from the intestine, = 24 this may be a limiting factor in delivery to the liver. Nevertheless, the highly hydrophilic nature of UDCA sulfates indicates that they may be ideal cytoprotective agents, and because UDCA has been shown to reduce the incidence of colonic tumors in animal models 16'Iv and, consequently, is being investigated as a tumor-suppressive agent, it may be of distinct advantage to prevent intestinal absorption of UDCA. Abbreviations used in this paper: GC-MS, gas chromatographymass spectrometry. © 1995 by the American Gastroenterological Association 0016-5085/95/$3.00

1836

RODRIGUES ET AL.

To our knowledge, the behavior of highly hydrophilic bile acid sulfates has not been studied previously; however, we hypothesized that the substitution of a sulfate moiety to the U D C A molecule would facilitate the sitespecific delivery of U D C A to the colon, where it may be of potential benefit in treating intestinal diseases. This article compares the intestinal metabolism and behavior of the individual bile acid sulfates of U D C A with the unconjugated bile acid and shows that the presence of the C-7 sulfate moiety protects against bacterial degradation and inhibits intestinal absorption of UDCA.

M a t e r i a l s and M e t h o d s Synthesis of Sulfated Esters of UDCA UDCA (99% pure) was obtained from Sigma Chemical Co. (St. Louis, MO). The monosulfate esters of UDCA were prepared by an improvement of the method adopted from Goto et al. 25 and described in detail elsewhere (Setchell and Yamashita, unpublished data). The synthesis involves selective protection of each of the ring hydroxyl groups in the UDCA molecule, followed by sulfation of the unprotected hydroxyl group and hydrolysis of the protecting group to release the monosulfate ester. The disulfate conjugate of UDCA was prepared by the reaction of UDCA with chlorosulfonic acid. Gas chromatography-mass spectrometry (GC-MS) was used to confirm the position of the sulfate groups by analysis of the products after oxidation, 26 solvolysis,27 and conversion to methyl ester-trimethylsilyl ether derivatives. 28'29 Chromatographic purity of the synthetic bile salts was found to be >97% for UDCA 7-sulfate and UDCA 3,7-disulfate and >95% for UDCA 3-sulfate, as determined by high-pressure liquid chromatography, thin-layer chromatography, and capillary-column gas chromatography (Setchell and Yamashita, unpublished data). Animal Studies

Male Sprague-Dawley rats (Harlan Sprague-Dawley Inc., Indianapolis, IN) weighing 210-290 g were maintained on a 12-hour light-dark cycle and fed standard laboratory chow ad libitum for 3 days. The animals were then transferred to metabolic cages in which they were housed individually and fed the same diet. UDCA, UDCA 3-sulfate, UDCA 7-sulfate, and UDCA 3,7-disulfate were administered by garage at a dose of 250 mg/day for 4 consecutive days. Each group comprised 3 - 6 rats. Body weights of the animals were measured each day. On day 5, the animals were killed by exsanguination under ether anesthesia. Plasma was collected and frozen at -20°C. The liver was removed, rinsed in normal saline, and flash-frozen in liquid nitrogen. The intestine was removed, divided into four sections (jejunum, ileum, cecum, and colon), and fash-frozen in liquid nitrogen. Urine and feces specimens were collected every 24 hours and frozen at -20°C. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the National

GASTROENTEROLOGY Vol. 109, No. 6

Academy of Sciences (National Institutes of Health publication no. 86-23, revised in 1985). Bile Acid A n a l y s i s Unconjugated and sulfate bile acids in intestinal contents and feces. Intestinal contents were weighed and dissected into small pieces. In each group, feces specimens (100 mg) from all animals were pooled on day 4 of the study and then ground into a fine paste. All samples were sonicated and sequentially refluxed in 80% methanol for 2 hours and chloroform/methanol (1:1) for 1 hour. 3° Samples were taken to dryness, and the dried extracts were resuspended in 80% methanol (20 mL). Fractions of the methanolic extract (1/40 of intestinal contents and 1/20 of feces samples) were removed and the internal standard nordeoxycholic acid (10 btg) was added. This extract was diluted with 0.01 mol/L acetic acid (20 mL) and passed first through a column of Lipidex 1000 (bed size, 4 × 1 cm; Packard Instrument Co., Groningen, The Netherlands) and then through a Bond-Elut C18 cartridge (Analytichem, Harbor City, CA). Bile acids were recovered by elution of the Lipidex 1000 column and the Bond-Elut cartridge with methanol (20 mL and 5 mL, respectively), and the combined extracts were taken to dryness. Unconjugated bile acids were isolated and separated from neutral sterols and conjugated bile acids by lipophilic anion-exchange chromatography on diethylaminohydroxypropyl Sephadex LH-203I (Lipidex-DEAP; Packard Instrument Co.). Recovery of unconjugated bile acids was achieved by elution with 0.1 mol/L acetic acid in 72% ethanol (7 mL) followed by evaporation of the solvents. Total conjugated bile acids were recovered with 9 mL of 0.3 mol/L acetic acid in 72% ethanol, pH 9.6. Salts were removed by passage through a Bond-Elut C** cartridge after addition of nordeoxycholic acid (10 btg), and conjugated bile acids were recovered by elution with 5 mL of methanol. Solvolysis was performed in a mixture of methanol (1 mL), distilled tetrahydrofuran (9 mL), and 1 mol/L trifluoroacetic acid in dioxane (0.1 mL) and heated to 45°C for 2 hours. 27 After solvolysis, unconjugated bile acids were isolated by chromatography on Lipidex-DEAP. Methyl ester derivatives were prepared by dissolving the sample in methanol (0.3 mL) and reacting with 2.7 mL of freshly distilled ethereal diazomethane. 2s After evaporation of the reagents, the methyl ester derivatives were converted to trimethylsilyl ethers by the addition of 50 ~tL of Tri-Sil reagent (Pierce Chemicals, Rockford, IL). A column of Lipidex 5000 (Packard Instrument Co.) was used to remove derivatizing reagents and to purify the sample. 29 Unconjugated and sulfate bile acids in plasma and urine. In each group, urine specimens from all animals were pooled on the individual days of collection. Day 1 represented the baseline sample, and samples from days 2, 3, and 4 were obtained during bile acid administration. After the addition of nordeoxycholic acid (1 /.tg), bile acids were quantitatively extracted from portions of urine (3 mL) and plasma (1 mL) using Bond-Elut C18 cartridges. 32 After liquid-solid extraction, isolation and separation of unconjugated and conjugated bile acids were achieved by lipophilic anion-exchange chromatogra-

December 1995

phy on Lipidex-DEAP. Solvolysis of bile acid conjugates and isolation of the hydrolyzed products were performed as described above, and bile acids were converted to volatile methyl ester-trimethylsilyl ether derivatives. Extent of bile acid conjugation in liver and jejunal contents. Samples of liver (100 mg) were ground to a fine

paste, and bile acids were extracted by reflm~ and passage through a column of Lipidex 1000 as described above for intestinal contents and feces. Group separation of bile acids, according to their mode of conjugation, was achieved by lipophilic anion-exchange chromatography. 3. Extracts from each animal were pooled, and bile acids and their conjugates were separated using Lipidex-DEAP and stepwise elution of the gel bed with the following buffers: 0.1 mol/L acetic acid in 72% ethanol (unconjugated bile acids); 0.3 mol/L acetic acid in 72% ethanol, pH 5.0 (glycine conjugates); 0.15 mol/L acetic acid in 72% ethanol, pH 6.5 (taurine conjugates); and 0.3 mol/L acetic acid in 72% ethanol, pH 9.6 (sulfate conjugates). After evaporation of the buffers, sulfated bile acids were solvolyzed, 2v whereas amidated conjugates were hydrolyzed with 50 U of cholylglycine hydrolase (Sigma Chemical Co.) in 2.5 mL of 0.2 mol/L phosphate buffer, pH 5.6, at 37°C overnight. 33 The resulting unconjugated bile acids were isolated on LipidexDEAP, and the methyl ester-trimethylsilyl ether derivatives were prepared as described above. After sotvolysis, the amidated bile acids from the jejunal portion of the rat intestine were isolated on Lipidex-DEAP by elution with 0.15 mol/L acetic acid in 72% ethanol, pH 6.5 (6 mL). Enzymic hydrolysis was performed as described above, 33and the resulting unconjugated bile acids were isolated and derivatized as described above. GC-MS. The methyl ester-trimethylsilyl ether derivatives were separated on a 30 m X 0.25 mm DB-l-fused silica capillary column (J & W Scientific, Folson, CA) using a temperature program from 225°C to 295°C with increments of 2°C/min and a final isothermal period of 30 minutes. GC-MS analysis was performed using a Finnigan 4635 mass spectrometer (Finnigan Inc., San Jose, CA) that housed an identical gas chromatography column operated under the same conditions. Electron ionization (70 eV) mass spectra were recorded over the mass range of 50-800 daltons by repetitive scanning of the eluting components. Identification of bile acids was made on the basis of the gas chromatography retention index relative to a homologous series of n-alkanes, referred to as the methylene unit value, and the mass spectrum compared with authentic standards. 34 Quantification of bile acids was achieved using gas chromatography by comparing the peak height response of the individual bile acids with the peak height response obtained from the internal standard. Liquid secondary ionization mass spectrometry. Liquid secondary ionization mass spectrometry negative ion spectra of urine samples were obtained after placing approximately 1 ~L of the methanolic extract onto a small drop of a glycerol/ methanol matrix spotted on a stainless steel probe. This probe was introduced into the ion source of a VG Autospec Q mass spectrometer (Fisons Instruments, Manchester, England), and

SULFATE CONJUGATES OF URSODEOXYCHOLATE 1837

a beam of fast atoms of cesium, generated from cesium iodine (35 KeV), was fired at the target containing the sample. Negative ion spectra were obtained over the mass range of 50-1000 daltons.

Statistical Analysis Data are expressed as mean + SEM or as mean values of all animals when extracts were pooled before analysis. Resuits from different groups were compared using paired and unpaired two-tailed Student's t test. P values of <0.05 were considered statistically significant.

Results Intraluminal Bile Acid Composition Along the Intestine The average weight of the resected segments of intestine for the animals in the control group and those administered the individual bile acids were similar. In control animals, the total amount of U D C A in the jejunum (0.03 - 0.01 mg) was negligible, accounting for only 0.3% of the total bile acids. However, the total mass of U D C A (and its percentage of the total bile acids) in the jejunum was greater in all animals 24 hours after the administration of the final dose of UDCA, U D C A 3-sulfate, U D C A 7~sulfate, and U D C A 3,7-disulfate, accounting for 9.95 -+ 0.49 m g (35.9%), 3.67 --- 0.45 mg (16.4%), 1.09 + 0.34 m g (4.7%), and 0.21 ___ 0.07 mg (2.0%), respectively. This result indicates very little conservation of U D C A when a sulfate group is conjugated in the C-7 position (Table 1). A22-UDCA, a specific metabolite of exogenously administered UDCA, 35 was detected in large proportions and amounts in the jejunum of animals administered U D C A and U D C A 3-sulfate. However, this metabolite was not detected in the control group and accounted for < 3 % of the total jejunal bile acids of the animals administered the C-7 sulfate conjugates of U D C A (Table 1). W h e n the conjugation pattern for U D C A in the jejunum was examined after separation of the bile acids by lipophilic anion-exchange chromatography (Figure 1), animals administered U D C A were found to have predominantly glyco- and tauro-UDCA, smaller amounts of unconjugated UDCA, and negligible amounts of sulfated UDCA. Although amidated and unconjugated forms of U D C A were found in the jejunum after administration of the C-7 sulfates, the total mass of U D C A in the jejunum was very small. In rats administered U D C A 3sulfate, the jejunum contained substantial proportions of amidared UDCA, indicating significant biotransformation by deconjugation and/or amidation. W i t h regard to endogenous bile acids, cholic acid was

1838

RODRIGUES ET AL.

GASTROENTEROLOGY Vol. 109, No. 6

Table 1, Mass and Percent Composition of the Principal Bile Acids in Jejunum From Control Rats and After Oral Administration of UDCA, UDCA 3-Sulfate, UDCA 7-Sulfate, or UDCA 3,7-Disulfate, Determined by GC-MS Control

UDCA 3-sulfate

UDCA 7-sulfate

UDCA 3,74Jisulfate

Mass

Percent

Mass

Percent

Mass

Percent

Mass

Percent

Mass

Percent

( mg)

(%)

( mg )

(%)

( mg)

(%)

( mg)

(%)

(rag)

(%)

Uthocholic acid Deoxycholic acid Chenodeoxycholic acid cz-Muricholic acid Cholic acid UDCA

0.02 ± 0,01 0.50 ± 0.03

&~2-UDCA

ND 0.64 ± 0.18

13-Muricholic acid &a2-~-Muricholic acid ~Muricho]ic acid

UDCA

0.25 1,48 6.82 0.03

_+ 0.05 + 0,24 + 1.10 _+ 0.01

0.2 _+ 0.1

0.26 _+ 0.01

1.0 -+ 0.1

0.07 + 0.01

0,3 + 0.1

0.01 + 0.01

0,1 _+ 0.1

ND

4.8 + 1.1

1.56 _+ 0.18

5,6 + 0.3

1.96 _+ 0.68

8.4 ± 1.5

0.76 _+ 0.08

3.3 _+ 0.1

0.53 _+ 0.05

48.5 ~+ 2.9 0.3 + 0.2 0

0.98 0.75 4.68 9.95 4.00 4.12

__+0.06 ± 0,02 ± 0.65 ± 0.49 ± 0.05 ± 0,25

0.43 0.18 8,49 3.67 2.04 3,02

16.8 ± 1.3 35.9 ± 0.4 14.5 ± 0.7

± ± ± ± + ±

0.10 0.02 1,22 0.45 0.08 0.80

37.7 ± 0,9 16.4 _+ 0.5 9.2 + 1.0

0.26 0.04 15,78 1,09 0.70 2.21

± ± + + + ±

0.06 0.01 1.30 0.34 0.05 0,17

0.12 ± 0.04 ND 6,78 + 1.03 0.21 + 0.07 0.21 ± 0.03 1,70 _+ 0.14

69.0 + 0.6 4,7 + 1.3 3.1 ± 0.1

3,44 + 0,57 0.10 _+ 0.01

0,85 ± 0.01 0.31 ± 0.01

2.09 __+0,16 0.37 __+0.03

1,49 + 0.22 0.31 __+0.04

0.34 + 0.09 0.24 ± 0.02

0,56 _+ 0.08 4.04 ± 2.40

0.30 +__0.01 27.77 __+1.69

0.24 + 0.01 22.57 ± 3.53

0.26 ± 0.01 22.91 ± 2.11

0,12 + 0,01 10,24 + 1.37

0 5.2 _+ 0,3

66.0 _+ 1.3 2.0 + 0.4 2.1 ± 0 . t

A22-o>Muricholic

acid Total

NOTE. Results are expressed as mean _+ SEM,

ND, not detected.

the major bile acid in the jejunum of the control animals, accounting for 48.5% + 2.9% (6.82 _+ 1.10 mg) of the total bile acids, and after UDCA administration, there was a significant decrease (P = 0.01) to 16.8% 4- 1.3% (4.68 + 0.65 mg). UDCA 3-sulfate also caused a decrease (P = 0.05) in the proportion ofcholic acid but to a lesser extent than UDCA, whereas administration of the C-7 sulfates of UDCA caused an increase (P = 0.01) in the proportion of cholic acid in the jejunum (Table 1). In the colon of the control animals, most of the bile acids were secondary and were identified mainly as deoxycholic and m-muricholic acids, but only small amounts of lithocholic acid were detected (Table 2). UDCA administration caused a decrease (P = 0.003) in the proportion of deoxycholic acid, and lithocholic acid became the

10

g E 3

A

8

Fecal Bile Acid Excretion No significant differences were found in the weight of feces excreted each day among the groups of animals. Total fecal bile acid excretion in animals administered UDCA, UDCA 3-sulfate, UDCA 7-sulfate, and UDCA disulfate was 14.85, 10.70, 12.65, and 10.88 mg/g feces, respectively. The feces of all animals became enriched with UDCA; however, for those animals administered the unconjugated bile acid, UDCA was almost

120 UDCA

E 100 >

:E 6 ¢,-

121 4 D

~

major bile acid present, accounting for 32.0% + 0.8% of the total colonic bile acids (P = 0.0004). In contrast, UDCA 7-sulfate and UDCA 3,7-disulfate administration led to substantial reductions in the mass and proportion of both deoxycholic acid and lithocholic acid in the colon.

=E

80

< 0 r~

60

B

UDCA

"6 U DCA-3S

8

40

~ 8

0

2 0 CONTROLt

UDCA-7S ==~UDCA-DS

00NTROL ~

m

UDCA-DS t~

Figure 1. Comparison of the (A) total mass of UDCA in the entire jejunum and (B) concentration in liver tissue in control rats and after oral administration of UDCA, UDCA 3-sulfate (UDCA-3S), UDCA 7-sulfate (UDCA-7S), and UDCA 3,7-disulfate (UDCA-DS). Bile acids were separated according to their mode of conjugation by anion-exchange chromatography before analysis of the individual fractions using GC-MS. Results are expressed as the mean values of all animals. 0, Unconjugated; [], glycine + taurine; I , sulfate.

December 1995

SULFATE CONJUGATES OF URSODEOXYCHOLATE 1839

Table 2. M a s s and Percent C o m p o s i t i o n of t h e Principal Bile Acids in Colon From Control Rats and After Oral Administration of UDCA, UDCA 3-Sulfate, UDCA 7-Sulfate, or UDCA 3,7-Disulfate, Determined by GC-MS Control

Lithocholic acid Deoxycholic acid Chenodeoxycholic acid c~-Muricholic acid Cholic acid UDCA A~2-UDCA ~-Muricholic acid A22-~-Muficholic

UDCA 3-sulfate

UDCA 7-sulfate

UDCA 3,7-disulfate

Mass

Percent

Mass

Percent

Mass

Percent

Mass

Percent

Mass

Percent

( mg )

(%)

(mg )

(%)

(mg )

(%)

(rag)

(%)

( mg)

(%)

0.08 + 0.02 0.94 +_ 0.34

3.4 ± 0.2 32.3 _+ 0.8

0.97 _+ 0.43 0.31 + 0.13

32.0 - 0,8 10.4 + 0.9

0.61 ± 0.19 0.58 +_ 0.07

18,4 ± 7.2 17.4 + 3.3

0,04 ± 0~02 0,36 + 0.16

1,3 ± 0.3 11,4 ± 2.1

0.02 ± 0.10 0.31 -- 0.04

0.03 0.15 0,47 0.02

± 0.01 ± 0.05 _+ 0.12 + 0.01 ND 0.31 ± 0.06

acid m-Muricholic acid ~=2-m-Muricholic acid Total

UDCA

15.5 + 1.7 0,6 + 0.2 0

0.05 0.01 0.24 0.31 0,02 0.13

+ 0,03 +_ 0,01 + 0.07 -- 0,26 ± 0.01 ± 0.06

8.9 + 2.8 8,3 ± 3.4 0,4 + 0.1

0.06 0.02 0.44 0.05 0.01 0.20

+ + + ± ± ±

0.01 0.01 0,12 0.02 0.01 0.03

13.8 ± 5,3 1.5 ± 0.2 0.2 ± 0,1

0.06 0.03 0.09 1.28

_+ 0.03 ± 0.02 ± 0.09 ± 0.38 ND 0.06 +- 0,04

2,5 ± 2.0 44,0 ± 9.5 0

0.07 0.03 0.23 0.20

± 0.05 ± 0.03_ + 0.15 ± 0.17 ND 0.06 _+ 0.20

0.04 ± 0.01 0,82 + 0.30

ND 0.83 ± 0.35

0.01 +_ 0.01 1.67 _+ 0.34

0.03 ± 0.01 0.88 ± 0.30

0,01 ± 0,01 0.24 ± 0.10

0.24 _+ 0.10 3.17 _+ 0.51

0.19 + 0,08 3.04 + 1,37

0.44 ± 0.08 4.07 ± 0.71

0.15 ± 0.02 2.98 ± 0.85

0.06 + 0.30 1.22 + 0,25

1.8 ± 0.6 27,2 ± 6.0

26.3 ± 9,8 14.7 ± 11.0 0

NOTE. Results are expressed as mean +_ SEM, ND, not detected.

exclusively found in the unconjugated form. In contrast, there were negligible concentrations of unconjugated UDCA in the feces of rats administered UDCA 7-sulfate and UDCA 3,7-disul~te; these two conjugates were excreted in feces virtually unchanged (Figure 2). The concentrations of the major secondary bile acids excreted in feces differed among the groups of animals. In the UDCA group, lithocholic acid increased markedly from control values, whereas the fecal excretion of lithocholic acid after UDCA 7-sulfate and UDCA 3,7-disulfate administration was reduced. A similar trend in deoxycholic acid excretion was found. Compared with normal rat feces, the ratio of lithocholic acid/deoxycholic acid increased more than 20-fold when UDCA was administered, increased l 1-fold with UDCA 3-sulfate administration, and did

not increase when the C-7 sulfates were administered (Figure 3). Bile Acid C o m p o s i t i o n of Liver T i s s u e

The concentrations and proportions of the individual bile acids in liver tissue are summarized in Table 3. UDCA concentration was 12.0 nmol/g in control animals and accounted for 2.8% of the total hepatic bile acids. Administration of UDCA and the C-3 sulfate caused increased concentrations and proportions of liver tissue UDCA (Figure 1). In addition, A22-IJDCA was found in the liver in relatively high proportions. In contrast, marked decreases in the total UDCA concentration and percent composition occurred when animals were administered the C-7 sulfated bile acids. UDCA administration resulted in increased hepatic lithocholic acid concentration, whereas decreases in lithocholic acid occurred after administration of the sulfate conjugates.

I ~;;,:{

~~; i~,~,>:>:;~:::,~lithocholate

~ ~

tO

qd

unconjugated ursodeoxycholate

X 0

D conjugated ursodeoxycholate 0

2

UDCA

.o_ 5

deoxycholate

: :;:::::' : ~:':~"~::::~ :

6

4 6 8 Bile acid concentration in feces (mg/g)

10

12

._~ e'-

4 3 2

1

o

CONTROL

e-

Figure 2, Fecal bile acid excretion in control rats and rats orally administered UDCA ([]), UDCA 3-sulfate ([]), UDCA 7-sulfate (11), and UDCA 3,7-disulfate (@). Bile acids were separated according to their mode of conjugation by anion-exchange chromatography before analysis of the individual fractions using GC-MS. Results are expressed as mean values of all animals. E3, Control; nd, net detected.

5

0

Figure 3. Ratio of lithocholic acid/deoxycholic acid in the feces of control rats and rats orally administered UDCA, UDCA 3-sulfate (UDCA3S), UDCA 7-sulfate (UDCA-7S), and UDCA 3,7-disulfate (UDCA-DS). Results are expressed as the mean values of all animals.

1840

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ET AL.

G A S T R O E N T E R O L O G Y Vol. 1 0 9 , No. 6

Table 3. Concentrations and Percent Composition of the Principal Bile Acids in Liver From Control Rats and After Oral Administration of UDCA, UDCA 3-Sulfate, UDCA 7-Sulfate, or UDCA 3,7-Disulfate, Determined by GC-MS Control

Lithocholic acid Deoxycholic acid Chenodeoxycholic acid c~-Muricholic acid Cholic acid UDCA &22-UDCA ~-Muficholic acid ~22-~Muricholic acid o>Muricholic acid A22-~Muricholic acid Total

UDCA 3-sulfate

UDCA

UDCA 7-sulfate

UDCA 3,7~isulfate

Concentration

Percent

Concentration

Percent

Concentration

Percent

Concentration

Percent

Concentration

Percent

(nmol/g)

(%)

(nmol/g)

(%)

( nmol/g)

(%)

(nmol/g)

(%)

(nmol/g)

(%)

4.6 29.0

1.1 6.7

8.0 22.5

2.4 6.8

3.2 52.6

0.9 14.5

3.2 14.9

1.0 4.5

2.2 17.3

0.8 6.1

17.2 39.7 157.5 12.0 ND 45.8 129,1 ND

4.0 9.1 36.2 2.8 0.0 10.5 29.7 0.0

12.4 16.1 42.3 99.6 37.4 60.6 20.9 6.3

3.7 4.8 12.7 30.0 11.3 18.3 6.3 1.9

9,2 6.0 86.6 56.6 31.3 65,4 36.3 8.1

2.5 1.6 23.9 15.6 8.6 18.1 10,0 2.2

6.4 0,4 186.1 6.1 8.7 36.1 59,8 4.0

1.9 0.1 56.0 1.8 2.6 10.8 18.0 1.2

0.7 6.5 163.2 4,9 5.8 50.1 23.0 5.0

0.2 2.3 57.7 1,7 2.0 17.7 8.1 1.8

ND 434.9

0.0

4.8 331.8

1.4

6.3 361.7

1.7

6.6 332.3

2.0

4.0 282.7

1.4

NOTE. Results are expressed as mean values. ND, not detected.

Deoxycholic acid concentration decreased in all groups with bile acid administration, and the reduction was greatest for the C-7 sulfates. Liver tissue cholic acid concentration decreased almost fourfold when UDCA was administered but increased slightly after administration of the C-7 sulfate conjugates. After administration of the individual bile acids, the conjugation of UDCA in liver tissue established that unconjugated UDCA and its C-3 sulfate were both biotransformed by conjugation, mainly with taurine. Irrespective of the administered bile acid, negligible concentrations and proportions of unconjugated UDCA and sulfated UDCA were found in the liver tissue of all animals (Figure 4).

A

2s

oll

2O 15 E v 10 :> = 5 0 a ~

25 B

25

2O 15

0

-

I/lllllFfllillmll

C-

.~ 20

20 15

~

o 0

10

1

-

-

II~HIl~lnllMIm|

Ounconj. glycine taurine sulfate

0/

- IIHHII~IBIK~I/- unconj, glycine taurine sulfate

Figure 4. UDCA concentration in liver tissue of rats orally administered (A) UDCA, (B) UDCA 3-sulfate, (C) UDCA 7-sulfate, and (D) UDCA 3,7-disulfate. Bile acids were separated according to their mode of conjugation by anion-exchange chromatography before analysis of the individual fractions using GC-MS. Results are expressed as mean values of all animals, unconj., Unconjugated.

Bile Acid Composition of Plasma and Urine Unconjugated plasma UDCA concentration in animals administered UDCA was 4.8 + 2.2 btmol/L, and this value was significantly greater than that found for animals administered UDCA 3-sulfate (0.9 + 0.4 ~mol/ L), UDCA 7-sulfate (0.7 + 0.5 btmol/L), and UDCA disulfate (1.0 _+ 0.2 btmol/L). Sulfated UDCA concentrations were similar and <0.3 btmol/L in all animal groups. The urinary excretion of UDCA was negligible (<0.4 nmol/day) before bile acid administration for all animals. After UDCA, urinary unconjugated UDCA excretion was 642.7 nmol/day. This was significantly greater than the concentration of UDCA excreted in the urine of the animals administered the sulfate conjugates (UDCA 3-sulfate, 5.2 nmol/day; UDCA 7-sulfate, 1.8 nmol/day; and UDCA 3,7-disulfate, 1.3 nmol/day). Sulfate conjugates of UDCA were also found in the urine after the administration of unconjugated UDCA (4.6 nmol/day), UDCA 3-sulfate (2.7 nmol/day), UDCA 7-sulfate (317.8 nmol/ day), and UDCA 3,7-disulfate (217.1 nmol/day). Although this represents <0.05 % of the daily dose administered, it was possible to detect these bile acid conjugates by liquid secondary ionization mass spectrometry analysis.

Discussion The cytotoxic or membrane-damaging effect of a bile acid is related to its physicochemical,properties. 36-3. Hydrophobic bile acids are markedly more membrane damaging than hydrophilic bile acids, and relative indices of cytotoxicity have been established based on the retention volume of the bile acid in reverse-phase high-

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pressure liquid chromatography systems ~9 or from partition coefficients in octanol/water. 4° It is paradoxical that the human liver synthesizes chenodeoxycholic acid, a hydrophobic molecule that is intrinsically hepatotoxic, as one of its primary bile acids; in cholestasis, the hepatic accumulation of this bile acid may initiate, contribute to, or exacerbate liver damage. In contrast, UDCA, the 7~-epimer of chenodeoxycholic acid, is highly hydrophilic and has been shown to counteract the membranedamaging effects of hydrophobic bile acids. 36-38'41'42 This is one rationale for the therapeutic use of UDCA in the treatment of a variety of liver diseases. After the oral or intravenous administration of UDCA, this bile acid is efficiently biotransformed in the liver, mainly by conjugation. Negligible concentrations and proportions of unconjugated UDCA are consequently found in human bile, 18'19 or in liver tissue of animals, 2° even after the administration of relatively high doses. Therefore, it could be argued that perhaps the beneficial effects of UDCA are the result of its conversion to more polarconjugated species, which being more hydrophilic42 should confer greater cytoprotective effects than unconjugated UDCA. Having found significant amounts of UDCA sulfate in the urine of patients administered UDCA orally,2~ we synthesized the individual sulfates of UDCA and showed that the C-7 sulfates stimulated bile flow and decreased lipid secretion to a greater extent than unconjugated UDCA. However, a disadvantage from the point of view of targeting the liver is the fact that bile acid sulfates are generally poorly absorbed from the intestine. 22'24 Nevertheless, UDCA sulfates may be appropriate candidates for intravenous administration because of their water solubility. Recent studies indicate that UDCA reduces tumor formation in animal models of colonic carcinogenesis.16'1v However, because this bile acid is absorbed from the small intestine and efficiently conjugated in the liver, 43 it is conserved and undergoes enterohepatic recycling, thus limiting its delivery to the colon and possibly reducing its effectiveness. The addition of a sulfate moiety to UDCA would be expected to restrict small intestinal absorption, thereby making the drug more available to the colon, whereas the increased hydrophilicity imparted by sulfation would predictably have greater cytoprotective and tumor-suppressive effects. Although the intestinal absorption of several sulfate conjugates of hydrophobic bile acids has been examined and shown to be poor, 24'44 we have described, for the first time, the fate of highly hydrophilic UDCA-sulfate conjugates, comparing their behavior with that of unconjugated UDCA. Consistent with our previous studies of adult Sprague-

SULFATE CONJUGATES OF URSODEOXYCHOLATE

1841

Dawley rats, 2° liver tissue UDCA concentrations increased markedly with oral administration of UDCA, and this bile acid was predominantly conjugated by amida= tion. Negligible amounts of unconjugated UDCA were found (Figure 4) and, in this regard, its metabolism 35 is similar to that of humans. 19 In contrast, when the C-7 sulfate and the disulfate conjugates were administered, hepatic concentrations of UDCA were low compared with the control animals, indicating that these conjugates were not absorbed from the intestine and, thus, there was negligible conservation of UDCA. Hepatic UDCA concentrations increased after administration of the C-3 sulfate, and the fact that it was mainly amidated indicated that significant desulfation and amidation of UDCA 3-sulfate had taken place. The pattern of conjugation of UDCA in the jejunum paralleled that of the liver tissue except that, in the UDCA and UDCA 3-sulfate administered groups, there was a higher proportion of unconjugated UDCA present (Figure 1). Previous studies of bile acid feeding had established that maximum enrichment of the bile acid pool is attained by 4 days,2° and prolonged feeding results in no further changes in bile acid composition. 3%45 The lack of intestinal absorption of the C-7 sulfates of UDCA is further reflected in the bile acid composition of feces. The fecal concentration of the total amount of UDCA in the animals administered UDCA 7-sulfate and UDCA 3,7-disulfate was markedly greater than that found in the feces of animals administered UDCA or UDCA 3-sulfate. In addition, the C-7 sulfates of UDCA were predominantly excreted unchanged in feces. These findings can be explained by the substrate specificity of bacterial sulfatases, which have been previously shown to be active only toward C-3 bile acid sulfates. 46'47 UDCA administration had a marked effect on the fecal excretion of the major secondary bile acids and lithocholic and deoxycholic acids. A large increase in the fecal lithocholic acid concentration occurred when unconjugated UDCA and UDCA 3-sulfate were administered, but UDCA 7sulfate and UDCA 3,7-disulfate administration had no significant effect on the fecal output of these secondary bile acids. These observations are supported by our previous findings (Setchell and Yamashita, unpublished data) in which the presence of a sulfate moiety at the C-7 position was shown to protect the bile acid from bacterial 7o~-dehydroxylation. Similar inhibition of intestinal biotransformation occurs when UDCA or chenodeoxycholic acid are substituted with a C-7 methyl group. 48'49 The increases in fecal and hepatic lithocholic acid concentrations can be explained by intestinal bacterial biotransformation of UDCA 5°'51 and, to a lesser extent, its C-3 sulfate (Setchell and Yamashita, unpublished data).

1842

RODRIGUES ET AL.

Biotransformation of UDCA to lithocholic acid occurs to a similar extent in both rats and humans, z9'2°'5°'51 An increase in deoxycholic acid in the feces of animals administered UDCA is consistent with the known competitive inhibition of cholic acid uptake at the terminal ileum, 52'53 which leads to an increased spillover into the colon and subsequent 70~-dehydroxylation to form deoxycholic acid. Interestingly, the UDCA C-7 sulfates seem to have the opposite effect; cholic acid concentrations in liver tissue were increased slightly compared with control animals, and fecal cholic acid and deoxycholic acid concentrations were decreased. In view of the fact that UDCA undergoes significant biotransformation to lithocholic acid and increases fecal deoxycholic acid concentration, both highly hydrophobic bile acids, it is perhaps surprising that beneficial effects of UDCA have been shown in animal models of chemically induced colon cancer. In these models, it has been established conclusively that hydrophobic bile acids promote tumor growth. Rectal and oral administration of bile acids, 5<55 bile diversion to the cecum, 56 cholestyramine feeding, 57 dietary fat, and certain fibers, 58-6° conditions that all increase the flux of bile acids through the colon, enhance tumor formation, consistent with a promoting effect. In vitro studies indicate that deoxycholic and lithocholic acids are comitogenic and increase the colonic epithelial cell proliferation rate. 61 Other effects on ornithine decarboxylase activity62 and HLA class I and II antigens have also been shown. 63 There are several possible explanations for the chemopreventive effect of UDCA. Any deleterious effects of increased lithocholic acid formation in the colon may be buffered by the presence of relatively high concentrations of UDCA in a manner similar to the cytoprotective effects of UDCA when coincubated in vitro or coinfused in vivo with hydrophobic bile acids that are membrane damaging. 36-38'41'42 Alternatively, the protective effects may be the result of decreased colonic deoxycholic acid concentration, which would imply that deoxycholic acid is of major importance in the promotion of colon cancer. Despite similar reductions in colonic deoxycholic acid with administration of the sulfated bile acids, the C-7 sulfates of UDCA may in principle be superior to UDCA because these conjugates are not biotransformed to more hydrophobic bile acids. Additionally, the lack of absorption of the C-7 sulfates in the small intestine may permit the use of lower doses to attain similar chemopreventive effects. The role of bile acids in human colonic carcinogenesis is less clear. Early studies indicate that fecal bile acid excretion, particularly lithocholic and deoxycholic acids, was increased in patients with colon cancer, adenomatous

GASTROENTEROLOGY Vol. 109, No. 6

polyps, and familial polyposis, 6<65 although these findings were not substantiated by several other investigators. 66-69 Compared with controls, patients with colonic cancer or adenomatous polyps have been reported to have increased aqueous-phase lithocholic and deoxycholic acids concentrations in feces, and these concentrations correlated with the extent of colonic cell proliferation] ° Despite preliminary data supporting a chemoprotective and/or cytoprotective effect of UDCA in animal models of colon cancer and in vitro cell systems, the increased lithocholic acid formation after UDCA administration may limit the overall effectiveness of UDCA in the colon. The lithocholate/deoxycholate ratio in feces is markedly increased after UDCA and UDCA 3-sulfate administration compared with controls (Figure 3). This may be less desirable because the ratio of fecal lithocholate/deoxycholate is increased in patients with colon cancer and in patients at high risk for the disease and is proposed to be of diagnostic value. 71 On the other hand, UDCA 7sulfate and UDCA 3,7-disulfate administration resulted in no change in the lithocholate/deoxycholate ratio. Although not statistically significant, a tendency towards a decrease in this ratio was observed, whereas the quantitative fecal excretion of these secondary bile acids was similar to control animals. In conclusion, the introduction of a sulfate group at the position C-7 of UDCA greatly increases the hydrophilicity of the molecule, which prevents intestinal absorption, thereby facilitating the site-specific delivery of UDCA to the colon. In contrast to unconjugated UDCA, which undergoes conversion to lithocholic acid and thereby increases the fecal lithocholate/deoxycholate ratio, which is considered a risk factor for colonic disease, the C-7 sulfates are metabolically inert. These conjugated bile acids may be more effective chemoprotective agents than UDCA in the colon.

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Received April 6, 1995. Accepted May 23, 1995. Address requests for reprints to: Kenneth D. R. Setchell, Ph.D., Department of Pediatrics, Clinical Mass Spectrometry Center, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229. Fax: (513) 559-7853. Supported in part by grant BD/2326/92-1D from Junta Nacional de Investigacao Cientifica e Tecnol6gica, Lisbon, Portugal (to C.M.P.R. for Ph.D. studies), and by a grant from the Pharmaceuticals Division of CIBA-GEIGY Corp., Summit, New Jersey (to C3.S). The Children's Hospital Research Foundation has applied for a patent for the use of ursodeoxycholic acid sulfate conjugates described in this report.