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Effects of the elevation of serum bile acids on gastric mucosal damage
Hepatology Research 14 (1999) 195 – 203
Effects of the elevation of serum bile acids on gastric mucosal damage Yohei Fukumoto *, Fujio Murakami, Masaya Andoh, Shuji Mizumachi, Kiwamu Okita First Di6ision, Department of Internal Medicine, Yamaguchi Uni6ersity School of Medicine, Ube, Yamaguchi Prefecture, 755 -8505, Japan Received 12 September 1998; received in revised form 20 November 1998; accepted 15 December 1998
Abstract In this study, we evaluated whether the elevated serum level of bile acids was a trigger of gastric mucosal damage in rats. A degree of mucosal injury was evaluated as an extent of ulcer formation due to HCl. That is, 0.6 N HCl was administered to the stomach via a tube in a rat model 3 h after ligation of the common bile duct, with additional infusion of four different bile acids. Taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA) were used as bile acid samples and saline was administered to rats as a control. Moreover, gastric mucosal blood flow and gastric content of epidermal growth factor (EGF), were evaluated in rats with severe mucosal damage. As a result, significant mucosal damage was detected in rats administered TDCA or TCDCA compared to controls. Gastric mucosal blood flow did not change, but the EGF level was significantly decreased by bile duct obstruction with TDCA infusion. These data indicate that gastric mucosal injury was readily induced by an elevated serum level of TDCA, TCDCA and their unconjugated bile salts and this effect was directly due to cytotoxicity of the bile acid. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gastric mucosal damage; Cytotoxic bile acid; Deoxycholic acid; Chenodeoxycholic acid
* Corresponding author. Present address: Total Care Unit, Yamaguchi University School of Medicine, Ube, Yamaguchi Prefecture, 755-8505, Japan. Tel.: + 81-836-22-2686; fax: +81-836-22-2687. E-mail address: [email protected] (Y. Fukumoto) 1386-6346/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 0 3 - 0
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1. Introduction Patients with liver cirrhosis demonstrate high rates of gastro-duodenal mucosal lesions and potential cytotoxic bile acids are accumulated in their peripheral blood [1,2]. In cases of portal hypertension, an increase of gastric blood flow has previously been reported, with morphologic changes of dilated microvessels in the gastric mucosa [3,4]. Moreover, in studies on the cytotoxic effects of different bile salts to isolated perfused rat liver, hepatocytes showed marked subcellular damage following administration of cholic acid or chenodeoxycholic acid and marked alterations of the canalicular membrane following that of lithocholic or taurolithocholic acid [5]. Concerning the relationship between gastric mucosal damage and bile salts, the direct detergent effect of bile salts to mucosa due to bile regurgitation from the duodenum to the stomach is presumed. On the other hand, the resistance of gastric mucosa to damage has also been observed after exposure to low levels of deoxycholic or glycodeoxycholic acid by stimulation of glycoprotein secretion in cultured rabbit gastric mucosal cells [6]. Although cytotoxic effects of bile acids against some organs have been observed in experimental studies in vivo and in vitro [7,8], no studies have investigated the effect of elevated bile acids in peripheral blood on damage to the gastric mucosa. In this study, we examined whether an elevation of serum bile acids caused the enhancement of HCl-induced ulcer formation in the rat stomach. Four different kinds of bile acids, commonly found in the serum, were applied to a rat model of common bile duct ligation and the gastric mucosal injury was investigated. In addition, changes in gastric mucosal blood flow and gastric levels of epidermal growth factor (EGF) were evaluated in this experimental model.
2. Materials and methods Male Wistar rats, weighing 2809 30 g obtained from Chiyoda-Kaihatsu (Kumamoto, Japan), were used. Animals were given free access to a standard solid diet and water until use. Food was withdrawn and only water was given 8 h before all animal examinations. To increase serum levels of bile acids, a bile acid sample was intravenously administered after ligation of the common bile duct. Since all bile acid samples, which were intravenously injected, were excreted into the bile within 15 min, ligation of the bile duct was necessary. Under anesthesia induced by sodium thiopental (2.5 mg/100 g, i.p.) and ether, a midline incision of the rat abdomen was performed and the common bile duct was doubly ligated close to the liver. After that, one of four different bile acid samples, taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA) (Sigma, St. Louis; purity 98% or higher) or tauroursodeoxycholic acid (TUDCA) (Sigma; purity : 90%), dissolved in 1.0 ml of distilled water, at a dose of 10 mmol/100 g body weight (b.w.), was injected once into the femoral vein of different rats. Then, the rat abdomen and skin were sutured. Control rats
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received saline instead of bile acid solution. After the operation, rats awoke from anesthesia and were given water only. All experimental groups consisted of five rats. This study was performed by the Guide of Animal Care Committee of this institute.
2.1. HCl-induced gastric mucosal damage The effect of an increased serum level of bile acid on gastric mucosa was investigated by the method of acid-induced mucosal disturbance. A total of 2 ml of 0.6 N HCl was administered to the rat stomach via a stomach tube, 3 h after common bile duct ligation and infusion of different bile acid samples or saline. In this examination, a sham-operated rat was used as another control. The rat was sacrificed following blood collection for serologic examinations 1 h later and the stomach was removed under ether anesthesia. The removed stomach was filled with 1% formaldehyde solution, by closing the duodenal side using a thread and the stomach was opened by an incision along the large curvature line. The mucosal surface of the stomach was macroscopically observed and photographed for examination. Mucosal damage was determined to calculate the total area of the gastric ulcer as an ulcer index (mm2) using photographs of the mucosa.
2.2. Serologic examination of li6er function and analysis of bile acid subfraction To evaluate biochemical hepatic dysfunction, serum levels of total bilirubin (T.BIL), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were determined with an automatic chemical analyzer (Toshiba, TBA-380, Tokyo, Japan). Total bile acid (T.BA) level was examined fluorimetrically by enzyme method (Hitachi 204, Tokyo, Japan) and bile acid subfraction was analyzed using high-performance liquid chromatography (HPLC) with a column of immobilized 3 a-hydroxysteroid dehydrogenase (Nippon Bunkou, Tokyo, Japan).
2.3. Gastric mucosal blood flow The effect of serum TDCA elevation on gastric mucosal blood flow was examined by the hydrogen gas clearance technique. Surgery was performed on the rat abdomen under anesthesia and a platinum electrode with a hook-shaped tip was inserted into the gastric lumen from the serosal side to gently fix the mucosa at the corpus and antrum of the stomach. Electrodes were connected with a VPS-400 (Unique Medical, Tokyo, Japan) as a power supply. Hydrogen gas (30% H2 in air) was inhaled for 20 s through a mask to saturate the gastric mucosa with hydrogen ions. Blood flow was demonstrated by the clearance of hydrogen ions from the tissue. After the first measurement of mucosal blood flow as a basal level, the procedure of common bile duct ligation and TDCA injection (10 mmol/100 g b.w.) was completed. Then, the rat abdominal wall was simply closed, leaving electrodes in the abdomen under continuous anesthesia. The rat abdomen was opened again 4 h after the operation. The second measurement of mucosal blood flow was taken by the same technique to evaluate the influence of TDCA on gastric mucosal blood
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flow. Changes in the blood flow were estimated by the difference between the first and second measurements. Five rats were used in this study.
2.4. Rat stomach EGF The contents of the rat stomach EGF were examined in the model for increased levels of serum TDCA. Ligation of the common bile duct and TDCA infusion (10 mmol/100 g b.w.) was performed by the same method. After 4 h, the stomach was removed at laparotomy after death under ether anesthesia and was immediately preserved in liquid nitrogen. After weighing the stomach, the extract was prepared by homogenation of the whole stomach in distilled water containing 500 U of aprotinin (Bayer, Leverkusen, Germany) per millilitre and centrifuged at 3000× g for 30 min at 4°C. The supernatant was obtained to study the EGF level using [125I] EGF reagent pack for radioimmunoassay (Amersham, Buckinghamshire, UK). The sham-operated rat was used as a control. Five rats were used in both experimental and control groups.
2.5. Data analysis All results are expressed as the mean 9 S.E. Paired and unpaired Student’s t-tests were used to analyze the data. A probability level of B 0.05 was considered significant.
3. Results
3.1. Effect of ele6ated serum le6el of bile acid subfractions on gastric mucosal damage Acid-induced gastric mucosal damage in rats administered different bile acids (TCA, TDCA, TCDCA or TUDCA) or saline after bile duct ligation and that in sham-operated rats are presented as ulcer indexes in Table 1. The ulcer index was significantly higher in TDCA and TCDCA infused rats than saline infused and sham-operated rats. However, there were no significant differences in TCA or TUDCA infused rats compared to the two controls. Additional administration of TDCA or TCDCA was considered to enhance HCl-induced ulcer formation in the stomach compared to controls.
3.2. Data of serum li6er dysfunction and pattern of serum bile acid subfractions in the rat model of bile duct ligation with different bile acid injections Serum data of liver function tests in rats 4 h after common bile duct obstruction with additional infusion of different bile acids or saline are shown in Table 2. Levels of T.BIL were significantly increased in TDCA and TCDCA administered rats compared to saline infused rats. ALT levels were also significantly higher in TCA,
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Table 1 Data of HCl-induced gastric mucosal damage shown by ulcer indexes (mm2) in sham-operated rats and common bile duct ligated rats administered saline, TCA, TDCA, TCDCA or TUDCAa Animal models
Ulcer index
Sham-operation Ligation+saline Ligation+TCA Ligation+TDCA Ligation+TCDCA Ligation+TUDCA
49.8 97.8 63.1 99.5 77.0 928.4 118.8 922.3 c ,* 198.3 925.7 c c ,** 88.1 913.7
a TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TUDCA, tauroursodeoxycholic acid. c PB0.05, compared to sham-operated rats. cc PB0.01 compared to sham-operated rats. * PB0.05, compared to saline administered rats. ** PB0.01 compared to saline administered rats.
TDCA and TCDCA infused rats. ALP levels were significantly elevated in TDCA and TCDCA rats and T.BA levels were significantly higher in TCDCA rats compared to saline administered rats. However, mean concentrations of serum total bile acid were almost the same in each bile acid administered group. Serum patterns of bile acid subfractions obtained from the same rat models are shown in Fig. 1. The technique for bile acid analysis using HPLC usually separates 15 peaks of bile acid subfraction, but in this study some undetermined fractions were included. However, they are supposed to be conjugated and unconjugated b-muricholic acids, because b-muricholic acid has been identified from rat bile at :13% [9]. In rats with bile duct ligation alone, taurine- and glycine-conjugated fractions of both cholic and deoxycholic acid were elevated 4 h after the model was
Table 2 Data of serum chemical examinations in common bile duct ligated rats administered saline, TCA, TDCA, TCDCA or TUDCAa Animal models
T.BIL (mg/dl)
ALT (U/l)
ALP (U/l)
T.BAa (mmol/l)
Ligation+saline Ligation+TCA Ligation+TDCA Ligation+TCDCA Ligation+TUDCA
2.50 9 0.52 2.589 0.44 7.18 9 0.32** 7.279 0.60** 1.309 0.49
221.8 9 68.4 428.2 9124.1* 1434.4 9 442.8* 2765.0 9120.4** 203.0 943.6
13.3 91.6 14.2 90.3 19.4 93.5* 21.29 1.7** 14.2 91.2
554.1 9 63.8 687.9 9 70.4 678.6 977.7 701.3 961.1* 601.5 9 92.5
a
T.BIL, total bilirubin; ALT, alanine aminotransferase; ALP, alkaline phosphatase; T.BA, total bile acid; TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TUDCA, tauroursodeoxycholic acid. * PB0.05, compared to saline administered rats. ** PB0.01, compared to saline administered rats.
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completed. In each bile acid administered model, the infused bile acid was the prevailing bile acid in the serum. Some unconjugated bile acid fractions were obtained and their actual levels are shown in Table 3. In models administered TCDA or TCDCA, unconjugated fractions were elevated in small levels.
3.3. Effect of ele6ated serum bile acids on gastric mucosal blood flow Data of the basal blood flow and the blood flow 4 h after obstruction of the common bile duct with infusion of TDCA are presented in Table 4. There was no significant difference between the basal and the experimental mucosal blood flows in either the corpus or antrum of the stomach. This indicated that the elevation of serum bile acids did not affect the gastric mucosal blood flow.
Fig. 1. Levels of serum bile acid subfractions in common bile duct obstructed rats administered saline, TCA, TDCA, TCDCA or TUDCA. *Undetermined fractions of bile acids. Bile acid fraction shown by vertical hatched square is the administered bile acid TUDCA, tauroursodeoxycholic acid; GCA, glycocholic acid; TCA, taurocholic acid; CA, cholic acid; GCDCA, glycochenodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; CDCA, chenodeoxycholic acid; GDCA, glycodeoxycholic acid; TDCA, taurodeoxycholic acid; DCA, deoxycholic acid.
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Table 3 Actual levels of unconjugated bile acid subfractions of cholic acid, deoxycholic acid and chenodeoxycholic acid as shown in Fig. 1 Animal models
Cholic acid
Deoxycholic acid
Chenodeoxycholic acid (mmol/l)
Ligation+saline Ligation+TCAa Ligation+TDCA Ligation+TCDCA Ligation+TUDCA
4.7 9 2.0 20.19 7.6 12.4 9 3.1 10.2 9 1.8 5.2 92.3
1.2 90.4 2.0 90.8 7.4 94.4 0.8 90.2 1.1 90.2
Trace 2.0 90.6 Trace 11.4 91.4 0.8 9 0.7
a
TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TUDCA, tauroursodeoxycholic acid. Underline shows unconjugated fraction of administered bile acid. Table 4 Gastric mucosal blood flow on the corpus and antrum of the stomach before the study (basal) and 4 h after TDCA administration with bile duct obstruction Blood flow
Corpus
Antrum (ml/min/100 g b.w.)
Basal TDCAa
101.499.5 110.79 13.0
109.7 9 12.2 93.7 9 16.0§
a §
TDCA, taurodeoxycholic acid. Not significant compared to the basal blood flow.
3.4. Effect of ele6ated serum bile acids on gastric EGF content Levels of gastric EGF in rats 4 h after bile duct ligation with TDCA administration and sham-operated rats are shown in Table 5. EGF content in the rat stomach was significantly decreased in the animal model compared to the control. The condition of obstructive cholestasis accompanied by an elevation of serum bile acids may have disturbed the EGF levels in the stomach. 4. Discussion In serum and livers of patients with advanced liver cirrhosis and end-stage of chronic cholestasis, accumulations of cholic, chenodeoxycholic and deoxycholic Table 5 Contents of epidermal growth factor (EGF) in the stomach in sham-operated rats and common bile duct ligated rats administered TDCA Models
Rat stomach EGF (ng/100 g stomach weight)
Sham-operation Ligation+TDCAa
2.03 90.21 0.989 0.25*
a
TDCA, taurodeoxycholic acid. * PB0.05, compared to sham-operated rats.
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acid have been reported to contribute to liver degeneration [1]. Although bile salts can be cytotoxic to the mucosal surface of the stomach, this effect is not normally observed under physiological circumstances of its defense mechanisms [6,10]. In the present study, significant gastric damage was observed in the bile duct ligated rats with additional administration of TDCA or TCDCA compared to saline infused rats. HCl-induced injury of the stomach was detected even in sham-operated rats and the ulcer index of this group was almost the same as that of bile duct ligated rats with saline infusion. This result indicates that an elevated pattern of serum bile acid subfractions due to bile duct ligation alone did not contribute to enhanced gastric mucosal damage. Additional infusion of TDCA or TCDCA to the obstructive model produced more prominent cytotoxic effects to the gastric mucosa than that of TCA or TUDCA. Moreover, serum levels of ALT and T.BIL were markedly increased in the groups administered TDCA or TCDCA compared to all other animal models in this study. Hepatic disturbance was produced more severely in these two rat models. As serum levels of total bile acid were almost the same in each model with different bile acid infusions, the pattern of serum bile acid subfractions is likely more important also for the induction of cytotoxicity to the liver than the serum level of total bile acid. Concerning the cause of gastric ulcer, gastric mucosal blood flow disturbance supports an ischemic etiology for mucosal injury in the stomach [11]. However, our results using TDCA did not reveal a significant change in gastric mucosal blood flow. On the other hand, the mean level of gastric EGF was significantly lower in rats administered TDCA than that in sham-operated rats. These data may indicate a direct cytotoxic effect of administered bile acid against gastric mucosa. As EGF in the stomach appears to act as a growth factor to accelerate cell proliferation and to promote ulcer healing on gastric epithelial cells [12,13], the decrease of gastric EGF may exert a secondary effect on ulcer formation of the stomach. Increased serum bile acids due to common bile duct ligation were the primary bile acid of cholic acid and the secondary bile acid of deoxycholic acid. As cholic acid is a main serum bile acid in animals, levels of the cholic acid group were considered to be increased in peripheral blood by obstruction of bile flow. In addition, serum elevation of the deoxycholic acid group was presumed to absorb residual deoxycholic acids in the intestine during 4 h after common bile duct ligation. In our rat models, the serum bile acid pattern largely reflected an enhanced bile acid. As hydrophobic unconjugated deoxycholic or chenodeoxycholic acids are more cytotoxic as compared to conjugated bile acids [5], both TDCA or TCDCA and their unconjugated bile acids are suspected to induce damage on the gastric mucosa. The mechanism for the presence of unconjugated bile acid in the serum after infusion of taurine conjugated bile acid is unknown, but highly concentrated serum bile acids may be thought to contrary spread though intestinal mucosa from the basement side to the lumen in the obstructive cholestasis. UDCA and TUDCA have been suggested to have a protective effect on hepatocyte structure and function and UDCA treatment for patients with chronic cholestatic liver disease has been reported to improve clinical and biological
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manifestations [14,15]. In this study, intravenous infusion of TUDCA to rat models of common bile duct ligation resulted in no significant disturbances of the gastric mucosa or the examined liver function tests compared to that in saline administered rats. Although the mechanism of the cytoprotective effect of UDCA has not been clearly established, it suggests that the hepatoprotective properties of UDCA may occurs through its effect on intestinal absorption and hepatocyte transportation of bile acids rather than a direct effect [14]. Results of the present study showed that an elevation of cytotoxic bile acids in serum enhanced acidification-induced gastric mucosal damage and decreased the gastric EGF level, one of gastric growth factors. Although many factors are probably involved in the pathogenesis of gastric mucosal injury, findings obtained in this study support the hypothesis that patients with liver cirrhosis and chronic cholestasis are at high risk for peptic ulcers and gastro-intestinal bleeding. References [1] Fischer S, Seuers U, Spengler U, Zwiebel FM, Koebe H-G. Hepatic levels of bile acids in end-stage chronic cholestatic liver disease. Clin Chim Acta 1996;251:173 – 86. [2] Greim H, Trulzsch D, Czygan P, et al. Mechanism of cholestasis. 6. Bile acids in human liver with or without biliary obstruction. Gastroenterology 1972;63:837 – 45. [3] Pique JM, Leung FW, Kitahora T, Sarfeh IJ, Tarnawski A, Guth PH. Gastric mucosal blood flow and acid secretion in portal hypertensive rats. Gastroenterology 1988;95:727 – 33. [4] Benoit JN, Womach WA, Korthuis RJ, Wilborn WH, Granger N. Chronic portal hypertension: effect on gastrointestinal blood flow distribution. Am J Physiol 1986;250:535 – 9. [5] Benedetti A, Alvaro D, Bassotti C, et al. Cytotoxicity of bile salts against biliary epithelium: a study in isolated bile ductile fragments and isolated perfused rat liver. Hepatology 1997;26:9 – 21. [6] Hata Y, Ota S, Kawabe T, Terano A, Razandi M, Ivey KJ. Bile salts stimulate mucous glycoprotein secretion from cultured rabbit gastric mucosal cells. J Lab Clin Med 1994;124:395 – 400. [7] Kanri R, Takiyama Y, Makini I. Effects of bile acids on iodine uptake and deoxyribonucleic acid synthesis in porcine thyroid cells in primary culture. Thyroid 1996;6:467 – 74. [8] Van Munster ID, Tangerman A, Haan AF, Nagengast FM. A new method for the determination of the cytotoxicity of bile acids and aqueous phase of stool: the effect of calcium. Eur J Clin Invest 1993;23:773–7. [9] Elliott WH. Metabolism of bile acids in liver and extrahepatic tissues; Hydroxylation of steroid nucleus. In: Danielsson H, Sjovall J, editors. Sterols and bile acids. Amsterdam: Elsevier, 1985:310 – 6. [10] Aihara N, Tazuma S, Kajiyama G. Hydrophilic bile salts and liposomes inhibit hydrophobic bile salt-induced release of glycoprotein by guinea-pig gall-bladder. J Gastroenterol Hepatol 1995;10:42 – 6. [11] Sarfoh IJ, Tarnawski A, Malki A, Mason GR, Mach T, Ivey KJ. Portal hypertension and gastric mucosal injury in rats. Effects of alcohol. Gastroenterology 1983;84:987 – 93. [12] Konturek SJ, Radecki T, Brozozowski T, et al. Gastric cytoprotection by epidermal growth factor. Gastroenterology 1981;81:438–43. [13] Konturek PK, Brozozowski T, Konturek SJ, et al. Role of epidermal growth factor, prostaglandin and sulfhydryls in stress-induced gastric lesions. Gastroenterology 1990;99:1607 – 15. [14] Hillaire S, Ballet F, Franco D, Setchell KDR, Poupon R. Effects of ursodeoxycholic acid and chenodeoxycholic acid on human hepatocytes in primary culture. Hepatology 1995;22:82 – 7. [15] Thibault N, Maurice M, Maratrat M, Cordier A, Feldmann G, Ballet F. Effect of tauroursodeoxycholate on actin filament alteration induced by cholestatic agents. a study in isolated rat hepatocyte couplets. J Hepatol 1993;19:367–76.
Effects of the elevation of serum bile acids on gastric mucosal damage Yohei Fukumoto *, Fujio Murakami, Masaya Andoh, Shuji Mizumachi, Kiwamu Okita First Di6ision, Department of Internal Medicine, Yamaguchi Uni6ersity School of Medicine, Ube, Yamaguchi Prefecture, 755 -8505, Japan Received 12 September 1998; received in revised form 20 November 1998; accepted 15 December 1998
Abstract In this study, we evaluated whether the elevated serum level of bile acids was a trigger of gastric mucosal damage in rats. A degree of mucosal injury was evaluated as an extent of ulcer formation due to HCl. That is, 0.6 N HCl was administered to the stomach via a tube in a rat model 3 h after ligation of the common bile duct, with additional infusion of four different bile acids. Taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA) were used as bile acid samples and saline was administered to rats as a control. Moreover, gastric mucosal blood flow and gastric content of epidermal growth factor (EGF), were evaluated in rats with severe mucosal damage. As a result, significant mucosal damage was detected in rats administered TDCA or TCDCA compared to controls. Gastric mucosal blood flow did not change, but the EGF level was significantly decreased by bile duct obstruction with TDCA infusion. These data indicate that gastric mucosal injury was readily induced by an elevated serum level of TDCA, TCDCA and their unconjugated bile salts and this effect was directly due to cytotoxicity of the bile acid. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gastric mucosal damage; Cytotoxic bile acid; Deoxycholic acid; Chenodeoxycholic acid
* Corresponding author. Present address: Total Care Unit, Yamaguchi University School of Medicine, Ube, Yamaguchi Prefecture, 755-8505, Japan. Tel.: + 81-836-22-2686; fax: +81-836-22-2687. E-mail address: [email protected] (Y. Fukumoto) 1386-6346/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 0 3 - 0
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1. Introduction Patients with liver cirrhosis demonstrate high rates of gastro-duodenal mucosal lesions and potential cytotoxic bile acids are accumulated in their peripheral blood [1,2]. In cases of portal hypertension, an increase of gastric blood flow has previously been reported, with morphologic changes of dilated microvessels in the gastric mucosa [3,4]. Moreover, in studies on the cytotoxic effects of different bile salts to isolated perfused rat liver, hepatocytes showed marked subcellular damage following administration of cholic acid or chenodeoxycholic acid and marked alterations of the canalicular membrane following that of lithocholic or taurolithocholic acid [5]. Concerning the relationship between gastric mucosal damage and bile salts, the direct detergent effect of bile salts to mucosa due to bile regurgitation from the duodenum to the stomach is presumed. On the other hand, the resistance of gastric mucosa to damage has also been observed after exposure to low levels of deoxycholic or glycodeoxycholic acid by stimulation of glycoprotein secretion in cultured rabbit gastric mucosal cells [6]. Although cytotoxic effects of bile acids against some organs have been observed in experimental studies in vivo and in vitro [7,8], no studies have investigated the effect of elevated bile acids in peripheral blood on damage to the gastric mucosa. In this study, we examined whether an elevation of serum bile acids caused the enhancement of HCl-induced ulcer formation in the rat stomach. Four different kinds of bile acids, commonly found in the serum, were applied to a rat model of common bile duct ligation and the gastric mucosal injury was investigated. In addition, changes in gastric mucosal blood flow and gastric levels of epidermal growth factor (EGF) were evaluated in this experimental model.
2. Materials and methods Male Wistar rats, weighing 2809 30 g obtained from Chiyoda-Kaihatsu (Kumamoto, Japan), were used. Animals were given free access to a standard solid diet and water until use. Food was withdrawn and only water was given 8 h before all animal examinations. To increase serum levels of bile acids, a bile acid sample was intravenously administered after ligation of the common bile duct. Since all bile acid samples, which were intravenously injected, were excreted into the bile within 15 min, ligation of the bile duct was necessary. Under anesthesia induced by sodium thiopental (2.5 mg/100 g, i.p.) and ether, a midline incision of the rat abdomen was performed and the common bile duct was doubly ligated close to the liver. After that, one of four different bile acid samples, taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA) (Sigma, St. Louis; purity 98% or higher) or tauroursodeoxycholic acid (TUDCA) (Sigma; purity : 90%), dissolved in 1.0 ml of distilled water, at a dose of 10 mmol/100 g body weight (b.w.), was injected once into the femoral vein of different rats. Then, the rat abdomen and skin were sutured. Control rats
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received saline instead of bile acid solution. After the operation, rats awoke from anesthesia and were given water only. All experimental groups consisted of five rats. This study was performed by the Guide of Animal Care Committee of this institute.
2.1. HCl-induced gastric mucosal damage The effect of an increased serum level of bile acid on gastric mucosa was investigated by the method of acid-induced mucosal disturbance. A total of 2 ml of 0.6 N HCl was administered to the rat stomach via a stomach tube, 3 h after common bile duct ligation and infusion of different bile acid samples or saline. In this examination, a sham-operated rat was used as another control. The rat was sacrificed following blood collection for serologic examinations 1 h later and the stomach was removed under ether anesthesia. The removed stomach was filled with 1% formaldehyde solution, by closing the duodenal side using a thread and the stomach was opened by an incision along the large curvature line. The mucosal surface of the stomach was macroscopically observed and photographed for examination. Mucosal damage was determined to calculate the total area of the gastric ulcer as an ulcer index (mm2) using photographs of the mucosa.
2.2. Serologic examination of li6er function and analysis of bile acid subfraction To evaluate biochemical hepatic dysfunction, serum levels of total bilirubin (T.BIL), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were determined with an automatic chemical analyzer (Toshiba, TBA-380, Tokyo, Japan). Total bile acid (T.BA) level was examined fluorimetrically by enzyme method (Hitachi 204, Tokyo, Japan) and bile acid subfraction was analyzed using high-performance liquid chromatography (HPLC) with a column of immobilized 3 a-hydroxysteroid dehydrogenase (Nippon Bunkou, Tokyo, Japan).
2.3. Gastric mucosal blood flow The effect of serum TDCA elevation on gastric mucosal blood flow was examined by the hydrogen gas clearance technique. Surgery was performed on the rat abdomen under anesthesia and a platinum electrode with a hook-shaped tip was inserted into the gastric lumen from the serosal side to gently fix the mucosa at the corpus and antrum of the stomach. Electrodes were connected with a VPS-400 (Unique Medical, Tokyo, Japan) as a power supply. Hydrogen gas (30% H2 in air) was inhaled for 20 s through a mask to saturate the gastric mucosa with hydrogen ions. Blood flow was demonstrated by the clearance of hydrogen ions from the tissue. After the first measurement of mucosal blood flow as a basal level, the procedure of common bile duct ligation and TDCA injection (10 mmol/100 g b.w.) was completed. Then, the rat abdominal wall was simply closed, leaving electrodes in the abdomen under continuous anesthesia. The rat abdomen was opened again 4 h after the operation. The second measurement of mucosal blood flow was taken by the same technique to evaluate the influence of TDCA on gastric mucosal blood
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flow. Changes in the blood flow were estimated by the difference between the first and second measurements. Five rats were used in this study.
2.4. Rat stomach EGF The contents of the rat stomach EGF were examined in the model for increased levels of serum TDCA. Ligation of the common bile duct and TDCA infusion (10 mmol/100 g b.w.) was performed by the same method. After 4 h, the stomach was removed at laparotomy after death under ether anesthesia and was immediately preserved in liquid nitrogen. After weighing the stomach, the extract was prepared by homogenation of the whole stomach in distilled water containing 500 U of aprotinin (Bayer, Leverkusen, Germany) per millilitre and centrifuged at 3000× g for 30 min at 4°C. The supernatant was obtained to study the EGF level using [125I] EGF reagent pack for radioimmunoassay (Amersham, Buckinghamshire, UK). The sham-operated rat was used as a control. Five rats were used in both experimental and control groups.
2.5. Data analysis All results are expressed as the mean 9 S.E. Paired and unpaired Student’s t-tests were used to analyze the data. A probability level of B 0.05 was considered significant.
3. Results
3.1. Effect of ele6ated serum le6el of bile acid subfractions on gastric mucosal damage Acid-induced gastric mucosal damage in rats administered different bile acids (TCA, TDCA, TCDCA or TUDCA) or saline after bile duct ligation and that in sham-operated rats are presented as ulcer indexes in Table 1. The ulcer index was significantly higher in TDCA and TCDCA infused rats than saline infused and sham-operated rats. However, there were no significant differences in TCA or TUDCA infused rats compared to the two controls. Additional administration of TDCA or TCDCA was considered to enhance HCl-induced ulcer formation in the stomach compared to controls.
3.2. Data of serum li6er dysfunction and pattern of serum bile acid subfractions in the rat model of bile duct ligation with different bile acid injections Serum data of liver function tests in rats 4 h after common bile duct obstruction with additional infusion of different bile acids or saline are shown in Table 2. Levels of T.BIL were significantly increased in TDCA and TCDCA administered rats compared to saline infused rats. ALT levels were also significantly higher in TCA,
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Table 1 Data of HCl-induced gastric mucosal damage shown by ulcer indexes (mm2) in sham-operated rats and common bile duct ligated rats administered saline, TCA, TDCA, TCDCA or TUDCAa Animal models
Ulcer index
Sham-operation Ligation+saline Ligation+TCA Ligation+TDCA Ligation+TCDCA Ligation+TUDCA
49.8 97.8 63.1 99.5 77.0 928.4 118.8 922.3 c ,* 198.3 925.7 c c ,** 88.1 913.7
a TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TUDCA, tauroursodeoxycholic acid. c PB0.05, compared to sham-operated rats. cc PB0.01 compared to sham-operated rats. * PB0.05, compared to saline administered rats. ** PB0.01 compared to saline administered rats.
TDCA and TCDCA infused rats. ALP levels were significantly elevated in TDCA and TCDCA rats and T.BA levels were significantly higher in TCDCA rats compared to saline administered rats. However, mean concentrations of serum total bile acid were almost the same in each bile acid administered group. Serum patterns of bile acid subfractions obtained from the same rat models are shown in Fig. 1. The technique for bile acid analysis using HPLC usually separates 15 peaks of bile acid subfraction, but in this study some undetermined fractions were included. However, they are supposed to be conjugated and unconjugated b-muricholic acids, because b-muricholic acid has been identified from rat bile at :13% [9]. In rats with bile duct ligation alone, taurine- and glycine-conjugated fractions of both cholic and deoxycholic acid were elevated 4 h after the model was
Table 2 Data of serum chemical examinations in common bile duct ligated rats administered saline, TCA, TDCA, TCDCA or TUDCAa Animal models
T.BIL (mg/dl)
ALT (U/l)
ALP (U/l)
T.BAa (mmol/l)
Ligation+saline Ligation+TCA Ligation+TDCA Ligation+TCDCA Ligation+TUDCA
2.50 9 0.52 2.589 0.44 7.18 9 0.32** 7.279 0.60** 1.309 0.49
221.8 9 68.4 428.2 9124.1* 1434.4 9 442.8* 2765.0 9120.4** 203.0 943.6
13.3 91.6 14.2 90.3 19.4 93.5* 21.29 1.7** 14.2 91.2
554.1 9 63.8 687.9 9 70.4 678.6 977.7 701.3 961.1* 601.5 9 92.5
a
T.BIL, total bilirubin; ALT, alanine aminotransferase; ALP, alkaline phosphatase; T.BA, total bile acid; TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TUDCA, tauroursodeoxycholic acid. * PB0.05, compared to saline administered rats. ** PB0.01, compared to saline administered rats.
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completed. In each bile acid administered model, the infused bile acid was the prevailing bile acid in the serum. Some unconjugated bile acid fractions were obtained and their actual levels are shown in Table 3. In models administered TCDA or TCDCA, unconjugated fractions were elevated in small levels.
3.3. Effect of ele6ated serum bile acids on gastric mucosal blood flow Data of the basal blood flow and the blood flow 4 h after obstruction of the common bile duct with infusion of TDCA are presented in Table 4. There was no significant difference between the basal and the experimental mucosal blood flows in either the corpus or antrum of the stomach. This indicated that the elevation of serum bile acids did not affect the gastric mucosal blood flow.
Fig. 1. Levels of serum bile acid subfractions in common bile duct obstructed rats administered saline, TCA, TDCA, TCDCA or TUDCA. *Undetermined fractions of bile acids. Bile acid fraction shown by vertical hatched square is the administered bile acid TUDCA, tauroursodeoxycholic acid; GCA, glycocholic acid; TCA, taurocholic acid; CA, cholic acid; GCDCA, glycochenodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; CDCA, chenodeoxycholic acid; GDCA, glycodeoxycholic acid; TDCA, taurodeoxycholic acid; DCA, deoxycholic acid.
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Table 3 Actual levels of unconjugated bile acid subfractions of cholic acid, deoxycholic acid and chenodeoxycholic acid as shown in Fig. 1 Animal models
Cholic acid
Deoxycholic acid
Chenodeoxycholic acid (mmol/l)
Ligation+saline Ligation+TCAa Ligation+TDCA Ligation+TCDCA Ligation+TUDCA
4.7 9 2.0 20.19 7.6 12.4 9 3.1 10.2 9 1.8 5.2 92.3
1.2 90.4 2.0 90.8 7.4 94.4 0.8 90.2 1.1 90.2
Trace 2.0 90.6 Trace 11.4 91.4 0.8 9 0.7
a
TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TUDCA, tauroursodeoxycholic acid. Underline shows unconjugated fraction of administered bile acid. Table 4 Gastric mucosal blood flow on the corpus and antrum of the stomach before the study (basal) and 4 h after TDCA administration with bile duct obstruction Blood flow
Corpus
Antrum (ml/min/100 g b.w.)
Basal TDCAa
101.499.5 110.79 13.0
109.7 9 12.2 93.7 9 16.0§
a §
TDCA, taurodeoxycholic acid. Not significant compared to the basal blood flow.
3.4. Effect of ele6ated serum bile acids on gastric EGF content Levels of gastric EGF in rats 4 h after bile duct ligation with TDCA administration and sham-operated rats are shown in Table 5. EGF content in the rat stomach was significantly decreased in the animal model compared to the control. The condition of obstructive cholestasis accompanied by an elevation of serum bile acids may have disturbed the EGF levels in the stomach. 4. Discussion In serum and livers of patients with advanced liver cirrhosis and end-stage of chronic cholestasis, accumulations of cholic, chenodeoxycholic and deoxycholic Table 5 Contents of epidermal growth factor (EGF) in the stomach in sham-operated rats and common bile duct ligated rats administered TDCA Models
Rat stomach EGF (ng/100 g stomach weight)
Sham-operation Ligation+TDCAa
2.03 90.21 0.989 0.25*
a
TDCA, taurodeoxycholic acid. * PB0.05, compared to sham-operated rats.
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acid have been reported to contribute to liver degeneration [1]. Although bile salts can be cytotoxic to the mucosal surface of the stomach, this effect is not normally observed under physiological circumstances of its defense mechanisms [6,10]. In the present study, significant gastric damage was observed in the bile duct ligated rats with additional administration of TDCA or TCDCA compared to saline infused rats. HCl-induced injury of the stomach was detected even in sham-operated rats and the ulcer index of this group was almost the same as that of bile duct ligated rats with saline infusion. This result indicates that an elevated pattern of serum bile acid subfractions due to bile duct ligation alone did not contribute to enhanced gastric mucosal damage. Additional infusion of TDCA or TCDCA to the obstructive model produced more prominent cytotoxic effects to the gastric mucosa than that of TCA or TUDCA. Moreover, serum levels of ALT and T.BIL were markedly increased in the groups administered TDCA or TCDCA compared to all other animal models in this study. Hepatic disturbance was produced more severely in these two rat models. As serum levels of total bile acid were almost the same in each model with different bile acid infusions, the pattern of serum bile acid subfractions is likely more important also for the induction of cytotoxicity to the liver than the serum level of total bile acid. Concerning the cause of gastric ulcer, gastric mucosal blood flow disturbance supports an ischemic etiology for mucosal injury in the stomach [11]. However, our results using TDCA did not reveal a significant change in gastric mucosal blood flow. On the other hand, the mean level of gastric EGF was significantly lower in rats administered TDCA than that in sham-operated rats. These data may indicate a direct cytotoxic effect of administered bile acid against gastric mucosa. As EGF in the stomach appears to act as a growth factor to accelerate cell proliferation and to promote ulcer healing on gastric epithelial cells [12,13], the decrease of gastric EGF may exert a secondary effect on ulcer formation of the stomach. Increased serum bile acids due to common bile duct ligation were the primary bile acid of cholic acid and the secondary bile acid of deoxycholic acid. As cholic acid is a main serum bile acid in animals, levels of the cholic acid group were considered to be increased in peripheral blood by obstruction of bile flow. In addition, serum elevation of the deoxycholic acid group was presumed to absorb residual deoxycholic acids in the intestine during 4 h after common bile duct ligation. In our rat models, the serum bile acid pattern largely reflected an enhanced bile acid. As hydrophobic unconjugated deoxycholic or chenodeoxycholic acids are more cytotoxic as compared to conjugated bile acids [5], both TDCA or TCDCA and their unconjugated bile acids are suspected to induce damage on the gastric mucosa. The mechanism for the presence of unconjugated bile acid in the serum after infusion of taurine conjugated bile acid is unknown, but highly concentrated serum bile acids may be thought to contrary spread though intestinal mucosa from the basement side to the lumen in the obstructive cholestasis. UDCA and TUDCA have been suggested to have a protective effect on hepatocyte structure and function and UDCA treatment for patients with chronic cholestatic liver disease has been reported to improve clinical and biological
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manifestations [14,15]. In this study, intravenous infusion of TUDCA to rat models of common bile duct ligation resulted in no significant disturbances of the gastric mucosa or the examined liver function tests compared to that in saline administered rats. Although the mechanism of the cytoprotective effect of UDCA has not been clearly established, it suggests that the hepatoprotective properties of UDCA may occurs through its effect on intestinal absorption and hepatocyte transportation of bile acids rather than a direct effect [14]. Results of the present study showed that an elevation of cytotoxic bile acids in serum enhanced acidification-induced gastric mucosal damage and decreased the gastric EGF level, one of gastric growth factors. Although many factors are probably involved in the pathogenesis of gastric mucosal injury, findings obtained in this study support the hypothesis that patients with liver cirrhosis and chronic cholestasis are at high risk for peptic ulcers and gastro-intestinal bleeding. References [1] Fischer S, Seuers U, Spengler U, Zwiebel FM, Koebe H-G. Hepatic levels of bile acids in end-stage chronic cholestatic liver disease. Clin Chim Acta 1996;251:173 – 86. [2] Greim H, Trulzsch D, Czygan P, et al. Mechanism of cholestasis. 6. Bile acids in human liver with or without biliary obstruction. Gastroenterology 1972;63:837 – 45. [3] Pique JM, Leung FW, Kitahora T, Sarfeh IJ, Tarnawski A, Guth PH. Gastric mucosal blood flow and acid secretion in portal hypertensive rats. Gastroenterology 1988;95:727 – 33. [4] Benoit JN, Womach WA, Korthuis RJ, Wilborn WH, Granger N. Chronic portal hypertension: effect on gastrointestinal blood flow distribution. Am J Physiol 1986;250:535 – 9. [5] Benedetti A, Alvaro D, Bassotti C, et al. Cytotoxicity of bile salts against biliary epithelium: a study in isolated bile ductile fragments and isolated perfused rat liver. Hepatology 1997;26:9 – 21. [6] Hata Y, Ota S, Kawabe T, Terano A, Razandi M, Ivey KJ. Bile salts stimulate mucous glycoprotein secretion from cultured rabbit gastric mucosal cells. J Lab Clin Med 1994;124:395 – 400. [7] Kanri R, Takiyama Y, Makini I. Effects of bile acids on iodine uptake and deoxyribonucleic acid synthesis in porcine thyroid cells in primary culture. Thyroid 1996;6:467 – 74. [8] Van Munster ID, Tangerman A, Haan AF, Nagengast FM. A new method for the determination of the cytotoxicity of bile acids and aqueous phase of stool: the effect of calcium. Eur J Clin Invest 1993;23:773–7. [9] Elliott WH. Metabolism of bile acids in liver and extrahepatic tissues; Hydroxylation of steroid nucleus. In: Danielsson H, Sjovall J, editors. Sterols and bile acids. Amsterdam: Elsevier, 1985:310 – 6. [10] Aihara N, Tazuma S, Kajiyama G. Hydrophilic bile salts and liposomes inhibit hydrophobic bile salt-induced release of glycoprotein by guinea-pig gall-bladder. J Gastroenterol Hepatol 1995;10:42 – 6. [11] Sarfoh IJ, Tarnawski A, Malki A, Mason GR, Mach T, Ivey KJ. Portal hypertension and gastric mucosal injury in rats. Effects of alcohol. Gastroenterology 1983;84:987 – 93. [12] Konturek SJ, Radecki T, Brozozowski T, et al. Gastric cytoprotection by epidermal growth factor. Gastroenterology 1981;81:438–43. [13] Konturek PK, Brozozowski T, Konturek SJ, et al. Role of epidermal growth factor, prostaglandin and sulfhydryls in stress-induced gastric lesions. Gastroenterology 1990;99:1607 – 15. [14] Hillaire S, Ballet F, Franco D, Setchell KDR, Poupon R. Effects of ursodeoxycholic acid and chenodeoxycholic acid on human hepatocytes in primary culture. Hepatology 1995;22:82 – 7. [15] Thibault N, Maurice M, Maratrat M, Cordier A, Feldmann G, Ballet F. Effect of tauroursodeoxycholate on actin filament alteration induced by cholestatic agents. a study in isolated rat hepatocyte couplets. J Hepatol 1993;19:367–76.