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Bile Acid Synthesis and Biliary Hydrophobicity during Obstructive Jaundice in Rats
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
65, 70–76 (1996)
0345
Bile Acid Synthesis and Biliary Hydrophobicity during Obstructive Jaundice in Rats TOKIO NAITO, M.D., SYOJI KUROKI, M.D.,1 KAZUO CHIJIIWA, M.D., FACS,
AND
MASAO TANAKA, M.D.
Department of Surgery I, Kyushu University Faculty of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan Submitted for publication April 19, 1996
INTRODUCTION Hepatic cholesterol 7a-hydroxylase, the rate-limiting enzyme for bile acid synthesis, is regulated by bile acid returning to the liver. In the bile duct-ligated rats, however, cholesterol 7a-hydroxylase activity is known to increase in spite of the elevated serum bile acids. The aim of this study was to investigate the relationship between the bile acid synthesis and biliary hydrophobicity to solve the paradoxical phenomenon. Male Wister rats (250–350 g) were divided into two groups, bile duct-ligated group and sham-operated group. Rats were sacrificed on the Days 1, 2, 4, 7, and 14 after the operation. Cholesterol 7a-hydroxylase activity, biliary bile acid composition, and biliary hydrophobicity were analyzed. Hepatic and serum total bile acid concentrations and serum 7a-hydroxycholesterol levels were also determined. Bile duct ligation caused significant increases in hepatic and serum bile acid concentrations on Day 1, which persisted for 14 days after the bile duct ligation. Activities of hepatic cholesterol 7a-hydroxylase increased to 3.2-fold of the preoperative value on Day 2, remained significantly high until Day 7, and then decreased to the basal value on Day 14. The serum 7a-hydroxycholesterol level essentially behaved in a similar fashion to that of hepatic cholesterol 7a-hydroxylase activity with a significant (P õ 0.01) positive correlation. b-Muricholic acid was predominant in bile until Day 7 (71 vs 10% in the controls on Day 4; P õ 0.05) with a concomitant decrease in the proportion of cholic acid. Biliary bile acid became less hydrophobic and hepatic cholesterol 7a-hydroxylase activity significantly correlated with the hydrophobicity of biliary bile acids (n Å 55, r Å 0.54, P õ 0.01). There were no significant correlations between the activity of cholesterol 7a-hydroxylase and total bile acid concentrations in serum, liver, or bile. The increased cholesterol 7a-hydroxylase activity is accompanied by the decreased biliary hydrophobicity, which may be a rationale for the paradoxical increase in bile acid synthesis in spite of the accumulation of bile acids in the serum and liver during obstructive jaundice in rats. q 1996 Academic Press, Inc.
Hepatic microsomal cholesterol 7a-hydroxylase catalyzes the first and rate-limiting step in the major pathway for bile acid biosynthesis from cholesterol. It has been believed that the activity of cholesterol 7a-hydroxylase is regulated by bile acids returning to the liver. Several investigators have reported that depletion of bile acids by biliary drainage or dietary administration of cholestyramine increases the enzyme activity and bile acid synthesis [1–5]. In patients with obstructive jaundice, it has been reported that the serum bile acid concentrations are elevated [6–10], and hepatic cholesterol 7a-hydroxylase activity [11] and bile acid synthesis [9, 12] are reduced. In bile duct-ligated rats, however, Danielsson [13] and Dueland et al. [14] showed a significant increase in the activity of cholesterol 7ahydroxylase. Recently, the concept of the bile acid feedback regulation mechanism has been questioned because of the failure to demonstrate the inhibition of bile acid synthesis in bile fistula rats [15] and in cultured hepatocytes. Heuman et al. [16] recently reported that hydrophobic bile acids suppressed the cholesterol 7ahydroxylase activity and this could be one of the reasons to explain the conflicting observations between humans and rats. We have previously reported that bile acid synthesis is not regulated directly by the portal bile acids returning to the liver [17]. The aim of the present study was to examine whether bile duct ligation increases biliary bile acid hydrophilicity and hence the activity of hepatic cholesterol 7ahydroxylase in the bile duct-ligated rats. Serum 7ahydroxycholesterol level was also monitored to confirm if the level could be the indicator of the hepatic enzyme activity [17–20] during obstructive jaundice. MATERIALS AND METHODS Materials. All solvents used were of analytical grade or distilled prior to use. Sephadex LH-20 was purchased from Pharmacia Fine Chemicals AB (Uppsala, Sweden) and dimethylethylsilylimidazole (DMESI) was from Tokyo Kasei Kogyo (Tokyo, Japan). Cholesterol, cholylglycinehydrolase, 3a-hydroxysteroid dehydrogenase, and dithiothreitol (DTT) were obtained from Sigma Chemical Co. (St. Louis, MO). 7a-Hydroxycholesterol and 5a-cholestane-3b,7b-diol were synthesized as described previously [21]. Bond Elut silica cartridge col-
1 To whom reprint request should be addressed. Fax: 81-92-6322478.
0022-4804/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Hepatic microsomal cholesterol 7a-hydroxylase activity in the bile duct-ligated rats (l) and the sham-operated rats (m). Values are means { SEM of 5–6 rats. *P õ 0.01 compared with the sham-operated rats.
umns were purchased from Analytichem International (Harbor City, CA). TMSI-H (hexamethyldisilazane-trimethylchlorosilane-pyridine, 2:1:10) was purchased from Gasukuro Kogyo (Tokyo, Japan). NADPH was obtained from Kojin (Tokyo, Japan). Equipments. A Shimadzu GC15A gas liquid chromatography (GC), equipped with a flame ionization detector, a van den Berg’s solventless injector, and a data processing system (Chromatopack CR3A; Shimadzu, Kyoto, Japan), and a Shimadzu Auto GC-MS 9020DF gas liquid chromatography-mass spectometry (GC-MS) system with a data processing system (SCAP 1123) were employed. A fused-silica capillary column (33 m 1 0.2 mm i.d.) coated with a 0.25-mm layer of cross-linked methylsilicon (Hicap CBP1; Shimadzu, Kyoto, Japan) was used. Conditions for GC were as follows: column oven temperature 2807C, injection port temperature 2907C, detector temperature 2907C, flow rate of helium carrier gas 2.7 ml/min. Conditions for determination of 7a-hydroxycholesterol by GC-MS were the same as described previously [21]. Animal experiment. Eight-week-old male Wister rats (Kyudo, Fukuoka, Japan), weighing between 250 and 300 g, were used. The animals were kept in individual cages and had free access to water and standard laboratory chow (Oriental Yeast Co., Ltd, Tokyo, Japan) under the controlled 12-hr light–dark cycle. After at least 1 week of acclimation period, rats were divided into two groups. The laparotomy was performed through a transverse skin incision under the intraperitoneal injection of pentobarbital anesthesia (35 mg/kg body weight). In the obstructive jaundice group, the common bile duct was doubly ligated and divided to prevent recanalization. In the sham-operated group, laparotomy and manipulation of the liver and the common bile duct except for the ligation and division of the common bile duct were performed. In the both groups, five rats were sacrificed between 10 and 12 AM on Days 1, 2, 4, 7, and 14 after the operation. The liver, bile, and blood were obtained for the following determinations. This protocol was reviewed and approved by the Committee of the Ethics on Animal Experiment in Faculty of Medicine, Kyushu University, and carried out under the control of the Guideline for Animal Experiment of the institute. Determination of hepatic microsomal cholesterol 7a-hydroxylase activity. Cholesterol 7a-hydroxylase activity was determined by the method previously described [21]. Liver homogenate was prepared in 50 mM Tris–HCl buffer (pH 7.4), containing 0.3 M sucrose, 10 mM DTT, and 10 mM EDTA. The homogenate was centrifuged at 20,000g for 15 min. The microsomal fraction was obtained by centrifugation of the supernatant fluid at 100,000g for 1 hr. The microsomal pellet was suspended in the homogenizing medium without DTT and recentrifuged at 100,000g for 1 hr. The resulting microsomal pellet was resuspended in 0.1 M potassium phosphate buffer (pH 7.4) con-
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taining 1 mM EDTA. An aliquot of microsomal suspension was used for protein determination by the method of Lowry et al. [22]. The standard assay system consisted of 0.5 ml of the microsomal preparation corresponding to 0.5–1.0 mg of protein and 0.1 M phosphate buffer (pH 7.4) containing 1 mM EDTA and 1 mM NADPH in a total volume of 1.0 ml. The enzyme reaction was conducted for 15 min at 377C. The reaction was terminated by addition of chloroform/methanol (2:1, v/v). 5a-Cholestane-3b,7b-diol was added as an internal standard. After extraction with chloroform/methanol (2:1, v/v), the organic phase was evaporated to dryness under a stream of nitrogen. Following purification with Bond Elut silica cartridge column [21], actual mass of 7a-hydroxycholesterol was analyzed by GC-MS. In the selected ion monitoring mode, the ion at m/z 456 (M-90) was scanned for trimethylsilyl ether derivative of 7a-hydroxycholerol and m/z 458 (M-90) for that of the internal standard. Assay of serum 7a-hydroxycholesterol. Serum concentration of 7a-hydroxycholesterol was determined as described previously [23]. In brief, two hundred pmol of 5a-cholestane-3b,7b-diol dissolved in 50 ml of ethanol was added to 0.2 ml of serum as the internal standard. After addition of 0.7 ml of 0.9% NaCl solution and 1.8 ml of absolute ethanol, sterols were extracted with n-hexane. The collected n-hexane layer was evaporated to dryness under a stream of nitrogen. Sample was then hydrolyzed in 2.0 ml of 3% KOH in 90% ethanol at 557C for 45 min. After addition of 1.2 ml of saline, the sterols were extracted with n-hexane and the solvent was evaporated under nitrogen. Following purification and derivatization, the serum total 7a-hydroxycholesterol level was quantitated as described above. Analysis of biliary bile acids. Bile obtained by the cannulation into the bile duct was deproteinized with 10 vol of ethanol. An aliquot was hydrolyzed by Clostridial cholylglycine hydrolase [24]. The hydrolyzate was acidified with HCl and extracted with ethyl acetate. The extracted free acids were esterified with 5% ethanolic hydrochloric acid and silylated with DMESI. The samples were passed through a Sephadex LH-20 column to remove excess reagent as reported previously [25]. The derivatives were analyzed by GC [26]. Nordeoxycholic acid was used as an internal standard. Determination of serum and liver bile acid concentration. One hundred to two hundred miligrams of liver was hydrolyzed with 10% KOH in 90% ethanol at 807C for 2 hr. Bile acids were extracted on Bond Elut C18 cartridge columns and the total bile acid concentration in the liver tissue was determined by the 3a-hydroxysteroid dehydrogenase method [27–29]. Serum bile acid concentration was also determined. Determination of microsomal free cholesterol concentration. Twenty micrograms of cholestanol dissolved in 50 ml of ethanol was added to 0.5 ml of microsomal suspension and microsomal-free cholesterol was extracted with chloroform/methanol (2:1) and purified on Bond-Elut C18 cartridge columns. After trimethylsilyl ether deri-
FIG. 2. Serum 7a-hydroxycholesterol level in the bile duct-ligated rats (l) and the sham-operated rats (m). Values are means { SEM of 5–6 rats. *P õ 0.05; **P õ 0.01 compared with the shamoperated rats.
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Hepatic Microsomal Cholesterol Level The concentrations of hepatic microsomal-free cholesterol are shown in Table 1. There were no significant differences in the microsomal-free cholesterol level between the two groups. Significant correlation between the hepatic cholesterol 7a-hydroxylase activity and the hepatic microsomal-free cholesterol level was absent (n Å 47, r Å 0.181). Concentrations of Hepatic and Serum Bile Acids
FIG. 3. Correlation between hepatic cholesterol 7a-hydroxylase activity and serum 7a-hydroxycholesterol level. Symbols indicate bile duct-ligated rats (l) and the sham-operated rats (m). y Å 33.6x / 299.5, n Å 54, r Å 0.589, P õ 0.01. vatization, cholesterol was quantitated by GC as described previously [26]. Hydrophobicity index of the biliary bile acids. Hydrophobicity indices of the biliary bile acids were calculated using the values reported by Heuman et al. [31]. Statistical analysis. Results are expressed as mean { SEM. The statistical difference between the group means was evaluated by Mann–Whitney U test. Probability values less than 0.05 were considered significant. Correlation between the parameters were examined by Spearman’s rank correlation test.
RESULTS
Hepatic Microsomal Cholesterol 7a-Hydroxylase Activity Changes in the cholesterol 7a-hydroxylase activities after bile duct ligation are shown in Fig. 1. In the shamoperated rats, the cholesterol 7a-hydroxylase activity was essentially constant for 14 days and did not differ significantly from the preoperative control value. In the bile duct-ligated rats, the cholesterol 7a-hydroxylase activity increased and reached the maximal level on Day 2 (75.0 { 6.9 pmol/min/mg protein), keeping the significantly higher value for 7 days, and then slowly decreased to the preoperative level on Day 14. Ligation of the common bile duct significantly increased the enzyme activity compared with the sham-operated rats on Days 2, 4, and 7. Serum 7a-Hydroxycholesterol Level Time course of the serum 7a-hydroxycholesterol level after the operation is shown in Fig. 2. In the shamoperated rats, the serum 7a-hydroxycholesterol level was essentially constant throughout the experimental period similar to that of the hepatic cholesterol 7ahydroxylase activity. In the bile duct-ligated rats, the serum 7a-hydroxycholesterol level increased with the maximal value on Day 4 and then decreased. There were significantly higher values in the obstructive jaundice group on Days 2, 4, 7, and 14. Positive correlation (n Å 54, r Å 0.589, P õ 0.01) between the hepatic cholesterol 7a-hydroxylase activity and serum 7a-hydroxycholesterol level was found (Fig. 3).
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In the sham-operated rats, total hepatic bile acid concentration did not differ significantly from the preoperative value. In the bile duct-ligated rats, concentration of hepatic bile acids was increased to 8.5-fold of that of the control on Day 1 and maintained a significantly high value for 14 days compared with the sham-operated rats. In both groups, the total serum bile acid concentration behaved in a fashion similar to those of the hepatic bile acids (Table 2). There were no significant correlations between the hepatic cholesterol 7ahydroxylase activity and either hepatic total bile acids (n Å 43, r Å 0.245) or serum total bile acids level (n Å 42, r Å 0.393). Biliary Bile Acid Concentration The change of biliary bile acid concentration is shown in Table 3. In the sham-operated group, total bile acid concentration did not differ significantly from the preoperative value. In the bile duct-ligated group, total bile acid concentration gradually increased by Day 2 and then decreased by Day 14. On Day 14, the value was significantly lower than the preoperative level. Biliary Bile Acid Composition Biliary bile acid composition is shown in Table 4. In the sham-operated group, the most abundant bile acid was cholic acid and b-muricholic acid, which constituted about 69 and 15%, respectively. Bile duct ligation caused a significant increase in the proportion of bmuricholic acid as early as on Day 1 and the value was kept high by Day 7. But on Day 14, the proportion of b-muricholic acid decreased to 30%. There was a significant positive correlation (n Å 55, r Å 0.597, P õ TABLE 1 Microsomal-Free Cholestrol Level (mg/mg Microsomal Protein) Days after surgery 0 1 2 4 7 14
Sham 20.3 21.5 11.4 16.2 22.8 18.0
{ { { { { {
5.2 2.6 1.4 3.0 2.5 5.6
BDL (4) (4) (4) (5) (4) (3)
20.3 21.6 18.1 25.8 19.4 34.6
{ { { { { {
5.2 4.0 2.1 4.6 1.7 3.1
(4) (4) (6) (4) (4) (4)
Note. Values are expressed as means { SEM. BDL, bile duct ligation. Number of animals in parenthesis.
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TABLE 2 Total Bile Acid Concentration in the Liver and Serum Hepatic bile acid (nmol/g liver) Days after surgery
Sham
0 1 2 4 7 14
40.1 16.9 99.5 66.1 29.4 47.4
{ { { { { {
18.9 4.7 38.8 35.3 10.0 36.6
Serum bile acid (nmol/ml)
BDL (5) (4) (4) (4) (4) (4)
40.1 { 18.9 (5) 341.5 { 84.3a (5) 232.3 { 47.9 a (6) 333.4 { 97.5a (5) 335.1 { 146.4a (4) 456.6 { 141.2 a (4)
Sham 31.9 19.2 50.4 56.8 95.8 38.5
{ { { { { {
8.4 4.0 16.5 35.3 30.7 16.5
BDL (4) (4) (4) (4) (4) (4)
31.9 { 8.4 (4) 269.1 { 62.4a (4) 205.7 { 7.5a (6) 360.0 { 38.0 a (4) 199.0 { 18.5a (4) 247.5 { 34.5a (4)
Note. Values are expressed as means { SEM; BDL, Bile duct ligation. Number of animals in parenthesis. a P õ 0.05 compared with the sham-operated controls.
0.01) between the activity of cholesterol 7a-hydroxylase and the proportion of b-muricholic acid. In contrast, there was a significant negative correlation (n Å 55, r Å 00.647, P õ 0.01) between the activity of cholesterol 7a-hydroxylase and the proportion of cholic acid. The proportion of deoxycholic acid decreased to a trace level by the bile duct ligation, and there was no significant correlation between the enzyme activity and the proportion of deoxycholic acid. Hydrophobicity Index of the Biliary Bile Acids Changes in hydrophobicity indices of the biliary bile acids are shown in Fig. 4. In the control group, the biliary hydrophobicity was unchanged throughout the experimental period; however, it was significantly decreased from Day 1 to Day 7 in the bile duct-ligated group and returned to the value observed in the shamoperated group on Day 14. There was a significant negative correlation (n Å 55, r Å 0.538, P õ 0.01) between the activity of cholesterol 7a-hydroxylase and the hydrophobicity index of the biliary bile acids (Fig. 5). DISCUSSION
The basic concept of the negative feedback regulation of bile acid synthesis is initially based on the experimental result by Eriksson, who demonstrated a several-fold increase in bile acid synthesis after complete biliary diversion [32], and followed by studies showing TABLE 3 Biliary Total Bile Acid Concentration Days after surgery
Sham
0 1 2 4 7 14
21.0 21.7 26.3 27.3 25.0 23.5
{ { { { { {
1.8 4.7 2.0 3.7 1.9 2.3
BDL (4) (4) (4) (4) (5) (4)
21.0 18.7 26.3 19.6 19.6 3.0
{ { { { { {
1.8 2.1 1.6 0.9 3.5 1.2a
(4) (4) (4) (5) (5) (5)
Note. Values (nmol/ml) are expressed as means { SEM. Number of animals in parenthesis. a P õ 0.01 compared with sham-operated group.
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enhanced activities of cholesterol 7a-hydroxylase and bile acid synthesis after depletion of bile acids pool by biliary drainage or oral administration of cholestyramine [33–35]. On the other hand, bile acid synthesis is decreased by oral or intravenous administration of taurocholate and taurochenodeoxycholate [36]. Thus, it has been believed that the amount of bile acids returning to the liver via portal vein regulates bile acid synthesis at the level of cholesterol 7a-hydroxylase. In patients with obstructive jaundice, it has been reported that the activity of hepatic cholesterol 7a-hydroxylase [11] and bile acid synthesis [12] are decreased. It is conceivable that accumulated serum and hepatic bile acids due to the disturbance of the bile acid excretion inhibit the enzyme activity. In contrast to humans, it has been reported in rats that the obstruction of the common bile duct leads to a two- to threefold increase in the cholesterol 7a-hydroxylase activity [13]. This was confirmed by the present study that the hepatic cholesterol 7a-hydroxylase activity significantly increased during the first 7 days after the bile duct ligation, when serum and hepatic bile acid concentrations were significantly increased. However, cholesterol 7a-hydroxylase activity had no correlation with either hepatic or serum total bile acid concentrations, and biliary bile acid concentration also had no correlation with cholesterol 7a-hydroxylase activity, indicating that the enzyme activity is not directly regulated by the bile acid concentration itself. We hypothesized that hydrophobicity of biliary bile acids, initially introduced by Heuman et al. [16], could explain the paradoxical effects. They fed seven different bile acids to rats with intact enterohepatic circulation and observed that the hydrophilic bile acids do not alter cholesterol 7a-hydroxylase activity, whereas hydrophobic bile acids down-regulate the enzyme activity [16]. A highly significant negative correlation was observed between the hydrophobicity index of the bile acid pool and the activity of cholesterol 7a-hydroxylase in their study. The present results showed an increased proportion of b-muricholic acid with a reciprocal decrease in cholic acid moiety resulted in decreased hydrophobicity indices of biliary bile acid and increased enzyme activities. Deoxycholic acid, a hydrophobic bile
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TABLE 4 Proportion of Biliary Bile Acids Days 0 1 2 4 7 14
b-MCA 11.5 5.8 51.2 8.2 66.7 10.0 71.0 13.3 56.0 13.8 34.6
Sham BDL Sham BDL Sham BDL Sham BDL Sham BDL
{ { { { { { { { { { {
2.3 0.5 4.5a 2.3 3.6a 3.2 8.3a 2.8 10.1b 2.1 11.4b
CA 68.4 67.1 39.5 74.4 24.6 68.6 17.5 56.3 23.7 63.3 31.2
{ { { { { { { { { { {
2.0 2.5 6.2a 2.0 3.9a 2.4 2.0a 3.5 7.6b 2.3 11.2b
CDCA 9.1 20.4 4.1 9.0 3.9 11.2 9.5 14.0 14.7 9.8 27.5
{ { { { { { { { { { {
DCA
2.1 3.1 1.3b 2.4 1.0 1.2 5.6 3.8 7.3 0.8 12.3
6.9 4.1 1.0 6.7 0.6 7.8 1.0 8.3 1.1 6.0 2.1
{ { { { { { { { { { {
0.9 0.9 0.5b 1.2 0.2a 1.2 0.7a 1.4 0.4a 0.7 0.8a
LCA 0.3 0.2 0.3 0.2 0.6 0.3 0.2 0.6 0.7 0.4 2.2
{ { { { { { { { { { {
0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.4 0.1 1.3
UDCA 3.0 1.3 0.8 1.4 0.5 1.6 0.6 3.7 1.0 1.8 2.3
{ { { { { { { { { { {
0.7 0.4 0.3 0.3 0.2a 0.3 0.2a 1.1 0.4 0.5 0.7
Note. Data (%) are expressed as mean { SEM of 5 rats. BDL, bile duct ligatrion; b-MCA, b-muricholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; UDCA, ursodeoxycholic acid. a P õ 0.01 vs sham group. b P õ 0.05 vs sham group.
acid, was a minor component of bile in rats and was decreased to a trace amount after the bile duct ligation, thus having little effect on total biliary hydrophobicity and the enzyme activity. A significant negative correlation was evident between the enzyme activities and biliary bile acids hydrophobicity indices. Proportion of cholic acid and b-muricholic acid also correlated with the enzyme activities. The results strongly suggest that the cholesterol 7a-hydroxylase activity is not modulated by the bile acid concentration but is regulated by hydrophobicity indices of biliary bile acids. It should be noted that the activity of cholesterol 7a-hydroxylase returned to the control level on Day 14 after the bile duct ligation, although serum and hepatic bile acid concentrations remained still high. The result suggests the presence of hepatocyte injury by prolonged biliary obstruction, as the mortality rate was high (approximately 40%) and most of the survived rats had ascites on Day 14. b-Muricholic acid is one of the 6b-hydroxylated prod-
ucts of chenodeoxycholic acid in the rat. It is a hydrophilic bile acid and is efficiently excreted in urine [37]. Thus, 6b-hydroxylation of bile acids are considered to be a bioprotection against cytotoxicities of hydrophobic bile acids, just like glucuronidation [38] and sulfation [39] of bile acids. The present results are consistent with that of Danielsson who reported increased bile acid 6b-hydroxylase activity in rats after bile duct ligation [13]. It is possible that chenodeoxycholic acid was effectively 6b-hydroxylated by increased activity of 6bhydroxylase, resulting in an increased proportion of bmuricholic acid and a decreased biliary hydrophobicity, which in turn increased cholesterol 7a-hydroxylase activity. In the present study, bile acid composition in the hepatocyte was not analyzed. However, we had previously shown that composition of bile acids in the hepatocytes is very similar to that of biliary bile acids by GC-MS [42]. It is thus probable that the hydrophobicity of bile acids in the hepatocytes may be similar to that in bile.
FIG. 4. Changes in hydrophobicity indices of the biliary bile acids in the bile duct-ligated rats (l) and the sham-operated rats (m). Values are means { SEM of 5 rats. *P õ 0.01 compared with the sham-operated rats.
FIG. 5. Correlation between hepatic cholesterol 7a-hydroxylase activity and hydrophobicity indices of the biliary bile acids. Symbols indicate bile duct-ligated rats (l) and the sham-operated rats (m). y Å 00.006x / 0.094, n Å 55, r Å 0.538, P õ 0.01.
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Steroid 12a-hydroxylase activity is reduced by bile acid feeding [40, 41]. Danielsson [13] also reported the decreased steroid 12a-hydroxylase activity in the rat after bile duct ligation. In the present study, proportion of cholic acid metabolites (cholic acid and deoxycholic acid) decreased significantly and that of chenodeoxycholic acid metabolites (chenodeoxycholic acid, b-muricholic acid, ursodeoxycholic acid, and lithocholic acid) increased by bile duct ligation. These results suggest that bile duct ligation also caused the inhibition of steroid 12a-hydroxylase activity as well as stimulation of cholesterol 7a-hydroxylase and bile acid 6b-hydroxylase activities in rats. Another possible mechanism to explain the paradoxical results is the recent observation by Pandak et al., showing that intravenous infusion of taurocholate failed to suppress the mRNA level, transcription rate, enzyme mass, and specific activity of cholesterol 7ahydroxylase [43]. Exclusion of bile acids from the intestine, whether by bile duct obstruction or biliary diversion, resulted in a loss of inhibitory effect of ‘‘intestinal factor(s)’’ and consequently in up-regulation of cholesterol 7a-hydroxylase [43]. Those ‘‘intestinal factor(s)’’ have not been identified so far and further studies should be required to answer this important problem. Serum 7a-hydroxycholesterol level was also increased by bile duct ligation and there was a significant positive correlation between the serum level of 7a-hydroxycholesterol and the hepatic cholesterol 7a-hydroxylase activity. We have shown that the serum level of 7a-hydroxycholesterol reflects hepatic cholesterol 7a-hydroxylase activity under various conditions in humans [19, 23, 33] and in experimental animals [17, 20]. The present result again confirmed the usefulness of serum 7a-hydroxycholesterol level as an indicator of hepatic bile acid synthesis. In conclusion, the increased activity of hepatic cholesterol 7a-hydroxylase is accompanied by the increased proportion of biliary b-muricholic acid and the decreased biliary hydrophobicity in rats with obstructive jaundice. This may be a rationale of the increased bile acid synthesis in spite of the markedly accumulated serum and hepatic bile acids levels in rats.
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ACKNOWLEDGMENTS The authors thank Drs. Yamashita, Makino, and Komura for their helpful suggestions and Miss Nishimura for her technical help. This work was supported in part by Grants-in-Aid 07671403 (S. Kuroki) and 07671406 (K. Chijiiwa) from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Myant, N. B., and Eder, H. A. The effect of drainage upon the synthesis of cholesterol in the liver. J. Lipid Res. 2: 363, 1961. 2. Heuman, D. M., Hernandes, C. R., Hylemon, P. B., Kubaska, W. M., Hartman, C., and Vlahcevic, Z. R. Regulation of bile acid synthesis. I. Effects of conjugated ursodeoxycholate and cholate on bile acid synthesis in chronic bile fistula rat. Hepatology 8: 358, 1988. ˚ kerland, J. E., and Bjo¨rkhem, I. The pool of 3. Einarsson, K., A
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free cholesterol is not of major importance for regulation of the cholesterol 7a-hydroxylase activity in rat liver microsomes. J. Lipid Res. 28: 253, 1987. Shefer, S., Hauser, S., Lapar, V., and Mosbach, E. H. Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7a-hydroxylase in the rat. J. Lipid Res. 14: 573, 1973. Mitropoulos, K. A., Balasubramaniam, S., and Myant, N. B. The effect of interruption of the enterohepatic circulation of bile acids and of cholesterol feeding on cholesterol 7a-hydroxylase in relation to the diurnal rhythm in its activity. Biochim. Biophys. Acta 326: 428, 1973. Sandberg, D. H., Sjo¨vall, J., Sjo¨vall, K., and Turner, D. A. Measurement of human serum bile acids by gas-liquid chromatography. J. Lipid Res. 6: 182, 1965. Murphy, G. M., Ross, A., and Billing, B. H. Serum bile acids in primary biliary cirrhosis. Gut 13: 201, 1972. Makino, I., Hashimoto, H., Shinozuka, K., Yoshino, K., and Nakagawa, S. Sulfated and nonsulfated bile acids in urine, serum, and bile of patients with hepatobiliary diseases. Gastroenterology 68: 545, 1975. Eklund, A., Norlander, A., and Norman, A. Bile acid synthesis and excretion following release of total extrahepatic cholestasis by percutaneous transhepatic drainage. Eur. J. Clin. Invest. 10: 349, 1980. Axelson, M., Mork, B., Aly, A., Wissen, O., and Sjo¨vall, J. Concentration of cholestenoic acids in plasma from patients with liver disease. J. Lipid Res. 30: 1877, 1989. Salen, G., Nicolau, G., Shefer, S., and Mosbach, E. H. Hepatic cholesterol metabolism in patients with gallstones. Gastroenterology 69: 676, 1975. Ichimiya, H., Yanagisawa, J., and Nakayama, F. Altered metabolism of bile alcohol and bile acid in complete extrahepatic cholestasis: Qualitative and quantitative aspects. J. Lipid Res. 28: 1028, 1987. Danielsson, H. Effect of biliary obstruction on formation and metabolism of bile acids in rat. Steroids 22: 567, 1973. Dueland, S., Reichen, J., Everson, G., and Davis, R. Regulation of cholesterol and bile acid homeostasis in bile obstructed rats. Biochem. J. 280: 373, 1991. Davis, R. A., Musso, C. A., Malone-McNeal, M., Lattier, G. R., Hyde, P. M., Archambault-Schexnayder, J., and Straka, M. Examination of bile acid negative feedback regulation in rats. J. Lipid Res. 29: 202, 1988. Heuman, D. M., Hylemon, P. B., and Vlahcevic, Z. R. Regulation of bile acid synthesis. III. Correlation between biliary bile salt hydrophobicity index and the activities of enzymes regulating cholesterol and bile acid synthesis in the rat. J. Lipid Res. 30: 1161, 1989. Fukushima, K., Ichimiya, H., Higashijima, H., Yamashita, Y., Kuroki, S., and Chijiiwa, K. Regulation of bile acid synthesis in the rat: Relationship between hepatic cholesterol 7a-hydroxylase activity and portal bile acids. J. Lipid Res. 36: 315, 1995.
18. Kuroki, S., Okamoto, S., Kosahara, K., Kishinaka, M., Yamashita, H., Ichimiya, H., Chijiiwa, K., and Oda, H. Effect of biliary drainage on serum 7a-hydroxycholesterol level in patients with obstructive jaundice. J. Surg. Res. 57: 352, 1994. 19. Kuroki, S., Okamoto, S., Naito, T., Oda, H., Nagase, S., et al. Serum 7a-hydroxycholesterol as a new parameter of liver function in patients with chronic liver diseases. Hepatology 22: 1182, 1995. 20. Nakano, K., Chijiiwa, K., Okamoto, S., Yamashita, H., Kuroki, S., and Tanaka, M. Hepatic cholesterol 7a-hydroxylase activity and serum 7a-hydroxycholesterol level during liver regeneration after partial hepatectomy in rats. Eur. Surg. Res. 27: 389, 1995. 21. Yamashita, H., Kuroki, S., and Nakayama, F. Assay of cholesterol 7a-hydroxylase utilizing a silica cartridge column and 5a-
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22. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265, 1951. 23. Oda, H., Yamashita, H., Kosahara, K., Kuroki, S., and Nakayama, F. Esterified and total 7a-hydroxycholesterol in human serum as an indicator for hepatic bile acid synthesis. J. Lipid Res. 31: 2209, 1990. 24. Nair, P. P., Gordon, M., and Rebach, J. The enzymatic cleavage of the carbon-nitrogen bond in 3a,7a,12a-trihydroxy-5b-cholan24-oylglycine. J. Biol. Chem. 242: 7, 1967. 25. Yanagisawa, J., Itoh, M., Ishibashi, M., Miyazaki, H., and Nakayama, F. Microanalysis of bile acid in human liver tissue by selected ion monitoring. Anal. Biochem. 104: 75, 1980.
33.
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35. 36.
26. Chijiiwa, K., and Nakayama, F. Simultaneous microanalysis of bile acids and cholesterol in bile by glass capillary column gas chromatography. J. Chromatogr. 431: 17, 1988.
37.
27. Talalay, P. In D. Glick (Ed.), Methods of Biochemical Analysis. New York: Interscience, 1960. Vol. 8, p. 119.
38.
28. Admirand, W. H., and Small, D. H. The physicochemical basis of cholesterol gallstone formation in man. J. Clin. Invest. 47: 1043, 1968.
39.
29. Turley, S. D., and Dietschy, J. M. Re-evaluation of the 3a-hydroxysteroid dehydrogenase assay for total bile acids in bile. J. Lipid Res. 19: 924, 1978. 30. Kishinaka, K., Kosahara, K., Okamoto, S., Oda, H., Ichimiya, H., Chijiiwa, K., and Kuroki, S. Metabolism of intravenously administered 7a-hydroxycholesterol-3b-stearate in hamsters. Biochim. Biophys. Acta 1165: 222, 1992. 31. Heuman, D. M. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J. Lipid Res. 30: 719, 1989. 32. Eriksson, S. Biliary excretion of bile acids and cholesterol in
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bile fistula rats. Bile acids and steroids. Proc. Soc. Exp. Biol. Med. 94: 578, 1957. Okamoto, S., Fukushima, K., Higashijima, H., Makino, I., Kishinaka, M., Oda, H., Yamashita, H., Ichimiya, H., Chijiiwa, K., and Kuroki, S. Serum 7a-hydroxycholesterol reflects hepatic bile acid synthesis in patients with obstructive jaundice after external biliary drainage. Hepatology 20: 95, 1994. Danielsson, H., Einarsson, K., and Johansson, G. Effect of biliary drainage on individual reactions in the conversion of cholesterol to taurocholic acid. Eur. J. Biochem. 2: 44, 1967. Shefer, S., Hauser, S., and Mosbach, E. H. 7a-Hydroxylation of cholestanol by rat liver microsomes. J. Lipid Res. 9: 328, 1968. Heuman, D., Vlahcevic, Z. R., Bailey, M. L., and Hylemon, P. B. Regulation of bile acid synthesis II. Effect of bile acid feeding on enzymes regulating hepatic cholesterol and bile acid synthesis in the rat. Hepatology 8: 892, 1988. Mahowald, T. A., Matschiner, J. T., and Hsia, S. L. Bile acid III. Acid I: The principal bile acid in urine of surgically jaundiced rats. J. Biol. Chem. 225: 795, 1957. Little, J. M., Chair, M. V., and Lester, R. Excretion of cholate glucuronide. J. Lipid Res. 26: 583, 1985. Palmer, R. H. The formation of bile acid sulfates: A new pathway of bile acid metabolism in humans. Proc. Natl. Acad. Sci. USA 58: 1047, 1967. Danielsson, H. Influence of dietary bile acids on formation of bile acids in rat. Steroids 22: 667, 1973. Kuroki, S., and Hoshita, T. Effect of bile acid feeding on hepatic steroid 12a-hydroxylase activity in hamsters. Lipids 18: 789, 1983. Akashi, Y., Miyazaki, H., and Nakayama, F. Correlation of bile acid composition between liver tissue and bile. Clin. Chim. Acta 133: 125, 1983. Pandak, W. M., Heuman, D. M., Hylemon, P. B., Chiang, J. Y., and Vlahcevic, Z. R. Failure of intravenous infusion of taurocholate to down regulate cholesterol 7a-hydroxylase in rats with biliary fistulas. Gastroenterology 108: 533, 1995.
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Bile Acid Synthesis and Biliary Hydrophobicity during Obstructive Jaundice in Rats TOKIO NAITO, M.D., SYOJI KUROKI, M.D.,1 KAZUO CHIJIIWA, M.D., FACS,
AND
MASAO TANAKA, M.D.
Department of Surgery I, Kyushu University Faculty of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan Submitted for publication April 19, 1996
INTRODUCTION Hepatic cholesterol 7a-hydroxylase, the rate-limiting enzyme for bile acid synthesis, is regulated by bile acid returning to the liver. In the bile duct-ligated rats, however, cholesterol 7a-hydroxylase activity is known to increase in spite of the elevated serum bile acids. The aim of this study was to investigate the relationship between the bile acid synthesis and biliary hydrophobicity to solve the paradoxical phenomenon. Male Wister rats (250–350 g) were divided into two groups, bile duct-ligated group and sham-operated group. Rats were sacrificed on the Days 1, 2, 4, 7, and 14 after the operation. Cholesterol 7a-hydroxylase activity, biliary bile acid composition, and biliary hydrophobicity were analyzed. Hepatic and serum total bile acid concentrations and serum 7a-hydroxycholesterol levels were also determined. Bile duct ligation caused significant increases in hepatic and serum bile acid concentrations on Day 1, which persisted for 14 days after the bile duct ligation. Activities of hepatic cholesterol 7a-hydroxylase increased to 3.2-fold of the preoperative value on Day 2, remained significantly high until Day 7, and then decreased to the basal value on Day 14. The serum 7a-hydroxycholesterol level essentially behaved in a similar fashion to that of hepatic cholesterol 7a-hydroxylase activity with a significant (P õ 0.01) positive correlation. b-Muricholic acid was predominant in bile until Day 7 (71 vs 10% in the controls on Day 4; P õ 0.05) with a concomitant decrease in the proportion of cholic acid. Biliary bile acid became less hydrophobic and hepatic cholesterol 7a-hydroxylase activity significantly correlated with the hydrophobicity of biliary bile acids (n Å 55, r Å 0.54, P õ 0.01). There were no significant correlations between the activity of cholesterol 7a-hydroxylase and total bile acid concentrations in serum, liver, or bile. The increased cholesterol 7a-hydroxylase activity is accompanied by the decreased biliary hydrophobicity, which may be a rationale for the paradoxical increase in bile acid synthesis in spite of the accumulation of bile acids in the serum and liver during obstructive jaundice in rats. q 1996 Academic Press, Inc.
Hepatic microsomal cholesterol 7a-hydroxylase catalyzes the first and rate-limiting step in the major pathway for bile acid biosynthesis from cholesterol. It has been believed that the activity of cholesterol 7a-hydroxylase is regulated by bile acids returning to the liver. Several investigators have reported that depletion of bile acids by biliary drainage or dietary administration of cholestyramine increases the enzyme activity and bile acid synthesis [1–5]. In patients with obstructive jaundice, it has been reported that the serum bile acid concentrations are elevated [6–10], and hepatic cholesterol 7a-hydroxylase activity [11] and bile acid synthesis [9, 12] are reduced. In bile duct-ligated rats, however, Danielsson [13] and Dueland et al. [14] showed a significant increase in the activity of cholesterol 7ahydroxylase. Recently, the concept of the bile acid feedback regulation mechanism has been questioned because of the failure to demonstrate the inhibition of bile acid synthesis in bile fistula rats [15] and in cultured hepatocytes. Heuman et al. [16] recently reported that hydrophobic bile acids suppressed the cholesterol 7ahydroxylase activity and this could be one of the reasons to explain the conflicting observations between humans and rats. We have previously reported that bile acid synthesis is not regulated directly by the portal bile acids returning to the liver [17]. The aim of the present study was to examine whether bile duct ligation increases biliary bile acid hydrophilicity and hence the activity of hepatic cholesterol 7ahydroxylase in the bile duct-ligated rats. Serum 7ahydroxycholesterol level was also monitored to confirm if the level could be the indicator of the hepatic enzyme activity [17–20] during obstructive jaundice. MATERIALS AND METHODS Materials. All solvents used were of analytical grade or distilled prior to use. Sephadex LH-20 was purchased from Pharmacia Fine Chemicals AB (Uppsala, Sweden) and dimethylethylsilylimidazole (DMESI) was from Tokyo Kasei Kogyo (Tokyo, Japan). Cholesterol, cholylglycinehydrolase, 3a-hydroxysteroid dehydrogenase, and dithiothreitol (DTT) were obtained from Sigma Chemical Co. (St. Louis, MO). 7a-Hydroxycholesterol and 5a-cholestane-3b,7b-diol were synthesized as described previously [21]. Bond Elut silica cartridge col-
1 To whom reprint request should be addressed. Fax: 81-92-6322478.
0022-4804/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Hepatic microsomal cholesterol 7a-hydroxylase activity in the bile duct-ligated rats (l) and the sham-operated rats (m). Values are means { SEM of 5–6 rats. *P õ 0.01 compared with the sham-operated rats.
umns were purchased from Analytichem International (Harbor City, CA). TMSI-H (hexamethyldisilazane-trimethylchlorosilane-pyridine, 2:1:10) was purchased from Gasukuro Kogyo (Tokyo, Japan). NADPH was obtained from Kojin (Tokyo, Japan). Equipments. A Shimadzu GC15A gas liquid chromatography (GC), equipped with a flame ionization detector, a van den Berg’s solventless injector, and a data processing system (Chromatopack CR3A; Shimadzu, Kyoto, Japan), and a Shimadzu Auto GC-MS 9020DF gas liquid chromatography-mass spectometry (GC-MS) system with a data processing system (SCAP 1123) were employed. A fused-silica capillary column (33 m 1 0.2 mm i.d.) coated with a 0.25-mm layer of cross-linked methylsilicon (Hicap CBP1; Shimadzu, Kyoto, Japan) was used. Conditions for GC were as follows: column oven temperature 2807C, injection port temperature 2907C, detector temperature 2907C, flow rate of helium carrier gas 2.7 ml/min. Conditions for determination of 7a-hydroxycholesterol by GC-MS were the same as described previously [21]. Animal experiment. Eight-week-old male Wister rats (Kyudo, Fukuoka, Japan), weighing between 250 and 300 g, were used. The animals were kept in individual cages and had free access to water and standard laboratory chow (Oriental Yeast Co., Ltd, Tokyo, Japan) under the controlled 12-hr light–dark cycle. After at least 1 week of acclimation period, rats were divided into two groups. The laparotomy was performed through a transverse skin incision under the intraperitoneal injection of pentobarbital anesthesia (35 mg/kg body weight). In the obstructive jaundice group, the common bile duct was doubly ligated and divided to prevent recanalization. In the sham-operated group, laparotomy and manipulation of the liver and the common bile duct except for the ligation and division of the common bile duct were performed. In the both groups, five rats were sacrificed between 10 and 12 AM on Days 1, 2, 4, 7, and 14 after the operation. The liver, bile, and blood were obtained for the following determinations. This protocol was reviewed and approved by the Committee of the Ethics on Animal Experiment in Faculty of Medicine, Kyushu University, and carried out under the control of the Guideline for Animal Experiment of the institute. Determination of hepatic microsomal cholesterol 7a-hydroxylase activity. Cholesterol 7a-hydroxylase activity was determined by the method previously described [21]. Liver homogenate was prepared in 50 mM Tris–HCl buffer (pH 7.4), containing 0.3 M sucrose, 10 mM DTT, and 10 mM EDTA. The homogenate was centrifuged at 20,000g for 15 min. The microsomal fraction was obtained by centrifugation of the supernatant fluid at 100,000g for 1 hr. The microsomal pellet was suspended in the homogenizing medium without DTT and recentrifuged at 100,000g for 1 hr. The resulting microsomal pellet was resuspended in 0.1 M potassium phosphate buffer (pH 7.4) con-
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taining 1 mM EDTA. An aliquot of microsomal suspension was used for protein determination by the method of Lowry et al. [22]. The standard assay system consisted of 0.5 ml of the microsomal preparation corresponding to 0.5–1.0 mg of protein and 0.1 M phosphate buffer (pH 7.4) containing 1 mM EDTA and 1 mM NADPH in a total volume of 1.0 ml. The enzyme reaction was conducted for 15 min at 377C. The reaction was terminated by addition of chloroform/methanol (2:1, v/v). 5a-Cholestane-3b,7b-diol was added as an internal standard. After extraction with chloroform/methanol (2:1, v/v), the organic phase was evaporated to dryness under a stream of nitrogen. Following purification with Bond Elut silica cartridge column [21], actual mass of 7a-hydroxycholesterol was analyzed by GC-MS. In the selected ion monitoring mode, the ion at m/z 456 (M-90) was scanned for trimethylsilyl ether derivative of 7a-hydroxycholerol and m/z 458 (M-90) for that of the internal standard. Assay of serum 7a-hydroxycholesterol. Serum concentration of 7a-hydroxycholesterol was determined as described previously [23]. In brief, two hundred pmol of 5a-cholestane-3b,7b-diol dissolved in 50 ml of ethanol was added to 0.2 ml of serum as the internal standard. After addition of 0.7 ml of 0.9% NaCl solution and 1.8 ml of absolute ethanol, sterols were extracted with n-hexane. The collected n-hexane layer was evaporated to dryness under a stream of nitrogen. Sample was then hydrolyzed in 2.0 ml of 3% KOH in 90% ethanol at 557C for 45 min. After addition of 1.2 ml of saline, the sterols were extracted with n-hexane and the solvent was evaporated under nitrogen. Following purification and derivatization, the serum total 7a-hydroxycholesterol level was quantitated as described above. Analysis of biliary bile acids. Bile obtained by the cannulation into the bile duct was deproteinized with 10 vol of ethanol. An aliquot was hydrolyzed by Clostridial cholylglycine hydrolase [24]. The hydrolyzate was acidified with HCl and extracted with ethyl acetate. The extracted free acids were esterified with 5% ethanolic hydrochloric acid and silylated with DMESI. The samples were passed through a Sephadex LH-20 column to remove excess reagent as reported previously [25]. The derivatives were analyzed by GC [26]. Nordeoxycholic acid was used as an internal standard. Determination of serum and liver bile acid concentration. One hundred to two hundred miligrams of liver was hydrolyzed with 10% KOH in 90% ethanol at 807C for 2 hr. Bile acids were extracted on Bond Elut C18 cartridge columns and the total bile acid concentration in the liver tissue was determined by the 3a-hydroxysteroid dehydrogenase method [27–29]. Serum bile acid concentration was also determined. Determination of microsomal free cholesterol concentration. Twenty micrograms of cholestanol dissolved in 50 ml of ethanol was added to 0.5 ml of microsomal suspension and microsomal-free cholesterol was extracted with chloroform/methanol (2:1) and purified on Bond-Elut C18 cartridge columns. After trimethylsilyl ether deri-
FIG. 2. Serum 7a-hydroxycholesterol level in the bile duct-ligated rats (l) and the sham-operated rats (m). Values are means { SEM of 5–6 rats. *P õ 0.05; **P õ 0.01 compared with the shamoperated rats.
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Hepatic Microsomal Cholesterol Level The concentrations of hepatic microsomal-free cholesterol are shown in Table 1. There were no significant differences in the microsomal-free cholesterol level between the two groups. Significant correlation between the hepatic cholesterol 7a-hydroxylase activity and the hepatic microsomal-free cholesterol level was absent (n Å 47, r Å 0.181). Concentrations of Hepatic and Serum Bile Acids
FIG. 3. Correlation between hepatic cholesterol 7a-hydroxylase activity and serum 7a-hydroxycholesterol level. Symbols indicate bile duct-ligated rats (l) and the sham-operated rats (m). y Å 33.6x / 299.5, n Å 54, r Å 0.589, P õ 0.01. vatization, cholesterol was quantitated by GC as described previously [26]. Hydrophobicity index of the biliary bile acids. Hydrophobicity indices of the biliary bile acids were calculated using the values reported by Heuman et al. [31]. Statistical analysis. Results are expressed as mean { SEM. The statistical difference between the group means was evaluated by Mann–Whitney U test. Probability values less than 0.05 were considered significant. Correlation between the parameters were examined by Spearman’s rank correlation test.
RESULTS
Hepatic Microsomal Cholesterol 7a-Hydroxylase Activity Changes in the cholesterol 7a-hydroxylase activities after bile duct ligation are shown in Fig. 1. In the shamoperated rats, the cholesterol 7a-hydroxylase activity was essentially constant for 14 days and did not differ significantly from the preoperative control value. In the bile duct-ligated rats, the cholesterol 7a-hydroxylase activity increased and reached the maximal level on Day 2 (75.0 { 6.9 pmol/min/mg protein), keeping the significantly higher value for 7 days, and then slowly decreased to the preoperative level on Day 14. Ligation of the common bile duct significantly increased the enzyme activity compared with the sham-operated rats on Days 2, 4, and 7. Serum 7a-Hydroxycholesterol Level Time course of the serum 7a-hydroxycholesterol level after the operation is shown in Fig. 2. In the shamoperated rats, the serum 7a-hydroxycholesterol level was essentially constant throughout the experimental period similar to that of the hepatic cholesterol 7ahydroxylase activity. In the bile duct-ligated rats, the serum 7a-hydroxycholesterol level increased with the maximal value on Day 4 and then decreased. There were significantly higher values in the obstructive jaundice group on Days 2, 4, 7, and 14. Positive correlation (n Å 54, r Å 0.589, P õ 0.01) between the hepatic cholesterol 7a-hydroxylase activity and serum 7a-hydroxycholesterol level was found (Fig. 3).
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In the sham-operated rats, total hepatic bile acid concentration did not differ significantly from the preoperative value. In the bile duct-ligated rats, concentration of hepatic bile acids was increased to 8.5-fold of that of the control on Day 1 and maintained a significantly high value for 14 days compared with the sham-operated rats. In both groups, the total serum bile acid concentration behaved in a fashion similar to those of the hepatic bile acids (Table 2). There were no significant correlations between the hepatic cholesterol 7ahydroxylase activity and either hepatic total bile acids (n Å 43, r Å 0.245) or serum total bile acids level (n Å 42, r Å 0.393). Biliary Bile Acid Concentration The change of biliary bile acid concentration is shown in Table 3. In the sham-operated group, total bile acid concentration did not differ significantly from the preoperative value. In the bile duct-ligated group, total bile acid concentration gradually increased by Day 2 and then decreased by Day 14. On Day 14, the value was significantly lower than the preoperative level. Biliary Bile Acid Composition Biliary bile acid composition is shown in Table 4. In the sham-operated group, the most abundant bile acid was cholic acid and b-muricholic acid, which constituted about 69 and 15%, respectively. Bile duct ligation caused a significant increase in the proportion of bmuricholic acid as early as on Day 1 and the value was kept high by Day 7. But on Day 14, the proportion of b-muricholic acid decreased to 30%. There was a significant positive correlation (n Å 55, r Å 0.597, P õ TABLE 1 Microsomal-Free Cholestrol Level (mg/mg Microsomal Protein) Days after surgery 0 1 2 4 7 14
Sham 20.3 21.5 11.4 16.2 22.8 18.0
{ { { { { {
5.2 2.6 1.4 3.0 2.5 5.6
BDL (4) (4) (4) (5) (4) (3)
20.3 21.6 18.1 25.8 19.4 34.6
{ { { { { {
5.2 4.0 2.1 4.6 1.7 3.1
(4) (4) (6) (4) (4) (4)
Note. Values are expressed as means { SEM. BDL, bile duct ligation. Number of animals in parenthesis.
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TABLE 2 Total Bile Acid Concentration in the Liver and Serum Hepatic bile acid (nmol/g liver) Days after surgery
Sham
0 1 2 4 7 14
40.1 16.9 99.5 66.1 29.4 47.4
{ { { { { {
18.9 4.7 38.8 35.3 10.0 36.6
Serum bile acid (nmol/ml)
BDL (5) (4) (4) (4) (4) (4)
40.1 { 18.9 (5) 341.5 { 84.3a (5) 232.3 { 47.9 a (6) 333.4 { 97.5a (5) 335.1 { 146.4a (4) 456.6 { 141.2 a (4)
Sham 31.9 19.2 50.4 56.8 95.8 38.5
{ { { { { {
8.4 4.0 16.5 35.3 30.7 16.5
BDL (4) (4) (4) (4) (4) (4)
31.9 { 8.4 (4) 269.1 { 62.4a (4) 205.7 { 7.5a (6) 360.0 { 38.0 a (4) 199.0 { 18.5a (4) 247.5 { 34.5a (4)
Note. Values are expressed as means { SEM; BDL, Bile duct ligation. Number of animals in parenthesis. a P õ 0.05 compared with the sham-operated controls.
0.01) between the activity of cholesterol 7a-hydroxylase and the proportion of b-muricholic acid. In contrast, there was a significant negative correlation (n Å 55, r Å 00.647, P õ 0.01) between the activity of cholesterol 7a-hydroxylase and the proportion of cholic acid. The proportion of deoxycholic acid decreased to a trace level by the bile duct ligation, and there was no significant correlation between the enzyme activity and the proportion of deoxycholic acid. Hydrophobicity Index of the Biliary Bile Acids Changes in hydrophobicity indices of the biliary bile acids are shown in Fig. 4. In the control group, the biliary hydrophobicity was unchanged throughout the experimental period; however, it was significantly decreased from Day 1 to Day 7 in the bile duct-ligated group and returned to the value observed in the shamoperated group on Day 14. There was a significant negative correlation (n Å 55, r Å 0.538, P õ 0.01) between the activity of cholesterol 7a-hydroxylase and the hydrophobicity index of the biliary bile acids (Fig. 5). DISCUSSION
The basic concept of the negative feedback regulation of bile acid synthesis is initially based on the experimental result by Eriksson, who demonstrated a several-fold increase in bile acid synthesis after complete biliary diversion [32], and followed by studies showing TABLE 3 Biliary Total Bile Acid Concentration Days after surgery
Sham
0 1 2 4 7 14
21.0 21.7 26.3 27.3 25.0 23.5
{ { { { { {
1.8 4.7 2.0 3.7 1.9 2.3
BDL (4) (4) (4) (4) (5) (4)
21.0 18.7 26.3 19.6 19.6 3.0
{ { { { { {
1.8 2.1 1.6 0.9 3.5 1.2a
(4) (4) (4) (5) (5) (5)
Note. Values (nmol/ml) are expressed as means { SEM. Number of animals in parenthesis. a P õ 0.01 compared with sham-operated group.
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enhanced activities of cholesterol 7a-hydroxylase and bile acid synthesis after depletion of bile acids pool by biliary drainage or oral administration of cholestyramine [33–35]. On the other hand, bile acid synthesis is decreased by oral or intravenous administration of taurocholate and taurochenodeoxycholate [36]. Thus, it has been believed that the amount of bile acids returning to the liver via portal vein regulates bile acid synthesis at the level of cholesterol 7a-hydroxylase. In patients with obstructive jaundice, it has been reported that the activity of hepatic cholesterol 7a-hydroxylase [11] and bile acid synthesis [12] are decreased. It is conceivable that accumulated serum and hepatic bile acids due to the disturbance of the bile acid excretion inhibit the enzyme activity. In contrast to humans, it has been reported in rats that the obstruction of the common bile duct leads to a two- to threefold increase in the cholesterol 7a-hydroxylase activity [13]. This was confirmed by the present study that the hepatic cholesterol 7a-hydroxylase activity significantly increased during the first 7 days after the bile duct ligation, when serum and hepatic bile acid concentrations were significantly increased. However, cholesterol 7a-hydroxylase activity had no correlation with either hepatic or serum total bile acid concentrations, and biliary bile acid concentration also had no correlation with cholesterol 7a-hydroxylase activity, indicating that the enzyme activity is not directly regulated by the bile acid concentration itself. We hypothesized that hydrophobicity of biliary bile acids, initially introduced by Heuman et al. [16], could explain the paradoxical effects. They fed seven different bile acids to rats with intact enterohepatic circulation and observed that the hydrophilic bile acids do not alter cholesterol 7a-hydroxylase activity, whereas hydrophobic bile acids down-regulate the enzyme activity [16]. A highly significant negative correlation was observed between the hydrophobicity index of the bile acid pool and the activity of cholesterol 7a-hydroxylase in their study. The present results showed an increased proportion of b-muricholic acid with a reciprocal decrease in cholic acid moiety resulted in decreased hydrophobicity indices of biliary bile acid and increased enzyme activities. Deoxycholic acid, a hydrophobic bile
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TABLE 4 Proportion of Biliary Bile Acids Days 0 1 2 4 7 14
b-MCA 11.5 5.8 51.2 8.2 66.7 10.0 71.0 13.3 56.0 13.8 34.6
Sham BDL Sham BDL Sham BDL Sham BDL Sham BDL
{ { { { { { { { { { {
2.3 0.5 4.5a 2.3 3.6a 3.2 8.3a 2.8 10.1b 2.1 11.4b
CA 68.4 67.1 39.5 74.4 24.6 68.6 17.5 56.3 23.7 63.3 31.2
{ { { { { { { { { { {
2.0 2.5 6.2a 2.0 3.9a 2.4 2.0a 3.5 7.6b 2.3 11.2b
CDCA 9.1 20.4 4.1 9.0 3.9 11.2 9.5 14.0 14.7 9.8 27.5
{ { { { { { { { { { {
DCA
2.1 3.1 1.3b 2.4 1.0 1.2 5.6 3.8 7.3 0.8 12.3
6.9 4.1 1.0 6.7 0.6 7.8 1.0 8.3 1.1 6.0 2.1
{ { { { { { { { { { {
0.9 0.9 0.5b 1.2 0.2a 1.2 0.7a 1.4 0.4a 0.7 0.8a
LCA 0.3 0.2 0.3 0.2 0.6 0.3 0.2 0.6 0.7 0.4 2.2
{ { { { { { { { { { {
0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.4 0.1 1.3
UDCA 3.0 1.3 0.8 1.4 0.5 1.6 0.6 3.7 1.0 1.8 2.3
{ { { { { { { { { { {
0.7 0.4 0.3 0.3 0.2a 0.3 0.2a 1.1 0.4 0.5 0.7
Note. Data (%) are expressed as mean { SEM of 5 rats. BDL, bile duct ligatrion; b-MCA, b-muricholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; UDCA, ursodeoxycholic acid. a P õ 0.01 vs sham group. b P õ 0.05 vs sham group.
acid, was a minor component of bile in rats and was decreased to a trace amount after the bile duct ligation, thus having little effect on total biliary hydrophobicity and the enzyme activity. A significant negative correlation was evident between the enzyme activities and biliary bile acids hydrophobicity indices. Proportion of cholic acid and b-muricholic acid also correlated with the enzyme activities. The results strongly suggest that the cholesterol 7a-hydroxylase activity is not modulated by the bile acid concentration but is regulated by hydrophobicity indices of biliary bile acids. It should be noted that the activity of cholesterol 7a-hydroxylase returned to the control level on Day 14 after the bile duct ligation, although serum and hepatic bile acid concentrations remained still high. The result suggests the presence of hepatocyte injury by prolonged biliary obstruction, as the mortality rate was high (approximately 40%) and most of the survived rats had ascites on Day 14. b-Muricholic acid is one of the 6b-hydroxylated prod-
ucts of chenodeoxycholic acid in the rat. It is a hydrophilic bile acid and is efficiently excreted in urine [37]. Thus, 6b-hydroxylation of bile acids are considered to be a bioprotection against cytotoxicities of hydrophobic bile acids, just like glucuronidation [38] and sulfation [39] of bile acids. The present results are consistent with that of Danielsson who reported increased bile acid 6b-hydroxylase activity in rats after bile duct ligation [13]. It is possible that chenodeoxycholic acid was effectively 6b-hydroxylated by increased activity of 6bhydroxylase, resulting in an increased proportion of bmuricholic acid and a decreased biliary hydrophobicity, which in turn increased cholesterol 7a-hydroxylase activity. In the present study, bile acid composition in the hepatocyte was not analyzed. However, we had previously shown that composition of bile acids in the hepatocytes is very similar to that of biliary bile acids by GC-MS [42]. It is thus probable that the hydrophobicity of bile acids in the hepatocytes may be similar to that in bile.
FIG. 4. Changes in hydrophobicity indices of the biliary bile acids in the bile duct-ligated rats (l) and the sham-operated rats (m). Values are means { SEM of 5 rats. *P õ 0.01 compared with the sham-operated rats.
FIG. 5. Correlation between hepatic cholesterol 7a-hydroxylase activity and hydrophobicity indices of the biliary bile acids. Symbols indicate bile duct-ligated rats (l) and the sham-operated rats (m). y Å 00.006x / 0.094, n Å 55, r Å 0.538, P õ 0.01.
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NAITO ET AL.: BILE ACID SYNTHESIS AND BILIARY HYDROPHOBICITY
Steroid 12a-hydroxylase activity is reduced by bile acid feeding [40, 41]. Danielsson [13] also reported the decreased steroid 12a-hydroxylase activity in the rat after bile duct ligation. In the present study, proportion of cholic acid metabolites (cholic acid and deoxycholic acid) decreased significantly and that of chenodeoxycholic acid metabolites (chenodeoxycholic acid, b-muricholic acid, ursodeoxycholic acid, and lithocholic acid) increased by bile duct ligation. These results suggest that bile duct ligation also caused the inhibition of steroid 12a-hydroxylase activity as well as stimulation of cholesterol 7a-hydroxylase and bile acid 6b-hydroxylase activities in rats. Another possible mechanism to explain the paradoxical results is the recent observation by Pandak et al., showing that intravenous infusion of taurocholate failed to suppress the mRNA level, transcription rate, enzyme mass, and specific activity of cholesterol 7ahydroxylase [43]. Exclusion of bile acids from the intestine, whether by bile duct obstruction or biliary diversion, resulted in a loss of inhibitory effect of ‘‘intestinal factor(s)’’ and consequently in up-regulation of cholesterol 7a-hydroxylase [43]. Those ‘‘intestinal factor(s)’’ have not been identified so far and further studies should be required to answer this important problem. Serum 7a-hydroxycholesterol level was also increased by bile duct ligation and there was a significant positive correlation between the serum level of 7a-hydroxycholesterol and the hepatic cholesterol 7a-hydroxylase activity. We have shown that the serum level of 7a-hydroxycholesterol reflects hepatic cholesterol 7a-hydroxylase activity under various conditions in humans [19, 23, 33] and in experimental animals [17, 20]. The present result again confirmed the usefulness of serum 7a-hydroxycholesterol level as an indicator of hepatic bile acid synthesis. In conclusion, the increased activity of hepatic cholesterol 7a-hydroxylase is accompanied by the increased proportion of biliary b-muricholic acid and the decreased biliary hydrophobicity in rats with obstructive jaundice. This may be a rationale of the increased bile acid synthesis in spite of the markedly accumulated serum and hepatic bile acids levels in rats.
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ACKNOWLEDGMENTS The authors thank Drs. Yamashita, Makino, and Komura for their helpful suggestions and Miss Nishimura for her technical help. This work was supported in part by Grants-in-Aid 07671403 (S. Kuroki) and 07671406 (K. Chijiiwa) from the Ministry of Education, Science and Culture of Japan.
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