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Biliary Lipid Output in the Early Stage of Acute Liver Failure Induced by 90% Hepatectomy in the Rat
Journal of Surgical Research 134, 81– 86 (2006) doi:10.1016/j.jss.2005.12.023
Biliary Lipid Output in the Early Stage of Acute Liver Failure Induced by 90% Hepatectomy in the Rat 1 Shun-Ichi Tanaka, M.D., Kazuo Chijiiwa, M.D., F.A.C.S.,2 and Yorio Maeda, M.D. Department of Surgery I, Miyazaki University School of Medicine, Miyazaki, Japan Submitted for publication February 12, 2005
supposed to be useful for early detection of hepatic failure after extensive hepatectomy. © 2006 Elsevier Inc. All
Background. Differences in biliary lipid output were compared in rats after 70% or 90% hepatectomy (Hx) to evaluate a possible index of the early stage of acute liver failure. Methods. Male Sprague-Dawley (SD) rats weighing 300 to 350 g were randomly divided into two groups for 70% Hx or 90% Hx, and animals were sacrificed at 0, 6, 24, and 48 h after Hx. Before sacrifice, a polyethylene tube was cannulated into the bile duct and bile was collected for 1 h. Outputs of total bile acids, phospholipid, and total cholesterol in serum and bile were determined. Biliary total cholesterol, bile acid concentrations, and bile acid component levels were determined using gas liquid chromatography. Hepatic microsomal cholesterol 7␣-hydroxylase and sterol 12␣hydroxylase activities were also determined using high performance liquid chromatography. Results. The 3-day survival rate after 90% Hx was 50%. In the 90% Hx group, the serum total bile acid concentration at each point was significantly higher than it was in the 70% Hx group. The bile flow rate and biliary outputs of cholesterol, phospholipid, and bile acids were significantly lower at 6 h after 90% Hx than after 70% Hx. Among bile acid species, cholic and chenodeoxycholic acid outputs into bile were significantly less at 6 h after 90% Hx. The activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase were decreased after 90% Hx. Conclusions. Our results suggest that determinations of the bile flow rate and biliary lipid outputs are
rights reserved.
Key Words: hepatectomy; liver failure; biliary lipid output; bile flow rate; bile acids. INTRODUCTION
It is known that bile flow, biliary bile acid secretion [1–5], and the activity of hepatic cholesterol 7␣-hydroxylase [6, 7] are different after partial (two-thirds) hepatectomy (Hx) compared to sham-operated rats. Alterations of the bile flow rate and biliary bile acid secretion rates after 50% and 75% Hx are reported to correlate with the regeneration rate of the remnant liver [8]. We and others have used 90% Hx in rats as a model of postoperative acute liver failure [9, 10], but the details of the changes in these factors in the early stage of acute liver failure induced by extensive Hx are not clear at present. In this study, we determined the bile flow rate, serum and biliary lipid concentrations, output of biliary lipid and each bile acid into bile, and the activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase of liver microsomes at 6, 24, and 48 h after 70% and 90% Hx. The aim of this study was to identify a possible index of the early stage of acute hepatic failure. MATERIALS AND METHODS Materials
1
A part of this study was supported by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Numbers 17591417 and 15591417 to Professor Chijiiwa). 2 To whom correspondence and reprint requests should be addressed at Kazuo Chijiiwa, M.D., F.A.C.S., Professor and Chairman, Department of Surgery I, Miyazaki University School of medicine, 5200 Kiyotake, Miyazaki 889-1692, Japan. E-mail: kazuochi@med. miyazaki-u.ac.jp.
The following materials were purchased: cholic, chenodeoxycholic, deoxycholic, lithocholic, and ursodeoxycholic acids (Sigma, St. Louis, MO), nor-cholic acid (Steraloids, Wilton, NH), 10% HCl-n-butanol (Nacalai Tesque, Kyoto, Japan), and Sil-Prep [hexamethyldisilazanetrimethylchlorosilane-pyridine ⫽ 3:1:9] (Alltech Associates, Deerfield, IL), All other chemicals were of the highest grade commercially available.
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Animals Male Sprague-Dawley rats, body weight 300 to 350 g, were obtained from SLC, Inc. (Shizuoka, Japan). All rats were housed in identical units in a room kept at 23°C with a 12-h light-dark cycle and were given free access to water and laboratory food for at least 1 week before the experiment.
Experimental Design After acclimation for one week, animals were randomly divided to two groups. Group 1 underwent 70% Hx, leaving the right anterior, right posterior, and caudate lobes as described by Higgins and Anderson [11]; group 2 underwent 90% Hx, leaving only the caudate lobes as described by Gaub and Iversen [9]. These operations were carried out under ether anesthesia. Food was withdrawn, and 20% glucose solution in tap water was given ad libitum for 24 h before Hx and until the end of the experiment. In a separate experiment to examine the postoperative survival rate for 3 days, 36 rats underwent 70% or 90% Hx under similar conditions. Only the survival rats were sacrificed at 0, 6, 24, and 48 h after Hx under anesthesia, and the bile, blood, and liver were obtained from six rats in each group at the same time. Bile samples were collected for 1 h before sacrifice by cannulating a PE-10 tube into the bile duct to determine the bile flow rate and concentrations of biliary lipids. Blood samples were drawn from the inferior vena cava with a syringe, and serum was used for measuring total bile acid, total cholesterol, and phospholipid concentrations. The enzyme activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase were determined using the liver. Animals were sacrificed after sample collection, and the liver weights were recorded. All surgical procedures were performed between 8 a.m. and 12 noon. The study was approved by the Ethical Committee of the Animal Laboratory Center, Miyazaki University.
Chemical Analysis Total bile acid, total cholesterol, and phospholipid concentrations in serum and bile were measured using an enzymatic and colorimeteric technique (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The protein concentration was determined by the method of Lowry et al. [12]. Bovine serum albumin was used as a standard.
Biliary Bile Acid Analysis Biliary bile acids were analyzed by capillary gas-liquid chromatography (GLC) as described by Batta et al. [13, 14]. A 20-l aliquot of bile was mixed with 400-l ethanol, boiled for 3 min, and then centrifuged at 400 ⫻ g for 10 min to precipitate the protein. There was 400 l of the supernatant evaporated under N 2, and enzymatic hydrolysis was performed. After hydrolysis, the samples were acidified to pH 1, and bile acids were extracted three times with 5 ml of ethyl acetate and evaporated. Ten percent HCl-n-butanol was added to the sample, and it was heated at 60°C for 4 h followed by evaporation under N 2. The bile acid esters were treated with 100 l of Sil-Prep at 55°C for 30 min and then evaporated under N 2. The trimethylsilyl ether derivatives obtained were dissolved in 100 l of hexane, centrifuged, and 1 to 2 l of the clear supernatant was injected into a GLC column (GC 14A, Shimazu, Kyoto, Japan, equipped with a fused silica column, CP-Sil-5 CB, 25 m X 0.25 mm I.D., Chrompack, Raritan, NJ) in the 20:1 split mode. The injector and detector temperature were 260° and 290°C, respectively. After injection, the oven temperature was kept at 150°C for 1 min, and then programmed to increase at a rate of 20°C/min to a final temperature of 295°C. Nor-cholic acid was used as the internal standard.
Enzyme Assay The activity of cholesterol 7␣-hydroxylase in the microsomal fraction of the liver was measured according to the method of Ogishima and Okuda [15] using internal cholesterol as the substrate. Briefly, the liver samples were homogenized on ice in 4 ml/g liver of the following buffer: 100 mM potassium phosphate, pH 7.2, containing 100 mM sucrose, 50 mM KCl, 1 mM EDTA, 3 mM dithiothreitol (DTT), 100 M leupeptin, 5 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride. Thus, the homogenates obtained were centrifuged at 9,000 ⫻ g for 20 min. The supernatant was then centrifuged at 100,000 ⫻ g for 60 min. The precipitated microsomal fraction was suspended in 1 ml of the same buffer as described above. Rat liver microsomes (400 g) were incubated at 37°C for 20 min in a buffer containing 0.1 M potassium phosphate buffer, pH 7.4, 0.1 mM EDTA, 20 mM cysteamine-HCl, 5 mM MgCl 2, 5 mM sodium isocitrate, 0.075 units of isocitrate dehydrogenase, and 0.5 mM NADPH in a final volume of 0.25 ml. The reaction was started by the addition of NADPH. At the end of reaction, to convert the 7␣-hydroxycholesterol formed into 7␣-hydroxy-4-cholesten-3-one, 50 l of 5% cholate (w/v) and 20 l of 0.1% cholesterol oxidase (0.244 units) dissolved in 10 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol and 1 mM DTT were added to the reaction mixture, and the mixture was incubated for another 10 min. The reaction was terminated by adding 1 ml of methanol, and the products were extracted from the mixture with 5 ml of n-hexane. After the solvents were evaporated, the products were dissolved in 20 l of 2-propanol. Aliquots were subjected to HPLC for quantification of 7␣-hydroxy-4-cholesten-3one by monitoring effluents at 240 nm. 7␣-hydroxy-4-cholesten-3-one sterol 12␣-hydroxylase was assayed as described by Noshiro et al. [16]. A typical reaction mixture contained 100 l of potassium phosphate buffer, pH 7.0, 0.1 mol of EDTA, 0.01 to 0.05 nmol of cytochrome P450, 0.5 units of NADPHcytochrome P450 reductase, 20 g of dilauroylglyceryl-3-phosphorylcholine, and a NADPH-generating system consisting of 0.5 mol of NADP, 5 mol of DL-isocitrate, 5 mol of MgCl 2, 0.44 units of isocitrate dehydrogenase, and 50 nmol of 7␣-hydroxy-4-cholesten3-one dissolved in 1.0 l of ethanol in a final volume of 1.0 ml. Incubation was carried out at 37°C for 10 min, and the reaction was terminated by the addition of 5.0 ml of benzene. The mixture was dispersed in a vortex mixer and centrifuged at 700 ⫻ g for 5 min. A 4.0-ml aliquot of the extract was evaporated under reduced pressure at room temperature, the residue was dissolved in 15-l chloroform, and an aliquot was assayed by HPLC. The column used was Fine Sil (4.6 ⫻ 250 mm), and the solvent system was isopropyl alcohol:nhexane, 18:82. The eluates were monitored by absorption at 240 nm with the use of a UV-spectrophotometric detector.
Statistical Analysis All data were expressed as means ⫾ SD. Stastical analysis was done by analysis of variance followed by Student’s unpaired t-test. A probability value less than 0.05 was considered statistically significant.
RESULTS Survival Rate After Hepatectomy
The survival rates for 3 days after 70% and 90% Hx were first examined in 36 rats in each group, and they were 96% and 50%, respectively. Post-operative death occurred at 24 h after 90% Hx. At autopsy, large amounts of ascites in the abdominal cavity were observed, suggesting the cause of the death was post-operative liver failure.
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TANAKA ET AL.: BILIARY LIPID METABOLISM AFTER 70% AND 90% HEPATECTOMY IN RATS
TABLE 1 Residual Liver Weight, Bile Flow per Hour per 100 g Body Weight, Serum Total Cholesterol, Phospholipid and Total Bile Acid Concentrations After 70% Hx and 90% Hx in Rats Hours after Hx
Residual liver weight (g) 70% Hx 90% Hx Bile flow rate (l/h/100g BW) 70% Hx 90% Hx Total cholesterol (mg/dl) 70% Hx 90% Hx Phospholipid (mg/dl) 70% Hx 90% Hx Total bile acid (mol/l) 70% Hx 90% Hx
Basal a
6h
24 h
48 h
7.3 ⫾ 0.7 7.3 ⫾ 0.7
2.6 ⫾ 0.4 0.8 ⫾ 0.1**
3.2 ⫾ 0.4 0.9 ⫾ 0.1**
4.2 ⫾ 1.1 1.4 ⫾ 0.2**
144 ⫾ 4 144 ⫾ 4
79 ⫾ 28 28 ⫾ 16*
95 ⫾ 14 82 ⫾ 28
116 ⫾ 16 91 ⫾ 43
27 ⫾ 3 27 ⫾ 3
40 ⫾ 10 27 ⫾ 4*
27 ⫾ 5 22 ⫾ 4
41 ⫾ 5 38 ⫾ 7
68 ⫾ 14 68 ⫾ 14
62 ⫾ 14 47 ⫾ 12
47 ⫾ 2 42 ⫾ 5*
75 ⫾ 12 70 ⫾ 14
32 ⫾ 25 32 ⫾ 25
75 ⫾ 40 267 ⫾ 120*
Basal value before Hx (n ⫽ 6). Mean ⫾ SD, n ⫽ 6; * P ⬍ 0.05 vs. 70% Hx; ** P ⬍ 0.01 vs. 70% Hx;
50 ⫾ 24 238 ⫾ 100**
117 ⫾ 88 275 ⫾ 66**
a
Body Weight, Liver Weight, Bile Flow, and Serum Lipid Concentration After Hepatectomy
The body weight at the time of Hx was 313 ⫾ 13 gram in the 70% Hx group and 317 ⫾ 10 g in the 90% Hx group. The actual percentage of liver resected was 74.6 ⫾ 3.1% in the 70% Hx group and 90.4 ⫾ 1.3% in the 90% Hx group. The remnant liver weight, bile flow rate, and serum lipid concentration are shown in Table 1. The residual liver weight was significantly lower after 90% Hx than it was after 70% Hx during the experimental period. It gradually increased in both groups. The bile flow rate (l/h/100 g BW) at 6 h after 90% Hx was significantly lower than after 70% Hx (28 ⫾ 16 versus 79 ⫾ 28, P ⬍ 0.05). The bile flow rate decreased significantly for 24 h after Hx in both groups and recovered thereafter. The serum total bile acid concentration was significantly elevated and was significantly higher at 6, 24, and 48 h after 90% Hx than after 70% Hx (Table 1). The higher values were maintained after 90% Hx during the observation period, and the values were elevated to eight-fold compared with the basal value. Biliary Lipid Output
Total bile acid, phospholipid, and cholesterol outputs into bile are depicted in Fig. 1. In general, they had declined at 6 h after Hx and gradually increased thereafter. The biliary outputs of total cholesterol and phospholipid at 6 and 24 h after 90% Hx were significantly lower than those after 70% Hx. The biliary output of total bile acid at 6 h after 90% Hx was significantly
P ⬍ 0.05 vs. basal value;
P ⬍ 0.01 vs. basal value.
lower than that after 70% Hx (1.5 ⫾ 0.6 versus 3.4 ⫾ 1.6 mmol/h/100 g BW, P ⬍ 0.05). Biliary Bile Acid Composition and Output
The biliary bile acid compositions at 6, 24, and 48 h after Hx were not significantly different between the 70% Hx and 90% Hx groups. The biliary outputs of each bile acid after 70% and 90% Hx are shown in Fig. 2. Biliary outputs of cholic and chenodeoxycholic acids at 6 h after 90% Hx were significantly lower than those after 70% Hx (196 ⫾ 80 versus 511 ⫾ 246 and 14.9 ⫾ 9 versus 46.5 ⫾ 25 mg/h/100 g BW, respectively, P ⬍ 0.05). Activities of Microsomal Cholesterol 7␣-hydroxylase and Sterol 12␣-Hydroxylase of the Liver
The enzyme activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase are shown in Fig. 3. While the activity of cholesterol 7␣-hydroxylase was decreased in both groups after Hx, the enzyme activity at 24 h after 90% Hx was significantly lower than that after 70% Hx (P ⬍ 0.05). The sterol 12␣-hydroxylase activity after 90% Hx was decreased, while it increased after 70% Hx. There was a significant difference in the enzyme activity between the two groups (P ⬍ 0.05) at 24 h after Hx. DISCUSSION
The most striking differences observed after 90% Hx were a significantly increased serum total bile acid concentration, and decreased bile flow and biliary outputs
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anion transporter (Mrp2), are markedly down-regulated after Hx [17, 19, 20]. It was recently shown that the bile acid concentrations in the liver after Hx are reduced [21]. Our result suggests that expressions of Ntcp and Oatp are more impaired after 90% Hx, resulting in higher serum total bile acid concentrations, than after 70% Hx. The serum total bile acid concentration at 6 h after aggressive Hx may be a useful parameter of the early stage of acute liver failure. Previous studies have shown increased bile flow and secretion of bile acids when expressed per gram of liver after two-thirds Hx [1, 2, 4, 17, 22]. Other studies have demonstrated that the bile flow rate and biliary bile acid secretion expressed as gram of body weight are decreased significantly after two-thirds Hx [2, 23]. Xu et al. demonstrated that the bile flow and biliary bile acid secretion rates are lower after 75% Hx than those after 50% Hx in rats but without statistically significant differences between the two groups [8]. In the present study, bile flow and biliary bile acid outputs expressed by body weight were significantly decreased,
FIG. 1. Rates of lipid secretion into biliary bile acids per gram of body weight after 70% and 90% Hx in rats. Bile was collected for 1 h at the indicated times. Total bile acid, total cholesterol, and phospholipid concentrations in biliary bile were measured by enzymatic and colorimeteric methods as described above. Solid circles stand for 70% Hx and squares for 90% Hx. Mean ⫾ SD, n ⫽ 6; *P ⬍ 0.05 versus 70% Hx; P ⬍ 0.05, 70% Hx versus basal value; P ⬍ 0.05, 90% Hx versus basal value.
of bile acids, phospholipids and cholesterol, compared with those after 70% Hx. Moreover, biliary outputs of cholic and chenodeoxycholic acids were significantly lower after 90% Hx. These differences were consistently observed at 6 h after Hx. Determination of these compounds may be useful for early detection of postoperative liver failure. Increased bile acid concentrations in the blood circulation after Hx have been reported [17, 18]. A similar result was observed after 70% and 90% Hx in the present study, and the serum bile acid concentration was significantly elevated in the early stage of acute liver failure induced by 90% Hx. The magnitude of its increase was eight times at 6 h after 90% Hx, while was two times after 70% Hx, compared with the basal value. It has been reported that the sinusoidal sodiumtaurocholate cotransporter (Ntcp) and the organic anion transporting polypeptides (Oatp1 and Oatp2), which maintain the expressions of the canalicular ATP-dependent bile salt export pump (Bsep) and multiorganic
FIG. 2. Outputs of different bile acid species as measured by GLC in biliary bile samples collected for 1 h at the indicated times during liver regeneration that after 70% Hx as compared with 90% Hx in rats. Solid circles indicate 70% Hx, and solid squares indicate 90% Hx. Mean ⫾ SD, n ⫽ 6; *P ⬍ 0.05 versus 70% Hx; P ⬍ 0.05, 70% Hx versus basal value; P ⬍ 0.05, 90% Hx versus basal value.
TANAKA ET AL.: BILIARY LIPID METABOLISM AFTER 70% AND 90% HEPATECTOMY IN RATS
FIG. 3. Changes of activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase after 70% and 90% Hx in the rat liver. Each specific activity of the remnant liver at fixed intervals after Hx was measured by HPLC as described above. Solid circles show the activities after 70% Hx, and solid squares show them after 90% Hx. Mean ⫾ SD, n ⫽ 6; *P ⬍ 0.05 versus 70% Hx; P ⬍ 0.05, 70% Hx versus basal value; P ⬍ 0.05, 90% Hx versus basal value.
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cholic acid/chenodeoxycholic acid is increased on day 2 [2]. The reason for the different cholic acid outputs in bile is not clear. In our study, biliary outputs of each bile acid species, including cholic acid and chenodeoxycholic acid, declined after 70% and 90% Hx. Interestingly, the biliary outputs of cholic acid and chenodeoxycholic acid at 6 h after 90% Hx were significantly lower than those after 70% Hx. The reason for the decreased total bile acid and cholic acid outputs in bile was examined by determination of cholesterol 7␣-hydroxylase and sterol 12␣-hydrozylase activities, enzymes for bile acid biosynthesis and cholic acid synthesis, respectively. We have previously shown that cholesterol 7␣-hydroxylase activity was suppressed for the first 2 days after Hx, but returned to the control level on the third day [7]. In the current study the activity of cholesterol 7␣-hydroxylase was decreased for 2 days after 70% and 90% Hx. Significantly lower activities after 90% Hx were observed at 24 h, whereas significantly lower biliary bile acid output was observed at 6 h after 90% Hx compared with those after 70% Hx. The reason for the delayed decrease in the cholesterol 7␣-hydroxylase activity compared with biliary bile acid output is not clear at present. In contrast to cholesterol 7␣-hydroxylase activity, sterol 12␣-hydroxylase was activated after 70% Hx but not after 90% Hx. Although biliary cholic acid output was decreased after both 70% and 90% Hx, the changes in sterol 12␣-hydroxylase activity were different. The lack of correlation between the enzyme activity and biliary cholic acid output needs further examination. It is estimated that determinations of the serum bile acid levels, bile flow rate, biliary cholesterol, phospholipids, and bile acid outputs are the most reliable early predictor of post-operative liver failure after extended Hx. REFERENCES
especially at 6 h after 70% and 90% Hx. The bile flow rate and biliary bile acid outputs at 6 h after 90% Hx were significantly lower than those after 70% Hx. Biliary cholesterol and phospholipid outputs were also decreased after 70% and 90% Hx. These values at 6 and 24 h after 90% Hx were significantly lower than those after 70% Hx. The decrease in biliary cholesterol and phospholipid outputs after 50% and 75% Hx has been reported to depend on the volume of excised liver [8]. Our result supports this report. In addition, our data suggest that the significantly lower bile flow rate and biliary cholesterol, phospholipid and bile acid outputs at 6 h after 90% Hx are useful for early detection of post-operative liver failure induced by extended Hx. It has been demonstrated that the concentration of cholic acid is increased on day 2, while that of chenodeoxycholic acid is decreased on day 1, compared with the control rats after two-thirds Hx and that the ratio of
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Ogishima T, Okuda K. An improved method for assay of cholesterol 7␣-hydroxylase activity. Anal Biochem 1986;158:228. Noshiro M, Ishida H, Hayashi S, Okuda K. Assays for cholesterol 7␣-hydroxylase and 12␣-hydroxylase using high performance liquid chromatography. Steroids 1985;45:539. Vos TA, Ros JE, Havinga R, et al. Regulation of hepatic transport systems involved in bile secretion during liver regeneration in rats. Hepatology 1999;29:1833. Zieve L, Anderson R, Lindblad S. Course of hepatic regeneration after 80% to 90% resection of normal rat liver. J Lab Clin Med 1985;105:331. Green RM, Gollan JL, Hagenbuch B, Meier PJ, Beier DR. Regulation of hepatocyte bile salt transporters during hepatic regeneration. Am J Physiol 1997;273:G621. Gerloff T, Geier A, Stieger B, et al. Differential expression of basolateral and canalicular organic anion transporters during regeneration of rat liver. Gastroenterology 1999;117:1408. Monte MJ, Martinez-Diez MC, El-Mir MY, et al. Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration. J Hepatol 2002;36:534. Monte MJ, El-Mir MY, Sainz GR, Bravo P, Marin JJ. Bile acid secretion during synchronized rat liver regeneration. Biochim Biophys Acta 1997;1362:56. Perez-Barriocanal F, Gonzalez J, Esteller A. Biliary lipid secretion in the regenerating liver of the rat. Br J Exp Pathol 1987; 68:1.
Biliary Lipid Output in the Early Stage of Acute Liver Failure Induced by 90% Hepatectomy in the Rat 1 Shun-Ichi Tanaka, M.D., Kazuo Chijiiwa, M.D., F.A.C.S.,2 and Yorio Maeda, M.D. Department of Surgery I, Miyazaki University School of Medicine, Miyazaki, Japan Submitted for publication February 12, 2005
supposed to be useful for early detection of hepatic failure after extensive hepatectomy. © 2006 Elsevier Inc. All
Background. Differences in biliary lipid output were compared in rats after 70% or 90% hepatectomy (Hx) to evaluate a possible index of the early stage of acute liver failure. Methods. Male Sprague-Dawley (SD) rats weighing 300 to 350 g were randomly divided into two groups for 70% Hx or 90% Hx, and animals were sacrificed at 0, 6, 24, and 48 h after Hx. Before sacrifice, a polyethylene tube was cannulated into the bile duct and bile was collected for 1 h. Outputs of total bile acids, phospholipid, and total cholesterol in serum and bile were determined. Biliary total cholesterol, bile acid concentrations, and bile acid component levels were determined using gas liquid chromatography. Hepatic microsomal cholesterol 7␣-hydroxylase and sterol 12␣hydroxylase activities were also determined using high performance liquid chromatography. Results. The 3-day survival rate after 90% Hx was 50%. In the 90% Hx group, the serum total bile acid concentration at each point was significantly higher than it was in the 70% Hx group. The bile flow rate and biliary outputs of cholesterol, phospholipid, and bile acids were significantly lower at 6 h after 90% Hx than after 70% Hx. Among bile acid species, cholic and chenodeoxycholic acid outputs into bile were significantly less at 6 h after 90% Hx. The activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase were decreased after 90% Hx. Conclusions. Our results suggest that determinations of the bile flow rate and biliary lipid outputs are
rights reserved.
Key Words: hepatectomy; liver failure; biliary lipid output; bile flow rate; bile acids. INTRODUCTION
It is known that bile flow, biliary bile acid secretion [1–5], and the activity of hepatic cholesterol 7␣-hydroxylase [6, 7] are different after partial (two-thirds) hepatectomy (Hx) compared to sham-operated rats. Alterations of the bile flow rate and biliary bile acid secretion rates after 50% and 75% Hx are reported to correlate with the regeneration rate of the remnant liver [8]. We and others have used 90% Hx in rats as a model of postoperative acute liver failure [9, 10], but the details of the changes in these factors in the early stage of acute liver failure induced by extensive Hx are not clear at present. In this study, we determined the bile flow rate, serum and biliary lipid concentrations, output of biliary lipid and each bile acid into bile, and the activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase of liver microsomes at 6, 24, and 48 h after 70% and 90% Hx. The aim of this study was to identify a possible index of the early stage of acute hepatic failure. MATERIALS AND METHODS Materials
1
A part of this study was supported by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Numbers 17591417 and 15591417 to Professor Chijiiwa). 2 To whom correspondence and reprint requests should be addressed at Kazuo Chijiiwa, M.D., F.A.C.S., Professor and Chairman, Department of Surgery I, Miyazaki University School of medicine, 5200 Kiyotake, Miyazaki 889-1692, Japan. E-mail: kazuochi@med. miyazaki-u.ac.jp.
The following materials were purchased: cholic, chenodeoxycholic, deoxycholic, lithocholic, and ursodeoxycholic acids (Sigma, St. Louis, MO), nor-cholic acid (Steraloids, Wilton, NH), 10% HCl-n-butanol (Nacalai Tesque, Kyoto, Japan), and Sil-Prep [hexamethyldisilazanetrimethylchlorosilane-pyridine ⫽ 3:1:9] (Alltech Associates, Deerfield, IL), All other chemicals were of the highest grade commercially available.
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Animals Male Sprague-Dawley rats, body weight 300 to 350 g, were obtained from SLC, Inc. (Shizuoka, Japan). All rats were housed in identical units in a room kept at 23°C with a 12-h light-dark cycle and were given free access to water and laboratory food for at least 1 week before the experiment.
Experimental Design After acclimation for one week, animals were randomly divided to two groups. Group 1 underwent 70% Hx, leaving the right anterior, right posterior, and caudate lobes as described by Higgins and Anderson [11]; group 2 underwent 90% Hx, leaving only the caudate lobes as described by Gaub and Iversen [9]. These operations were carried out under ether anesthesia. Food was withdrawn, and 20% glucose solution in tap water was given ad libitum for 24 h before Hx and until the end of the experiment. In a separate experiment to examine the postoperative survival rate for 3 days, 36 rats underwent 70% or 90% Hx under similar conditions. Only the survival rats were sacrificed at 0, 6, 24, and 48 h after Hx under anesthesia, and the bile, blood, and liver were obtained from six rats in each group at the same time. Bile samples were collected for 1 h before sacrifice by cannulating a PE-10 tube into the bile duct to determine the bile flow rate and concentrations of biliary lipids. Blood samples were drawn from the inferior vena cava with a syringe, and serum was used for measuring total bile acid, total cholesterol, and phospholipid concentrations. The enzyme activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase were determined using the liver. Animals were sacrificed after sample collection, and the liver weights were recorded. All surgical procedures were performed between 8 a.m. and 12 noon. The study was approved by the Ethical Committee of the Animal Laboratory Center, Miyazaki University.
Chemical Analysis Total bile acid, total cholesterol, and phospholipid concentrations in serum and bile were measured using an enzymatic and colorimeteric technique (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The protein concentration was determined by the method of Lowry et al. [12]. Bovine serum albumin was used as a standard.
Biliary Bile Acid Analysis Biliary bile acids were analyzed by capillary gas-liquid chromatography (GLC) as described by Batta et al. [13, 14]. A 20-l aliquot of bile was mixed with 400-l ethanol, boiled for 3 min, and then centrifuged at 400 ⫻ g for 10 min to precipitate the protein. There was 400 l of the supernatant evaporated under N 2, and enzymatic hydrolysis was performed. After hydrolysis, the samples were acidified to pH 1, and bile acids were extracted three times with 5 ml of ethyl acetate and evaporated. Ten percent HCl-n-butanol was added to the sample, and it was heated at 60°C for 4 h followed by evaporation under N 2. The bile acid esters were treated with 100 l of Sil-Prep at 55°C for 30 min and then evaporated under N 2. The trimethylsilyl ether derivatives obtained were dissolved in 100 l of hexane, centrifuged, and 1 to 2 l of the clear supernatant was injected into a GLC column (GC 14A, Shimazu, Kyoto, Japan, equipped with a fused silica column, CP-Sil-5 CB, 25 m X 0.25 mm I.D., Chrompack, Raritan, NJ) in the 20:1 split mode. The injector and detector temperature were 260° and 290°C, respectively. After injection, the oven temperature was kept at 150°C for 1 min, and then programmed to increase at a rate of 20°C/min to a final temperature of 295°C. Nor-cholic acid was used as the internal standard.
Enzyme Assay The activity of cholesterol 7␣-hydroxylase in the microsomal fraction of the liver was measured according to the method of Ogishima and Okuda [15] using internal cholesterol as the substrate. Briefly, the liver samples were homogenized on ice in 4 ml/g liver of the following buffer: 100 mM potassium phosphate, pH 7.2, containing 100 mM sucrose, 50 mM KCl, 1 mM EDTA, 3 mM dithiothreitol (DTT), 100 M leupeptin, 5 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride. Thus, the homogenates obtained were centrifuged at 9,000 ⫻ g for 20 min. The supernatant was then centrifuged at 100,000 ⫻ g for 60 min. The precipitated microsomal fraction was suspended in 1 ml of the same buffer as described above. Rat liver microsomes (400 g) were incubated at 37°C for 20 min in a buffer containing 0.1 M potassium phosphate buffer, pH 7.4, 0.1 mM EDTA, 20 mM cysteamine-HCl, 5 mM MgCl 2, 5 mM sodium isocitrate, 0.075 units of isocitrate dehydrogenase, and 0.5 mM NADPH in a final volume of 0.25 ml. The reaction was started by the addition of NADPH. At the end of reaction, to convert the 7␣-hydroxycholesterol formed into 7␣-hydroxy-4-cholesten-3-one, 50 l of 5% cholate (w/v) and 20 l of 0.1% cholesterol oxidase (0.244 units) dissolved in 10 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol and 1 mM DTT were added to the reaction mixture, and the mixture was incubated for another 10 min. The reaction was terminated by adding 1 ml of methanol, and the products were extracted from the mixture with 5 ml of n-hexane. After the solvents were evaporated, the products were dissolved in 20 l of 2-propanol. Aliquots were subjected to HPLC for quantification of 7␣-hydroxy-4-cholesten-3one by monitoring effluents at 240 nm. 7␣-hydroxy-4-cholesten-3-one sterol 12␣-hydroxylase was assayed as described by Noshiro et al. [16]. A typical reaction mixture contained 100 l of potassium phosphate buffer, pH 7.0, 0.1 mol of EDTA, 0.01 to 0.05 nmol of cytochrome P450, 0.5 units of NADPHcytochrome P450 reductase, 20 g of dilauroylglyceryl-3-phosphorylcholine, and a NADPH-generating system consisting of 0.5 mol of NADP, 5 mol of DL-isocitrate, 5 mol of MgCl 2, 0.44 units of isocitrate dehydrogenase, and 50 nmol of 7␣-hydroxy-4-cholesten3-one dissolved in 1.0 l of ethanol in a final volume of 1.0 ml. Incubation was carried out at 37°C for 10 min, and the reaction was terminated by the addition of 5.0 ml of benzene. The mixture was dispersed in a vortex mixer and centrifuged at 700 ⫻ g for 5 min. A 4.0-ml aliquot of the extract was evaporated under reduced pressure at room temperature, the residue was dissolved in 15-l chloroform, and an aliquot was assayed by HPLC. The column used was Fine Sil (4.6 ⫻ 250 mm), and the solvent system was isopropyl alcohol:nhexane, 18:82. The eluates were monitored by absorption at 240 nm with the use of a UV-spectrophotometric detector.
Statistical Analysis All data were expressed as means ⫾ SD. Stastical analysis was done by analysis of variance followed by Student’s unpaired t-test. A probability value less than 0.05 was considered statistically significant.
RESULTS Survival Rate After Hepatectomy
The survival rates for 3 days after 70% and 90% Hx were first examined in 36 rats in each group, and they were 96% and 50%, respectively. Post-operative death occurred at 24 h after 90% Hx. At autopsy, large amounts of ascites in the abdominal cavity were observed, suggesting the cause of the death was post-operative liver failure.
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TANAKA ET AL.: BILIARY LIPID METABOLISM AFTER 70% AND 90% HEPATECTOMY IN RATS
TABLE 1 Residual Liver Weight, Bile Flow per Hour per 100 g Body Weight, Serum Total Cholesterol, Phospholipid and Total Bile Acid Concentrations After 70% Hx and 90% Hx in Rats Hours after Hx
Residual liver weight (g) 70% Hx 90% Hx Bile flow rate (l/h/100g BW) 70% Hx 90% Hx Total cholesterol (mg/dl) 70% Hx 90% Hx Phospholipid (mg/dl) 70% Hx 90% Hx Total bile acid (mol/l) 70% Hx 90% Hx
Basal a
6h
24 h
48 h
7.3 ⫾ 0.7 7.3 ⫾ 0.7
2.6 ⫾ 0.4 0.8 ⫾ 0.1**
3.2 ⫾ 0.4 0.9 ⫾ 0.1**
4.2 ⫾ 1.1 1.4 ⫾ 0.2**
144 ⫾ 4 144 ⫾ 4
79 ⫾ 28 28 ⫾ 16*
95 ⫾ 14 82 ⫾ 28
116 ⫾ 16 91 ⫾ 43
27 ⫾ 3 27 ⫾ 3
40 ⫾ 10 27 ⫾ 4*
27 ⫾ 5 22 ⫾ 4
41 ⫾ 5 38 ⫾ 7
68 ⫾ 14 68 ⫾ 14
62 ⫾ 14 47 ⫾ 12
47 ⫾ 2 42 ⫾ 5*
75 ⫾ 12 70 ⫾ 14
32 ⫾ 25 32 ⫾ 25
75 ⫾ 40 267 ⫾ 120*
Basal value before Hx (n ⫽ 6). Mean ⫾ SD, n ⫽ 6; * P ⬍ 0.05 vs. 70% Hx; ** P ⬍ 0.01 vs. 70% Hx;
50 ⫾ 24 238 ⫾ 100**
117 ⫾ 88 275 ⫾ 66**
a
Body Weight, Liver Weight, Bile Flow, and Serum Lipid Concentration After Hepatectomy
The body weight at the time of Hx was 313 ⫾ 13 gram in the 70% Hx group and 317 ⫾ 10 g in the 90% Hx group. The actual percentage of liver resected was 74.6 ⫾ 3.1% in the 70% Hx group and 90.4 ⫾ 1.3% in the 90% Hx group. The remnant liver weight, bile flow rate, and serum lipid concentration are shown in Table 1. The residual liver weight was significantly lower after 90% Hx than it was after 70% Hx during the experimental period. It gradually increased in both groups. The bile flow rate (l/h/100 g BW) at 6 h after 90% Hx was significantly lower than after 70% Hx (28 ⫾ 16 versus 79 ⫾ 28, P ⬍ 0.05). The bile flow rate decreased significantly for 24 h after Hx in both groups and recovered thereafter. The serum total bile acid concentration was significantly elevated and was significantly higher at 6, 24, and 48 h after 90% Hx than after 70% Hx (Table 1). The higher values were maintained after 90% Hx during the observation period, and the values were elevated to eight-fold compared with the basal value. Biliary Lipid Output
Total bile acid, phospholipid, and cholesterol outputs into bile are depicted in Fig. 1. In general, they had declined at 6 h after Hx and gradually increased thereafter. The biliary outputs of total cholesterol and phospholipid at 6 and 24 h after 90% Hx were significantly lower than those after 70% Hx. The biliary output of total bile acid at 6 h after 90% Hx was significantly
P ⬍ 0.05 vs. basal value;
P ⬍ 0.01 vs. basal value.
lower than that after 70% Hx (1.5 ⫾ 0.6 versus 3.4 ⫾ 1.6 mmol/h/100 g BW, P ⬍ 0.05). Biliary Bile Acid Composition and Output
The biliary bile acid compositions at 6, 24, and 48 h after Hx were not significantly different between the 70% Hx and 90% Hx groups. The biliary outputs of each bile acid after 70% and 90% Hx are shown in Fig. 2. Biliary outputs of cholic and chenodeoxycholic acids at 6 h after 90% Hx were significantly lower than those after 70% Hx (196 ⫾ 80 versus 511 ⫾ 246 and 14.9 ⫾ 9 versus 46.5 ⫾ 25 mg/h/100 g BW, respectively, P ⬍ 0.05). Activities of Microsomal Cholesterol 7␣-hydroxylase and Sterol 12␣-Hydroxylase of the Liver
The enzyme activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase are shown in Fig. 3. While the activity of cholesterol 7␣-hydroxylase was decreased in both groups after Hx, the enzyme activity at 24 h after 90% Hx was significantly lower than that after 70% Hx (P ⬍ 0.05). The sterol 12␣-hydroxylase activity after 90% Hx was decreased, while it increased after 70% Hx. There was a significant difference in the enzyme activity between the two groups (P ⬍ 0.05) at 24 h after Hx. DISCUSSION
The most striking differences observed after 90% Hx were a significantly increased serum total bile acid concentration, and decreased bile flow and biliary outputs
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anion transporter (Mrp2), are markedly down-regulated after Hx [17, 19, 20]. It was recently shown that the bile acid concentrations in the liver after Hx are reduced [21]. Our result suggests that expressions of Ntcp and Oatp are more impaired after 90% Hx, resulting in higher serum total bile acid concentrations, than after 70% Hx. The serum total bile acid concentration at 6 h after aggressive Hx may be a useful parameter of the early stage of acute liver failure. Previous studies have shown increased bile flow and secretion of bile acids when expressed per gram of liver after two-thirds Hx [1, 2, 4, 17, 22]. Other studies have demonstrated that the bile flow rate and biliary bile acid secretion expressed as gram of body weight are decreased significantly after two-thirds Hx [2, 23]. Xu et al. demonstrated that the bile flow and biliary bile acid secretion rates are lower after 75% Hx than those after 50% Hx in rats but without statistically significant differences between the two groups [8]. In the present study, bile flow and biliary bile acid outputs expressed by body weight were significantly decreased,
FIG. 1. Rates of lipid secretion into biliary bile acids per gram of body weight after 70% and 90% Hx in rats. Bile was collected for 1 h at the indicated times. Total bile acid, total cholesterol, and phospholipid concentrations in biliary bile were measured by enzymatic and colorimeteric methods as described above. Solid circles stand for 70% Hx and squares for 90% Hx. Mean ⫾ SD, n ⫽ 6; *P ⬍ 0.05 versus 70% Hx; P ⬍ 0.05, 70% Hx versus basal value; P ⬍ 0.05, 90% Hx versus basal value.
of bile acids, phospholipids and cholesterol, compared with those after 70% Hx. Moreover, biliary outputs of cholic and chenodeoxycholic acids were significantly lower after 90% Hx. These differences were consistently observed at 6 h after Hx. Determination of these compounds may be useful for early detection of postoperative liver failure. Increased bile acid concentrations in the blood circulation after Hx have been reported [17, 18]. A similar result was observed after 70% and 90% Hx in the present study, and the serum bile acid concentration was significantly elevated in the early stage of acute liver failure induced by 90% Hx. The magnitude of its increase was eight times at 6 h after 90% Hx, while was two times after 70% Hx, compared with the basal value. It has been reported that the sinusoidal sodiumtaurocholate cotransporter (Ntcp) and the organic anion transporting polypeptides (Oatp1 and Oatp2), which maintain the expressions of the canalicular ATP-dependent bile salt export pump (Bsep) and multiorganic
FIG. 2. Outputs of different bile acid species as measured by GLC in biliary bile samples collected for 1 h at the indicated times during liver regeneration that after 70% Hx as compared with 90% Hx in rats. Solid circles indicate 70% Hx, and solid squares indicate 90% Hx. Mean ⫾ SD, n ⫽ 6; *P ⬍ 0.05 versus 70% Hx; P ⬍ 0.05, 70% Hx versus basal value; P ⬍ 0.05, 90% Hx versus basal value.
TANAKA ET AL.: BILIARY LIPID METABOLISM AFTER 70% AND 90% HEPATECTOMY IN RATS
FIG. 3. Changes of activities of cholesterol 7␣-hydroxylase and sterol 12␣-hydroxylase after 70% and 90% Hx in the rat liver. Each specific activity of the remnant liver at fixed intervals after Hx was measured by HPLC as described above. Solid circles show the activities after 70% Hx, and solid squares show them after 90% Hx. Mean ⫾ SD, n ⫽ 6; *P ⬍ 0.05 versus 70% Hx; P ⬍ 0.05, 70% Hx versus basal value; P ⬍ 0.05, 90% Hx versus basal value.
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cholic acid/chenodeoxycholic acid is increased on day 2 [2]. The reason for the different cholic acid outputs in bile is not clear. In our study, biliary outputs of each bile acid species, including cholic acid and chenodeoxycholic acid, declined after 70% and 90% Hx. Interestingly, the biliary outputs of cholic acid and chenodeoxycholic acid at 6 h after 90% Hx were significantly lower than those after 70% Hx. The reason for the decreased total bile acid and cholic acid outputs in bile was examined by determination of cholesterol 7␣-hydroxylase and sterol 12␣-hydrozylase activities, enzymes for bile acid biosynthesis and cholic acid synthesis, respectively. We have previously shown that cholesterol 7␣-hydroxylase activity was suppressed for the first 2 days after Hx, but returned to the control level on the third day [7]. In the current study the activity of cholesterol 7␣-hydroxylase was decreased for 2 days after 70% and 90% Hx. Significantly lower activities after 90% Hx were observed at 24 h, whereas significantly lower biliary bile acid output was observed at 6 h after 90% Hx compared with those after 70% Hx. The reason for the delayed decrease in the cholesterol 7␣-hydroxylase activity compared with biliary bile acid output is not clear at present. In contrast to cholesterol 7␣-hydroxylase activity, sterol 12␣-hydroxylase was activated after 70% Hx but not after 90% Hx. Although biliary cholic acid output was decreased after both 70% and 90% Hx, the changes in sterol 12␣-hydroxylase activity were different. The lack of correlation between the enzyme activity and biliary cholic acid output needs further examination. It is estimated that determinations of the serum bile acid levels, bile flow rate, biliary cholesterol, phospholipids, and bile acid outputs are the most reliable early predictor of post-operative liver failure after extended Hx. REFERENCES
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