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Serum level of ornithine carbamoyltransferase is influenced by the state of Kupffer cells
Clinica Chimica Acta 380 (2007) 170 – 174 www.elsevier.com/locate/clinchim
Serum level of ornithine carbamoyltransferase is influenced by the state of Kupffer cells Hiroshi Murayama a,⁎, Masaki Ikemoto b , Yoshihiro Fukuda b , Shoji Tsunekawa c , Atsuo Nagata a a
Immunology Laboratory, Diagnostics Department, YAMASA Corporation, 2-10-1 Araoi-cho, Choshi, Chiba, 288-0056, Japan b School of Health Sciences, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan c Department of Surgery, Kansai Denryoku Hospital, Osaka 553-0003, Japan Received 6 December 2006; received in revised form 5 February 2007; accepted 7 February 2007 Available online 15 February 2007
Abstract Background: The ratio of ornithine carbamoyltransferase (OCT) to alanine aminotransferase (ALT) or glutamate dehydrogenase (GDH) in serum has been suggested as an indicator for the diagnosis of hepatocellular carcinoma and alcoholic liver disease, respectively. However, the mechanisms responsible for the increase in these ratios are still unclear. Methods: Wistar rats were pretreated with lipopolysaccharide (LPS) or gadolinium chloride (GD) before being administered with thioacetamide (TAA, 200 mg/kg, ip). Serum OCT and ALT levels were compared with control values. Half-lives of the enzymes in circulation were evaluated after the intravenous injection of the purified enzymes into rats with or without the pretreatment. Results: The serum level of OCT at 24 h after the administration of TAA was significantly lower in the LPS-treated group, and not influenced by pretreatment with GD. The half-life of OCT was prolonged from 1.06 ± 0.14 to 2.07 ± 0.29 h (p b 0.05) by the pretreatment with GD, but not influenced by the administration of LPS. No change was observed in the clearance of GDH or ALT among the pretreatments. Conclusions: Leakage into and clearance from the circulation of OCT are influenced by whether Kupffer cells are activated or not. OCT alone or in combination with other markers may be a useful indicator for Kupffer cell activation as well as mitochondrial damage in hepatic cells. © 2007 Elsevier B.V. All rights reserved. Keywords: Alanine aminotransferase; Glutamate dehydrogenase; Hepatocellular carcinoma; Kupffer cell; Ornithine carbamoyltransferase
1. Introduction Ornithine carbamoyltransferase (OCT) mainly distributes in mitochondria in the periportal region of the hepatic lobule [1–3]. With its restricted distribution in the liver, OCT has been used as a marker for liver diseases [4–8]. To date, OCT alone and in combination with other markers of hepatocellular damage has been shown to be a relatively specific marker for some liver diseases. The ratio of OCT to alanine aminotransferase (ALT) was reported to be increased in patients with hepatocellular
Abbreviations: CH, chronic hepatitis; GD, gadolinium chloride; GDH, glutamate dehydrogenase; HCC, hepatocellular carcinoma; LC, liver cirrhosis; LPS, lipopolysaccharide; OCT, ornithine carbamoyltransferase; TAA, thioacetamide. ⁎ Corresponding author. Tel.: +81 479 22 9840; fax: +81 479 22 9845. E-mail address: [email protected] (H. Murayama). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.02.006
carcinoma [8]. Similarly, the ratio of glutamate dehydrogenase (GDH) to OCT (GDH/OCT) has been shown to be a marker for alcoholic liver damage [9]. However, applications have been limited by the lack of a satisfactory explanation for the increase in these indicators. The Kupffer cell is a resident macrophage in the liver and has important roles in the pathogenesis of liver diseases. The activation of Kupffer cells has been reported to influence the severity of the damage in some liver diseases through the production of inflammatory cytokines such as tumor necrosis factor (TNF)-α [10,11]. Generally, changes in serum activities of injury markers in those conditions have been considered as a consequence of the change in the extent of liver damage. However, it has been reported that the modulation of the reticuloendothelial system alters the rate at which proteins are cleared from the circulation and influences the serum activities of some enzyme markers [12,13]. Further, it is unclear whether
H. Murayama et al. / Clinica Chimica Acta 380 (2007) 170–174
the state of the Kupffer cell itself, activated or inactivated, influences the leakage of markers given that it does not contribute to the course of liver damage. In this report, we studied the mechanism behind the rise in the level of OCT in serum from the viewpoint of Kupffer cell activation and found that the leakage and clearance of OCT is greatly influenced by the state of Kupffer cells. The meaning of the modified serum level of OCT from a diagnostic perspective was discussed. 2. Materials and methods 2.1. Materials Thioacetamide (TAA) was from Sigma Chem Co. (St. Louis, MO). ALT purified from pig heart and GDH purified from bovine liver were from Roche Diag. (Switzerland). Lipopolysaccharide (LPS) from Escherichia coli O128: B12 and other reagents were from Nacalai tesque (Kyoto, Japan).
2.2. Animals and treatments Male Wistar rats were from CLEA Japan Co. (Tokyo, Japan). The rats were housed in wire-bottom cages with a 12-h light/dark cycle and fed a normal CE-2 diet (CLEA Japan Co.). In the TAA-induced acute injury model, 9-week old rats were divided into 3 groups of 9 each and were intraperitoneally administered TAA (200 mg/kg) with or without pretreatment with LPS (5 mg/kg, ip) or gadolinium chloride (GD) (7 mg/kg, iv). In the TAA-induced chronic liver injury model, 9 rats (8 weeks old) were orally administered TAA (20 mg/kg/day) for 24 weeks. After 24 weeks, the administration of TAA was stopped and 8 weeks was allowed to pass before the administration of GD (7 mg/kg, iv). All the pretreatments with LPS and GD were made 48 and 24 h before the start of the experiments, respectively. All blood samples were collected from a tail vein under anesthesia and allowed to clot for 2 h and then centrifuged at 1000 ×g. Sera were stored at − 80 °C until use. All experiments were performed according to the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society (Tokyo, Japan).
2.3. Purification of OCT OCT was partially purified from rat liver mitochondria. The mitochondria were prepared by the method of Johnson and Lardy [14] and kept at − 80 °C until use. The following procedures were carried out at 4 °C throughout. Thawed mitochondria obtained from 3 rats were suspended in 20 ml of purification buffer (10 mmol/l sodium phosphate buffer, pH 7.2) and centrifuged at 15,000 ×g for 15 min at 4 °C. The supernatant was applied to a column of Q-sepharose (10 × 100 mm) equilibrated with purification buffer. The passed fraction was directly applied to a column of hydroxyapatite (10 × 50 mm) equilibrated with the same buffer. The column was washed thoroughly with 30 mmol/l sodium phosphate buffer (pH 7.2) and then the OCT fraction was eluted with 100 mmol/l sodium phosphate buffer (pH 7.2). The eluted fraction was concentrated with an ultrafiltration membrane (Amicon Ultra, Millipore) and applied to a Sephadex G-150 gelfiltration column (16 × 950 mm) equilibrated with purification buffer with 50 mmol/l NaCl. The fractions were assayed for OCT by ELISA, pooled, and concentrated with the ultrafiltration membrane. OCT was detected as a main band by SDS-PAGE, its purity being no less than 50%. The final concentration of OCT used in the experiment was about 500 μg/ml.
2.4. Injection experiments OCT (50 μg), ALT (15 units), and GDH (10 units) were dissolved in saline, mixed, and intravenously injected into 9-week-old rats with or without the preadministration of LPS (5 mg/kg, ip) or GD (7 mg/kg, iv). Pretreatments with LPS and GD were made 48 h and 24 h before the start of the experiments, respectively. Blood samples were collected 5, 15, and 30 min, and 1, 2, 3, 4, 5, 6,
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8, 10, 12, 24, 30, 36, and 48 h after the injection of the enzyme. All blood samples were collected and stored as mentioned above. Most enzymes disappear from circulation in a biphasic exponential manner and the slower latter phases following the rapid primary phases have been considered to be the actual clearances [15]. Half-lives of the enzymes were calculated by fitting to the equation A = a1e − λ1t + a2e − λt + A∞, where A represents the serum activity at any time, t, and A∞ represents the activity in the steady state [16].
2.5. Measurement of ALT, GDH, OCT, and TNF-α Serum ALT activity was measured with a commercially available test kit, Transaminase CII-test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Serum GDH activity was measured as reported previously [17]. Serum OCT levels were measured by ELISA as reported previously [18]. Serum TNFα was measured with a commercially available ELISA kit (BioSource International, Camarillo, CA).
2.6. Statistical analysis A statistical comparison among groups was made using the Dunnett test for parametric data or non-parametric Dunn test for others, with p b 0.05 considered significant. The comparisons between before and after the administration to rats with TAA-induced chronic hepatitis in Table 2 were made using the Wilcoxon matched-pairs signed-ranks test.
3. Results 3.1. Effect of pretreatment with LPS or GD before the administration of TAA The effect of LPS or GD-pretreatment on the leakage of OCT was investigated in the TAA-induced acute liver injury model. In the TAA-induced acute liver injury model, the peaks of the increase in serum levels of injury marker enzymes were at 24 h after the administration of TAA, regardless of the pretreatment. The serum level of OCT at 24 h after the administration of TAA (200 mg/kg, ip) was significantly reduced by the preadministration of LPS (Table 1). Conversely, the administration of GD before that of TAA increased the peak levels of OCT, although the difference was not significant. The extent of the hepatocellular damage evaluated in the histological study appeared to be the same among all treatments (data not shown). The serum level of TNF-α was evaluated at 24 h after the administration of TAA and was found to be significantly higher in LPS-treated rats than control rats (Table 1).
Table 1 Effect of the pretreatment with LPS or GD on serum levels of OCT, ALT, and TNF-α at 24 h after TAA administration in rats
Control LPS pretreatment GD pretreatment
OCT (ng/ml)
ALT (IU/l)
OCT/ALT
(pg/ml)
6120±4046 2773 ± 2144⁎
229.2 ± 170.3 238.9 ± 136.8
22.9 ± 13.9 10.5 ± 6.3⁎
32.9 ± 39.7 173.0 ± 371.4⁎
5452 ± 12516
257.8 ± 278.0
20.5 ± 10.9
24.5 ± 32.3
LPS (5 mg/kg, ip), GD (7 mg/kg, iv), or saline was administered to rats (n = 9) before the administration of TAA (200 mg/kg, ip). Serum samples were taken at 24 h after TAA administration. Values are medians ± interquartile ranges and asterisks indicate a significant difference from the corresponding control groups using non-parametric Dunn test (p b 0.05).
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Table 2 Effect of the administration of GD to TAA-treated rats on serum levels of OCT and ALT
Before GD-administration 48 h after GD-administration
OCT (ng/ml)
ALT (IU/l)
OCT/ALT
97.5 ± 58.9 139.4 ± 58.9⁎
21.7 ± 7.4 20.2 ± 4.3
4.3 ± 1.5 6.8 ± 2.2⁎
GD (7 mg/kg, iv) was administered to rats (n = 8) which had been treated with TAA (20 mg/kg/day, po) for 24 weeks and then kept for 8 weeks after the cessation of TAA-administration. Serum samples were taken at 48 h after the administration of GD. Values are means ± S.D. and asterisks indicate a significant difference from the corresponding control groups (p b 0.05).
When LPS (5 mg/kg, ip) was administered to un-treated normal rats, serum levels of OCT and ALT were slightly, but not significantly, increased just after and decreased significantly 48 h after the administration. The serum level of TNF-α was markedly increased at 1–2 h after the administration of LPS, suggesting activation of Kupffer cells, and decreased to the initial level at 8 h. Administration of a higher dose of LPS (20 mg/kg, ip) induced severe liver damage resulting in marked increase of ALT and OCT. The administration of GD had no effect on the serum levels of the enzymes in normal rats (data not shown). 3.2. Effect of the administration of GD to TAA-treated rats To further investigate the influence of Kupffer cells on the serum level of OCT in circulation, the cells were inactivated by GD in a TAA-induced chronic liver injury model. Thioacetamide was administered to the rats for 24 weeks and then the administration was ceased for 8 weeks to eliminate the effect of TAA. Hepatitis was reported to continue for at least 2 months after the cessation of TAA administration [19]. The serum OCT
Fig. 1. Disappearance of OCT from the circulation. Fifty micrograms of OCT was injected intravenously into control rats (♦), or rats pretreated with LPS (×) or GD (□). LPS (5 mg/kg, ip) or GD (7 mg/kg, iv) was pre-administered to rats 48 h or 24 h before the injection of OCT. The concentrations of OCT are shown relative to the values at 5 min after the injection. Error bars mean the S.D. for 3 to 5 experiments and asterisks indicate a significant difference from the control (p b 0.05).
Table 3 Effect of pretreatment with LPS or GD on serum half-lives of OCT, ALT, and GDH in rats Half-life in circulation (h)
Un-treated (n = 3) LPS pretreatment (n = 5) GD pretreatment (n = 3)
OCT
ALT
GDH
1.06 ± 0.14 1.31 ± 0.35 2.07 ± 0.29⁎
10.5 ± 0.5 9.88 ± 1.10 10.1 ± 0.4
8.27 ± 0.25 8.58 ± 0.93 8.17 ± 0.76
LPS (5 mg/kg, ip) and GD (7 mg/kg, iv) were administered to rats 48 and 24 h, respectively, before the injection of the enzymes. Values are means ± S.D. and asterisk indicates a significant difference from the corresponding control (p b 0.05).
level was increased at 48 h after the administration of GD (7 mg/ kg, iv), while the ALT activity showed no increase (Table 2). 3.3. Injection experiments From the results shown above, we speculated that the clearance of OCT in circulation is modulated by the state of Kupffer cells. In order to confirm this idea, partially purified OCT together with ALT and GDH was intravenously injected into rats and the clearance of the enzymes was monitored (Fig. 1). The half-lives of OCT, ALT, and GDH in circulation are shown in Table 3. Prolonged clearance of OCT was observed in GD-pretreated rats (p b 0.05), while no change was observed in the clearance of ALT and GDH. Pre-administration of LPS seemed to have no effect on the clearance of the enzymes tested in this experiment. 4. Discussion In the present study, we demonstrated that the level of OCT in serum is greatly influenced by whether Kupffer cells are activated or not. The activation of Kupffer cells by LPS caused significant decreases in serum OCT levels in TAA-treated rats. Further, the serum level of OCT was increased in the chronic liver injury model and the half-life of OCT in circulation was prolonged by the inactivation of Kupffer cells by GD. The state of Kupffer cells does not seem to contribute to the course of liver damage in TAA-induced toxicity since the serum activity of ALT was not significantly affected and the extent of the damage evaluated in a histological study was the same among treatments. Therefore, it is speculated that lowering of the serum OCT level by the activation of Kupffer cells is a consequence not of the suppression of liver damage but of the modulation of serum OCT levels by some mechanisms. There are 2 possible explanations for the lowering of serum OCT levels; inhibition of the leakage into the circulation or increased clearance from the circulation. Since the clearance of OCT from the circulation was not affected, the leakage of OCT into the blood stream is more likely to be inhibited by activated Kupffer cells. Still, the reason why OCT, but not ALT, in serum was influenced should be investigated. The difference in the distribution of both enzymes, predominant distribution of OCT in mitochondria and localization of ALT mainly in
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cytosol, may affect leakage into the circulation under this condition. In addition, the effect of the activation of Kupffer cells on the clearance of OCT from the circulation should be studied further, since the effect of LPS on the clearance of enzymes might be limited within a short period. By contrast, the cause of the increase in the serum OCT level after the inactivation of Kupffer cells by GD was simply demonstrated by the prolongation of the half-life of OCT in serum in the injection experiment. Since the clearance of neither ALT nor GDH was affected by the modulation of Kupffer cell activity, the ratio of OCT to those enzyme markers would be a useful indicator for the activation of Kupffer cells. Though the mechanisms for the elimination of enzymes in serum are not fully understood [15], the reticuloendothelial system has been reported to be involved in the clearance of some enzymes including lactate dehydrogenase and aspartate aminotransferase [12,13]. The modulation of the reticuloendothelial system by the administration of some agents or infection by Riley virus altered the rate of clearance of these enzymes, but not that of ALT. This observation appears to be similar to the findings made here and support the idea that the mechanism for the elimination of OCT from the circulation is different from that of ALT and GDH. It has been reported that GDH/OCT is a useful marker for the diagnosis of alcoholic liver injury [9]. The increase of GDH/ OCT in patients with alcoholic liver injury has been explained by the differential distribution of the enzymes within the liver lobule; a centrilobular dominant distribution for GDH and a limited localization in the periportal region for OCT [20,21]. This assumption is based on the observation that the damage to hepatic cells is greater in the centrilobular region than periportal region [22]. In addition to this zonal difference, inhibition of the leakage of OCT into the circulation might also contribute to the increase of GDH/OCT, since the activation of Kupffer cells has been reported in patients with alcoholic liver injury [23]. OCT/ALT has been reported to be higher in patients with hepatocellular carcinoma (HCC) than those with liver cirrhosis (LC) or chronic hepatitis (CH) [8]. This observation could not be explained by the distribution of OCT and ALT, since both of them are located in the periportal region. It is speculated that there are some differences in the mechanisms of leakage or clearance between the 2 enzymes. It has been reported that OCT is a less sensitive marker than ALT in patients with virus-related CH, while OCT is a much more sensitive indicator in rats with CH induced by TAA [18,24]. Activation of Kupffer cells has been reported in patients with hepatitis C [25], while Kupffer cells are thought to be inactivated in patients with HCC or LC considering the Th1/Th2 imbalance in those patients [26–28]. These differences in the state of Kupffer cells between CH and HCC may partly explain the increase of OCT/ALT in HCC, since the serum OCT level is greatly influenced by the state of Kupffer cells. Of course, the increase in the level of OCT does not fully explain this phenomenon. The cause of the decrease in the ALT activity in HCC still remains to be elucidated. In the present study, we have demonstrated that the serum level of OCT is influenced by whether Kupffer cells are activated or not. Serum OCT levels were lower when Kupffer
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cells were activated, and vice versa. Comparisons of OCT with other markers may be useful for the interpretation of the state of Kupffer cells as well as mitochondrial damage in hepatic cells. Acknowledgments We thank Ms. Yasuko Kikuchi for the technical assistance, and Mr. Masaru Hamaoki and Mr. Shuichi Miyazaki for the helpful discussions. References [1] Reichard H. Ornithine carbamyl transferase activity in human tissue homogenates. J Lab Clin Med 1960;56:218–21. [2] Mizutani A. Cytochemical demonstration of ornithine carbamoyltransferase activity in liver mitochondria of rat and mouse. J Histochem Cytochem 1988;16:172–80. [3] Miyanaka K, Gotoh T, Nagasaki A, et al. Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver. Histochem J 1998;30:741–51. [4] Reichard H. Serum ornithine carbamoyl transferase activity in man. A highly specific test of liver and biliary tract involvement observation on 695 patients. Acta Med Scand 1962;172:723–38. [5] Oya R, Matsushima T, Kasai M, et al. Clinical meaning of serum ornithine carbamyl transferase (OCT) activity in liver diseases (in Japanese). Sougourinshou 1976;25:2250–4. [6] Mehrotra MP, Pursnani ML, Singh MM, Sharma RD. Serum ornithine carbamyl transferase (OCT) activity in viral hepatitis. J Assoc Phys India 1981;29:457–62. [7] Koike T, Hirakawa H, Miyake K, Tanimizu I. Serum mitochondrial glutamic–oxaloacetic transaminase activities in various liver diseases, especially in relation to other biochemical liver function tests (in Japanese). Curr Lab Med 1991;9:93–9. [8] Watanabe Y, Mori S, Fujiyama S, Sato T, Mori M. Clinical evaluation of serum ornithine carbamoyltransferase by enzyme-linked immunosorbent assay in patients with liver diseases. Enzyme Protein 1994–95; 48:18–26. [9] Takase S, Takada A, Tsutsumi M, Matsuda Y. Biochemical markers of chronic alcoholism. Alcohol 1985;2:405–10. [10] Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci 2002;65:166–76. [11] Su GL. Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation. Am J Physiol Gastrointest Liver Physiol 2002;283: G256–65. [12] Mahy BWJ, Rowson KEK, Parr CW. Studies on the mechanism of action of Riley virus IV. The reticuloendothelial system and impaired plasma enzyme clearance in infected mice. J Exp Med 1967;125:277–88. [13] Wakim KG, Fleisher GA. Fate of enzymes in body fluids — an experimental study. IV. Relationship of the reticuloendothelial system to activities and disappearance rates of various enzymes. J Lab Clin Med 1963;61:107–19. [14] Johnson D, Lardy H. Isolation of liver or kidney mitochondria. In: Estabrook RW, Pullman M, editors. Methods in enzymology. New York: Academic Press; 1967. p. 94–6. [15] Posen S. Turnover of circulating enzymes. Clin Chem 1970;16:71–84. [16] Fleisher GA, Wakim KG. The fate of enzymes in body fluids — an experimental study. I. Disappearance rate of glutamic–pyruvic transaminase under various conditions. J Lab Clin Med 1963;61:76–85. [17] Working Group on Enzymes. Proposal of standard methods for the determination of enzyme catalytic concentrations in serum and plasma at 37 °C. III. Glutamate dehydrogenase (L-glutamate: NAD(P)+oxidoreductase (deaminating), EC 1.4.1.3). Eur J Clin Chem Clin Biochem 1992;30:493–502. [18] Murayama H, Igarashi M, Mori M, Fukuda Y, Ikemoto M, Nagata A. A sensitive ELISA for serum ornithine carbamoyltransferase utilizing the enhancement of immunoreactivity at alkaline pH. Clin Chim Acta 2006;368:125–30.
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[19] Muller A, Machnik F, Zimmermann T, Schubert H. Thioacetamideinduced cirrhosis-like liver lesions in rats — usefulness and reliability of this animal model. Exp Pathol 1988;34:229–36. [20] Maly P, Sasse D. Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver. J Histochem Cytochem 1991;39:1121–4. [21] Miyanaka K, Gotoh T, Nagasaki A, et al. Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver. Histochem J 1998;30:741–51. [22] French SW. Biochemical basis for alcohol-induced liver injury. Clin Biochem 1989;22:41–9. [23] Hines IN, Wheeler MD. Recent advances in alcoholic liver disease III. Role of the innate immune response in alcoholic hepatitis. Am J Physiol Gastrointest Liver Physiol 2004;287:G310–4. [24] Murayama H, Ikemoto M, Fukuda Y, Tsunekawa S, Nagata A. Advantage of serum type-I arginase and ornithine carbamoyltransferase in the
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evaluation of acute and chronic liver damage induced by thioacetamide in rats. Clin Chim Acta 2006;375:63–8. Royer C, Steffan AM, Navas MC, Fuchs A, Jaeck D, Stoll-Keller F. A study of susceptibility of primary human Kupffer cells to hepatitis C virus. J Hepatol 2003;38:250–6. Chau GY, Wu CW, Lui WY, et al. Serum interleukin-10 but not interleukin6 is related to clinical outcome in patients with resectable hepatocellular carcinoma. Ann Surg 2000;231:552–8. Sakaguchi E, Kayano K, Segawa M, Okamoto M, Sakaida I, Okita K. Th-1 down-regulation at the single-lymphocyte level in HCV-related liver cirrhosis and the effect of TGF-beta on Th1 response: possible implications for the development of hepatoma. Hepatol Res 2002;24:282. Hattori E, Okumoto K, Adachi T, et al. Possible contribution of circulating interleukin-10 (IL-10) to anti-tumor immunity and prognosis in patients with unresectable hepatocellular carcinoma. Hepatol Res 2003;27:309–14.
Serum level of ornithine carbamoyltransferase is influenced by the state of Kupffer cells Hiroshi Murayama a,⁎, Masaki Ikemoto b , Yoshihiro Fukuda b , Shoji Tsunekawa c , Atsuo Nagata a a
Immunology Laboratory, Diagnostics Department, YAMASA Corporation, 2-10-1 Araoi-cho, Choshi, Chiba, 288-0056, Japan b School of Health Sciences, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan c Department of Surgery, Kansai Denryoku Hospital, Osaka 553-0003, Japan Received 6 December 2006; received in revised form 5 February 2007; accepted 7 February 2007 Available online 15 February 2007
Abstract Background: The ratio of ornithine carbamoyltransferase (OCT) to alanine aminotransferase (ALT) or glutamate dehydrogenase (GDH) in serum has been suggested as an indicator for the diagnosis of hepatocellular carcinoma and alcoholic liver disease, respectively. However, the mechanisms responsible for the increase in these ratios are still unclear. Methods: Wistar rats were pretreated with lipopolysaccharide (LPS) or gadolinium chloride (GD) before being administered with thioacetamide (TAA, 200 mg/kg, ip). Serum OCT and ALT levels were compared with control values. Half-lives of the enzymes in circulation were evaluated after the intravenous injection of the purified enzymes into rats with or without the pretreatment. Results: The serum level of OCT at 24 h after the administration of TAA was significantly lower in the LPS-treated group, and not influenced by pretreatment with GD. The half-life of OCT was prolonged from 1.06 ± 0.14 to 2.07 ± 0.29 h (p b 0.05) by the pretreatment with GD, but not influenced by the administration of LPS. No change was observed in the clearance of GDH or ALT among the pretreatments. Conclusions: Leakage into and clearance from the circulation of OCT are influenced by whether Kupffer cells are activated or not. OCT alone or in combination with other markers may be a useful indicator for Kupffer cell activation as well as mitochondrial damage in hepatic cells. © 2007 Elsevier B.V. All rights reserved. Keywords: Alanine aminotransferase; Glutamate dehydrogenase; Hepatocellular carcinoma; Kupffer cell; Ornithine carbamoyltransferase
1. Introduction Ornithine carbamoyltransferase (OCT) mainly distributes in mitochondria in the periportal region of the hepatic lobule [1–3]. With its restricted distribution in the liver, OCT has been used as a marker for liver diseases [4–8]. To date, OCT alone and in combination with other markers of hepatocellular damage has been shown to be a relatively specific marker for some liver diseases. The ratio of OCT to alanine aminotransferase (ALT) was reported to be increased in patients with hepatocellular
Abbreviations: CH, chronic hepatitis; GD, gadolinium chloride; GDH, glutamate dehydrogenase; HCC, hepatocellular carcinoma; LC, liver cirrhosis; LPS, lipopolysaccharide; OCT, ornithine carbamoyltransferase; TAA, thioacetamide. ⁎ Corresponding author. Tel.: +81 479 22 9840; fax: +81 479 22 9845. E-mail address: [email protected] (H. Murayama). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.02.006
carcinoma [8]. Similarly, the ratio of glutamate dehydrogenase (GDH) to OCT (GDH/OCT) has been shown to be a marker for alcoholic liver damage [9]. However, applications have been limited by the lack of a satisfactory explanation for the increase in these indicators. The Kupffer cell is a resident macrophage in the liver and has important roles in the pathogenesis of liver diseases. The activation of Kupffer cells has been reported to influence the severity of the damage in some liver diseases through the production of inflammatory cytokines such as tumor necrosis factor (TNF)-α [10,11]. Generally, changes in serum activities of injury markers in those conditions have been considered as a consequence of the change in the extent of liver damage. However, it has been reported that the modulation of the reticuloendothelial system alters the rate at which proteins are cleared from the circulation and influences the serum activities of some enzyme markers [12,13]. Further, it is unclear whether
H. Murayama et al. / Clinica Chimica Acta 380 (2007) 170–174
the state of the Kupffer cell itself, activated or inactivated, influences the leakage of markers given that it does not contribute to the course of liver damage. In this report, we studied the mechanism behind the rise in the level of OCT in serum from the viewpoint of Kupffer cell activation and found that the leakage and clearance of OCT is greatly influenced by the state of Kupffer cells. The meaning of the modified serum level of OCT from a diagnostic perspective was discussed. 2. Materials and methods 2.1. Materials Thioacetamide (TAA) was from Sigma Chem Co. (St. Louis, MO). ALT purified from pig heart and GDH purified from bovine liver were from Roche Diag. (Switzerland). Lipopolysaccharide (LPS) from Escherichia coli O128: B12 and other reagents were from Nacalai tesque (Kyoto, Japan).
2.2. Animals and treatments Male Wistar rats were from CLEA Japan Co. (Tokyo, Japan). The rats were housed in wire-bottom cages with a 12-h light/dark cycle and fed a normal CE-2 diet (CLEA Japan Co.). In the TAA-induced acute injury model, 9-week old rats were divided into 3 groups of 9 each and were intraperitoneally administered TAA (200 mg/kg) with or without pretreatment with LPS (5 mg/kg, ip) or gadolinium chloride (GD) (7 mg/kg, iv). In the TAA-induced chronic liver injury model, 9 rats (8 weeks old) were orally administered TAA (20 mg/kg/day) for 24 weeks. After 24 weeks, the administration of TAA was stopped and 8 weeks was allowed to pass before the administration of GD (7 mg/kg, iv). All the pretreatments with LPS and GD were made 48 and 24 h before the start of the experiments, respectively. All blood samples were collected from a tail vein under anesthesia and allowed to clot for 2 h and then centrifuged at 1000 ×g. Sera were stored at − 80 °C until use. All experiments were performed according to the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society (Tokyo, Japan).
2.3. Purification of OCT OCT was partially purified from rat liver mitochondria. The mitochondria were prepared by the method of Johnson and Lardy [14] and kept at − 80 °C until use. The following procedures were carried out at 4 °C throughout. Thawed mitochondria obtained from 3 rats were suspended in 20 ml of purification buffer (10 mmol/l sodium phosphate buffer, pH 7.2) and centrifuged at 15,000 ×g for 15 min at 4 °C. The supernatant was applied to a column of Q-sepharose (10 × 100 mm) equilibrated with purification buffer. The passed fraction was directly applied to a column of hydroxyapatite (10 × 50 mm) equilibrated with the same buffer. The column was washed thoroughly with 30 mmol/l sodium phosphate buffer (pH 7.2) and then the OCT fraction was eluted with 100 mmol/l sodium phosphate buffer (pH 7.2). The eluted fraction was concentrated with an ultrafiltration membrane (Amicon Ultra, Millipore) and applied to a Sephadex G-150 gelfiltration column (16 × 950 mm) equilibrated with purification buffer with 50 mmol/l NaCl. The fractions were assayed for OCT by ELISA, pooled, and concentrated with the ultrafiltration membrane. OCT was detected as a main band by SDS-PAGE, its purity being no less than 50%. The final concentration of OCT used in the experiment was about 500 μg/ml.
2.4. Injection experiments OCT (50 μg), ALT (15 units), and GDH (10 units) were dissolved in saline, mixed, and intravenously injected into 9-week-old rats with or without the preadministration of LPS (5 mg/kg, ip) or GD (7 mg/kg, iv). Pretreatments with LPS and GD were made 48 h and 24 h before the start of the experiments, respectively. Blood samples were collected 5, 15, and 30 min, and 1, 2, 3, 4, 5, 6,
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8, 10, 12, 24, 30, 36, and 48 h after the injection of the enzyme. All blood samples were collected and stored as mentioned above. Most enzymes disappear from circulation in a biphasic exponential manner and the slower latter phases following the rapid primary phases have been considered to be the actual clearances [15]. Half-lives of the enzymes were calculated by fitting to the equation A = a1e − λ1t + a2e − λt + A∞, where A represents the serum activity at any time, t, and A∞ represents the activity in the steady state [16].
2.5. Measurement of ALT, GDH, OCT, and TNF-α Serum ALT activity was measured with a commercially available test kit, Transaminase CII-test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Serum GDH activity was measured as reported previously [17]. Serum OCT levels were measured by ELISA as reported previously [18]. Serum TNFα was measured with a commercially available ELISA kit (BioSource International, Camarillo, CA).
2.6. Statistical analysis A statistical comparison among groups was made using the Dunnett test for parametric data or non-parametric Dunn test for others, with p b 0.05 considered significant. The comparisons between before and after the administration to rats with TAA-induced chronic hepatitis in Table 2 were made using the Wilcoxon matched-pairs signed-ranks test.
3. Results 3.1. Effect of pretreatment with LPS or GD before the administration of TAA The effect of LPS or GD-pretreatment on the leakage of OCT was investigated in the TAA-induced acute liver injury model. In the TAA-induced acute liver injury model, the peaks of the increase in serum levels of injury marker enzymes were at 24 h after the administration of TAA, regardless of the pretreatment. The serum level of OCT at 24 h after the administration of TAA (200 mg/kg, ip) was significantly reduced by the preadministration of LPS (Table 1). Conversely, the administration of GD before that of TAA increased the peak levels of OCT, although the difference was not significant. The extent of the hepatocellular damage evaluated in the histological study appeared to be the same among all treatments (data not shown). The serum level of TNF-α was evaluated at 24 h after the administration of TAA and was found to be significantly higher in LPS-treated rats than control rats (Table 1).
Table 1 Effect of the pretreatment with LPS or GD on serum levels of OCT, ALT, and TNF-α at 24 h after TAA administration in rats
Control LPS pretreatment GD pretreatment
OCT (ng/ml)
ALT (IU/l)
OCT/ALT
(pg/ml)
6120±4046 2773 ± 2144⁎
229.2 ± 170.3 238.9 ± 136.8
22.9 ± 13.9 10.5 ± 6.3⁎
32.9 ± 39.7 173.0 ± 371.4⁎
5452 ± 12516
257.8 ± 278.0
20.5 ± 10.9
24.5 ± 32.3
LPS (5 mg/kg, ip), GD (7 mg/kg, iv), or saline was administered to rats (n = 9) before the administration of TAA (200 mg/kg, ip). Serum samples were taken at 24 h after TAA administration. Values are medians ± interquartile ranges and asterisks indicate a significant difference from the corresponding control groups using non-parametric Dunn test (p b 0.05).
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Table 2 Effect of the administration of GD to TAA-treated rats on serum levels of OCT and ALT
Before GD-administration 48 h after GD-administration
OCT (ng/ml)
ALT (IU/l)
OCT/ALT
97.5 ± 58.9 139.4 ± 58.9⁎
21.7 ± 7.4 20.2 ± 4.3
4.3 ± 1.5 6.8 ± 2.2⁎
GD (7 mg/kg, iv) was administered to rats (n = 8) which had been treated with TAA (20 mg/kg/day, po) for 24 weeks and then kept for 8 weeks after the cessation of TAA-administration. Serum samples were taken at 48 h after the administration of GD. Values are means ± S.D. and asterisks indicate a significant difference from the corresponding control groups (p b 0.05).
When LPS (5 mg/kg, ip) was administered to un-treated normal rats, serum levels of OCT and ALT were slightly, but not significantly, increased just after and decreased significantly 48 h after the administration. The serum level of TNF-α was markedly increased at 1–2 h after the administration of LPS, suggesting activation of Kupffer cells, and decreased to the initial level at 8 h. Administration of a higher dose of LPS (20 mg/kg, ip) induced severe liver damage resulting in marked increase of ALT and OCT. The administration of GD had no effect on the serum levels of the enzymes in normal rats (data not shown). 3.2. Effect of the administration of GD to TAA-treated rats To further investigate the influence of Kupffer cells on the serum level of OCT in circulation, the cells were inactivated by GD in a TAA-induced chronic liver injury model. Thioacetamide was administered to the rats for 24 weeks and then the administration was ceased for 8 weeks to eliminate the effect of TAA. Hepatitis was reported to continue for at least 2 months after the cessation of TAA administration [19]. The serum OCT
Fig. 1. Disappearance of OCT from the circulation. Fifty micrograms of OCT was injected intravenously into control rats (♦), or rats pretreated with LPS (×) or GD (□). LPS (5 mg/kg, ip) or GD (7 mg/kg, iv) was pre-administered to rats 48 h or 24 h before the injection of OCT. The concentrations of OCT are shown relative to the values at 5 min after the injection. Error bars mean the S.D. for 3 to 5 experiments and asterisks indicate a significant difference from the control (p b 0.05).
Table 3 Effect of pretreatment with LPS or GD on serum half-lives of OCT, ALT, and GDH in rats Half-life in circulation (h)
Un-treated (n = 3) LPS pretreatment (n = 5) GD pretreatment (n = 3)
OCT
ALT
GDH
1.06 ± 0.14 1.31 ± 0.35 2.07 ± 0.29⁎
10.5 ± 0.5 9.88 ± 1.10 10.1 ± 0.4
8.27 ± 0.25 8.58 ± 0.93 8.17 ± 0.76
LPS (5 mg/kg, ip) and GD (7 mg/kg, iv) were administered to rats 48 and 24 h, respectively, before the injection of the enzymes. Values are means ± S.D. and asterisk indicates a significant difference from the corresponding control (p b 0.05).
level was increased at 48 h after the administration of GD (7 mg/ kg, iv), while the ALT activity showed no increase (Table 2). 3.3. Injection experiments From the results shown above, we speculated that the clearance of OCT in circulation is modulated by the state of Kupffer cells. In order to confirm this idea, partially purified OCT together with ALT and GDH was intravenously injected into rats and the clearance of the enzymes was monitored (Fig. 1). The half-lives of OCT, ALT, and GDH in circulation are shown in Table 3. Prolonged clearance of OCT was observed in GD-pretreated rats (p b 0.05), while no change was observed in the clearance of ALT and GDH. Pre-administration of LPS seemed to have no effect on the clearance of the enzymes tested in this experiment. 4. Discussion In the present study, we demonstrated that the level of OCT in serum is greatly influenced by whether Kupffer cells are activated or not. The activation of Kupffer cells by LPS caused significant decreases in serum OCT levels in TAA-treated rats. Further, the serum level of OCT was increased in the chronic liver injury model and the half-life of OCT in circulation was prolonged by the inactivation of Kupffer cells by GD. The state of Kupffer cells does not seem to contribute to the course of liver damage in TAA-induced toxicity since the serum activity of ALT was not significantly affected and the extent of the damage evaluated in a histological study was the same among treatments. Therefore, it is speculated that lowering of the serum OCT level by the activation of Kupffer cells is a consequence not of the suppression of liver damage but of the modulation of serum OCT levels by some mechanisms. There are 2 possible explanations for the lowering of serum OCT levels; inhibition of the leakage into the circulation or increased clearance from the circulation. Since the clearance of OCT from the circulation was not affected, the leakage of OCT into the blood stream is more likely to be inhibited by activated Kupffer cells. Still, the reason why OCT, but not ALT, in serum was influenced should be investigated. The difference in the distribution of both enzymes, predominant distribution of OCT in mitochondria and localization of ALT mainly in
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cytosol, may affect leakage into the circulation under this condition. In addition, the effect of the activation of Kupffer cells on the clearance of OCT from the circulation should be studied further, since the effect of LPS on the clearance of enzymes might be limited within a short period. By contrast, the cause of the increase in the serum OCT level after the inactivation of Kupffer cells by GD was simply demonstrated by the prolongation of the half-life of OCT in serum in the injection experiment. Since the clearance of neither ALT nor GDH was affected by the modulation of Kupffer cell activity, the ratio of OCT to those enzyme markers would be a useful indicator for the activation of Kupffer cells. Though the mechanisms for the elimination of enzymes in serum are not fully understood [15], the reticuloendothelial system has been reported to be involved in the clearance of some enzymes including lactate dehydrogenase and aspartate aminotransferase [12,13]. The modulation of the reticuloendothelial system by the administration of some agents or infection by Riley virus altered the rate of clearance of these enzymes, but not that of ALT. This observation appears to be similar to the findings made here and support the idea that the mechanism for the elimination of OCT from the circulation is different from that of ALT and GDH. It has been reported that GDH/OCT is a useful marker for the diagnosis of alcoholic liver injury [9]. The increase of GDH/ OCT in patients with alcoholic liver injury has been explained by the differential distribution of the enzymes within the liver lobule; a centrilobular dominant distribution for GDH and a limited localization in the periportal region for OCT [20,21]. This assumption is based on the observation that the damage to hepatic cells is greater in the centrilobular region than periportal region [22]. In addition to this zonal difference, inhibition of the leakage of OCT into the circulation might also contribute to the increase of GDH/OCT, since the activation of Kupffer cells has been reported in patients with alcoholic liver injury [23]. OCT/ALT has been reported to be higher in patients with hepatocellular carcinoma (HCC) than those with liver cirrhosis (LC) or chronic hepatitis (CH) [8]. This observation could not be explained by the distribution of OCT and ALT, since both of them are located in the periportal region. It is speculated that there are some differences in the mechanisms of leakage or clearance between the 2 enzymes. It has been reported that OCT is a less sensitive marker than ALT in patients with virus-related CH, while OCT is a much more sensitive indicator in rats with CH induced by TAA [18,24]. Activation of Kupffer cells has been reported in patients with hepatitis C [25], while Kupffer cells are thought to be inactivated in patients with HCC or LC considering the Th1/Th2 imbalance in those patients [26–28]. These differences in the state of Kupffer cells between CH and HCC may partly explain the increase of OCT/ALT in HCC, since the serum OCT level is greatly influenced by the state of Kupffer cells. Of course, the increase in the level of OCT does not fully explain this phenomenon. The cause of the decrease in the ALT activity in HCC still remains to be elucidated. In the present study, we have demonstrated that the serum level of OCT is influenced by whether Kupffer cells are activated or not. Serum OCT levels were lower when Kupffer
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cells were activated, and vice versa. Comparisons of OCT with other markers may be useful for the interpretation of the state of Kupffer cells as well as mitochondrial damage in hepatic cells. Acknowledgments We thank Ms. Yasuko Kikuchi for the technical assistance, and Mr. Masaru Hamaoki and Mr. Shuichi Miyazaki for the helpful discussions. References [1] Reichard H. Ornithine carbamyl transferase activity in human tissue homogenates. J Lab Clin Med 1960;56:218–21. [2] Mizutani A. Cytochemical demonstration of ornithine carbamoyltransferase activity in liver mitochondria of rat and mouse. J Histochem Cytochem 1988;16:172–80. [3] Miyanaka K, Gotoh T, Nagasaki A, et al. Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver. Histochem J 1998;30:741–51. [4] Reichard H. Serum ornithine carbamoyl transferase activity in man. A highly specific test of liver and biliary tract involvement observation on 695 patients. Acta Med Scand 1962;172:723–38. [5] Oya R, Matsushima T, Kasai M, et al. Clinical meaning of serum ornithine carbamyl transferase (OCT) activity in liver diseases (in Japanese). Sougourinshou 1976;25:2250–4. [6] Mehrotra MP, Pursnani ML, Singh MM, Sharma RD. Serum ornithine carbamyl transferase (OCT) activity in viral hepatitis. J Assoc Phys India 1981;29:457–62. [7] Koike T, Hirakawa H, Miyake K, Tanimizu I. Serum mitochondrial glutamic–oxaloacetic transaminase activities in various liver diseases, especially in relation to other biochemical liver function tests (in Japanese). Curr Lab Med 1991;9:93–9. [8] Watanabe Y, Mori S, Fujiyama S, Sato T, Mori M. Clinical evaluation of serum ornithine carbamoyltransferase by enzyme-linked immunosorbent assay in patients with liver diseases. Enzyme Protein 1994–95; 48:18–26. [9] Takase S, Takada A, Tsutsumi M, Matsuda Y. Biochemical markers of chronic alcoholism. Alcohol 1985;2:405–10. [10] Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci 2002;65:166–76. [11] Su GL. Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation. Am J Physiol Gastrointest Liver Physiol 2002;283: G256–65. [12] Mahy BWJ, Rowson KEK, Parr CW. Studies on the mechanism of action of Riley virus IV. The reticuloendothelial system and impaired plasma enzyme clearance in infected mice. J Exp Med 1967;125:277–88. [13] Wakim KG, Fleisher GA. Fate of enzymes in body fluids — an experimental study. IV. Relationship of the reticuloendothelial system to activities and disappearance rates of various enzymes. J Lab Clin Med 1963;61:107–19. [14] Johnson D, Lardy H. Isolation of liver or kidney mitochondria. In: Estabrook RW, Pullman M, editors. Methods in enzymology. New York: Academic Press; 1967. p. 94–6. [15] Posen S. Turnover of circulating enzymes. Clin Chem 1970;16:71–84. [16] Fleisher GA, Wakim KG. The fate of enzymes in body fluids — an experimental study. I. Disappearance rate of glutamic–pyruvic transaminase under various conditions. J Lab Clin Med 1963;61:76–85. [17] Working Group on Enzymes. Proposal of standard methods for the determination of enzyme catalytic concentrations in serum and plasma at 37 °C. III. Glutamate dehydrogenase (L-glutamate: NAD(P)+oxidoreductase (deaminating), EC 1.4.1.3). Eur J Clin Chem Clin Biochem 1992;30:493–502. [18] Murayama H, Igarashi M, Mori M, Fukuda Y, Ikemoto M, Nagata A. A sensitive ELISA for serum ornithine carbamoyltransferase utilizing the enhancement of immunoreactivity at alkaline pH. Clin Chim Acta 2006;368:125–30.
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