Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease

Transcription

1 Journal of Hepatology 38 (2003) Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease Yoshio Sumida 1, Toshiaki Nakashima 1, *, Takaharu Yoh 1, Masanori Furutani 1, Akihisa Hirohama 1, Yuko Kakisaka 1, Yoshiki Nakajima 1, Hiroki Ishikawa 1, Hironori Mitsuyoshi 1, Takeshi Okanoue 1, Kei Kashima 1, Hajime Nakamura 2, Junji Yodoi 2 1 Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto , Japan 2 Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan Background/Aims: Thioredoxin (TRX) is a stress-inducible thiol-containing protein. The aim of this study was to evaluate the clinical significance of serum TRX in patients with nonalcoholic steatohepatitis (NASH) or simple steatosis. Methods: Serum TRX levels were determined using an enzyme-linked immunosorbent assay kit in 25 patients with NASH, 15 patients with simple steatosis, and 17 healthy volunteers. Results: Serum TRX levels (medians and (ranges), ng/ml) were significantly elevated in patients with NASH (60.3 ( )), compared to those in patients with simple steatosis (24.6 ( ), P ¼ ) and in healthy controls (23.5 ( ), P, ). Serum ferritin levels in patients with NASH were also significantly higher than the levels in patients with simple steatosis. The receiver operating characteristic curve confirmed that serum TRX and ferritin levels were predictors for distinguishing NASH from simple steatosis. Higher grades of histological iron staining were observed in NASH than in simple steatosis. Serum TRX tended to increase in accordance with hepatic iron accumulation and the histological severity in patients with NASH. Conclusions: The pathogenesis of NASH may be associated with iron-related oxidative stress. The serum TRX level is a parameter for discriminating NASH from simple steatosis as well as a predictor of the severity of NASH. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Ferritin; Iron; Nonalcoholic steatohepatitis; Oxidative stress; Thioredoxin 1. Introduction A wide spectrum of fatty liver diseases has been documented in nondrinkers, ranging from simple steatosis to steatohepatitis. Nonalcoholic steatohepatitis (NASH), which is characterized by a necroinflammatory disorder with the fatty infiltration of hepatocytes [1], has been increasingly recognized as an important clinical entity because it may progress to fibrosis or cirrhosis, with a fatal outcome [2,3]. Diagnosis is confirmed by liver histology, which typically shows macrovesicular steatosis and lobular inflammation with or without fibrosis. Mallory bodies are occasionally seen in the absence of a history of Received 4 April 2002; received in revised form 3 September 2002; accepted 18 September 2002 * Corresponding author. Tel.: ; fax: address: nakashim@koto.kpu-m.ac.jp (T. Nakashima). excessive ethanol ingestion [1]. Though the exact pathogenesis of NASH has not been clarified, several lines of studies have suggested that oxidative stress may play an important role in its pathological mechanism [4 7]. On the other hand, thioredoxin (TRX), a redox-active protein with two cysteine residues (-Cys-Gly-Pro-Cys-) at its active site, has a variety of biological activities, including the scavenging of active oxygen radicals and the regulation of redox-sensitive molecules [8]. Since TRX is induced by many forms of oxidative stress [9,10], serum TRX levels are believed to be a clinically useful indicator of oxidative stress. Serum or plasma levels of TRX are reported to be elevated in patients with acquired immunodeficiency syndrome [11], rheumatoid arthritis [12], and neoplasms [13], but the source of the serum TRX remains unknown. Immunohistochemical analysis has demonstrated that TRX is distributed throughout the human body, including the liver, kidney, gastrointestinal epithelium, and gonads [9] /02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S (02)

2 Y. Sumida et al. / Journal of Hepatology 38 (2003) We previously reported that serum TRX levels were elevated in patients with chronic hepatitis C or other liver diseases [14 16]. In the present study, we examined the serum TRX levels, clinical laboratory data, and liver histology findings in NASH patients to ascertain whether oxidative stress is associated with the pathogenesis of NASH and to determine the potential value of using serum TRX levels to differentiate NASH from simple steatosis. 2. Patients and methods 2.1. Patients A total of 25 patients with NASH and 15 patients with simple steatosis were enrolled in the study (Table 1), in addition to 17 healthy volunteers as controls (12 males and five females, median age 42 years, range years). Informed written consent was obtained from all patients at the time of their liver biopsy, and the study was conducted in conformance with the Helsinki Declaration. Patients consuming more than 20 g of alcohol per day and patients with evidence of liver cirrhosis or hepatocellular carcinoma were excluded from the present study. None of the patients had ingested drugs known to produce hepatic steatosis (including corticosteroids, highdose estrogens, methotrexate, tetracycline, calcium channel blockers, or amiodarone) in the previous 6 months. One patient with NASH had a history of gastrointestinal surgery. Another patient with NASH had been occupationally exposed to unidentified solvents [17]. Two other NASH patients had been treated with polyenephosphatidylcholine. None of the patients with simple steatosis had received medication. A diagnosis of NASH was established if a combination of the following clinical and histopathological features were present: (i) a persistent abnormal liver biochemistry for more than 3 months; (ii) a liver biopsy showing steatosis (.10%) in the presence of lobular and/or portal inflammation, with or without Mallory bodies or fibrosis; and (iii) the exclusion of other diseases, such as viral hepatitis, autoimmune hepatitis, drug-induced liver disease, primary biliary cirrhosis, biliary obstruction, hemochromatosis, Wilson s disease, and a-1-antitrypsin-deficiency-associated liver disease. Hemochromatosis was excluded on the basis of the clinical symptoms, the family history, laboratory data, magnetic resonance imaging, and histological findings. Simple steatosis was also diagnosed by liver biopsy. Body mass index (BMI) was calculated using the following formula: weight in kilograms/(height in meters) 2. Obesity was defined as a BMI of greater than 25, according to the criteria of the Japan Society for the Study of Obesity [18]. Patients were assigned a diagnosis of diabetes mellitus if a documented use of oral hypoglycemic medication or insulin, a random glucose level in excess of 200 mg/dl, or a fasting glucose level of greater than 126 mg/dl on at least two occasions was present [19]. Hyperlipidemia was diagnosed if the cholesterol level was higher than 220 mg/dl and/or the triglyceride level was over 160 mg/dl. Hypertension was diagnosed if the patient was on antihypertensive medication and/or had a resting recumbent blood pressure of greater or equal to 140/90 mmhg on at least two occasions Laboratory evaluation and serum TRX measurement Venous blood samples were taken in the morning after a 12 h overnight fast. The laboratory evaluation in all patients included a blood cell count and the measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), cholinesterase (ChE), gamma globulin, total cholesterol, triglyceride, fasting blood glucose, and ferritin levels. These parameters were measured using the standard techniques of clinical chemistry laboratories. For the TRX measurements, the serum was stored at 230 8C until use. The serum TRX level was then measured using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit (Fujirebio Inc., Tokyo, Japan), as described previously [14,16]. This ELISA showed a highly specific reactivity on TRX with no cross-reactivity to several proteins with homologue sequence on the active center. The detection limit of the assay was 2.0 ng/ml. The intra- and interassay coefficients of variation were % and %, respectively [20]. All measurements Table 1 Clinical characteristics of patients with NASH and simple steatosis a Variable NASH (n ¼ 25) Simple steatosis (n ¼ 15) P value Gender (female) 11 (44.0%) 5 (33.3%) Age (years) 48 (15 72) 50 (23 68) BMI (kg/m 2 ) 25.3 ( ) 24.7 ( ) Obesity 13 (52.0%) 6 (40.0%) Type II diabetes 5 (20.0%) 2 (13.3%) Hyperlipidemia 20 (80.0%) 14 (93.3%) Hypertension 3 (12.0%) 5 (33.3%) Hemoglobin (g/dl) 15.2 ( ) 14.5 ( ) Platelet count (10 4 /ml) 22.0 ( ) 20.8 ( ) AST (IU/l) 43 (21 179) 34 (19 88) ALT (IU/l) 71 (26 312) 49 (21 173) GGT (IU/l) 105 (21 548) 86 (36 539) ChE (IU/l) 359 (67 504) 404 ( ) Gamma globulin (g/dl) 1.14 ( ) 1.11 ( ) Cholesterol (mg/dl) 209 ( ) 217 ( ) Triglyceride (mg/dl) 114 (45 664) 174 (67 459) Glucose (mg/dl) 104 (74 176) 106 (86 132) Serum ferritin (ng/ml) 161 (24 675) 113 (17 224) a Results are presented as numbers with percentages in parentheses for qualitative data and as medians and (ranges) for quantitative data. P values for qualitative data were calculated using Fisher s exact probability test, and the P values for quantitative data were calculated using the Mann Whitney U-test. Abbreviations and normal values: BMI, body mass index; hemoglobin, g/dl; platelet count, /ml; AST, aspartate aminotransferase, IU/l; ALT, alanine aminotransferase, 6 33 IU/l; GGT, gamma-glutamyl transpeptidase, 3 54 IU/l; ChE, cholinesterase, IU/l; gamma globulin, g/dl; cholesterol, mg/dl; triglyceride, mg/dl; glucose, mg/dl; and serum ferritin, ng/ml.

3 34 Y. Sumida et al. / Journal of Hepatology 38 (2003) were made in duplicate, and the average values were used in the statistic analyses Histological analysis All patients enrolled in this study underwent a percutaneous liver biopsy under ultrasonic guidance. The liver specimens were embedded in paraffin and stained with hematoxylin and eosin, Masson-trichrome, reticulin silver stain, and Perls Prussian Blue. A semiquantitative histological grading was used for the histological assessment of hepatic iron accumulation [21]. Briefly, grade 0 indicates no detectable iron, grade 1 indicates granules of iron resolved only at a magnification of 400, grade 2 indicates discrete iron granules resolved at a magnification of 100, grade 3 indicates iron granules resolved at a magnification of 25, and grade 4 indicates masses of iron visible to the naked eye or at a magnification of 10. The histological findings of NASH were interpreted and scored according to the classification proposed by Brunt et al. [22]. The activity of hepatitis (necroinflammatory grade) was determined by the presence of hepatocellular steatosis, ballooning and inflammation (acinar and portal) features as follows: grade 1, mild; grade 2, moderate; and grade 3, severe. The severity of hepatic fibrosis (stage) was defined as follows: stage 1, zone 3 perisinusoidal fibrosis; stage 2, zone 3 perisinusoidal fibrosis with portal fibrosis; stage 3, zone 3 perisinusoidal fibrosis and portal fibrosis with bridging fibrosis; and stage 4, cirrhosis. All grading and staging were performed by a single pathologist without knowledge of the patients clinical or laboratory data Statistical analysis Results are presented as the medians and ranges for quantitative data or as numbers with percentages in parentheses for qualitative data. Statistical differences in quantitative data were determined using the Mann Whitney U-test and the Kruskal Wallis test, when applicable; Fisher s exact probability test was used for qualitative data (Table 1 and Fig. 1). To assess the use of clinical parameters in differentiating NASH from simple steatosis, we calculated the sensitivity and the specificity for each value of each test and then constructed receiver operating characteristic (ROC) curves by plotting the sensitivity against the reverse specificity (1 minus specificity) at each value [23] (Fig. 2). In this assessment, a larger area under the ROC curve corresponds to a more useful marker for the diagnosis of NASH. The best cutoff point for the diagnosis of NASH is the point at which the sum of the sensitivity and the specificity is maximized. Correlation coefficients were calculated by Spearman rank correlation analysis (Table 2 and Fig. 3). Differences were considered statistically significant at all P values less than serum ferritin (232 (94 675) ng/ml) than those with simple steatosis (120 (22 224) ng/ml, P ¼ 0:023), while the serum ferritin levels of female patients with NASH (127 (24 472) ng/ml) tended to be higher than those with simple steatosis (46 (17 153) ng/ml) but the difference was not statistically significant (P ¼ 0:089) Serum TRX levels Serum TRX levels (medians and (ranges), ng/ml) were significantly elevated in patients with NASH (57.7 ( )), compared to those in patients with simple steatosis (26.8 ( )) and in the control group (23.5 ( )); however, the difference between patients with simple steatosis and the control group was not statistically significant (Fig. 1). No difference in the serum TRX levels between sexes was observed for either patient group (NASH patients, P ¼ 0:805; simple steatosis patients, P. 0:999) ROC curve analysis for serum ferritin and TRX The ROC curves for serum ferritin and TRX used to discriminate NASH from simple steatosis are shown in Fig. 2. The serum ferritin threshold for the prediction of NASH was 124 ng/ml; at this threshold, the sensitivity was 75.0% and the specificity was 64.3% (Fig. 2A). On 3. Results 3.1. Patient demographics and laboratory evaluation The main demographic and clinical laboratory features of the NASH and simple steatosis patients are compared in Table 1. The prevalence of obesity, type II diabetes, hyperlipidemia, and hypertension, and the levels of all clinical parameters other than serum ferritin were similar in both groups. Serum ferritin levels were found to be significantly elevated in the NASH patients, compared with those of the simple steatosis patients. When data from both patient groups were combined, a significant difference in serum ferritin levels was observed between sexes (males, 183 (22 675) ng/ml vs. females, 102 (17 402) ng/ml, P ¼ 0:029). Male patients with NASH had significantly higher levels of Fig. 1. Serum TRX levels in NASH (n ¼ 25), simple steatosis (n ¼ 15), and healthy controls (Control, n ¼ 17). The box contains the values between the 25th and 75th percentiles and the bold horizontal line is the median; the error bars stretch from the 10th to the 90th percentiles, and individual data are represented by closed circles. The overall significance of the differences between the three groups according to a non-parametric Kruskal Wallis analysis of variance was P, Therefore, the significance of the differences between the groups was determined using Scheffe s method. *P ¼ and **P, , compared to the values for simple steatosis and the control group, respectively.

4 Y. Sumida et al. / Journal of Hepatology 38 (2003) Fig. 2. Receiver operating characteristic (ROC) curves for serum ferritin (A) and TRX (B) used to discriminate NASH from simple steatosis. The area under the ROC curve for serum TRX (0.785) was larger than that of serum ferritin (0.660). the other hand, the serum TRX threshold was 34.7 ng/ml; at this threshold, the sensitivity was 72.0% and the specificity was 86.7% (Fig. 2B). The areas under the ROC curves for serum ferritin and TRX were and 0.785, respectively. The differences were not statistically significant Comparison of histological iron staining patterns between NASH and simple steatosis patients A histological iron analysis was performed in 22 NASH patients (nine females and 13 males) and 13 simple steatosis patients (four females and nine males). Positive iron staining was observed in 15 (five females and ten males) of the 22 patients with NASH: nine patients (four females and five males) exhibited grade 1 staining, three patients (one female and two males) exhibited grade 2 staining, and three patients (three males) exhibited grade 3 staining. Five (one female and four males) of the 13 patients with simple steatosis exhibited grade 1 iron staining. As a result, the grade of iron staining was significantly higher in the NASH patients than in the simple steatosis patients (P ¼ 0:034, Mann Whitney U-test). The serum TRX levels of patients with each grade of iron staining are shown in Table 2. A significant correlation between serum TRX level and iron grade was observed when all of the patients were evaluated together. The serum TRX levels of the NASH patients increased in proportion to the grade of iron staining, but this correlation was not significant. NASH patients with iron grades of 0 or 1 tended to have higher serum TRX levels than simple steatosis patients with the same iron grades, but the difference was not statistically significant Correlation between serum TRX levels and liver histology findings in NASH patients Among the 25 NASH patients, 15 patients (60.0%) had grade 1 activity, eight patients (32.0%) had grade 2 activity, and only two patients (8.0%) had grade 3 activity. Fibrosis was observed in 23 NASH patients (92.0%): eight patients (32.0%) with stage 1 fibrosis, ten patients (40.0%) with stage 2, and five patients (20.0%) with stage 3. As shown in Fig. 3, the serum TRX level tended to increase in parallel with the severity of NASH; serum TRX levels were significantly correlated with both the grade of activity (r ¼ 0:442, P ¼ 0:031) and the stage of fibrosis (r ¼ 0:503, P ¼ 0:014). Table 2 Relation between serum TRX levels and histological iron grade in patients with NASH and simple steatosis a Iron grade P value b NASH (n ¼ 22) 40.2 ( ) (n ¼ 7) c 50.7 ( ) (n ¼ 9) d 64.6 ( ) (n ¼ 6) Simple steatosis (n ¼ 13) 24.6 ( ) (n ¼ 8) c 30.7 ( ) (n ¼ 5) d Total (n ¼ 35) 26.1 ( ) (n ¼ 15) 35.8 ( ) (n ¼ 14) 64.6 ( ) (n ¼ 6) a Data from patients with an iron grade of 2 or 3 were combined because of the small number of patients. Results are presented as the medians and (ranges) of serum TRX levels. b P values were calculated by using Spearman correlation analysis. c P ¼ 0:355 (Mann Whitney U-test). d P ¼ 0:053 (Mann Whitney U-test).

5 36 Y. Sumida et al. / Journal of Hepatology 38 (2003) Fig. 3. Comparison between serum TRX levels and liver histology findings in patients with NASH. The box contains the values between the 25th and 75th percentiles and the bold horizontal line is the median; the error bars stretch from the 10th to the 90th percentiles, and individual data are represented by closed circles. The TRX levels (medians and (ranges), ng/ml) were 37.2 ( ) for grade 1 (n ¼ 15), 65.9 ( ) for grade 2 (n ¼ 8), 69.0 ( ) for grade 3 (n ¼ 2), 22.1 ( ) for stage 0 (n ¼ 2), 32.2 ( ) for stage 1 (n ¼ 8), 64.2 ( ) for stage 2 (n ¼ 10), and 66.4 ( ) for stage 3 (n ¼ 5). Furthermore, the stage of hepatic fibrosis was significantly correlated with the grade of activity (r ¼ 0:767, P ¼ 0:0002) but not with the grade of iron staining (r ¼ 0:149, P ¼ 0:496). 4. Discussion Liver biopsy remains not only the best diagnostic tool for confirming NASH, but also the most sensitive and specific means of providing prognostic information [24,25]. To the best of our knowledge, no clinical or laboratory parameters have been used to predict NASH that do not require a liver biopsy. The present study clearly shows that serum ferritin and TRX levels were significantly higher in NASH patients than in simple steatosis patients. The utility of serum ferritin and TRX levels for discriminating between NASH and simple steatosis was subsequently analyzed using ROC curves. By calculating the area under the ROC curve, which is an indicator of the utility of the threshold, we found that the serum ferritin and TRX levels were parameters for discriminating NASH from simple steatosis. The pathological mechanisms of NASH have not been clarified, but one concept for the initiation of necroinflammation suggests a two-hit process: first, the accumulation of excessive fat in the liver, and second, the development of oxidative stresses [26]. These two steps result in the production of lipid peroxides that are probably involved in the stimulation of collagen synthesis in hepatic stellate cells [27,28]. The elevated serum TRX level in NASH patients supports the hypothesis that oxidative stress may contribute to the pathogenesis of NASH. Potential sources of oxidative stress in NASH include (1) the cytochrome P-450, CYP2E1 [29], which is induced by ketones and fatty acids, (2) peroxisomal b-oxidation of fatty acids, which increases when mitochondrial b-oxidation is inhibited [7,30], (3) excessive free hepatic iron [31], and (4) various drugs that inhibit the respiratory chain [32]. Consistent with several previous findings [3,33,34], the present data showing that serum ferritin levels and the grade of hepatic iron staining are higher in NASH patients than in simple steatosis patients suggest that hepatic iron may be responsible for the pathogenesis of NASH by possibly generating hydroxyl radicals via the Fenton reaction. The underlying mechanisms for hepatic iron accumulation in NASH remain unknown, but recent studies in western countries have indicated that the hemochromatosis Cys282Tyr mutation contributes to iron overloading in many of these patients [33,34]. In the Japanese population, however, the frequency of this mutation is known to be extremely rare [35]. Another plausible explanation is that insulin resistance, which is often associated with NASH [7], may be directly responsible for the accumulation of iron in the liver [36]. High levels of circulating insulin have been reported to produce a rapid up-regulation in the expression of transferrin receptors on hepatocyte surfaces [37]. On the other hand, most (93.0%) of the present NASH patients exhibited some degree of hepatic fibrosis. Hepatic stellate cells, which are the principal collagen-producing cells in the liver [27], appear to be activated by oxidative stresses [28,38]. This was supported by the present data showing that the serum TRX level was positively correlated with the degree of hepatic fibrosis in NASH patients. In contrast to the finding by George et al. [34] that an increase in iron stores is significantly associated with an increase in hepatic fibrosis in patients with NASH, the present data reveal that the grade of iron staining has no correlation with the degree of fibrosis, in agreement with several previous studies [39,40]. One research group has asserted that significant iron accumulation is not seen in most

6 Y. Sumida et al. / Journal of Hepatology 38 (2003) patients with nonalcoholic fatty liver diseases, including NASH [25,31]. In this way, the role of hepatic iron in the pathogenesis of NASH remains controversial and unresolved at this time. In the present study, patients with NASH and without iron deposition (iron grade 0) tended to have a higher serum TRX level than patients with simple steatosis and without iron deposition. This finding suggests that not only iron-related but also iron-unrelated oxidative stress may be involved in the pathogenesis of NASH. Considering that TRX controls the activity of NF-kB, a nuclear factor that regulates the transcription of several genes involved in the inflammatory response [41], and that extracellular TRX can exhibit chemokine-like and cytokine-like activities [11], elevated serum TRX levels seem likely to have an active role in the production of oxidative stress in NASH. In conclusion, the increases in serum TRX levels and hepatic iron concentrations suggest that iron-induced oxidative stress may play an important role in the pathogenesis of NASH. The serum TRX level can help to discriminate NASH from simple steatosis and may be a predictor of the disease severity in patients with NASH. Determining the serum TRX level may be useful for selecting steatosis patients in whom a liver biopsy should be performed and may assist in the development of further therapeutic options. Acknowledgements Part of this work was presented at the Annual Meeting of the Digestive Disease Week (DDW), held in Atlanta, Georgia, on May 23, 2001 [42]. References [1] Ludwig J, Viggiano TR, McGill DB, Ott BJ. Non-alcoholic steatohepatitis. Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 1980;55: [2] Powell EE, Cooksley WGE, Hanson R, Searle J, Halliday JW, Powell LW. The natural history of nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years. Hepatology 1990;11: [3] Bacon BR, Farahvash MJ, Janney CG, Neuschwander-Tetri BA. Nonalcoholic steatohepatitis: an expanded clinical entity. Gastroenterology 1994;107: [4] Day CP, James OFW. Hepatic steatosis: innocent bystander or guilty party? Hepatology 1998;29: [5] Sheth SG, Gordon FD, Chopra S. Non alcoholic steatohepatitis. Ann Intern Med 1997;126: [6] Ludwig J, McGill DB, Lindor KD. Nonalcoholic steatohepatitis. J Gastroenterol Hepatol 1997;12: [7] Sanyal AJ, Campbell-Sargent C, Mirshahi F, Rizzo WB, Contos MJ, Sterling RK, et al. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 2001;120: [8] Hirota K, Matsui M, Iwata S, Nishiyama A, Mori K, Yodoi J. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc Natl Acad Sci USA 1997;94: [9] Nakamura H, Nakamura K, Yodoi J. Redox regulation of cellular activation. Annu Rev Immunol 1997;15: [10] Holmgren A. Thioredoxin. Annu Rev Biochem 1985;54: [11] Nakamura H, De Rosa SC, Yodoi J, Holmgren A, Ghezzi P, Herzenberg LA, et al. Chronic elevation of plasma thioredoxin: inhibition of chemotaxis and curtailment of life expectancy in AIDS. Proc Natl Acad Sci USA 2001;27: [12] Yoshida S, Katoh T, Tetsuka T, Uno K, Matsui N, Okamoto T. Involvement of thioredoxin in rheumatoid arthritis: its costimulatory roles in the TNF-a-induced production of IL-6 and IL-8 from cultured synovial fibroblasts. J Immunol 1999;163: [13] Nakamura H, Bai J, Nishinaka Y, Ueda S, Sasada T, Ohshio G, et al. Expression of thioredoxin and glutaredoxin, redox-regulating proteins, in pancreatic cancer. Cancer Detect Prevent 2000;24: [14] Sumida Y, Nakashima T, Yoh T, Nakajima Y, Ishikawa H, Mitsuyoshi H, et al. Serum thioredoxin levels as an indicator of oxidative stress in patients with hepatitis C virus infection. J Hepatol 2000;33: [15] Sumida Y, Nakashima T, Yoh T, Kakisaka Y, Nakajima Y, Ishikawa H, et al. Serum thioredoxin elucidates the significance of serum ferritin as a marker of oxidative stress in chronic liver diseases. Liver 2001;21: [16] Nakashima T, Sumida Y, Yoh T, Kakisaka Y, Nakajima Y, Ishikawa H, et al. Thioredoxin levels in the sera of untreated hepatitis patients and those treated with glycyrrhizin or ursodeoxycholic acid. Antiox Redox Signal 2000;2: [17] Cotrim HP, Andrade ZA, Parana R, Portugal M, Lyra LG, Freitans LAR. Nonalcoholic steatohepatitis: a toxic liver disease in industrial workers. Liver 1999;19: [18] Japanese Society for the Study of Obesity. New criteria of obesity (in Japanese). J Jpn Soc Study Obes 2000;6: [19] American Diabetes Association. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 1997;20: [20] Kogaki H, Fujiwara Y, Yoshiki A, Kitajima S, Tanimoto T, Mitsui A, et al. Sensitive enzyme-linked immunosorbent assay for adult T-cell leukemia-derived factor and normal value measurement. J Clin Lab Anal 1996;10: [21] Di Bisceglie AM, Axiotis CA, Hoofnagle JH, Bacon BR. Measurement of iron status in patients with chronic hepatitis. Gastroenterology 1992;102: [22] Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Non-alcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94: [23] Zweig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem 1993;39: [24] Oliver FWJ, Christopher PD. Non-alcoholic steatohepatitis (NASH): a disease of emerging identity and importance. J Hepatol 1998;29: [25] Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver diseases: a spectrum of clinical and pathological severity. Gastroenterology 1999;116: [26] Day CP, James OFW. Steatohepatitis: a tale of two hits? Gastroenterology 1998;114: [27] Friedman S. The cellular basis of hepatic fibrosis. N Engl J Med 1993;328: [28] Lee KS, Buck M, Houglum K, Chojkier M. Activation of hepatic stellate cells by TGFa and collagen type I is mediated by oxidative stress through c-myb expression. J Clin Invest 1995;96: [29] Weltman MD, Farrell GC, Hall P, Ingelman-Sundberg M, Liddle C. Hepatic cytochrome P450 2E1 is increased in patients with non-alcoholic steatohepatitis. Hepatology 1998;27: [30] James O, Day C. Non-alcoholic steatohepatitis: another disease of affluence. Lancet 1999;353: [31] Younossi ZM, Gramlich T, Bacon BR, Matteoni CA, Boparai N, O Neill R, et al. Hepatic iron and nonalcoholic fatty liver disease. Hepatology 1999;30:

7 38 Y. Sumida et al. / Journal of Hepatology 38 (2003) [32] Berson A, De Beco V, Letterson P, Robin MA, Moreau C, El Kahwaji J, et al. Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes. Gastroenterology 1998;114: [33] Bonkovsky HL, Jawaid Q, Tortorelli K, LeClair P, Cobb J, Lambrecht RW, et al. Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis. J Hepatol 1999;31: [34] George DK, Goldwurm S, Macdonald GA, Cowley LL, Walker NI, Ward PJ, et al. Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis. Gastroenterology 1998;114: [35] Sohda T, Yanai J, Soejima H, Tamura K. Frequencies in the Japanese population of HFE gene mutations. Biochem Genet 1999;37: [36] Mendler MH, Turlin B, Moirand R, Jouanolle AM, Sapey T, Guyader D, et al. Insulin resistance-associated hepatic iron overload. Gastroenterology 1999;117: [37] Davis RJ, Corvera S, Czech MP. Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane. J Biol Chem 1986;261: [38] Pietrangelo A. Metals, oxidative stress and hepatic fibrogenesis. Semin Liver Dis 1996;16: [39] Washington K, Wright K, Shyr Y, Hunter EB, Olson S, Raiford DS. Hepatic stellate cell activation in nonalcoholic steatohepatitis and fatty liver. Hum Pathol 2000;31: [40] Angulo P, Keach JC, Batts KP, Lindor KD. Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology 1999;30: [41] Hirota K, Murata M, Sachi Y, Nakamura H, Takeuchi J, Mori K, et al. Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step mechanism of redox regulation of transcription factor NFkB. J Biol Chem 1999;274: [42] Nakashima T, Sumida Y, Yoh T, Kakisaka Y, Mitsuyoshi H, Okanoue T, et al. Serum thioredoxin levels as an indicator of oxidative stress in patients with nonalcoholic steatohepatitis. Gastroenterology 2001;120(Suppl 1):A107.