Ferritin levels in chronic hepatitis C virus infection with and without Diabetes.

Mahmoud M.A Yossof1; Ehab Abd El-Khalek1; Mohammad Abd El-Halim El-Helaly1; Sabry El-Azhary2; Farag El-Shahat3; Soma S. Abd El-Gawad4 1Internal medicine, 3Biochemistry, 4Clinical Pathology departments, Mansoura University. 2Internal medicine department, El-Azhar University - Dumiatta Faculty of Medicine.

Abstract Background: Increasing evidence exists suggesting an association between hepatitis C Virus (HCV) infection and diabetes. Both diabetes and HCV infection are associated with high serum ferritin levels. AIM: To evaluate serum ferritin levels and iron levels in HCV-chronic hepatitis patients with or without diabetes and to study the relationship between them and insulin resistance (HOMA-IR), B-cell function (HOMA-B), some markers of oxidative stress and degree of hepatic affection. Patients and methods: A total of 70 individuals divided into 3 groups: 25 nondiabetic patients with chronic HCV infection, 25 diabetic patients with chronic HCV infection, and 20 subjects as a control group. All were subjected to through clinical evaluation, abdominal ultrasonography, line function tests, serum ferritin, serum iron, assessment of insulin resistance (HOMA-IR) and B-cell function (HOMA-B), some markers of oxidative stress [total thiol & malondialdehyde (MDA)] and liver biopsy with grading of chronic hepatitis activity using modified knodell score. Results: High ferritin, total thiol, MDA, HOMA-IR and HOMA-B, were detected in both patients groups but more in presence of diabetes. The serum iron increase as the degree of hepatic inflammation increases but statistically insignificant there was a positive correlation between ferritin and other mentioned markers. Conclusion: High ferritin in chronic HCV patients may be related to the inflammatory process. In diabetic patients, high ferritin may be a cause of a result of hyper insulinemia. This raise the idea of expected benefits from blood letting and iron chelation in these patients. Introduction The prevalence of type 2 diabetes mellitus in liver diseases associated with chronic hepatitis C virus (HCV) infection is higher than in other liver diseases, including chronic hepatitis B(1-3). Although an association between liver cirrhosis (LC) and diabetes mellitus has been clearly recognized for at least 30 years (4), some studies have revealed that the higher prevalence of diabetes mellitus in HCV-infected patients is not exclusively related to cirrhosis(1,5). On the other hand, Insulin resistance and bcell dysfunction are generally recognized as risk factors for the development of impaired glucose tolerance and overt diabetes mellitus(6), but how insulin resistance and ?-cell function contribute to the pathogenesis of impaired glucose tolerance in patients with HCV is not well understood. Iron deposition in the liver is known to be a comm-on histological feature of HCV-related liver disease(7,8) , therefore glucose metabolism may be related to hepatic iron overload(9). It is increasingly recognized

that iron influences glucose metabolism, even in the absence of significant iron overload. In the general population, body iron stores are positively associated with the development of glucose intolerance, type 2 diabetes(10), and gestational diabetes (11). Hyperferritininemia is present in 6.6% of unselected patients with type 2 diabetes(12). Serum concentrations of ferritin are usually increased in poorly controlled type 1 and type 2 diabetic subject, and ferritin has been shown to predict HbA1c independently of glucose, probably reflecting increased oxidat-ive stress. Short-time improvement in glyce-mic control is followed by variable decreases in serum ferritin concentration(13). This study was conducted to investigate whether the high prevalence of diabetes observed in patients in-fected by the hepatitis C virus (HCV) is related to iron stores. Also, the present work was conducted to clarify the contribution of insulin resistance estimated by HOMA-IR and b-cell function estimated by HOMA-b to the HCV-infected liver and to investigate the relation-ship between these markers and iron overload estimated on the basis of serum ferritin levels in patients with chronic HCV infection wheth-er diabetic or non diabetic. Patients and methods The present work included: 1) 25 non diabetic patients with chronic HCV infection (18 males and 7 females), aged 47.36 5 years. 2) 25 diabetic patients with chronic HCV infection (19 males and 6 females), aged 46.12 5.47 years. 3) 20 subjects as a control group (14 males and 6 females), aged 47.6 5.09 years. All patients were positive for serum HCV antibody and HCVRNA. Patients were classified according to the criteria of the American Diabetes Association (1997)(14) as diabetic if an Fasting Plasma Glucose FPG of 126 mg/dl was documented and as non-diabetic if their FPG was < 126 mg/dl. Diabetic patients was on controlled by diet regimen. Diagnosis and classification of the patients was based upon: 1. Full history taking including personal hist-ory, symptoms attributable to hepatitis or schi-stosomiasis and past history of jaundice, anti-bilharzial treatment, transfusion of blood or its components or any dental manipulation. 2. Complete clinical assessment stressing on the characters of the liver and spleen and the presence or absence of ascites. 3.Complete blood picture. 4.Liver function tests (biliru-bin, ALT, AST, serum proteins, albumin and prothrombin time and concentration). 5.Abdominal ultrasonography. 6.Hepatitis markers (HCV antibody HCV- RNA by PRC and HBS Ag). 7.Serum ferritin. 8. Serum iron. 9.Fasting plasma glucose, immunoreactive insulin. 10.Blood lipids, total thiol and malon-dialdehyde (MDA). 11.Grading of chronic hepatitis activity using the most widely used scoring system, the histological activity index (HAI) which is also known as modified Knodell score(15). Patients with decompensat-ed LC who exhibited ascites, jaundice, or hepatic encephalopathy and patients with hepatocellular carcinoma were excluded from the present study. Patients with major cardiac and pulmonary disease, uraemia, inflammat-ory conditions (blood leucocytes count above 8000/mm3), hyperthyroidism, anemia, hem-olysis, recent blood transfusion, recent iron therapy were also excluded.

Methods Samples: Fasting blood samples were colle-cted from each subject incubated at 37. After centrifuge at 3000 rpm for 15, serum was separated from each sample and derided into aliquots. All samples were kept at 80 C o until time of assay. Biochemical analysis 1. Lipid profile: a. Triacyl of lycerol according to Wahlefeld, 1974(17) (High density lipopro-tein cholesterol). b.Total cholesterol accord-ing to Allain et al, 1974(16). c. Increase HDL-C according to Bachrik and Alerbs, 1986(18) (Low density lipoprotein cholesterol). d. Increase HDL-C according to Bachrik and Alerbs, 1986(18). 2.Serum total thial estimated by sepectro photometric assay according to Hu, 1994(19). 3. Lipid peroxidation product (MDA) was measured in serum using thio barbituric acid (TBA) assay according to Dyaper and Hadley, 1990(20). 4. Serum ferritin levels were estimated using ELISA kit, according to Bentely and William, 1974(21). 5. Serum insulin was estimated using ELISA kit according to Bentely and William, 1974(21). 6. Serum insulin level was assayed by specific enzyme linked immune sorbet assay (ELISA) using kits obtained from Biosource - Europe S.A (8 rue DEL, Industries, D1900, Nivelles. Belgium) and they were used according to manufacture instructions. HOMA-IR and b-cell function (i.e. insulin secretion) (HOMA-B) were calculated as FPG (mg/dl) IRI U/ml) 405 and (IRI (U/ml) 360) (FPG (mg/dl) 63 [3]), respectively(22). Statistics Results are reported as median values and ranges for quantitative data or as numbers with percentages in parentheses for qualitative data. Differences in quantitative data between groups were tested for statistical significance by the Mann-Whitney U-test and the Kruskal-Wallis non-parametric analysis of variance (ANOVA) when applicable. When the results of the Kruskal-Wallis analysis were signifi-cant, the significance between-group differ-ence was determined using Scheffe's method. Correlation coefficients were calculated by Spearman rank correlation analysis. Differ-ences were considered to be statistically signi-ficant at all P-Values less than 0.05.

Discussion It is well known that cirrhosis of the liver and increasing fibrosis in the pancreas, caused by excessive accumulation of body iron, can lead to the development of type 2 diabetes mellitus. However, although it has been assumed that iron accumulation has to be severe to cause organ damage sufficient to lead to diabetes, recent studies have suggested that a lesser accumulation of iron, as expressed by serum ferritin levels can alter carbohydrate homeostasis of the body(10). Also, iron over-load is associated with liver injury, although the mechanism also remains to be elucida-ted(23). However, the major hepatic toxicities of iron overload include damage to multiple cell types (hepatocytes, Kupffer cells, hepatic cells) and to multiple subcellular organelles (mitochondria, lysosomes, and smooth endoplasmic reticulum). Heavy iron overload, as occurs in primary (hereditary) or secondary forms of hemochromatosis may cause cirrho-sis, liver failure, and hepatocellular carci-noma. In addition, iron has been shown to be a contributory factor in the development or progression of alcoholic liver disease, non-alcoholic liver steatohepatitis, chronic viral hepatitis, prophyria cutanea tarda and perhaps, in alpha 1-antitrypsin deficiency and end stage liver disease regardless of cause(24). In the present work, ferritin level was found to be significantly higher in hepatic group than control group, in diabetic hepatic patients than control group, and in diabetic hepatic than hepatic group (table 1). Ferritin is a high molecular weight iron storage protein occur-ring mainly in the cells of the liver and reticuloendothelial system(25). Although serum ferritin levels could increase to a degree disproportionate to that of iron stores in some forms of inflammation, liver disease, and increased red-cell turnover, it closely reflects the size of the total iron burden(26) and ferritin iron(27). DiBiseegle et al(28), found that 46% of patients with chronic hepatitis had elevated serum iron, ferritin or transferrin saturation. The increased serum ferritin may be attributed to a chronic inflammatory process perse, as a part of an acute phase reaction unrelated to hepatocellular necrosis(29). Also, other studies(30,31) reported a significantly higher serum iron and transferrin saturation in HCV patients than others. They suggested that this blunts the action of interferon due to the suppression of the immune system. In another study, it was found that iron enhances HCV replication in hepatocyte, suggesting that iron deposition in

hepatocytes could facilitate HCV infection the liver(32). In diabetes, glycation of transferrin decreases its ability to bind ferrous from, and by increasing the pool of free iron, stimulates ferritin synthesis. The serum iron in this study was found to be increased as the grade of histopathological score increased in both groups but this did not reach statistical significance (tables 6, 7). This may reflect somewhat sever liver inflamm-ation in HCV patients. The reasons for increased iron status in chronic hepatitis C may be attributed to a variety of factors. First, hepatocellular damage may cause release of iron in hepatocytes(33), as supported by the finding of positive correlation between ferritin level and transaminase level and by increase level of serum iron as degree of hepatic infla-mmation increases. Second, chronic liver disease may often be associated with an increased iron absorption in the gastrointe-stinal tract with marginally elevated hepatic iron(34). As regards, iron and diabetes mellitus there are five scientific evidences favor the hypothesis that iron plays a role in type 2 diabetes. First, increased prevalence of hemo-chromatosis was found among unselected patients with type 2 diabetes(35). Second, frequent blood donations, leading to decreas-ing iron stores, have been demonstrated to constitute a protective factor for the develop-ment of diabetes(36,37,38). Third, a recent random-ized study also suggests that iron stores may influence insulin action in type 2 diabetes(39). In this report, a statistically significant incr-ease in insulin sensitivity was observed in blood letting group with decrease of blood HBA1c Fourth, a novel syndrome of hepatic iron overload has been described. This condition is know as insulin resistanceasso-ciated hepatic iron overload (IR-HIO) and combines abnormality in iron metabolism (isolated hyperferritinemia with normal transferrin saturation), steatohepatitis, and the insulin resistance syndrome (obesity, hyperli-pidemia, abnormal glucose metabolism, and hypertension)(40). Fifth, insulin resistance features are frequently seen in patients chronically infected with hepatitis C virus(41). In these subjects, body-mass index (BMI), elderliness, iron stores, and family history of diabetes and advanced liver fibrosis were found to predict the development of diabetes (42,43). This is the evidence but what about interacting pathways linking glucose and iron metabolism. Insulin is known to cause a rapid and marked stimulation of iron uptake by fat cells, redistributing transferrin receptors from an intracellular membrane compartment to the cell surface(44). Insulin is also responsible for the increased ferritin synthesis in cultured rat glioma cells(45). Importantly, transferrin receptors have been shown to colocalize with insulinresponsive glucose transporters and insulin-like growth factor II receptors in the microsomal membranes of cultured adipo-cytes, suggesting that regulation of iron uptake by insulin occurs in parallel with its effects on glucose transport(46). Reciprocally, iron influences insulin action. Iron interferes with insulin inhibition of glucose production by the liver. Hepatic extraction and metabo-lism of insulin is reduced with increasing iron stores, leading to peripheral hyperinsul-inemia(47). In fact, the initial and most common abnormality seen in iron overload conditions is liver insulin resistance(48). There is some evidence that iron overload also affects skeletal muscle(49), the main effector of insulin action. Also, increased accumulation of iron affects insulin synthesis and secretion in the pancreas(50). Oxidative stress influences both glucose and iron metabolism. In the present work, MDA and thiol level was found to be significantly higher in hepatic group than control group, in diabetic hepatic patients than control group, and in diabetic hepatic than hepatic group (table 1). Oxidative stress induces both insulin resistance [by decreasing internalization of insulin] and increased ferritin synthesis(51). Also, recent reports have suggested that oxidative stress may be involved in viral diseases, such as acquired immunodeficiency

syndrome and chronic hepatitis C (53,54). We measured the serum total thiol and malondialdehyde levels, which have recently been established as indicators of oxidative stress. In patients with HCV infection, and found that they increased in parallel with their serum ferritin levels. Also, a significant correlation between ferritin and MDA and total thiol were detected, (table 5). Since iron ions are well known to generate active oxygen radicals, such as hydroxyl radicals, via the Fenton reaction in the liver, iron-induced oxidative stress may be involved in HCV - related liver diseases(54,55). Regarding, Lipids in our study, there was non significant difference between the studied groups (table4). However, a positive correlation has been documented between ferritin and cholesterol and low density lipoprotein cholesterol (table 5). Iron chelation blocks oxidation of LDL, and iron released from heme and ferritin favour oxidation of this lipoprotein(56). Both the hyperinsulinemic euglycemic clamp method of assessing insulin resistance and the euinsulinemic hypergly-cemic clamp method of assessing b-cell function (insulin secretion) are time-consuming and difficult to use in routine clinical studies Laakso(57) has shown that fasting plasma insulin concentrations provide a good surrogate for insulin resistance. Matthews et al(22). have also proposed the homeostasis model assessment for insulin resistance (HOMA-IR) and the homeostasis model assessment for b-cell function (HOMA -b). These assessments are carried out by using the steady-state fasting plasma glucose (FPG) and insulin concentrations (immunor-eactive insulin, IRI). In the present work, we found that immuno reactive insulin resistance expressed as IRI & HOMA IR and b-cell function expressed as HOMA-B were signifi-cantly higher in hepatic patients than control group, in hepatic diabetic than control group, and in hepatic diabetic group than hepatic patients (table 3). Also there was positive correlation between IRI, HOMA-IR, HOMA-B and ferritin in the studied groups (table 5). These results suggest that hepatic iron or iron-induced oxidative stress is closely related to insulin resistance(48). Cross-talk between iron metabolism and insulin-glucose metabolism has recently been documented(9), for example, iron has been found to reduce hepatic extraction/metabolism of insulin and to interfere with insulin action on the liver, leading to peripheral hyperinsulinemia(58) and insulin resistance. The finding that the HOMA-b value increased in the hepatic group indicates the hypersecretion of insulin by islet b-cell probably as compensation to insulin resistance. These results raise the idea of benefits expected from iron depletion in some groups of patients. First diabetic metabolic control was found to be improved in 35-45% of patients with hemochromatosis after iron depletion(59). Also, iron chelators seem benef-icial in optimizing diabetic metabolic control(60), and treatment with either insulin or oral hypoglycemic agents could be disconti-nued in some patients(61). Second, iron depletion may have good effect on diabetic complication. Iron chelation by desferroxa-mine inhibits vascular smooth muscle cell proliferation(62). Long-term use of the modified iron chelator hydroxyethyl starch conjugated-desferroxamine prevented endothelial dysfun-ction associated with experimental diabetes(63). In type 2 diabetic patients, coronary artery responses to cold stress testing improved substantially after desferroxamine administra-tion(64). Similarly, iron chelation was shown to ameliorate the endothelial dysfunction of patients with coronary heart disease(65). Also, several studies have reported that iron chelators restored motor and sensory nerve conduction velocity in diabetics with neuro-pathy(66). Third, repeated phlebotomy had reported to normalize ALT level and cause disappearance of iron from liver tissue(67). fourth decreased hepatic iron content was found in patients with chronic hepatitis C who respond to interferon therapy(31). Thus, large-scale clinical trials, are needed to search for the usefulness

and cost effectiveness of thera-peutic measures that decrease iron toxicity and to evaluate simple and inexpensive therapies, such as blood letting and iron chela-tors, are emerging as alternative and effective treatments for insulin resistance and for improving response to interferon therapy. Several limitations applied to our study. First, a single measurement of serum ferritin may not be representative of the 'true' exposure to stored iron (if, for example, recent blood loss or recent use of aspirin has occurred). Thus, an optimal indictor of exposure would be serial measurements of the ferritin level over a given period of time. Second although measurement of insulin resistance by the HOMA index has been demonstrated to correlate with results obtained using the glucose clamp technique or minimal model analysis, its validity in different populations has never been tested. Third, we did not collect dietary records that would enable us to understand why relatively higher serum ferritin levels were observed in our study subjects. Fourth, in our study, it was not possible to measure hepatic iron content, however it was reported by DiBiseegle et al(28), that there was no significant correlation between hepatic iron and both serum iron and transerrin saturation in chronic hepatitis patients. References 1. Mason AL, Lau JY, Hoang N, et al: Association of diabetes mellitus and chronic hepatitis C virus infection. Hepatology 1999; 29: 328-33. 2. Caronia S, Taylor K, Pagliaro L, et al: Further evidence for an association between non-insulin-dependent diabetes mellitus and chronic hepatitis C virus infection. Hepatology 1999; 30: 1059-63. 3. Mehta SH, Brancati FL, Sulkowski MS, et al: Prevalence of type 2 diabetes mellitus amng persons with hepatitis C virus infection in the Unites States. Ann Intern Med 2000; 133: 592-9. 4. Muting D, Lackas N, Reikowski H, Rishmond S: Cirrhosis of the liver and diabetes mellitus. A study of 140 combined cases. Geriatr Med Mon 1996; 11: 38590. 5. Knobler H, Schihmanter R, Zifroni A, et al: Increased risk of type 2 diabetes in noncirrhotic patients with chronic hepatitis C virus infection. Mayo Clin Proc 2000; 75:355-9. 6. Martin BC, Warram JH, Krolewski AS, et al: Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study. Lancet 1992; 340: 925-9. 7. Lecube A, Hernandez C, Genesca J, et al: High prevalence of glucose abnormalities in patients with hepatitis C virus infection: a multivariate analusis considering the liver injury. Diabetes Care 2004; 27: 1171-1175. 8. Riggio O, Montagnese F, Fiore P, et al: Iron overload in patients with chronic viral hepatitis: how common is it? Am J Gastroenterol 1997; 92: 1298-301. 9. Fernandez-Real JM, Lopez-Bermero A, Ricart W: Cross-talk between iron metabolism and diabetes. Diabetes 2002; 51: 2348-54. 10. Salonen JT, Tuomainen T-P, Nyyssenen K, et al: Relation between iron stores and non-insulin-depen-dent diabetes in men: case-control study. Br Med J1999; 317: 727-730. 11. Lao TT CP, Tam KF: Gestational diabetes mellitus in the last trimester: a feature of maternal iron excess? Diabetes Med 2001; 18: 218-223. 12. Lao TT, Tam KF:Maternal serum ferritin and gestational impaired glucose tolerance. Diabetes Care 1997; 20: 1368-1369.

13. Bertelsen M, Nggard EE, Carrier MJ: Oxidative stress impairs insulin internalization in endothelial cells in vitro. Diabetologia 2001; 44: 605-613. 14. Report of the expert committee on the diagnosis and classification of diabetes mellitus: Diabetes care 1997; 20: 1183-97. 15. Knodell RG, Ishak KG, Black WC, et al: Formul-ation a, and application of numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. The French MERAVIR Cooperation Study Group. Hepatology 1994;20:15-20. 16. Allain CA, Poon LS, Chan CGS, et al: Enzymatic determination of total serum cholesterol. Clinical Chemistry 1974; 20: 470-475. 17. Wahlefeld AW: Triglyceride: determination after enzymatic hydrolysis. In Method of Enzymatic Alysis 1974; (ed H.U. Bergmyer) pp 1831-1835. Academic Press, New York, NY. 18. Bachrik PS, Alerbs JJ: Precipitation methods for quantification of lipoprotein. In method in Enzymology 1986; vol. 129 (eds J.J. Albers & J.P. Segrest), pp 78-100. Academic Press, London. 19. Hu ML: Measurement of thiol groups and glutath-ione in plasma. Methods Enzymol 1988; 233: 380-5. 20. Drapper H, Hadley M: Assessment of MDA Method. Enzymol; 1990: 186: 421431. 21. Bentley DP, Williams P: Serum ferritin concen-tration as an index of storage iron in rheumatoid arthritis. J Clin Pathol 1974; Oct, 27(10): 786-8. 22. Matthews DR, Hosker JP, Rudenski AS, et al: Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentration in man. Diabetologia 1985; 28: 412-29. 23. Bacon BR, Britton RS: The pathology of hepatic iron overload: a free radicalmediated process? Hepatol. 1990; 11: 127-137. 24. Bonkovsky HL and Lambrecht RW: Iron-induced liver injury. Clin Liver Dis 2000 May; 4(2): 409-29. 25. Lipschitz DA, Cook JD, Finch A: Clinical evalua-tion of serum ferritin as an index of iron stores. N Engl J Med 1974; 290: 1213-1216. 26. Prieto J, Barry M, Sherlock S: Serum ferritin in patients with iron overload and with acute and chronic liver disease. Gastroenterology 1975; 68: 525-533. 27. Nielsen P, Gunther U, Durken M, et al: Serum ferritin iron overload and liver damage: correlation to body iron stores and diagnostic relevance. J Lab Clin Med 2000; 135: 413-418. 28. DiBisceglie AM, Axiotis CA, Lioofiagle JH, et al: Measurement of iron status in patients with chronic hepatitis. Gastroenterology 1992; 102: 2108-2113. 29. Konijin AM, Hershko C: Ferritin synthsis in inflammation. Pathogenesis of impaired iron release. Br. J. Haematol 1977; 37: 7-16. 30. Rber N, Konikoff F, Moshkowitz M, et al: Increa-sed serum iron saturation without liver iron accum-ulation distinguish chronic hepatitis C from other chronic liver disease. Dig Dis Science 1994; 39: 2656-2659. 31. Rber N, Moshkowitz M, Konikoff F, et al: Elevated serum iron predicts poor response to interferon treatment in patients with chronic hepatitis C. Dig Dis Science 1995; 40: 2431-2422. 32. Kakizaki S, Takagi H, Horiguchi N, et al: Iron enhances hepatitis C virus replication in cultured human hepatocytes. Liver 2000 Apr; 20(2) 125-8. 33. Acon BR, Britton RS: The role of the liver in normal iron metabolism. Semin Liver Dis 1984; 4: 181-192.

34. Farinati F, Cardin R, De Maria N et al: HCV infec-tion correlates with increased serum ferritin, hepatic iron & lipoperoxidation. Gastroenterol. 1993; 104: 23A. 35. Conte D, Manachino D, Colli A, et al: Prevalence of genetic hemochromatosis in a cohort of Italian patients with diabetes mellitus.Ann intern Med 1998; 128: 370-373. 36. Ascherio A, Rimm EB, Giovannucci E, et al: Blood donations and risk of coronary heart disease in men. Circulation 2001; 103: 52-57. 37. Fleming DJ, Jacques PF, Tucker KL, et al: Iron status of the free-living, elderly Framingham Heart Study cohort: an iron-replete population with a high prevalence of elevated iron stores. Am J Clin Nutr 2001; 73: 638-646. 38. Yale JF, Grosee M, Seemayer TA, et al: Diabetes prevention in BB rats by frequent blood withdrawal started at a young age. Diabetes 1988; 37: 327-333. 39. Fernandez-Real JM, Castro A, Garcia-Bragado F, et al: Blood letting in highferritin type 2 diabetes: effects on insulin sensitivity and [beta]-cell function. Diabetes 2002; 51: 1000-1004. 40. Moirand R, Mortaji A, Loral O, et al: A new syndrome of liver iron overload with normal transf-erring saturation. Lancet 1997; 349: 95-97. 41. Mendler MH, Turlin B, Moirand R, et al: Insulin resistance-associated hepatic iron overload. Gastroenterology 1999; 117: 1155-1163. 42. Petit JM, Bour JB, Galland-Jos C, et al: Risk factors for diabetes mellitus and early insulin resistance in chronic hepatitis C. J Hepatol 2001; 35: 279-83. 43. Marchesini G, Brizi M, Bianchi G, et al: Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes 2001; 50: 1844-1850. 44. Davis RJ, Corvera S, Czech MP: Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferring receptors to the plasma membrane. J Biol Chem 1986; 261: 8708-8711. 45. Yokomori N, Iwasa Y, Aida K, et al: Transcriptional regulation of ferritin messenger ribonucleic acid level by insulin in cultured rat glioma cells. Endocrinology 1991; 128: 1474-1480. 46. Tanner LI, Lienhard GE: Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters. J Cell Biol 1989; 108: 1537-1545. 47. Niederau C, Berger M, Stremmel W, et al: Hyper-insulinemia in non-cirrhotic haemochromatosis: impaired hepatic insulin degreadation? Diabetologia 1984; 26: 441-444. 48. Dandona P, Hussain MAM, Varghese Z, et al: Insulin resistance and iron overload. Ann clin Biochem 1983; 20: 77-79. 49. Shafer AI, Cheron RG, Dluhy R, et al:Clinical consequences of acquired transfusional iron overload in adults. N Engl J Med 1981; 304: 319-324. 50. Tuomainen TP, Nyyssonen K, Salonen R, et al:Body iron stores are associated with serum insulin and blood glucose concentrations. Diabetes Care 1997; 20: 426428. 51. Bertelsen M, Nggard EE, Carrier MJ:Oxidative stress impairs insulin internalization in endothelial cells in vitro. Diabetologia 2001; 44: 605-613. 52. Nakamura H, De Rosa S, Roederer M, et al: Elevation of plasma thoiredoxin levels in HIV-infected individuals. Int Immunol 1996; 8: 603-11. 53. Farinati F, Cardin R, De Maria N, et al: Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis. J Hepatol 1995; 22: 4495. 54. Larrea E, Beloqui O, Munoz-Navas MA, et al: Sup-eroxide dismutase in patients with chronic hepatitis C virus infection. Free Rad Biol Med 1998; 24: 1235-41.

55. Sumida Y, Nakashima T, Yoh T, et al: Serum thioredoxin levels as an indicator of oxidative stress in patients with hepatitis C virus infection. J Hepatol 2000; 33: 61622. 56. Abdalla DSP, Campa A, Monteiro MP: Low density lipoprotein oxidation by stimulated neutrophils and ferritin. Atherosclerosis 1992; 97: 149-159. 57. Laakso M: How good a marker is insulin level for insulin resistance. Am J Epidemiol 1993; 137: 959-65. 58. Niederau C, Berger M, Stremmel W, et al: Hyperin-sulinemia in non-cirrhotic haemochromatosis: impa-ired hepatic insulin degradation. Diabetologia 1984; 26: 441-4. 59. Davis WD, Arrowsmith WR: The treatment of heamochromatosis by massive venesection. Ann Intern Med 1953; 39: 723-734. 60. Cutler P: Deferoxamine therapy in high-ferritin diabetes. Diabetes 1989; 38: 1207-1210. 61. Redmon JB, Pyzdrowski KL, Robertson RP: No effect of deferoximine therapy on glucose homeostasis and insulin secretion in individuals with NIDDM and elevated serum ferritin. Diabetes 1993; 42: 544-549. 62. Porreca E, Ucchino S, Di Febbo C, et al: Antipro-liferative effect of desferrioxamine on vascular smooth muscle cells in vitro and in vivo. Arterioscler Thromb Vasc Biol 1994; 14: 299-304. 63. Pieper GM, Siebeneich W: Diabetes-induced endothelial dysfunction is prevented by long-term treat-ment with the modified iron chelator, hydroxyethyl starch conjugated-deferoxamine. J Cardiovasc Pharmacol 1997; 30: 734-738. 64. Nitenberg A, Ledoux S, Valensi P, et al: Coronary microvascular adaptation to myocardial metabolic demand can be restored by inhibition of iron-catalyzed formation of oxygen free radicals in type 2 diabetic patients. Diabetes 2002; 51: 813818. 65. Duffy SJ, Biegelsen ES, Holbrook M, et al: Iron chelation improves endothelial function in patients with coronary artery disease. Circulation 2001; 103: 2799-2804. 66. Cameron NE, Cotter MA: Effects of an extracellular metal chelator on neurovascular function in diabetic rats. Diabetologia 2001; 44: 621-628. 67. Hayashi H, Takikawa T, Nishimura N, et al: Impro-vement of serum aminotransferase levels after phlebo-tomy in patients with chronic hepatitis C and excess hepatic iron. Am J Gastroenterol 1994; 89: 986-988.