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Iron and Ribavirin Increased Hepatic Iron Deposition Resulting From Treatment of Chronic Hepatitis C With  Ribavirin

M. Isabel Fiel, MD,1 Thomas D. Schiano, MD,2 Maria Guido, MD,1 Swan N. Thung, MD,1 Karen L. Lindsay, MD,3 Gary L. Davis, MD, 4 James H. Lewis, MD,5 Leonard B. Seeff, MD,6 and Henry C. Bodenheimer, Jr, MD2


Key Words: Hemosiderosis; Viral hepatitis; Hepatitis C virus, Interferon
Abstract

Increased levels of hepatic iron may impair the response of patients with chronic hepatitis C to treatment with interferon-alfa, but combination therapy with ribavirin has demonstrated efficacy in the treatment of hepatitis C. When used alone or with interferon-alfa, ribavirin may cause a dose-dependent reversible hemolytic anemia. We compared the extent and cellular localization of iron deposition in liver tissue from biopsy specimens obtained before and after 36 weeks of therapy with ribavirin or placebo for 59 patients with chronic hepatitis C. Paired slides were available for review from 26 ribavirin and 27 placebo recipients. Iron deposition was assessed using coded slides stained with Perls Prussian blue and was semiquantitated in hepatocytes, Kupffer cells, and areas of fibrosis. The overall iron score fell by 0.96 in the placebo group and increased 1.69 in the ribavirin recipients. Iron was deposited mainly in hepatocytes; the hepatocyte iron score increased from 2.19 to 3.81 in the ribavirin group. The amount of iron staining in Kupffer cells declined in the placebo group and increased slightly in the ribavirin group. Iron changes in areas of fibrosis were minor and did not differ between groups. Increased total hepatic iron deposition occurred during a 9-month course of ribavirin. Ribavirin-associated hemolysis deposits iron preferentially in hepatocytes. This increased deposition of hepatic iron does not seem to affect the biochemical or histologic response to ribavirin therapy but may have implications for hepatocyte susceptibility to future injury.


Chronic hepatitis C virus (HCV) infection is a major causative factor responsible for the development of cirrhosis. Significant iron deposition occurs in cirrhosis secondary to any cause, but especially in HCV and alcohol-related chronic liver disease.1 Increased iron deposition, possibly related to the release of iron from injured hepatocytes, also is observed in patients with HCV who do not have cirrhosis.2 High levels of hepatic iron may negatively influence the response to interferon-alfa therapy. 3-5 Lower hepatic iron concentration favors a beneficial response to treatment. 3 Phlebotomy decreases serum aminotransferase activities in patients with HCV and may improve the response to interferon-alfa in some patients.6 Ribavirin, an oral purine nucleoside analog, has a broad spectrum of antiviral activity.7,8 This drug, however, does not lower hepatitis C viral titers.8-12 When used in combination with interferon-alfa in the treatment of HCV, the rates of normalization of aminotransferase values, viral eradication, and histologic improvement are higher than when interferon-alfa is used alone.13-16 One major adverse effect of ribavirin is a dose-dependent reversible hemolysis.8,9,15,17-20 Chronic intravascular hemolysis typically leads to increased reticuloendothelial cell iron deposition with little increase in hepatocyte iron. However, ribavirin-induced hemolysis is postulated to occur extravascularly within the reticuloendothelial system.21,22 Hepatic iron accumulation may predispose liver cells to injury from oxidative stress. In addition, a beneficial response to interferon-alfa may be blunted.15 We evaluated stainable hepatic iron before and after ribavirin therapy of HCV. The changes noted were correlated with the clinical response to ribavirin as measured by alanine aminotransferase (ALT) activity. Materials and Methods

This project was part of a multicenter, double-blind, randomized, controlled trial comparing ribavirin and placebo therapy in 59 patients with HCV.12 Twenty-nine patients received ribavirin at a dose of 1,200 mg/d, and 30 patients received placebo for a duration of 36 weeks. Liver biopsies were performed within 6 months before enrollment and at the end of therapy. Patients were not taking iron supplements and had no history of hemochromatosis or porphyria cutanea tarda. No patient had a history of chronic or hemolytic anemia, and none were using alcohol. Fifty-three paired slides were available and coded, 26 from the ribavirin group and 27 from the placebo group. Perls Prussian blue staining for trivalent iron was applied on 5-µm-thick sections of the liver biopsy specimens. Two pathologists (M.I.F. and M.G.), blinded to the identity of the treatment group and to whether biopsy specimens were obtained before or after treatment, reviewed the slides. Quantitative and qualitative scores for hepatic iron deposition were determined by consensus. Stainable iron was assessed by using the semiquantitative grading system proposed by Tirmann-Schmelzer with modifications.23 Brissot and coworkers23 demonstrated this method to be reflective of hepatic iron concentration because it takes into account the different iron storage areas of the hepatic lobule. Briefly, hepatocyte iron, Kupffer cell iron, and areas of fibrosis were assessed and given scores ranging from 0 to 4 arriving at an elementary score A. The hepatocyte score is multiplied by a coefficient of 3, and all 3 scores are then cumulated (score B). The scores are based on the number and percentage of cells containing iron and the areas of iron deposits. The histologic iron estimation included determination of the intensity of iron load according to the following scores: 1, sparse, fine granules; 2, patchy, fine granules; 3, diffuse, fine granules; and 4, diffuse, coarse granules. Total iron scores (score B), hepatocyte iron scores, and Kupffer cell iron scores before and after treatment were compared within each group and then between the 2 treatment groups by using Fisher exact test for statistical analysis. The ALT response at the end of therapy was correlated with change in iron scores. The definition of a positive therapeutic response was normalization of ALT activity at the end of therapy. Other parameters evaluated included mean hemoglobin and reticulocyte count before and after treatment.Results are expressed as mean ± SD with range (minimum and maximum).Results

Hemoglobin levels decreased by a mean of 13% (range, 0-4 g/dL [0-40 g/L]) in the ribavirin treated group. A decline in the hemoglobin level of more than 2 g/dL (20 g/L) occurred in 79% of patients receiving ribavirin; a decline of 4 g/dL (40 g/L) or more was observed in 21% of patients. No appreciable decrease in the hemoglobin level was noted in the placebo group. The reticulocyte count increased by a mean of 6.9% at the end of treatment in the ribavirin-treated patients.The semiquantitative evaluation of iron deposition in stained slides is shown in [ Table 1 ] . The overall iron score at week 0 in the ribavirin-treated group was 3.04 ± 3.75 (range, 0-12), and the overall iron score at week 36 was 4.73 ± 4.03 (range, 0-12). The difference in total iron scores increased from week 0 to week 36 by 1.69. In the placebo group, the overall iron score at week 0 was 3.74 and at week 36 was 2.77, demonstrating a slight decline of 0.97 by the conclusion of the study.The hepatocyte iron score in the ribavirin-treated group at the time of enrollment was 2.19 ± 3.75 (range, 0-2), and at week 36, the score was 3.81 ± 4.03 (range, 0-12) (Table 1). The difference between week 0 and week 36 was 1.62. In the placebo group, the hepatocyte iron score at week 0 was 2.56 ± 4.07 (range, 0-12), and at week 36, the hepatocyte iron score was 2.0 ± 3.43 (range, 0-11). The score of Kupffer cell iron deposition in the ribavirin treated group at baseline was 0.81 ± 1.06 (range, 0-3), and at week 36, it was 0.92 ± 1.06 (range, 0-3). In the placebo group, the Kupffer cell iron score at week 0 was 0.89 ± 0.97 (range, 0-3), and at week 36, the score was 0.52 ± 0.70 (range, 0-2) (Table 1). [ Image 1 ] and [ Image 2 ] demonstrate the change in iron deposition before and after treatment. Normalization of ALT at week 36 did not correlate with the total amount of iron deposition nor change in iron scores [ Table 2 ] and [ Table 3 ].Discussion

HCV infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. At present, interferon-alfa is the only therapy approved for treatment of HCV infection. 24 However, approximately 50% of those treated have no response, and of the patients who show response, 50% to 80% will experience relapse within 6 months of discontinuation of therapy.25 Combined treatment with interferon-alfa and ribavirin has shown promise for normalizing serum aminotransferase values and decreasing the degree of histologic damage in patients with HCV.13,14,26,27Distribution of iron in the liver and the amount of iron deposition may have an influence on the patient's response to interferon-alfa therapy.3-5 In a study by Barton et al,4 total hepatic iron scores were higher in patients with incomplete response. In addition, Olynyk et al3 reported that hepatic iron concentration has an influence on the response to therapy for chronic hepatitis C with interferon-alfa. Ribavirin has been used for many years as a treatment for respiratory syncytial virus infection.28 Its predominant adverse effect is dose-dependent hemolysis and resulting anemia.16-20 Declines in hematocrit may range from 10% to 13% (0.10-0.13) with a compensatory increase in reticulocyte counts8,9 and an increase in total serum bilirubin. 10 The mechanism of this hemolysis is thought to relate to the RBC accumulation of ribavirin triphosphate.21,22 The increased hepatic iron deposition likely results from uptake of iron released from destroyed RBCs. Di Bisceglie and coworkers2 also have postulated that ribavirin may cause increased iron absorption from the gastrointestinal tract. In their study of ribavirin effects, iron staining was noted to increase in hepatocytes and Kupffer cells after ribavirin treatment, but there was no change in the relative cellular distribution of iron. Iron released intravascularly due to hemolysis likely would be taken up mainly by Kupffer cells. In the present study and work by others,2 iron staining also was seen within hepatocytes. The degree of increased iron deposition seems greater within hepatocytes than other liver cells. Ferritin that accumulates in Kupffer cells as a consequence of RBC uptake and destruction subsequently may be transferred to adjacent hepatocytes. An elegant study done by Sibille et al29 showed iron, in the form of ferritin, being released by the phagocytosing Kupffer cells and rapidly taken up by hepatocytes. We believe this may explain why, in the present study, more abundant iron deposition was found in hepatocytes. Piperno and colleagues30 postulated that iron overload may contribute to persistent HCV infection by supplying the virus with essential nutrients and by producing immune alteration that impairs recovery. This may become important over time in ribavirin-treated patients if hepatic iron deposition continues to increase.2 Hepatic iron stores increased significantly in patients treated with ribavirin compared with those treated with placebo. This increase was demonstrated by using an evaluation of histologic hepatic iron that correlates with the hepatic iron concentration.2,23 Change in ALT levels during ribavirin therapy was not related to iron deposition before treatment or to liver iron levels after therapy. The long-term effects of increased hepatic iron stores secondary to ribavirin treatment remain to be evaluated. As longer courses of treatment and long-term maintenance therapy with ribavirin alone or in combination with interferon-alfa are contemplated, excessive amounts of iron in the liver could limit the usefulness of ribavirin. Accumulated iron deposition within the liver might reach excessive levels during prolonged treatment, especially in patients with high preexisting concentrations of hepatic iron.
 
From the 1 The Lillian and Henry M. Stratton-Hans Popper Department of Pathology and the 2Division of Liver Diseases, Department of Medicine, The Mount Sinai Medical Center of the City University of New York, New York, NY; the 3Division of Gastrointestinal and Liver Diseases, Department of Medicine, University of Southern California, Los Angeles, CA; the 4Section of Hepatobiliary Diseases, Department of Medicine, University of Florida, Gainesville; the 5Division of Liver Diseases, Division of Gastroenterology, Department of Medicine, Georgetown University Medical Center, Washington, DC; and the 6National Institutes of Health, Bethesda, MD.Presented at The American Association for the Study of Liver Diseases Annual Meeting, Chicago, IL, November 1995. Published in abstract form in Hepatology. 1995;22:291A. Address reprint requests to Dr Bodenheimer: Division of Liver Diseases, Dept of Medicine, The Mount Sinai Medical Center, One Gustave Levy Place, New York, NY 10029.
References
 
Bacon BR. Diagnosis and management of hemochromatosis. Gastroenterology. 1997;13:995-999.
Di Bisceglie AM, Bacon BR, Kleiner DE, et al. Increase in hepatic iron stores following prolonged therapy with ribavirin in patients with chronic hepatitis C. J Hepatol. 1994;21:1109-1112.
Olynyk JK, Reddy KR, Di Bisceglie AM, et al. Hepatic iron concentration as a predictor of response to interferon alfa therapy in chronic hepatitis C. Gastroenterology. 1995;108:1104-1109.
Barton AL, Banner BF, Cable EE, et al. Distribution of iron in the liver predicts the response of chronic hepatitis C infection to interferon therapy. Am J Clin Pathol. 1995;103:419-424.
Clemente MG, Congia M, Lai ME, et al. Effect of iron overload on the response to recombinant interferon-alfa treatment in transfusion-dependent patients with thalassemia major and chronic hepatitis C. J Pediatr. 1994;125:123-128.
Hayashi H, Takikawa T, Nishimura N, et al. Improvement of serum aminotransferase levels after phlebotomy in patients with chonic active hepatitis C and excess hepatic iron. Am J Gastroenterol. 1994;89:986-988.
Patterson JL, Fernandez-Larsson R. Molecular mechanisms of action of ribavirin. Rev Infect Dis. 1990;12:1139-1146.
Fried MW, Fong T-L, Swain MG, et al. Therapy of chronic hepatitis B with a 6-month course of ribavirin. J Hepatol. 1994;21:145-150.
Di Bisceglie AM, Shindo M, Fong T-L, et al. A pilot study of ribavirin therapy for chronic hepatitis C. Hepatology. 1992;16:649-654.
Di Bisceglie AM, Conjeevaran HS, Fried MW, et al. Ribavirin as therapy for chronic hepatitis C. Ann Intern Med. 1995;123:897-903.
Dusheiko G, Main J, Thomas H, et al. Ribavirin treatment for patients with chronic hepatitis C: results of a placebo-controlled study. J Hepatol. 1996;25:591-598.
Bodenheimer HC, Lindsay KL, Davis GL, et al. Tolerance and efficacy of oral ribavirin treatment of chronic hepatitis C: a multicenter trial. Hepatology. 1997;26:473-477.
Brillanti S, Garson J, Mauro F, et al. A pilot study of combination therapy with ribavirin plus interferon-alpha for interferon-alpha resistant chronic hepatitis C. Gastroenterology. 1994;107:812-817.
Schvarcz R, Yun ZB, Sonnenberg A, et al. Combined treatment with interferon alpha 2b and ribavirin for chronic hepatitis C in patients with a previous non-response or non-sustained response to interferon alone. J Med Virol. 1995;46:43-47.
Bizollon T, Palazzo U, Ducerf C, et al. Pilot study of the combination of interferon alfa and ribavirin therapy of recurrent hepatitis C after liver transplantation. Hepatology. 1997;26:500-504.
Reichard O, Schvarcz R, Weiland O. Therapy of hepatitis C: alpha-interferon and ribavirin. Hepatology. 1997;26(suppl):108S-111S.
Camps J, Garcia N, Riezu-Boz JI, et al. Ribavirin in the treatment of chronic hepatitis C unresponsive to alfa interferon. J Hepatol. 1993;19:408-412.
Kakumu S, Yoshioka K, Wakita T, et al. A pilot study of ribavirin and interferon beta for the treatment of chronic hepatitis C. Gastroenterology. 1993;105:507-512.
Reichard O, Andersson J, Schvarcz R, et al. Ribavirin treatment for chronic hepatitis C. Lancet. 1991;337:1058-1061.
Schulman NR. Assessment of hematologic effects of ribavirin in humans. In: Smith RA, Knight V, Smith JA, eds. Clinical Applications of Ribavirin. Orlando, FL: Academic Press; 1984:79-92.
Canonico PG, Kende M, Huggins JW. The toxicology and pharmacology of ribavirin in experimental animals. In: Smith RA, Knight V, Smith JA, eds. Clinical Applications of Ribavirin. Orlando, FL: Academic Press; 1984:65-77.
Canonico PG, Kastello MD, Cosgriff TM, et al. Hematological and bone marrow effects of ribavirin in Rhesus monkeys. Toxicol Appl Pharmacol. 1984;74:163-172.
Brissot P, Bourel M, Herry D, et al. Assessment of liver iron content in 271 patients: a reevaluation of direct and indirect methods. Gastroenterology. 1981;80:557-565.
Davis GL, Lau JJ, Lim HL. Therapy for chronic hepatitis C. Gastroenterol Clin North Am. 1994;23:603-613.
Davis GL, Balant LA, Schiff ER, et al. Treatment of chronic hepatitis C with recombinant interferon alfa: a multicenter randomized controlled trial. N Engl J Med. 1989;321:1501-1506.
Chemello L, Cavaletto L, Bernardinello E, et al. The effect of interferon alfa and ribavirin combination therapy in naive patients with chronic hepatitis C. J Hepatol. 1995;23(suppl):8-12.
Braconer J, Paulsen O, Engman K, et al. Combined alpha-interferon and ribavirin treatment for chronic hepatitis C virus infection. Scand J Infect Dis. 1995;27:325-329.
Gilbert BE, Knight V. Biochemistry and clinical applications of ribavirin. Antimicrob Agents Chemother. 1986;30:201-205.
Sibille J-C, Kondo H, Aisen P. Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein. Hepatology. 1988;8:296-301.
Piperno A, Fargion S, D'Alba R, et al. Liver damage in Italian patients with hereditary hemochromatosis is highly influenced by hepatitis B and C virus infection. J Hepatol. 1992;16:364-368.    
   

Liver iron accumulation in chronic hepatitis C patients without HFE mutations: relationships with histological damage, viral load and genotype and -glutathione S-transferase levels 

EUROPEAN JOURNAL OF GASTROENTEROLOGY & HEPATOLOGY 2001;13:1355-1361 November 2001 Edoardo Gianninia; Luca Mastraccib; Federica Bottaa; Paola Romagnolia; Alberto Fasolia; Domenico Rissoc; Francesca Faravellid; Paola Ceppab; Pasquale B. Lantieric; Gian Carlo Icardic; Roberto Testaa Gastroenterology Unit, aDepartment of Internal Medicine, bDepartment of Pathology and cDepartment of Health Sciences, University of Genoa, and dDivision of Human Genetics, Galliera Hospital, Genoa, Italy   

Background Host and viral factors have been suggested as possible causative factors for the presence of liver iron accumulation in chronic hepatitis C. However, there is no agreement regarding the influence of liver iron accumulation on the biochemical and histological severity of chronic hepatitis C.
Moreover, data concerning the relationships between both viral load and genotype and liver iron accumulation are scanty. Aims: To evaluate the biochemical, histological and virological assessment of a group of chronic hepatitis C patients without risk factors for iron overload, on the basis of the presence, degree and distribution of liver iron accumulation. Methods: Fifty-three chronic hepatitis C patients (34 men, 19 women; age 44 ± 11 years) with no risk factors for liver iron accumulation and showing no HFE mutations were chosen from a broader cohort of chronic hepatitis C patients. The presence, degree and distribution of liver iron accumulation were assessed using Deugnier's score. Relationships between the presence of liver iron accumulation and grading and staging were carried out separately. Hepatitis C virus RNA serum levels and viral genotype were compared in patients with or without liver iron accumulation. Alpha glutathione S-transferase serum levels were assessed in all patients. Results: Overall, liver iron accumulation was mild and was present in 19 patients (36%). It was associated with male gender (P = 0.0358), and was reflected by high serum iron levels (P = 0.001) and high ferritin levels (P < 0.0001). Hepatitis C virus RNA levels and genotype were not associated with the presence of liver iron accumulation. In multivariate analysis, ferritin was the only variable significantly associated with liver iron accumulation (P < 0.0001). Grading was higher in patients with liver iron accumulation regardless of the site of iron deposition. Fibrosis was present in all patients with iron overload; these patients were more frequently cirrhotic. Moreover, patients with mesenchymal or mixed deposition had higher staging than patients with hepatocytic or no iron deposition. This feature was reflected by higher -glutathione S-transferase levels. Conclusions:
Liver iron accumulation is mild in chronic hepatitis C patients without HFE mutations and is mainly reflected by serum ferritin levels.
Viral characteristics do not seem to play a role in iron deposition. Liver iron accumulation is associated with higher grading, advanced fibrosis and cirrhosis. Moreover, higher staging is associated with mesenchymal or mixed iron deposition. In these patients, higher -glutathione S-transferase levels seem to reflect more complex damage. http://www.transplantjournal.com/

Iron as a Comorbid Factor in Chronic Viral Hepatitis

Herbert L. Bonkovsky, M.D. American Journal of  Gastroenterology January 2002 Volume 97, Number 1
Pages 1-4

Iron is an abundant element of the earth, and it has served an essential role in the emergence of oxygen-based plant and animal life on our planet.
Deficiency of iron is the most common cause of anemia and, when severe, is associated with many symptoms and signs. To help prevent such occurrences, virtually all forms of life on earth have developed schemes and means to assure their acquisition and retention of iron. In fact, we humans have virtually no natural means of excreting excess iron, probably because, for most of our natural history as an evolving species, we were more prone to problems related to iron deficiency than to iron excess.

The situation for some of us has changed dramatically during the past couple of millennia, with the emergence of iron overload or hemochromatosis as a common condition.
Indeed primary, or hereditary, hemochromatosis is the most common inborn error of metabolism among whites from central and northern Europe.
Most causes of hereditary hemochromatosis are due to a single homozygous mutation (nt: g845a) of the HFE gene, which produces the now familiar substitution of tyrosine for cysteine at amino acid 282 of the HFE protein [the C282Y mutation. Most men and at least one third of women who are C282Y +/+, if undiscovered and untreated, will develop pathological iron overload.
Hereditary or acquired hemochromatosis may also occur as a result of other mutations of HFE [especially H63D and S65C, as a result of mutations in other genes involved in iron metabolism [e.g., ferroprotein or hepcidin, or as a result of dyserythropoietic anemias, the most important of which are the thalassemias.

Regardless of cause, excess iron is toxic and potentially fatal, and the liver, which in all forms of hemochromatosis is the major organ for iron storage, is the principal site of iron-mediated toxicity.

Thus, iron overload per se may cause hepatic fibrosis, cirrhosis, decompensation, and hepatocellular carcinoma. Indeed, the latter complication of cirrhosis is especially common in hemochromatosis

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