Iron overload and genotype 3 are associated with liver steatosis in chronic
hepatitis C

L. I. Fernández Salazar, T. Álvarez Gago¹, R. Aller de la Fuente, A. Orduña Domingo², T. Arranz Santos,
F. de la Calle Valverde, L. del Olmo Martín, D. de Luis Román³ and J. M. González Hernández

Services of Digestive Diseases, ¹Pathology and ²Microbiology. Hospital Clínico Universitario. Valladolid.
³Service of Endocrinology. Hospital del Río Hortega. Valladolid. Spain

 

ABSTRACT

Objective: to determine epidemiological, biochemical, virological, and histological factors associated with liver steatosis in chronic hepatitis C.
Subjects: the medical histories of 53 patients biopsied for chronic hepatitis C diagnosis between June 2000 and December 2002 were retrospectively studied. Epidemiological, biochemical, and virological data were collected. Patients with hepatitis B virus or human immunodeficiency virus coinfection were excluded. Liver biopsy specimens were reviewed and scored by one pathologist. Weight and height were measured at liver biopsy time. The statistic association between qualitative and quantitative variables and the presence of liver steatosis was studied.
Results: steatosis was identified in 52% of biopsies. There was no statistic association with age, sex, method of transmission, duration of infection, alcohol consumption, other diseases, body mass index, glucose, triglycerides, cholesterol, AST, ALT, GGT, alkaline phosphatase, bilirubin, or viral load. Liver steatosis was associated with serum iron, transferrin saturation, and ferritin. Genotype 3 was also associated with steatosis. Piecemeal necrosis, hepatocellular injury, Kupffer cell hyperplasia, liver iron, and portal fibrosis were also associated with steatosis. A multivariate analysis showed that genotype 3, Kupffer cell hyperplasia, and liver iron were associated with the presence of steatosis.
Conclusions: liver steatosis in chronic hepatitis C associates with genotype 3, Kupffer cell hyperplasia, and iron overload. Hepatic steatosis also associates with greater inflammation and fibrosis, and must be considered to contribute to disease progression.

Key words: Chronic hepatitis. Hepatitis C virus. Lipids. Metabolic syndrome. Iron overload. Prognosis.

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Fernández Salazar LI, Álvarez Gago T, Aller de la Fuente R, Orduña Domingo A, Arranz Santos T, de la Calle Valverde F, del Olmo Martín L, de Luis Román D, González Hernández JM. Iron overload and genotype 3 are associated with liver steatosis in chronic hepatitis C. Rev Esp Enferm Dig 2004; 96: 818-828.

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Recibido: 03-12-03.
Aceptado: 28-04-04.

Correspondencia: L. Fernández Salazar. C/ Gamazo, 4 - 3º B. 47004 Valladolid. Telf.: 983 212 398.

 

INTRODUCTION

Hepatic steatosis is a frequent finding in hepatic biopsies of patients with chronic hepatitis C (CHC) (1). The role of hepatic steatosis in the natural history of CHC is unclear, but several authors link it to fibrosis (2-6).

Fat deposits in the liver can be due to factors that are independent of infection with hepatitis C virus (HCV), such as alcohol consumption, diabetes mellitus, hypertrigylceridemia, or overweight (4,5). They may also be a consequence of a cytopathic effect of HCV (6).

Our objective was to determine the frequency and severity of steatosis in CHC, and epidemiological, biochemical, virological, and histological factors associated with its presence. We attempted to determine whether the presence of steatosis is related to factors independent of HCV infection, and whether its presence implies a more severe liver disease.

MATERIAL AND METHODS

Patients. The case histories of 53 patients biopsied for CHC between June 2000 and December 2002 were retrospectively studied. CHC diagnosis was established from ALT (alanine aminotransferase) elevation for over six months, the presence of HCV antibodies, and the detection of HCV RNA in the serum. Patients with hepatitis B virus (HBV) or human immunodeficiency virus (HIV) coinfection were excluded; the final number of patients was 50. Data on method of transmission, length of evolution (known for only 14 patients), associated diseases, and alcohol consumption were obtained. Patients were classified into three groups based on alcohol consumption during the previous 12 months: a) non-drinkers; b) less than 40 g of alcohol per day; and c) more than 40 g alcohol per day. Patients were weighted and measured for height during their admission for a liver biopsy.

Biochemistry and hemogram. The following were obtained: glucose, triglycerides, cholesterol, aspartate aminotransferase (AST), ALT, alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGT), bilirubin, serum iron, transferrin, transferrin saturation index (TSI), ferritin, platelets.

Histological study. Hepatic biopsies were performed under ultrasonographic guidance using a modified Menghini aspiration-biopsy needle (Surecut^®). Liver tissue was fixed in formol and then in paraffin. A single pathologist was in charge of the study of biopsy samples as shown in table I, using standard staining: hematoxylin-eosin, trichrome, and Perls. Portal/periportal activity and lobular activity gave the degree based on Scheuer's criteria (7).

Virology. HCV genotype and viral load were determined within the three months prior to the liver biopsy. As a screening test for serologic diagnosis, an enzymoimmunoanalysis test (EIA, Abbott Chicago, USA) was used, and then confirmed by immunoblot (Deciscan HCV, Sanofi Diagnostic Pasteur, Marnes la Coquette, France). PCR before reverse transcription (RT-PCR) (Amplicor HCV, Roche Branchburg, USA) was used for viral RNA detection. Genotyping was performed based on reverse hybridization of amplified fragments from RT-PCR (Inno-Lipa 2nd generation HCV, Innogenetics Zarijndecht, Belgium).

Immunology. The positivity of autoantibodies (antinuclear, anti-DNA, anti-mitochondrial, anti-smooth muscle, and anti-parietal cell) was studied. Titres equal to or greater than 1/40 were included in the study.

Statistical analysis. Demographic, histological, and laboratory parameters were compared to the presence or absence of steatosis and its severity. Categorical variables were expressed as percentages, and continual variables as averages plus standard deviation. The Chi squared tests by Pearson, Mann-Whitney, and Kruskal-Wallis were used. Finally, variables significantly associated with steatosis in the univariate analysis (p < 0.05) were included in a multivariate model of logistic regression.

RESULTS

Table II shows the general characteristics of patients.

Steatosis presence and severity

Steatosis was observed in 52% (26/50) of liver biopsies. It was mild in 65.3% (17/26), moderate in 23% (6/26), and severe in 11.5% (3/26). Steatosis was macrovacuolar in 38.4% (10/26), microvacuolar in 3.8% (1/26), and mixed in 57.6% (15/26).

Factors associated with the presence of steatosis

1. Epidemiological (Table III): neither steatosis nor its severity were associated with differences in age, sex, time of evolution, method of infection, alcohol consumption, or associated diseases. Steatosis was not associated with a greater body mass index (BMI). In patients with a BMI above the average value for all patients, steatosis was not found more frequently. No relation between BMI and steatosis was found in patients infected with HCV genotype-3 or a HCV of a different genotype.

2. Biochemical and immunological (Table III): patients with hepatic steatosis were found to have elevated serum iron, TSI, and ferritin. A relationship between serum ferritin and steatosis severity was also found (Table IV).

3. Virological (Table III): genotype 3 was clearly associated with the presence of steatosis. Hepatic steatosis was found in 100% of hepatic biopsies from patients infected with HCV genotype-3, as compared to 35% of patients infected with genotype-1 viruses. Table IV shows the differences in steatosis for the various genotypes. Hepatic steatosis was not associated with viral load in the total number of patients or in genotype-3 or non-genotype-3 subgroups.

4. Histological (Table V): the presence of steatosis was associated with piecemeal necrosis and hence with higher activity levels, as well as with greater portal fibrosis, greater hepatocellular degeneration, presence of detectable hepatic iron, and hyperplasia of Kupffer cells. Steatosis severity was related to iron deposits (Table IV).

Genotype, piecemeal necrosis, hepatocellular degeneration, Kupffer cell hyperplasia, hepatic iron, grade, and portal fibrosis were included within the multivariate analysis. Three variables were independently associated with hepatic steatosis: hepatic iron (OR 12.884; 95% CI: 2.197-75.536; p = 0.005), genotype 3 (OR 43.48; 95% CI: 3.64-50; p = 0.003), and Kupffer cell hyperplasia (OR 16.622; 95% CI: 1.570-176.03; p = 0.020).

DISCUSSION

The frequency and severity of liver steatosis in our patients is similar to that described in other studies (2,4,8-13). We did not find any of the associations between steatosis and coexistence of metabolic syndrome such as diabetes or hyperlipidemia, or overweight based on BMI that other authors have found (4,5,11,12,14-16). There are several possible explanations for this: a) our patient group is very homogenous in BMI; b) 65% of patients have only mild steatosis, and therefore low severity may prevent differences to be significant; c) considering fatty liver and non-alcoholic steatohepatitis (NASH), the percentage of patients in whom is found only insulin resistance but not diabetes, hyperlipemia, or obesity is not negligible (17,18); and d) in NASH, and possibly in steatosis associated with CHC (6), visceral obesity is more important than peripheral obesity (17,19), and BMI is not an adequate indicator of visceral obesity.

In our patients, hepatic iron is clearly associated with hepatic steatosis. Such association has not been found in other studies (4,6), but there is reliable evidence that hepatic steatosis, metabolic syndrome, and iron overload are interrelated (17,18,20-30). CHC steatosis has been related to factors involved in non-alcoholic fatty liver disease, such as leptin, TNF-alpha (tumour necrosis factor-alpha), and CYP2E1 (cytochrome P450 2E1) (11,31-33) levels, and nonalcoholic steatosis is known to be associated with high iron indexes.

A possible cytopathic role of HCV itself, inducing steatosis, is supported by different studies. Although there are studies that do not show any relationship between steatosis and HCV genotype or viral load (8,34), a correlation between steatosis, serum and intrahepatic titres of viral RNA, and hepatic expression of HCV core protein in CHC has been demonstrated (6,11,12,35,36). Transgenic rats that express HCV proteins develop hepatic steatosis, mitochondrial disorders, and problems in very-low-density-lipoprotein (VLDL) secretion by hepatocytes (37-39). HCV entry into hepatocytes is mediated by the receptor of low-density lipoproteins, and interactions between HCV core proteins and apolipoproteins Apo1 and Apo2 have been shown (40,41). HCV infection, most frequently by genotype 3, also induces hypolipobetaproteinemia (42,43) and hypocholesterolemia (12,16,43,44), and sustained response to antiviral treatment makes steatosis disappear and reverts hypocholesterolemia (13,43,44). The steatosis in our patients is more frequent and severe in those infected with genotype 3. The role of HCV, above all with genotype 3, in the genesis of hepatic steatosis in CHC therefore appears probable. Just as other authors have done, we found no association between viral load and presence of steatosis or its severity (4).

We have found no association between steatosis and age, sex, or method of transmission either. Other authors generally report this lack of association also (4-6), although age has been associated with steatosis (12). A greater alcohol intake was not found among steatosis patients, although it is true that the CHC patients we biopsied are patients with great possibilities of receiving treatment who are not habitual drinkers. The role that alcohol plays in relation to CHC steatosis has been studied by several authors (3-5,9). Alcohol intake does not appear to be an important factor in the development of steatosis in CHC (4, 5), although a relationship is found in drinkers (10). Alcohol and steatosis may have a synergic action in CHC fibrogenesis (3). Among serum biochemical factors, only iron, TSI, and ferritin are associated with steatosis in the univariate analysis in our patients. It is possible that if the group of patients with severe steatosis had been larger, a greater level of hepatic cytolysis would have been found (35).

Steatosis is a condition that makes the liver vulnerable to other aggressions (32), and can be considered an aggravating factor in CHC. In our patients we found Kupffer cell hyperplasia associated with steatosis; this may also be explained by disorders in the phagocytic system as described in hepatic steatosis (45). Just as other authors (6), we have found steatosis associated with greater piecemeal necrosis and greater hepatocellular degeneration, which is typical of both alcoholic and nonalcoholic steatosis, although it does not associate with greater centrolobular fibrosis (5,46). Other authors have found an association between steatosis and perisinusoidal fibrosis (47). The relationship between steatosis, or its progression, and CHC fibrogenesis has been demonstrated (2-6,8,9), but we could only show an association between steatosis and portal fibrosis in the univariate analysis.

REFERENCES

1. Goodman ZD, Ishak KG. Histopathology of hepatitis C virus infection. Semin Liver Dis 1995; 15: 70-81.        [ Links ]

2. Castéra L, Hézode C, Roudor Thoraval F, Bastie A, Zafrani ES, Pawlotsky JM. Worsening of steatosis is an independent factor of fibrosis progression in untreated patients with chronic hepatitis C and paired biopsies. Gut 2003; 52: 288-92.        [ Links ]

3. Sefarty L, Poujol-Robert A, Carbonell N, Chazouillieres O, Poupon RE, Poupon R. Effect of the interaction between steatosis and alcohol intake on liver fibrosis progression in chronic hepatitis C. Am J Gastroenterol 2002; 97: 1807-12.        [ Links ]

4. Hourigan LF, Macdonald GA, Purdie D, Whitehall VH, Shorthouse C, Closton A, et al. Fibrosis in chronic hepatitis C correlates significantly with body mass index and steatosis. Hepatology 1999; 29: 1215-9.        [ Links ]

5. Monto A, Alonzo J, Watson JJ, Grunfeld C, Wright TL. Steatosis in chronic hepatitis C: relative contributions of obesity, diabetes mellitus, and alcohol. Hepatology 2002; 36: 729-36.        [ Links ]

6. Adinolfi LE, Gambardella M, Andreana A, Tripodi MF, Utili R, Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology 2001; 33: 1358-64.        [ Links ]

7. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991; 13: 372-4.        [ Links ]

8. Hwang SJ, Luo JC, Chu CW, Lai CR, Lu CL, Tsay SH, et al. Hepatic steatosis in chronic hepatitis C virus infection: prevalence and clinical correlation. J Gastroenterol Hepatol 2001; 16: 190-5.         [ Links ]

9. Ong JP, Younossi ZM, Speer C, Olano A, Gramlich T, Boparai N. Chronic hepatitis C and superimposed nonalcoholic fatty liver disease Liver 2001; 21: 266-71.        [ Links ]

10. Rubbia-Brandt l, Leandro G, Spahr L, Giostra E, Quadri R, Male PJ, et al. Liver steatosis in chronic hepatitis C: a morphological sign suggesting infection with HCV genotype 3. Histopathology 2001; 39: 119-24.        [ Links ]

11. Romero-Gómez M, Castellano-Megias VM, Grande L, Irles JA, Cruz M, Nogales MC, et al. Serum leptin levels correlate with hepatic steatosis in chronic hepatitis C. Am J Gastroeterol 2003; 98: 1135-41.        [ Links ]

12. Poynard T, Ratziu V, Mc Huntchison J, Manns M, Goodman Z, Zeuzem S, et al. Effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C. Hepatology 2003; 38: 75-85.        [ Links ]

13. Kumar D, Farell GC, Fung C, George J. Hepatitis C virus genotype is cytopathic to hepatocytes: reversal of hepatic steatosis after sustained therapeutic response. Hepatology 2002; 36: 1266-72.        [ Links ]

14. Czaja AJ, Carpenter HA, Santrach PJ, et al. Host- and disease-specific factors affecting steatosis in chronic hepatitis C. J Hepatol 1998; 29: 198-206.        [ Links ]

15. Hickman IJ, Clouston AD, Macdonald GA, Purdie DM, Prins JB, Ash S, et al. Effect of weight reduction on liver histology and biochemistry in patients with chronic hepatitis C. Gut 2002; 51: 89-94.        [ Links ]

16. Hui JM, Kench J, Farrell GC, Lin R, Samarasinghe D, Liddle C, et al. Genotype-specific mechanisms for hepatic steatosis in chronic hepatitis C infection. J Gastroenterol Hepatol 2002; 17: 873-81.        [ Links ]

17. Chitturi S, Abeygunasekera S, Farrell GC, Homes-Walker J, Hui JM, Fung C, et al. NASH and insulin resistance: Insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology 2002; 35: 373-9.        [ Links ]

18. Pagano G, Pacini G, Musso G, Gambino R, Mecca F, Depetris N, et al. Nonalcoholic steatohepatitis, insulin resistance, and metabolic syndrome: further evidence for an etiologic association. Hepatology 2002; 35: 367-72.        [ Links ]

19. Marchesini G, Brizi M, Bianchi G, Tomassetti S, Bugianesi E, Lenzi M, et al. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes 2001; 50: 1844-50.        [ Links ]

20. Salonen JT, Tuomainen TP, Nyyssönen K, Lakka HM. Relation between iron stores and non-insulin dependent diabetes in men: case-control study. BMJ 1998; 317: 727.        [ Links ]

21. Moirand R, Mortaji AM, Loréal O, Paillard F, Brissot P, Deugnier Y. A new syndrome of liver iron overload with normal transferrin saturation. Lancet 1997: 349: 95-7.        [ Links ]

22. Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, et al. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology 2003; 37: 917-23.        [ Links ]

23. Fergion S, Mattioli M, Fracanzani AL, Sampietro M, Tavazzi D, Fociani P, et al. Hyperferritinemia, iron overload, and multiple metabolic alterations identify patients at risk for nonalcoholic steatohepatitis. Am J Gastroenterol 2001; 96: 2448-55.        [ Links ]

24. Mendler MH, Turlin B, Moirand R, Jouanolle AM, Sapey T, Guyader D, et al. Insulin resistance-associated hepatic iron overload. Gastroenterology 1999; 117: 1155-63.        [ Links ]

25. Fernández-Real JM, Ricart-Engel W, Arroyo E, Balança R, Casamitjana-Abella R, Cabrero D, et al. Serum ferritin as a component of the insulin resistance syndrome. Diabetes Care 1998; 21: 62-8.        [ Links ]

26. 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: 421-9.        [ Links ]

27. George DK, Godwurm 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: 311-8.        [ Links ]

28. Hernández C, Genescà J, Esteban I, García L, Simó R. Relación entre los depósitos de hierro y la diabetes mellitus en pacientes infectados por el virus de la hepatitis C: Estudio de casos y controles. Med Clin (Barc) 2000; 115: 23-4.        [ Links ]

29. Petit JM, Bour JB, Galland-Jos C, Minello A, Verges B, Guiguet M, et al. Risk factors for diabetes mellitus and early insulin resistance in chronic hepatitis C. J Hepatol 2001; 35: 279-83.        [ Links ]

30. García González N, Prieto Valtueña J. Virus C, hierro y azúcar. Med Clin (Barc) 2000; 115: 25-6.        [ Links ]

31. Gochee PA, Jonsson JR, Clouston AD, Pandeya N, Purdie DM, Powell EE. Steatosis in chronic hepatitis C: association with increased messenger RNA expression of collagen I, tumor necrosis factor-alpha and cytochrome P450 2E1. J Gastroenterol Hepatol 2003; 18: 386-92.        [ Links ]

32. Day CP. Pathogenesis of steatohepatitis. Best Pract Res Clinical Gastroenterol 2002; 16: 663-78.        [ Links ]

33. Giannini E, Botta F, Cataldi A, Tenconi GL, Ceppa P, Barecca T, et al. Leptin levels in nonalcoholic steatohepatitis and hepatitis C. Hepatogastroenterology 1999; 46: 2422-5.        [ Links ]

34. Giannini E, Ceppa P, Botta F, Mastracci L, Romagnoli P, Comino I, et al. Leptin has no role in determining severity of steatosis and fibrosis in patients with chronic hepatitis C. Am J Gastroenterol 2000; 95: 3211-7.        [ Links ]

35. Fujie H, Yotsuyanagi H, Moriya K, Shintani Y, Tsutsumi T, Takayama T, et al. Steatosis and intrahepatic hepatitis C virus in chronic hepatitis. J Med Virol 1999; 59: 141-5.        [ Links ]

36. Rubbia-Brandt L, Quadri R, Abid K, Giostra E, Male PJ, Mentha G, et al. Hepatocyte steatosis is a cytopathic effect of hepatitic C virus genotype 3. J Hepatol 2000; 33: 106-15.        [ Links ]

37. Lerat H, Honda M, Beard MR, Loesch K, Sun J, Yang Y, et al. Steatosis and liver cancer in transgenic mice expressing the structural and non-structural proteins of hepatitis C virus. Gastroenterology 2002; 122: 352-65.        [ Links ]

38. Okuda M, Li K, Beard M, Showalter L, Schoille F, Lemon SM, et al. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology 2002; 122: 366-75.        [ Links ]

39. Perlemuter G, Sabile A, Letteron P, Vona G, Topilco A, Chretien Y, et al. Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis. FASEB J 2002: 16: 85-94.        [ Links ]

40. Angello V, Abel G, Elfahal M, Knight G, Zhang QX. Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci USA 1999; 96: 12766-71.        [ Links ]

41. Shi ST, Polyak SJ, Tu H, Taylor DR, Gretch DR, Lai M. Hepatitis C virus NS5A colocalizes with the core protein on lipids droplets and interacts with apolipoproteins. Virology 2002; 292: 198-210.        [ Links ]

42. Petit JM, Benichou M, Duvillard L, Jooste V, Bour JB, Minello A, et al. Hepatitis C virus-associated hypobetalipoproteinemia is correlated with plasma viral load, steatosis, and liver fibrosis. Am J Gastroenterol 2003; 98: 1150-4.        [ Links ]

43. Sefarty L, Andreani T, Giral P, Carbonell N, Chazouilleres O, Poupon R. Hepatitis C virus induced hypobetalipoproteinemia: a possible mechanism for steatosis in chronic hepatitis C. J Hepatol 2001: 34: 428-34.        [ Links ]

44. Hofer H, Bankl HC, Wrba F, Steindl-Munda P, Peck-Radosavljevic M, Osterreicher C, et al. Hepatocellular fat accumulation and low serum cholesterol in patients infected with HCV-3 a. Am J Gastroenterol 2002; 97: 2880-5.        [ Links ]

45. Diehl AM. Nonalcoholic steatosis and steatohepatitis IV. Nonalcoholic fatty liver disease abnormalities in macrophage function and cytokines. Am J Physiol Gastrointest Liver Physiol 2000; 282: 91-5.        [ Links ]

46. Zaitoun AM, Al Mardini H, Awad S, et al. Quantitative assessment of fibrosis and steatosis in liver biopsies from patients with chronic hepatitis C. J Clin Pathol 2001; 54: 461-5.        [ Links ]

47. Clouston AD, Jonsson JR, Purdie DM, et al. Steatosis and chronic hepatitis C: analysis of fibrosis and stellate cell activation. J Hepatol 2001, 34: 314-20.        [ Links ]