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Revista Española de Enfermedades Digestivas

versión impresa ISSN 1130-0108

Rev. esp. enferm. dig. vol.96 no.12 Madrid dic. 2004



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



Objective: to determine epidemiological, biochemical, virological, and histological factors associated with liver steatosis in chronic hepatitis C.
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.
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.

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.

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

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



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.


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.


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).


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.


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