We studied one hundred seven-four naive patients with chronic hepatitis C infection (CHC) from the Northeast of Brazil (91 male, 83 female). All patients enrolled had increased aminotransferase levels for at least six months and tested positive for anti-HCV antibodies (third-generation enzyme immunoassay) and HCV-RNA (RT-PCR, Roche Cobas Amplicor 2.0, Roche Diagnostics, Basel, Switzerland).

The HCV genotype, determined by LiPA assay (Innolipa HCV II; Immunogenetics, Ghent, Belgium). All patients were enrolled at the Liver Institute of Pernambuco in Brazil from February 2007 to October 2009.

This cross-sectional study was conducted according with the Helsinki declaration of 1975. Specific informed consent was obtained for the study and the protocol was approved by the Internal Review Board of the University of Pernambuco- Brazil. The investigations performed to exclude other causes of liver disease included a hepatobiliary system ultrasound, viral serology, autoantibody titers, serum iron, ferritin and transferrin saturation, ceruloplasmin and copper levels, and alpha1-antitrypsin. Patients who had a > 100 g/week alcohol intake determined by a detailed personal history, questioning of family members, and an investigation of previous medical records, were excluded. Also it was excluded treatment with immunosuppressive drugs or drugs causing steatosis (corticosteroids, antiepileptic agents, tamoxifen and amiodarone).

The diagnosis of diabetes type II, hypertension, and dyslipidemia were based on the criteria of the American Diabetes Association (Alexandria, VA, USA); fasting glucose ≥ 100 mg/dL; triglyceride (Tg) ≥ 150 mg/dL; high density lipoprotein (HDL) < 40 mg/dL in men or < 50 mg/dL in women; and ≥ 130 mmHg systolic or ≥ 85 mmHg diastolic) 18 . The folic acid and B12 vitamin reference were 3.1-17.5 ng/mL and 197-866 pg/mL respectively.

Normative reference Hcy levels were considered to be 12 or less (μmol/L) in males and 10 or less in females 10 . The homocysteine cut-off level in this study was 9 μmol/L determined by ROC curve.

The MTHFR polymorphism was analyzed in all 174 patients, however only in 138 of these patients the serum samples were collected at the time of liver biopsy. Thus, we used 138 serum samples to determine total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (Tg), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (γGT), alkaline phosphatase (AP), fasting glucose, fasting insulin, and insulin resistance (homeostasis model assessment-insulin resistance [HOMA-IR]: fasting insulin (U/mL) fasting glucose (mmol/L)/22.5) 19 . For insulin resistance the cut-off value was considered to be ≥ 2.5. Blood samples were centrifuged within 60 min to separate plasma, serum and leukocyte cells and storaged at-80 °C.

The homocysteine levels were determined by chemiluminescence method 20 ; Fasting Glucose, total cholesterol and fractions, triglycerides, ALT, AST, AP, γGT and fasting insulin were performed by standard methods using automated techniques (Cobas, Roche). The LDL cholesterol was determined by Friedwald equation 21 .

The C677T polymorphism was determined by a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. The C→T transition creates a restriction site for the enzyme Hinf I and the digested product was isolated electrophoretically in 2% agarose gel and the fragments were visualized in ultraviolet light (UV) after being stained with ethidium bromide. Wild type (CC) shows a single fragment of 198 bp; heterozygote (CT) shows fragments of 198, 175 and 23 bp; and mutant homozygote (TT) shows two fragments with 175 and 23 bp 22 .

The liver tissue was fixed in 4% formaldehyde and processed for hematoxylin-eosin and Masson trichrome stains for histological analysis. Histological analyses were evaluated by only one pathologist who was unaware of the HCV genotype and of the patient's clinical characteristics. Stages of fibrosis and grades of inflammation were scored according to METAVIR, that it consists of F0 (no fibrosis), F1 (portal fibrosis without septa), F2 (portal fibrosis with few septa), F3 (numerous septa without cirrhosis), F4 (cirrhosis). Steatosis was graded 0-3 based on percentages of hepatocytes harbouring lipid droplets in the biopsy (0 reflecting none; 1 equalling 40-33%; 2 referring to 33-66%; and 3 representing > 66% steatotic hepatocytes).

Data analysis was performed with SPSS 15.0 software. Distribution normality of the groups considered was preliminary evaluated by Kolmogorov-Sminorv test. Differences between groups were analyzed by analysis of variance (ANOVA) when variables were normally distributed. Chisquare test or Fisher's exact test were used to compare categorical variables. Logistic regression analysis was used to identify independent predictors for MTHFR polymorphism, gender, triglyceride, fibrosis and steatosis. The proportion of MTHFR alleles were distributed in patients in accordance with the Hardy-Weinberg equilibrium. Results were considered significant when the p value was less than 0.05.

In the present study 174 patients with CHC were included. There were 52.3% (91/174) males and 47.7% (83/174) females. The biochemical characteristics according genotype and histological classification were only analyzed in 138 patients with CHC and the patients were stratified according to viral genotype 1 (n = 93) and non-1 (n = 45).

The biochemical characteristics according to the genotype classification demonstrated Hcy levels and concentrations of total cholesterol differ significantly between patients with genotype 1 and genotype non-1 (9.96 versus 9.39 μmol/L and 158.01 versus 138.58 mg/dL, respectively, p = 0.01) (Table 1). The Hcy level differs significantly between no steatosis and steatosis (9.0 versus 10.3 μmol/L respectively, p = 0.03) (Table 2). Although neither folate and vitamin B12 nor triglycerides, total cholesterol, HDL, LDL, HOMA, glucose and Hcy level differ between genotypes frequencies of the 677C/T (MTHFR) polymorphism (p > 0.05) (Table 3).

The MTHFR polymorphism was analyzed from peripheral blood of 174 patients. The genotype TT was more frequent in HCV non-1 genotype than genotype 1 (9.8% versus 4.4% respectively, p = 0.01) (Table 4). Associated with this no relation was observed in the genotype frequencies of the 677C/T (MTHFR) polymorphism according to HCV genotype and histopathological classification (p > 0.05) (Table 5). Hence, a significant difference was observed in the genotype TT+CT frequencies according to histological grades of fibrosis (1+2 [n = 116] versus 3+4 [n = 58]) (p = 0.001) and of steatosis (No Steatosis [n = 70] versus Steatosis [n = 104]) (p = 0.04) regardless of HCV genotype (Table 6).

In multi regression analysis no relation were observed among MTHFR polymorphism, Hcy level, HCV genotype and lipid profile as a independent variables for steatosis and fibrosis (Table 7 and 8).

The heterogeneity of Brazilian population regarding racial definition mixed with social economic factors may represent a confounding factor herein. The absence of information on the reported genetic risk factors in the Northeast of Brazil population, which is considered to be genetically very heterogeneous, led us to design the present study. In our data we reported that the genotype TT was more frequent in the HCV genotype non-1 without association with histological grades of fibrosis and of steatosis. We also observed significant difference in the genotype TT+CT frequencies according to histological grades of fibrosis and steatosis regardless of HCV genotype.

Several biological and clinical implications have been suggested to occur in relationship with the MTHFR 677C/T polymorphism. The MTHFR polymorphisms were found to be associated with increased cardiovascular risk in several populations including Lebanese, Japanese and French Canadians 23 24 25 . Toniutto et al., also found a relation between MTHFR 677C/T polymorphism and liver fibrosis in patients who underwent liver transplantation with recurrent hepatitis C and also speculates that the MTHFR polymorphism could play a direct profibrogenic effect, modulating the action of proteins involved in collagen degradation 26 .

Otherwise Borgia et al. did not find association with polymorphisms of MTHFR in the outcome of pegylated-IFNα plus ribavirin treatment in patients with chronic hepatitis C, only the homocysteine levels 27 . Silva et al., only confirms the association between increased plasma homocysteine concentration in Alzheimer's disease and suggests that C677T MTHFR polymorphisms not contributed to genetic susceptibility for Alzheimer's disease in elderly individuals in the Northeast of Brazil 28 .

However, another study conducted in Northeast of Brazil, Couto et al., screened 843 neonates for MTHFR C677T polymorphism. The T677 allele frequency and TT677 genotype was higher than those observed in other studies of African-descent populations. The T allele frequency was 0.23 and the C/T and T/T genotypes prevalence were 36.2 and 5.3 percent, respectively 29 .

The present study provides evidence that a genetic background, such as the MTHFR polymorphism through hyperhomocysteinemia induced derangement of lipid metabolism, may contribute to the development of higher degrees of steatosis, which in turn accelerates the progression of liver fibrosis in chronic HCV infection. Potential mechanisms of this effect may include the increased sensitivity of steatotic livers to oxidative stress, cytokine-mediated injury, and steatosis-related hepatic insulin resistance 30 .

Other important finding of our study was the higher Hcy level in patients with steatosis, although the MTHFR polymorphism was not identified as a risk factor for steatosis in the whole population (HCV genotypes 1+ non-1) in the multi regression analysis. It should be understood because the high plasma levels of Hcy have been reported to negatively influence normal cell function in many different tissues, such as vascular endothelium and smooth muscle cells and the liver. These effects, in turn, may explain the association of hyperhomocysteinaemia with vascular disease (thrombosis of arterial and venous districts and atherosclerosis) 31 32 33 34 35 36 and more recently, have been advocated for a possible role in pathogenesis and evolution of chronic liver disease 36 37 .

Hcy is a toxic non-protein sulfur containing amino acids in humans. It is formed exclusively upon demethylation of the essential amino acid- methionine. The Hcy metabolism occurs through the junction of the remethylation and transsulfuration pathways. These pathways are strongly influenced by enzymes polymorphisms. MTHFR enzyme has fundamental importance in plasmatic Hcy regulation 38 39 .

Hcy decreases the expression of a wide range of antioxidant enzymes 40 41 42 and impairs endothelial nitric oxide (NO) bioavailability by inhibiting glutathione peroxidase activity raise the possibility that Hcy sensitizes cells to the cytotoxic effects of agents or conditions known to generate reactive oxygen species (ROS). Decreased NO bioavailability has also been shown in vitro to increase the expression of monocyte chemoattractant protein 1 (MCP-1) which may enhance intravascular monocyte recruitment and lead to accelerated lesion formation 43 .

There is evidence that Hcy induced endoplasmic reticulum (ER) stress causes dysregulation of the endogenous sterol response pathway, thereby leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides without impairing the hepatic export of lipids 44 . Similar result was observed in Adinolfi et al., studies that investigated the role of these factors in the development of HCV related steatosis and in the progression of chronic hepatitis C in 116 patients, 50% had a body mass index (BMI) of 25 or higher; 58% were infected with HCV genotype 1, and 65.5% showed steatosis. According to multivariate analysis, steatosis was independently associated with hyperhomocysteinemia (OR = 7.1) 10 .

We also observed lower concentrations of serum total cholesterol in CHC patients genotype non-1. Similar results have been described by Corey at al., who demonstrated that serum lipids play a role in hepatitis C virion circulation and hepatocyte entry. In a cohort of 179 patients with CHC this author showed that patients with HCV had lower concentrations of total cholesterol than in the control group. These data support the hypothesis that the lipo-viral particles use the LDL-C receptors of hepatocytes as points of entry of the virus. Once inside into hepatocyte, the replication dependents of the lipid environment of the host 45 46 47 .

In summary, our study had important implications. According to our data, Hcy levels were highly prevalent in subjects with chronic hepatits C with steatosis regardless of HCV genotype and vitamin deficiency. The presence of genotype TT of MTHFR C677T polymorphism was more common in CHC genotype non-1 infected patient regardless of histopathological classification and genotype TT+CT frequencies were significant in the presence of fibrosis grade 1+2 and of steatosis in CHC infected patients from the northeast of Brazil. The genetic susceptibility of MTHFR C677T polymorphism should be confirmed in a large population.