This study comprised 35 patients with a diagnosis of NAFLD based on liver biopsy findings (09 males and 26 females, age mean 49years) and 51 healthy subjects, without NAFLD (16 males and 35 females, age mean 39years), according to ultrasound findings at the Liver Institute of Pernambuco – Brazil between 2005 to 2008. In addition, all patients had elevated alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) levels at least on two occasions, over 6months prior to enrollment. The study protocol was approved by the Ethics Committee for Human Hesearch of the University of Pernambuco and a written consent was obtained from every individual participating in the study. This transversal study was conducted in accordance with the Helsinki declaration of 2008.
All patients were negative for markers of Wilson’s disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake kept under 100g/week. Patients who were hepatitis B surface antigen– and/or HIV-positive and had other potential causes of liver disease were excluded. Patients with clinically decompensated cirrhosis or contraindications for liver biopsy were not included in the study.
None of the patients were taking medication that could cause steatosis (salicylates, nonsteroidal anti-inflammatory drugs, corticosteroids, valproic acid, amiodarone, perhexiline maleate) or modify serum levels of homocysteinemia (folate, vitamin B12).
Diagnosis of type 2 diabetes and dyslipidemia were based on the criteria of the American Diabetes Association (fasting glucose ≥ 100mg/dL; Triglyceride ≥ 150mg/dL HDL < 40mg/dl in man or < 50mg/dL in woman) . Overweight corresponded to Body Mass Index (BMI) > 25kg/m2 and obesity to BMI ≥ 30kg/m2.
Blood samples were collected after fasting overnight and centrifuged within 60min to separate plasma, serum and leukocyte cells and storaged at – 80°C.
Fasting Glucose, total cholesterol and fractions, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), γGT were performed by standard methods using automated techniques (Modular P800, Hitach/Roche) in all patients at basal line and at the end of the study.
The homocysteine levels were determined by HPLC (high performance liquid cromatography) with fluorimetric detection . The folic acid and B12 vitamin were determined by standard methods using automated techniques (Elecsys and COBAS analyzers/Roche).
The insulin resistance index was calculated based on fasting insulin and fasting glucose according to homeostasis model assessment (HOMA -IR) . The Body Mass Index (BMI) is defined as the individual’s body mass divided by the square of his or her height.
For MTHFR polymorphism identification, the DNA was extracted from leukocytes by the salting out method. The C677T and A1298C MTHFR polymorphisms were determined by PCR-RFLP (Hinf I) and PCR-ASA, respectively [22, 23]. The amplified and digested fragments were analyzed in 3% agarose gel and the fragments were visualized in ultraviolet light (UV) after being stained with ethidium bromide. The 677 wild type (CC) shows a single fragment of 198bp; heterozygote (CT) shows fragments of 198, 175 and 23bp; and mutant homozygote (TT) shows two fragments with 175 and 23bp . The polymorphism MTHFR A1298C wild type and mutated alleles yield fragments of 77-bp and 120-bp, respectively .
A single liver pathologist scored all specimens with expertise in NAFLD: macro and microvacuolar fatty change, zonal distribution, foci of necrosis, portal and perivenular fibrosis, inflammatory and fibrotic infiltrate with zonal distribution. Macrovesicular steatosis was classified in low steatosis (<33% of hepatocytes with steatosis), moderate (34-66%) and intense (>66%) .
Data analysis was performed with BioEstat 5.0 software. The quantitative variables were described by mean values ± SD. T-test and Mann–Whitney U test were used in variables with normal and without normal distribution. Spearman’s r coefficient was used to discover a correlation between continuous variables (folate and B12 vitamin status and homocysteine). The frequencies of each allele were calculated as q = (2a + b)/n, where a corresponded to the number of homozygotes, b to the number of heterozygotes, and n to the number of alleles analyzed, respectively. Hardy-Weinberg equilibrium was tested for the SNP by comparing observed frequencies with expected frequencies and using a χ2 test. The differences in genotypes from each polymorphic position between cases and controls were assessed by Fisher’s exact tests. In all statistical evaluations, P < 0.05 was taken as significant.