Enhanced serum concentrations of transforming growth factor-beta1 in simple fatty liver: is it really benign?

One hundred and forty six adult Caucasian patients from the beginning of 2005 to the end of 2007 were consecutively investigated at our Department (Figure 1) in a cross-sectional fashion.

Every patient gave his or her informed consent to this study, which had been approved by the local Ethics Committee.

We enrolled 108 patients who fulfilled the following inclusion criteria: presence of overweight/obesity and visceral adiposity, associated with recent US features of "bright liver", with or without aminotransferases increase of unknown origin.

Subjects were classified as being overweight or as having first degree obesity on the basis of body mass index (BMI) cut-off points of ≥ 25.0 and ≤ 29.9, or > 29.9 and ≥ 34.9 kg/m², respectively. Central obesity was identified by waist circumference (WC) > 102 cm in men or > 88 cm in women, measured at the midpoint between the lower border of the rib cage and the iliac crest. Metabolic syndrome (MS) was defined according to the revised Adults Treatment Panel III (2001), and three or more criteria were considered: plasma glucose concentration of at least 100 mg dL^-1, WC > 102 cm in men and > 88 cm in women, serum high-density lipoprotein (HDL)-cholesterol concentration < 40 mg dL^-1 in men and < 50 mg dL^-1 in women, blood pressure of at least 130/85 mm Hg, and serum triglyceride concentration of at least 150 mg dL^-1.

IR was calculated by modified homeostasis model assessment-index (HOMA), with the following formula: fasting insulin (μU/mL) * plasma glucose (mg/dL)/405 [11].

Exclusion criteria were a recent history of acute inflammation (very high, ≥ 4 times the upper limit of normality, values of C reactive protein, CRP); presence of hepatitis B and C, neoplastic and/or haematological diseases, autoimmune and storage diseases; prior (at least 3 months) use of drugs inducing hepatic steatosis or affecting inflammation or angiotensin-converting enzyme inhibitors/angiotensin II type 1 receptor blockers. Alcohol abuse was ruled out according to the DSM-IV diagnostic criteria, by means of screening tests such as MAST (Michigan Alcohol Screening Test) and CAGE (Cut down, Annoyed, Guilty, and Eye opener) [12], as well as random tests for blood alcohol concentration and the use of a surrogate marker, e.g., mean corpuscular volume. Patients on antihypertensive therapy maintained a balanced medical regimen throughout the study.

Eighty-seven out of 108 patients initially selected agreed to perform liver biopsy. On the basis of the results of hepatic histology, 42 patients (19 females) were assigned to the NASH group and 45 (21 females) to the FL one. Steatohepatitis was graded on the basis of the degree of macrovesicular steatosis, mixed lobular inflammation and hepatocyte ballooning, using a composite NAFLD activity score (NASH, > 5) [13]. The presence of perisinusoidal fibrosis was noted and scored as none, rare, mild or moderate. Early fibrosis was distinguished from advanced fibrosis based on the presence of bridging fibrosis (score of 3 or more).

Thirty-eight individuals were diagnosed as to have elevated values of serum alanine aminotransferase (ALT) for at least six months. These subjects possessed detectable serum HCV-RNA (COBAS AmpliScreen HCV Test, v2.0, with automated amplification and detection using polymerase chain reaction method on the COBAS AMPLICOR Analyzer, Roche; the lower detection limit was 200 IU/mL), before starting antiviral treatment. Thirty-six patients (17 females) underwent liver biopsy. Histological features were evaluated using the Ishak scoring system for inflammation and fibrosis [14]. In brief, inflammation was scored using four parameters (periportal or periseptal interface hepatitis, confluent necrosis, focal lytic necrosis and portal inflammation) to obtain a histological activity index (HAI, maximum score 18), and fibrosis was scored as 0 – 6. The selected patients' tissue specimens were considered adequate for evaluation when at least four portal (or septal) areas were available for review and if they had length superior to 1.5 cm.

Determinations were made by two expert operators, blinded to each other, using an ultrasound (US) diagnostic system (ESAOTE, Genoa, Italy) with a 3.5-MHz convex probe. The classification of "bright liver" was based on the following scale of hyperechogenity: 0 = absent, 1 = light, 2 = moderate, 3 = severe.

CRP was dosed by an enzyme immunoassay kit of BioCheck, Inc, Foster City, CA, USA.

TGF-β1 was dosed by using Quantikine immunoassay kit from R&D Systems, Inc. Minneapolis, MN, USA. Serum separator tubes were used to allow samples to clot for 30 minutes at room temperature. For complete release of TGF-β1, samples were incubated overnight at 2 – 8°C before centrifugation for 15 minutes at 1000 × g. Removed serum was stored at -70°C.

The intra-assay and inter-assay precision coefficient variation was 3.4% and 8.4%, respectively. TGF-β1 levels in 15 controls showed a median of 26.9 ng/mL, and 5^th – 95^th percentile 23.0 – 34.0. The mean minimum detectable was 4.87 pg/mL.

For the determination of ferritin in serum was used the Ferritin ELISA Quantitation kit by GenWay Biotech, Inc. San Diego, CA 92121. The minimum detectable ferritin concentration by the assay was 5.0 ng/mL. Normal values were in males 20–370 ng/mL and in females 10–150 ng/mL. The inter-assay coefficients of variation were 4.2%, 5.1% and 6.6% at the concentrations of 37, 221 and 340 ng/mL, respectively, whereas the intra-assay coefficients of variation were 3.5%, 5.7% and 3.6% at the same concentrations, respectively.

Data collection of sonographic parameters was done before the histological classification, whereas serum ferritin and growth factor concentrations were obtained on stored samples.

The liver biopsy, blood samples and US parameters were strictly carried out within two months in order to lessen potentially confounding lifestyle changes or intercurrent illnesses.

Variables normally distributed (Kolmogorov-Smirnov test) such as age (P = 0. 13), ferritin (P = 0.12), ALT (P = 0.1), CRP (P = 0.07) and TGF-β1 (P = 0.054) were expressed as mean (SD). BMI, not normally distributed (P = 0.003), and ordinals, i.e., US and histology scores, were expressed as median and range.

The t test or ANOVA and the Mann-Withey test or Kruskal-Wallis were adopted to compare means or median, respectively. The pairwise analysis of subgroups, post-hoc comparisons after ANOVA, was obtained by the Tukey test. Furthermore, the ANCOVA was used to control for factors. The chi square was performed to look for differences in the classification system. Tracking the degree of association between single parameters in each group, Pearson's r or Spearman's rho was chosen according to the variable distribution (normal or not normal as well as being ordinals, respectively). Statistical analysis was performed operating on Systat 12 and MedCalc Version 9.4. software packages.