The mechanism(s) determining the progression from fatty liver to steatohepatitis is currently unknown. Our goal was to define the relative impact of iron overload, genetic mutations of HFE, and insulin resistance on the severity of liver fibrosis in a population of subjects with nonalcoholic fatty liver disease (NAFLD) who had low prevalence of obesity and no overt symptoms of diabetes. In a cohort of 263 prospectively enrolled patients with NAFLD, 7.4% of patients had signs of peripheral iron overload and 9% had signs of hepatic iron overload, but 21.1% had hyperferritinemia. The prevalence of C282Y and H63D HFE mutations was similar to the general population and mutations were not associated with iron overload. Although subjects were on average only moderately overweight, insulin sensitivity, measured both in the fasting state and in response to oral glucose, was lower. Univariate analysis demonstrated that the presence of severe fibrosis was independently associated with older age, female sex, overweight, aspartate/alanine aminotransferase ratio, serum ferritin level, fasting glucose and insulin levels, decreased insulin sensitivity, and with histologic features (degree of necroinflammation and steatosis). After adjustment for body mass index (BMI), age, sex, and degree of steatosis, ferritin levels (odds ratio [OR] ‫؍‬ 1.77; 95% CI ‫؍‬ 1.21-2.58; P ‫؍‬ .0032) and the oral glucose insulin sensitivity (OR ‫؍‬ 0.53; CI ‫؍‬ 0.33-0.87; P ‫؍‬ .0113) were independent predictors of severe fibrosis. In conclusion, the current study indicates that insulin resistance is a major, independent risk factor for advanced fibrosis in patients with NAFLD. Increased ferritin levels are markers of severe histologic damage, but not of iron overload. Iron burden and HFE mutations do not contribute significantly to hepatic fibrosis in the majority of patients with NAFLD. (HEPATOLOGY 2004;39:179 -187.) N onalcoholic fatty liver disease (NAFLD) is growing in importance, causing in a wide spectrum of hepatic injury, ranging from fatty liver to steatohepatitis and cirrhosis. 1 Whereas pure fatty liver has a benign clinical course, nonalcoholic steatohepatitis (NASH) is a recognized cause of progressive liver fibrosis that eventually leads to cirrhosis, liver failure, and hepatocellular carcinoma. 2-4 NAFLD and NASH are the two most common chronic liver diseases in Western countries, with an estimated prevalence in the general population of 10% to 20% and 2% to 3%, respectively. 2,5-7 A two-hit theory 8 has been proposed to explain the progression from simple steatosis to NASH, fibrosis, or cirrhosis. The first hit is represented by excessive hepatic fat accumulation primarily owing to insulin resistance and is supported by the close relation observed between NAFLD and several features of the metabolic syndrome (visceral obesity, type II diabetes mellitus, and dyslipidemia). 9 -11 The second hit is related mainly to increased intrahepatic oxidative stress, which promotes hepatic fibrosis through the production of reactive oxygen sp...

Given that phlebotomy reduces insulin resistance, which is associated with liver tissue damage, future studies should evaluate the effect of iron depletion on liver histology and cardiovascular end points.

Vesicular-mediated communication between cells appears critical in many biological processes. Extracellular vesicles (EVs) released from healthy and diseased cells are involved in a network of exchange of biologically active molecules. Since EVs present in biological fluids carry the signature of the cell of origin, they are potential biomarkers for ongoing physiological or pathological processes. Despite the knowledge on EV biology accrued in recent years, techniques of EV purification remain a challenge and all the described methods have some advantages and disadvantages. In the present study, we described a method based on charge precipitation of EVs from biological fluids and from cell supernatants in comparison with the differential ultracentrifugation, which is considered the gold standard for EV purification. The analysis of ζ-potential revealed that EVs have a negative charge that allows the interaction with a positively charged molecule, such as protamine. Protamine was shown to induce EV precipitation from serum and saliva and from cell culture media without the need for ultracentrifugation. EV resuspension was facilitated when protamine (P) precipitation was performed in the presence of PEG 35,000 Da (P/PEG precipitation). The recovery of precipitated EVs evaluated by NanoSight analysis was more efficient than that obtained by ultracentrifugation. By electron microscopy the size of EVs was similar after both methods were used, and the expression of CD63, CD9 and CD81 exosomal markers in the P/PEG-precipitated EVs indicated an enrichment in exosomes. The RNA recovery of P/PEG-precipitated EVs was similar to that of EVs isolated by ultracentrifugation. In addition, P/PEG-precipitated EVs retained the biological activity in vitro as observed by the induction of wound closure by keratinocytes and of proliferation of tubular epithelial cells. In conclusion, charge-based precipitation of EVs has the merit of simplicity and avoids the requirement of expensive equipments and may be used for the efficient isolation of EVs from small biological samples.

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ID-NAT and serology are complementary in detecting HBV infection in first-time donors, but HBV-DNA is superior to HBsAg detection in repeat donors.