Comparison of the severity of metabolic, liver and cardiovascular damage in NAFLD patients attending the hepatology clinic over the last three decades.

BACKGROUND AND AIMS: NAFLD prevalence is increasing worldwide. AIM: to assess whether severity of hepatic, metabolic and cardiovascular (CV) disease changed over time. METHODS: 422 NAFLD patients (388 biopsy proven and 34 clinical cirrhosis) diagnosed between 1990 and 2021 and subdivided according to decade of presentation. Metabolic parameters, early atherosclerosis (carotid plaques at Doppler ultrasound), severity of liver damage (NAS score, NASH, significant fibrosis (≥2) and cirrhosis) and PNPLA3 genotyping were assessed. RESULTS: No difference in age, sex and prevalence of dyslipidemia and hypertension was found across decades (p for trend), whereas a higher prevalence of diabetes (p = 0.02), obesity (p<0.001), histological severe steatosis (p<0.001), NASH (p<0.001), fibrosis ≥2 (p<0.001), cirrhosis (p<0.001) and carotid plaques (p = 0.05) was observed in the last decade compared to the others. A higher prevalence of PNPLA3 GG polymorphism was found over time (p = 0.02). In the whole cohort, age, metabolic alterations and PNPLA3 G homozygosity were independent risk factors for hepatic fibrosis and carotid plaques, independently of the decade considered. CONCLUSION: Over the past 10 years compared to previous decades, NAFLD patients presented to observation with more severe liver disease and subclinical atherosclerosis, paralleling the spread of diabetes and obesity. PNPLA3 unfavorable genotype became more prevalent over time.

Interaction between Lifestyle Changes and PNPLA3 Genotype in NAFLD Patients during the COVID-19 Lockdown.

The coronavirus disease 2019 (COVID-19) lockdown dramatically changed people's lifestyles. Diet, physical activity, and the PNPLA3 gene are known risk factors for non-alcoholic fatty liver disease (NAFLD). Aim: To evaluate changes in metabolic and hepatic disease in NAFLD patients after the COVID-19 lockdown. Three hundred and fifty seven NAFLD patients were enrolled, all previously instructed to follow a Mediterranean diet (MD). Anthropometric, metabolic, and laboratory data were collected before the COVID-19 lockdown in Italy and 6 months apart, along with ultrasound (US) steatosis grading and information about adherence to MD and physical activity (PA). In 188 patients, PNPLA3 genotyping was performed. After the lockdown, 48% of patients gained weight, while 16% had a worsened steatosis grade. Weight gain was associated with poor adherence to MD (p = 0.005), reduced PA (p = 0.03), and increased prevalence of PNPLA3 GG (p = 0.04). At multivariate analysis (corrected for age, sex, MD, PA, and PNPLA3 GG), only PNPLA3 remained independently associated with weight gain (p = 0.04), which was also associated with worsened glycemia (p = 0.002) and transaminases (p = 0.02). During lockdown, due to a dramatic change in lifestyles, half of our cohort of NAFLD patients gained weight, with a worsening of metabolic and hepatologic features. Interestingly, the PNPLA3 GG genotype nullified the effect of lifestyle and emerged as an independent risk factor for weight gain, opening new perspectives in NAFLD patient care.

Metabolic comorbidities and male sex influence steatosis in chronic hepatitis C after viral eradication by direct-acting antiviral therapy (DAAs): Evaluation by the controlled attenuation parameter (CAP).

BACKGROUND: Chronic hepatitis C (CHC) is associated with hepatic steatosis, related to both a direct viral action and metabolic features. Vice-versa data on hepatic steatosis after viral eradication by direct-acting antiviral agents (DAA) are undefined although the presence of metabolic alterations could strongly influence the occurrence of steatosis as in NAFLD. The controlled attenuation parameter (CAP) (FibroscanⓇ) allows the qualitative and quantitative evaluation of fatty liver. AIM: to evaluate in patients with CHC whether hepatic steatosis diagnosed by CAP modifies after DAAs-induced sustained virologic response (SVR). METHODS: Data were collected the day of DAAs therapy starting and six months after SVR. CAP ≥ 248 dB/m defined the presence of steatosis. RESULTS: 794 CHC SVR patients referring to 2 Italian Units were enrolled. Mean age was 64 ± 16 ys, 50% males, BMI 25.4 ± 4 kg/m2, genotype type-1 in 73%, type-3 in 8%. Prevalence of hepatic steatosis at baseline was 32% by US and 46% by CAP. De novo steatosis developed in 125 (29%), resolution in 122 (30%). At multivariate analysis de novo steatosis was independently associated with male sex (OR 1.7, CI 95% 1.09-2.67; p = 0.02) and baseline BMI (for unit increase OR 1.19, CI 95%1.11-1.29; p < 0.001). Baseline BMI (for unit increase OR 0.47, CI 95% 0.25-0.89; p = 0.02) and triglycerides (for unit increase OR 0.93, CI 95% 0.87-0.99; p = 0.03) prevented steatosis resolution after therapy. CONCLUSIONS: after SVR de novo steatosis and resolution of baseline steatosis are closely related to the presence of metabolic comorbidities.

Hepcidin resistance in dysmetabolic iron overload.

BACKGROUND & AIMS: Dysmetabolic iron overload syndrome (DIOS) is a frequent condition predisposing to metabolic, cardiovascular and hepatic damage, whose pathogenesis remains poorly defined. Aim of this study was to characterize iron metabolism in DIOS. METHODS: We evaluated 18 patients with DIOS, compared to 18 with nonalcoholic fatty liver and 23 healthy individuals with normal iron status, and 10 patients with hereditary haemochromatosis by a 24-h oral iron tolerance test with hepcidin measurement and iron metabolism modelling under normal iron stores. RESULTS: Dysmetabolic iron overload syndrome patients had higher peak transferrin saturation and area under the-curve of transferrin saturation than subjects with normal iron status, but lower values than haemochromatosis patients (P < 0.05 for all). Conversely, they had higher peak circulating hepcidin levels and area under the curve of hepcidin than the other groups (P < 0.05 for all). This was independent age, sex, haemoglobin, ferritin, and transferrin saturation levels (P = 0.0002). Hepcidin increase in response to the rise in transferrin saturation (hepcidin release index) was not impaired in DIOS patients. Viceversa, the ability of the hepcidin spike to control the rise in transferrin saturation at the beginning of the test (hepcidin resistance index) was impaired in DIOS (P = 0.0002). In DIOS patients, the hepcidin resistance index was correlated with ferritin levels at diagnosis (P = 0.016). CONCLUSIONS: Dysmetabolic iron overload syndrome is associated with a subtle impairment in the ability of the iron hormone hepcidin to restrain iron absorption following an iron challenge, suggesting a hepcidin resistance state. Further studies are required to better characterize the molecular mechanism underpinning this new iron metabolism alteration.

A randomized trial of iron depletion in patients with nonalcoholic fatty liver disease and hyperferritinemia.

AIM: To compare iron depletion to lifestyle changes alone in patients with severe nonalcoholic fatty liver disease (NAFLD) and hyperferritinemia, a frequent feature associated with more severe liver damage, despite at least 6 mo of lifestyle changes. METHODS: Eligible subjects had to be 18-75 years old who underwent liver biopsy for ultrasonographically detected liver steatosis and hyperferritinemia, ferritin levels ≥ 250 ng/mL, and NAFLD activity score > 1. Iron depletion had to be achieved by removing 350 cc of blood every 10-15 d according to baseline hemoglobin values and venesection tolerance, until ferritin < 30 ng/mL and/or transferrin saturation (TS) < 25%. Thirty-eight patients were randomized 1:1 to phlebotomy (n = 21) or lifestyle changes alone (n = 17). The main outcome of the study was improvement in liver damage according to the NAFLD activity score at 2 years, secondary outcomes were improvements in liver enzymes [alanine aminotransferases (ALT), aspartate aminotransferase (AST), and gamma-glutamyl-transferases (GGT)]. RESULTS: Phlebotomy was associated with normalization of iron parameters without adverse events. In the 21 patients compliant to the study protocol, the rate of histological improvement was higher in iron depleted vs control subjects (8/12, 67% vs 2/9, 22%, P = 0.039). There was a better improvement in steatosis grade in iron depleted vs control patients (P = 0.02). In patients followed-up at two years (n = 35), ALT, AST, and GGT levels were lower in iron-depleted than in control patients (P < 0.05). The prevalence of subjects with improvement in histological damage or, in the absence of liver biopsy, ALT decrease ≥ 20% (associated with histological improvement in biopsied patients) was higher in the phlebotomy than in the control arm (P = 0.022). The effect of iron depletion on liver damage improvement as assessed by histology or ALT decrease ≥ 20% was independent of baseline AST/ALT ratio and insulin resistance (P = 0.0001). CONCLUSION: Iron depletion by phlebotomy is likely associated with a higher rate of improvement of histological liver damage than lifestyle changes alone in patients with NAFLD and hyperferritinemia, and with amelioration of liver enzymes.

Risk of nonalcoholic steatohepatitis and fibrosis in patients with nonalcoholic fatty liver disease and low visceral adiposity.

BACKGROUND & AIMS: Increased visceral adiposity is considered the hallmark of the metabolic syndrome, whose hepatic manifestation is nonalcoholic fatty liver disease (NAFLD), although a subset of patients does not have visceral obesity. Our study aimed to compare metabolic alterations and liver damage in patients with NAFLD with and without visceral obesity. METHODS: Four hundred and thirty one consecutive patients with liver biopsy-confirmed NAFLD were divided in three groups according to waist circumference, the simplest surrogate marker of visceral obesity. One hundred and thirty three patients (31%) had a waist circumference ≤94 (males) and ≤80 cm (females) (group A), 157 (36%) between 94 and 102, and 80 and 88 (B), and the remaining 141 (33%) had values higher than 102 and 88 cm (C). RESULTS: Significant trends for older age, higher prevalence of female gender, lower HDL, higher triglycerides, altered glucose metabolism, hypertension, and metabolic syndrome were observed with increasing visceral adiposity. In contrast, non-alcoholic steatohepatitis (NASH) detected in 55% and 72% of patients with normal and increased waist circumference, respectively, and the presence of fibrosis ≥2 were not associated with visceral adiposity. Alanine aminotransferase (ALT), ferritin, HOMA-IR >4, and severe steatosis were independently associated with NASH, whereas ferritin and impaired glucose tolerance were associated with fibrosis ≥2. CONCLUSIONS: Patients with normal waist circumference, despite milder metabolic alterations, may have NASH and are at risk of developing fibrosis, suggesting that once NAFLD is present, visceral obesity is not a major determinant of liver damage severity.

Risk of severe liver disease in nonalcoholic fatty liver disease with normal aminotransferase levels: a role for insulin resistance and diabetes.

UNLABELLED: It is uncertain whether patients with nonalcoholic fatty liver disease (NAFLD) and normal alanine aminotransferase (ALT) have a milder disease and should undergo liver biopsy. We reviewed the histological data of 458 Italian patients with NAFLD in whom liver biopsy was indicated by altered liver enzymes (395 cases, 86%), or persistently elevated ferritin or long-lasting severe steatosis (63 cases). Factors associated with nonalcoholic steatohepatitis (NASH) and fibrosis >/= 2 were identified by multivariate analysis. Patients with normal ALT were significantly older, had lower body mass index, fasting triglycerides, insulin resistance according to homeostasis model assessment (HOMA-IR), ALT, and gamma-glutamyltransferase, but a higher prevalence of hypertension. NASH was diagnosed in 59% and 74% of the patients with normal and increased ALT, respectively (P = 0.01). In the overall series of patients, NASH was independently predicted by ALT (odds ratio [OR], 1.11; 95% confidence interval [CI], 1.04-1.19 per 10-IU/mL increase) and diabetes (OR, 1.5; 95% CI, 1.1-2.0). The same variables were selected in patients with increased ALT, whereas in those with normal ALT, HOMA-IR and ALT were independent predictors. Severe fibrosis was independently predicted by serum ferritin (OR, 1.04; 95% CI, 1.001-1.08 per 50-ng/mL increase), ALT (OR, 1.07; 95% CI, 1.02-1.14), and diabetes (OR, 1.8; 95% CI, 1.4-2.3) in the overall series, serum ferritin and diabetes in those with increased ALT, and only HOMA-IR (OR, 1.97; 95% CI, 1.2-3.7) in patients with normal ALT. CONCLUSION: Normal ALT is not a valuable criterion to exclude patients from liver biopsy. Alterations in glucose metabolism and insulin resistance in subjects with normal ALT should also be considered in the selection of NAFLD cases for histological assessment of disease severity and progression.

Association between heterozygosity for HFE gene mutations and hepatitis viruses in hepatocellular carcinoma.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are strong and independent risk factors for hepatocellular carcinoma (HCC) development. Patients with hereditary hemochromatosis (HH) are considered at risk of developing cancer. However, the interaction between HFE gene mutations and hepatitis viruses for HCC development has not been systematically searched for. To assess the interaction between HFE gene mutations and exogenous risk factors in the risk of HCC occurrence, a case-only approach, in which just a series of patients is enrolled, was used. Three hundred three cirrhotic patients (231 males, 72 females) from five liver units in different geographic areas of Italy, who developed HCC during regular follow-up between January 1999 and March 2003, and whose blood DNA was available, were analyzed. In all subjects, hepatitis B surface antigen (HBsAg), anti-HCV and HFE gene mutations were assayed; alcohol intake was recorded by history. The interaction between HFE genotypes and hepatitis viruses for HCC was estimated by multivariate analysis adjusting for the confounding effect of alcohol intake, area of residence and months of follow-up. Of the 303 HCC cases, 12 (4.0%) were heterozygous for the C282Y mutation, 93 (30.7%) for the H63D, and 198 (65.3%) homozygous for the wild allele. Multivariate analysis showed that C282Y heterozygous males were 3.8-fold (95% CI=1.0-15.2) more likely to be HBV positive and that H63D heterozygous females were 6.0-fold (95% CI=1.2-113.8) more likely to be HCV positive than wild type subjects. In conclusion, given the association between C282Y mutation and HBV infection in male patients with HCC, a careful evaluation and follow-up should be considered in the C282Y-positive subjects with hepatitis B virus related liver disease. The interaction between the H63D mutation and HCV, observed only in women, may reflect a higher sensitivity to H63D-induced iron metabolism abnormalities and a reduced antioxidant capability in the presence of an even minor increase of iron which may occur as a consequence of the coexistence of hepatitis C infection and heterozygosity for HH.

Cytotoxic T-lymphocyte antigen-4 A49G polymorphism is associated with susceptibility to and severity of alcoholic liver disease in Italian patients.

AIMS: To determine whether the functional A49G polymorphism of cytotoxic T-lymphocyte antigen-4 (CTLA-4), a T-cell surface molecule that modulates T-lymphocyte activation and influences the risk of developing alcohol-induced autoantibodies, plays a role in susceptibility to alcoholic liver disease (ALD) and influences disease severity in Italian alcohol abusers. METHODS: One hundred and eighty-three patients with chronic ALD (61 cirrhosis), 115 end-stage HCV cirrhosis, 102 non-alcoholic fatty liver disease (NAFLD), 93 healthy subjects and 43 heavy drinkers without liver disease were studied. CTLA-4 gene polymorphism was analysed by restriction analysis. RESULTS: The frequency of the CTLA-4 polymorphism was higher in patients with ALD than in patients with HCV chronic hepatitis and NAFLD, healthy subjects (P < 0.0001), and heavy drinkers without liver disease (P = 0.02). In patients with ALD, homozygosity for the CTLA-4 polymorphic allele (G/G genotype) was more represented in subjects with cirrhosis (P = 0.047), and independently associated with the risk of cirrhosis (OR 3.5; P = 0.03). CONCLUSIONS: The CTLA-4 polymorphic G allele, probably by interfering with the immune response, may confer susceptibility to ALD and, in homozygous state, to alcoholic cirrhosis.