Dariusz Marek Lebensztejn

Omega-3 fatty acids for treatment of non-alcoholic fatty liver disease: design and rationale of randomized controlled trial

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome since obesity and insulin resistance are the main pathogenic contributors for both conditions. NAFLD carries increased risk of atherosclerosis and cardiovascular diseases. There is an urgent need to find effective and safe therapy for children and adults with NAFLD. Data from research and clinical studies suggest that omega-3 fatty acids may be beneficial in metabolic syndrome-related conditions and can reduce the risk of cardiovascular disease.Methods/designWe are conducting a randomized, multicenter, double-blind, placebo-controlled trial of treatment with omega-3 fatty acids in children with NAFLD. Patients are randomized to receive either omega-3 fatty acids containing docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) or placebo for 24 weeks. The dose of omega-3 (DHA+ EPA) ranges from 450 to 1300 mg daily. Low calorie diet and increased physical activity are advised and monitored using validated questionnaires. The primary outcome of the trial is the number of patients who decreased ALT activity by ≥ 0,3 of upper limit of normal. The main secondary outcomes are improvement in the laboratory liver tests, liver steatosis on ultrasound, markers of insulin resistance and difference in fat/lean body mass composition after 6 months of intervention.DiscussionPotential efficacy of omega-3 fatty acids in the treatment of NAFLD will provide needed rationale for use of this safe diet supplement together with weight reduction therapy in the growing population of children with NAFLD.Trial registrationNCT01547910

Omega-3 Fatty Acids Therapy in Children with Nonalcoholic Fatty Liver Disease: A Randomized Controlled Trial

OBJECTIVE To evaluate the efficacy and safety of omega-3 fatty acid supplementation in children with nonalcoholic fatty liver disease (NAFLD). STUDY DESIGN Overweight/obese children with NAFLD (n = 76; median age, 13 years; IQR, 11.1-15.2 years) were eligible to participate in the study. The diagnosis of NAFLD was based on elevated alanine aminotransferase (ALT) to ≥ 30% of the upper limit of normal (ULN) and liver hyperechogenicity on ultrasound. Patients were randomized to receive omega-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid, 450-1300 mg/day) or placebo (omega-6 sunflower oil). The primary outcome was the number of patients who demonstrated decreased ALT activity by ≥ 0.3 times the ULN. Secondary outcomes included alterations in liver function tests, liver hyperechogenicity, insulin resistance, and other metabolic markers after 6 months of intervention. RESULTS Out of 76 enrolled patients, 64 completed the trial and were analyzed. After 6 months, we found no significant differences between the omega-3 and placebo groups in the number of patients with decreased ALT by ≥ 0.3 times the ULN (24 vs 23) or in median (IQR) ALT activity (48.5 [31-62] U/L vs 39 [27-55] U/L), liver hyperechogenicity, insulin resistance, or serum lipid levels. However, patients in the omega-3 group had lower levels of aspartate aminotransferase (28 [25-36] U/L vs 39 [27-55] U/L; P = .04) and gamma-glutamyl transpeptidase (26 [17.5-36.5] U/L vs 35 [22-52] U/L; P = .04), and significantly higher levels of adiponectin. CONCLUSION Omega-3 fatty acid supplementation did not increase the number of patients with decreased ALT levels and it did not affect liver steatosis on ultrasound, but it improved aspartate aminotransferase and gamma-glutamyl transpeptidase levels in children with NAFLD compared with placebo. TRIAL REGISTRATION Registered with ClinicalTrials.gov: NCT01547910.

Serum concentration of adiponectin, leptin and resistin in obese children with non-alcoholic fatty liver disease

PURPOSE Obesity, insulin resistance and dyslipidemia are the most significant risk factors of non-alcoholic fatty liver disease (NAFLD) but the role of adipokines in patomechanism of this disease is not clear. The aim of the study was to evaluate the serum levels of leptin, adiponectin and resistin in obese children with NAFLD. MATERIAL/METHODS The fasting serum levels of adipokines were determined in 44 consecutive obese children with suspected liver disease and in 24 lean controls. The degree of the ultrasound liver steatosis was graded according to Saverymuttu. RESULTS The fatty liver was confirmed in 33 children by ultrasonography (16 of them also showed an increased ALT activity). The serum leptin level was significantly higher and adiponectin level was lower in the obese children with NAFLD when compared to controls. Only adiponectin correlated with homeostasis model assessment of insulin resistance (HOMA-IR). Significant negative correlations were found between the ultrasonographic grades of liver steatosis and adiponectin and resistin levels. Serum adiponectin and resistin levels were lower in children with an advanced liver steatosis (grade 3, n=10) compared to patients with a mild steatosis (grade 1-2, n=23). The ability of serum adiponectin and resistin to differentiate children with an advanced liver steatosis from those with mild steatosis was significant. CONCLUSIONS These data suggest a role of both adiponectin and resistin in the pathogenesis of NAFLD in obese children and confirm the association between adiponectin and insulin resistance. Adiponectin and resistin may be suitable serum markers in predicting an advanced liver steatosis in children with NAFLD.

Serum fibrosis markers as predictors of an antifibrotic effect of interferon alfa in children with chronic hepatitis B.

Objective Interferon alfa (IFN-&agr;) may retard hepatic fibrogenesis in adults with chronic hepatitis C. We evaluated prospectively four selected serum fibrosis markers before, immediately after and 12 months after IFN treatment of children with chronic hepatitis B (CHB). Methods Forty-seven children (mean age 8 years, range 4–16) with CHB underwent IFN-&agr; treatment (3 MU t.i.w.) for 5 months. Fibrosis and inflammation were assessed blindly before and 12 months after the end of treatment. Serum laminin-2, collagen IV, MMP-2 and MMP-9/TIMP-1 complex were determined using automated assays. Results Twelve months after treatment had been discontinued levels of laminin-2, collagen IV and MMP-2 were decreased, and serum MMP-9/TIMP-1 complex was increased. Levels did not differ between sustained responders (42.5%) and non-responders. Similarly, fibrosis did not progress in both groups, whereas histological inflammation improved only in responders. Conclusions A 5 month IFN-&agr; treatment has no marked effect on histological liver fibrosis in children with CHB, irrespective of virological response. The evolution of serum fibrosis markers suggests they may be more sensitive to detect minor antifibrotic effects than semiquantitative follow-up histology.

Ultrastructure of hepatocyte mitochondria in nonalcoholic steatohepatitis in pediatric patients: Usefulness of electron microscopy in the diagnosis of the disease

Ultrastructure of hepatocyte mitochondria in nonalcoholic steatohepatitis in pediatric patients: usefulness of electron microscopy in the diagnosis of the disease

Current drugs in early development for treating hepatitis C virus-related hepatic fibrosis

Introduction: More than 100 million people worldwide are infected with hepatitis C virus (HCV), which is responsible for chronic liver disease accompanied by progressive fibrosis of hepatic tissue, often leading to liver cirrhosis. Novel therapeutic options are able to clear the virus in almost all diagnosed patients. However, even after successful treatment, hepatic fibrosis may persist in many of them. There is no registered therapy specific for liver fibrosis, but numerous molecules are currently in development. Areas covered: In this review, the authors look at drugs in early development for the treatment of HCV-related hepatic fibrosis. Their mechanism of action is based on the inhibition of hepatic inflammation, the modulation of cellular sources of extracellular matrix (ECM), the stimulation of ECM degradation and prevention of collagen crosslinking. Importantly, significant antifibrotic effects have been demonstrated with both IFN-based and IFN-free anti-HCV regimens. Expert opinion: Successful future therapy is likely to be based on sequential administration of drugs leading initially to HCV clearance, followed by treatment for the possible reversal of liver fibrosis. The primary consideration with clinical trials carried out in patients with advanced liver disease is safety. Indeed, the evaluation of anti-fibrotic therapy depends on reliable noninvasive techniques for quantification of liver fibrosis, such as transient or shear-wave elastography or serologic tests which are able to replace liver biopsy.

Pediatric non-alcoholic steatohepatitis: The first report on the ultrastructure of hepatocyte mitochondria

AIM To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric non-alcoholic steatohepatitis (NASH). METHODS Ultrastructural investigations were conducted on biopsy liver specimens obtained from 10 children (6 boys and 4 girls) aged 2-14 years with previously clinicopathologically diagnosed NASH. The disease was diagnosed if liver biopsy revealed steatosis, inflammation, ballooned hepatocytes, Mallory hyaline, or focal necrosis, varying degrees of fibrosis in the absence of clinical, serological, or histological findings of infectious liver diseases, autoimmune hepatitis, metabolic liver diseases, or celiac disease. For ultrastructural analysis, fresh small liver blocks (1 mm(3) volume) were fixed in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer. The specimens were postfixed in osmium tetroxide, subsequently dehydrated through a graded series of ethanols and propylene oxide, and embedded in Epon 812. The material was sectioned on a Reichert ultramicrotome to obtain semithin sections, which were stained with methylene blue in sodium borate. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined using an Opton EM 900 transmission electron microscope. RESULTS Ultrastructural analysis of bioptates obtained from children with non-alcoholic steatohepatitis revealed characteristic repetitive mitochondrial abnormalities within hepatocytes; mainly mitochondrial polymorphisms such as megamitochondria, loss of mitochondrial cristae, and the presence of linear crystalline inclusions within the mitochondrial matrix of an increased electron density. The crystalline inclusions were particularly evident within megamitochondria (MMC), which seemed to be distributed randomly both within the hepatic parenchymal cell and the zones of hepatic lobule, without special variations in abundance. The inclusions appeared as bundles viewed longitudinally, or as an evenly spaced matrix in cross section, and frequently caused mitochondrial deformation. The average diameter of these linear structures was 10 nm and the average space between them 20 nm. Sometimes enlarged intramitochondrial granules were seen in their vicinity. Foamy cytoplasm of hepatocytes was found, resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. The perivascular space of Disse was frequently dilated, and contained transitional hepatic stellate cells, as well as mature and/or newly forming collagen fiber bundles. CONCLUSION Marked ultrastructural abnormalities observed in hepatocyte mitochondria, especially their polymorphism in the form of MMC and loss of mitochondrial cristae, accompanied by foamy cytoplasm, clearly indicate a major role of these organelles in the morphogenesis of pediatric NASH. Our findings seem to prove the high effectiveness of electron microscopy in the diagnosis of the disease.

Chemerin as a novel non-invasive serum marker of intrahepatic lipid content in obese children

BackgroundEctopic hepatic lipid accumulation is closely related to the development of insulin resistance, which is regarded as one of the most significant risk factors of non-alcoholic fatty liver disease (NAFLD). The current study has shown that fat tissue constitutes an important endocrine organ with its own production and metabolism of many biologically active substances, among which adipokines play an important role. Classic adipokines (e.g. leptin, adiponectin, resistin) are fat-derived hormones which serum level is altered in patients with NAFLD. The role of novel adipokines in the pathomechanism of this disease is not clear. Therefore, the aim of our study was to evaluate the serum concentrations of chemerin, omentin and vaspin in obese children with NAFLD.MethodsForty-five obese children, aged 7-17 years old, were admitted to our Department with suspected liver disease (hepatomegaly, and/or ultrasonographic liver brightness, and/or increased ALT activity). Viral hepatitides, as well as autoimmune and metabolic liver diseases were excluded. Fasting serum levels of chemerin, omentin and vaspin were determined. The grade of liver steatosis in ultrasound was graded according to Saverymuttu. 1HMR spectroscopy was performed with a 1.5 T scanner and with PRESS sequencing.ResultsFatty liver was confirmed in 39 children by ultrasound and in 33 patients by 1HMRS (19 of them also had increased ALT activity /NAFLD/). Chemerin and vaspin levels were significantly higher in children with NAFLD compared to the control group (n = 30). The concentration of chemerin was significantly higher in children with advanced liver steatosis compared to non-hepatopathic patients (p = 0,02). Significant positive correlations were found between the total liver lipids in 1HMRS and chemerin (r = 0,33; p = 0,02) and vaspin (r = 0,4; p = 0,006). The ability of serum chemerin (cut-off = 190 ng/ml, Se = 75%, Sp = 58%) to differentiate children with fatty liver in 1HMRS from those without steatosis was significant (AUC = 0,7, p = 0,04). Omentin and vaspin did not allow a useful prediction to be made.ConclusionChemerin seems to be the most suitable non-invasive biomarker in predicting both intrahepatic lipid content in obese children and advanced liver steatosis in children with NAFLD.

Serum fetuin A concentration is elevated in children with non-alcoholic fatty liver disease

PURPOSE To assess the serum fetuin A concentration as a potential marker of subclinical atherosclerosis in obese children with NAFLD. MATERIAL/METHODS A prospective analysis of 45 obese children initially diagnosed with liver pathology (elevated serum ALT activity and/or ultrasonographic liver brightness and/or hepatomegaly) was conducted. The diagnosis of NAFLD was established in the children with elevated serum ALT activity and liver steatosis on ultrasound examination. Viral hepatitis, autoimmune, metabolic liver diseases (Wilson disease, alpha-1-antitrypsin deficiency, cystic fibrosis) and drug and toxin-induced liver injury were excluded in all children. The degree of liver steatosis was graded according to Saverymuttu scale and the total liver lipids concentration was assessed using proton magnetic resonance spectroscopy ((1)H MRS). RESULTS Serum fetuin A concentration was significantly higher in examined children compared to the control group (n=30) (p=0.00002). Higher serum fetuin A concentration was also observed in children with NAFLD (n=19) in comparison to the controls (p=0.000026). Additionally, higher BMI values, waist circumferences, ALT and GGT activity, intensity of liver steatosis on ultrasound and total concentration of lipids in the liver in (1)H MRS were found in children with NAFLD compared to the rest of the examined obese patients (n=26). There was not found any correlation of the investigated glycoprotein with any other assessed parameters both in children with NAFLD and obese children without NAFLD. CONCLUSION Higher serum fetuin A concentration found in children with NAFLD compared to the control group support the hypothesis that atherosclerotic processes may develop faster in hepatopatic obese patients.

Tumor necrosis factor alpha and its soluble receptors in obese children with NAFLD.

PURPOSE To evaluate the serum levels of tumor necrosis factor (TNF) alpha and its soluble receptors in obese children with non-alcoholic fatty liver disease (NAFLD). MATERIAL/METHODS Fasting serum levels of TNFalpha and its receptors were determined in 45 consecutive obese children with suspected liver disease and 20 lean controls. The degree of liver steatosis was graded in ultrasound according to Saverymuttu. 1H-MR spectroscopy was performed with 1.5T scanner with PRESS sequence. RESULTS A fatty liver was confrmed in 32 children by ultrasonography (group I); 16 of them also had increased ALT activity (group Ia - NAFLD). Serum concentrations of TNFalpha and its receptors were significantly higher in obese children with NAFLD compared to controls. Significant correlation was found between ultrasonographic grade of liver steatosis and TNFalpha level but serum level of this adipokine was not significantly different in children with advanced liver steatosis (grade 2-3, n=13) compared to patients with mild steatosis (grade 1, n=19). The ability of TNFalpha and its receptors (R1, R2) to differentiate children with advanced liver steatosis from those with mild steatosis was insignificant. However, the ability of serum TNFalpha to differentiate obese children with liver steatosis from those without steatosis was significant (AUC=0.7448, p=0.0291). CONCLUSION Although TNFalpha does not predict advanced liver steatosis, it may be suitable serum marker in predicting liver steatosis in obese children.