Fiorella Piemonte

Analysis of glutathione: implication in redox and detoxification

BACKGROUND Glutathione is a ubiquitous thiol-containing tripeptide, which plays a central role in cell biology. It is implicated in the cellular defence against xenobiotics and naturally occurring deleterious compounds, such as free radicals and hydroperoxides. Glutathione status is a highly sensitive indicator of cell functionality and viability. Its levels in human tissues normally range from 0.1 to 10 mM, being most concentrated in liver (up to 10 mM) and in the spleen, kidney, lens, erythrocytes and leukocytes. In humans, GSH depletion is linked to a number of disease states including cancer, neurodegenerative and cardiovascular diseases. The present review proposes an analysis of the current knowledge about the methodologies for measuring glutathione in human biological samples and their feasibility as routine methods in clinical chemistry. Furthermore, it elucidates the fundamental role of glutathione in pathophysiological conditions and its implication in redox and detoxification process. TESTS AVAILABLE Several methods have been optimised in order to identify and quantify glutathione forms in human biological samples. They include spectrophotometric, fluorometric and bioluminometric assays, often applied to HPLC analysis. Recently, a liquid chromatography-mass spectrometry technique for glutathione determination has been developed that, however, suffers from the lack of total automation and the high cost of the equipment. CONCLUSION Glutathione is a critical factor in protecting organisms against toxicity and disease. This review may turn useful for analysing the glutathione homeostasis, whose impairment represents an indicator of tissue oxidative status in human subjects.

Lifestyle intervention and antioxidant therapy in children with nonalcoholic fatty liver disease: A randomized, controlled trial†

No proven treatment exists for nonalcoholic fatty liver disease (NAFLD) in children and adolescents. We sought to determine the efficacy of lifestyle intervention with or without antioxidant therapy in pediatric NAFLD. A total of 53 patients (age 5.7‐18.8 years, 37 boys) were included. Lifestyle intervention consisting of a diet tailored to the patients calorie needs, and increased physical activity was prescribed in all. Patients were concomitantly randomized to alpha‐tocopherol 600 IU/day plus ascorbic acid 500 mg/day (n = 25) or placebo (n = 28), and treated for 24 months. The study was an extension of a previous study aimed at evaluating the effect of 12‐month lifestyle intervention and antioxidant therapy on serum levels of aminotransferases. The primary end point of the present study was change in liver histology on repeated biopsy at 24 months. Secondary end points were changes in body weight, liver enzymes, and insulin sensitivity indices on 2‐hour oral glucose tolerance test. The amount of weight lost at 24 months was similar in the placebo and antioxidant groups (−4.75 [range, −16‐4.0] versus −5.5 [range, −12.2‐0.4] kg, respectively, P = 0.9). A significant improvement occurred in the grade of steatosis, lobular inflammation, and hepatocyte ballooning, and in the NAFLD activity score in both groups. Levels of aminotransferases, triglycerides, cholesterol, fasting glucose, and insulin, and insulin sensitivity indices improved significantly as well. The improvement in all these parameters was not significantly different between the two groups. Conclusion: Lifestyle intervention with diet and increased physical activity induces weight loss and is associated with a significant improvement in liver histology and laboratory abnormalities in pediatric NAFLD. Alpha‐tocopherol plus ascorbic acid does not seem to increase the efficacy of lifestyle intervention alone. (HEPATOLOGY 2008.)

NAFLD in children: A prospective clinical‐pathological study and effect of lifestyle advice

Nonalcoholic fatty liver disease (NAFLD), a common cause of chronic liver disease in adults, is incompletely characterized in children. We conducted a prospective study to better characterize the clinical presentation of NAFLD in children and to determine the effect of lifestyle advice in the management of pediatric NAFLD. From June 2001 to April 2003, 84 children (age 3‐18.8 yr) who had elevated aminotransferases and the diagnosis of NAFLD confirmed via liver biopsy underwent a 2‐hour oral glucose tolerance test and a 12‐month program of lifestyle advice consisting of diet and physical exercise. Thirty‐four (40.5%) patients were obese (body mass index [BMI] >97th percentile), and 43 (51.2%) were overweight (BMI 85th‐97th percentile). Ten (12%) had abnormal glucose tolerance; 10 (12%) had elevated triglycerides, cholesterol, or both; and all had normal blood pressure. Most children (67/84, 80%) were insulin‐resistant, including the 7 children with normal BMI (<85th percentile). Increased liver fibrosis was present in 49 (58.1%) patients and was independently associated with obesity (OR 2.7, 95% CI 1.2‐6.2) and age (1‐year increase; OR 1.2, 95% CI 1.04‐1.5). A 12‐month program with diet and physical exercise resulted in a significant decrease in BMI, and levels of fasting glucose, insulin, lipids, and liver enzymes, as well as liver echogenicity on ultrasonography. In conclusion, children with NAFLD are almost always insulin‐resistant regardless of BMI. Obesity and older age are independently associated with increased liver fibrosis. A simple lifestyle advice program significantly improves insulin resistance, and the liver disease in pediatric NAFLD. (HEPATOLOGY 2006;44:458–465.)

COQ2 Nephropathy: A Newly Described Inherited Mitochondriopathy with Primary Renal Involvement

Primary coenzyme Q(10) (CoQ(10)) deficiency includes a group of rare autosomal recessive disorders primarily characterized by neurological and muscular symptoms. Rarely, glomerular involvement has been reported. The COQ2 gene encodes the para-hydroxybenzoate-polyprenyl-transferase enzyme of the CoQ(10) synthesis pathway. We identified two patients with early-onset glomerular lesions that harbored mutations in the COQ2 gene. The first patient presented with steroid-resistant nephrotic syndrome at the age of 18 months as a result of collapsing glomerulopathy, with no extrarenal symptoms. The second patient presented at five days of life with oliguria, had severe extracapillary proliferation on renal biopsy, rapidly developed end-stage renal disease, and died at the age of 6 months after a course complicated by progressive epileptic encephalopathy. Ultrastructural examination of renal specimens from these cases, as well as from two previously reported patients, showed an increased number of dysmorphic mitochondria in glomerular cells. Biochemical analyses demonstrated decreased activities of respiratory chain complexes [II+III] and decreased CoQ(10) concentrations in skeletal muscle and renal cortex. In conclusion, we suggest that inherited COQ2 mutations cause a primary glomerular disease with renal lesions that vary in severity and are not necessarily associated with neurological signs. COQ2 nephropathy should be suspected when electron microscopy shows an increased number of abnormal mitochondria in podocytes and other glomerular cells.

Mirnome analysis reveals novel molecular determinants in the pathogenesis of diet-induced nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is an emerging disease with a broad spectrum of liver conditions. The complex molecular pathogenesis of NAFLD is still unclear. In this study, we conducted an analysis of microRNA (miRNA) expression profiles in liver of rats made NAFLD by different diets. To this aim, Sprague-Dawley rats were fed ad libitum for 3 months with different diets: standard diet (SD), diet enriched in fats and low in carbohydrates (HFD), SD with high fructose (SD-HF) and diet with high levels of fats and fructose (HFD-HF). Our results demonstrated that the treatment with different dietetic regimens caused a significant increase of the body weight and the alteration of some metabolic parameters compared with control animals, as well as various liver injuries. The miRNAs analysis showed the significant downregulation of three miRNAs (miR-122, miR-451 and miR-27) and the upregulation of miR-200a, miR-200b and miR-429 in HFD, SD-HF and HFD-HF rats. Besides, miR-21 expression was significantly decreased only in fructose-enriched diets. These miRNAs target molecules involved in the control of lipid and carbohydrate metabolism, signal transduction, cytokine and chemokine-mediated signaling pathway and apoptosis. Western blot analysis of PKCδ, LITAF, ALDOLASE-A, p38MAPK, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 showed a diet-induced deregulation of all these proteins. Interestingly, the expression pattern of LITAF, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 might be well explained by the trend of their specific mRNAs, by potentially regulatory miRNAs, or both. In conclusion, we highlight for the first time the potential involvement of novel determinants (miRNAs and proteins) in the molecular pathogenesis of diet-induced NAFLD.

Glutathione in blood of patients with Friedreich's ataxia

Oxidative stress and mitochondrial dysfunction have long been considered to play a role in Friedreichs ataxia, a neurodegenerative disease due to a GAA expansion in a gene coding for a mitochondrial protein (frataxin), implicated in the regulation of iron metabolism. Since glutathione is an important antioxidant whose role has been recently proposed in the pathogenesis of some neurodegenerative diseases, we investigated glutathione metabolism in the blood of 14 patients with Friedreichs ataxia by measuring total, free and protein‐bound glutathione concentrations.

Effect of vitamin E on aminotransferase levels and insulin resistance in children with non‐alcoholic fatty liver disease

Background  Few data are available on the effect of antioxidants in paediatric non‐alcoholic fatty liver disease (NAFLD).

EPI-743 reverses the progression of the pediatric mitochondrial disease—Genetically defined Leigh Syndrome

BACKGROUND Genetically defined Leigh syndrome is a rare, fatal inherited neurodegenerative disorder that predominantly affects children. No treatment is available. EPI-743 is a novel small molecule developed for the treatment of Leigh syndrome and other inherited mitochondrial diseases. In compassionate use cases and in an FDA Expanded Access protocol, children with Leigh syndrome treated with EPI-743 demonstrated objective signs of neurologic and neuromuscular improvement. To confirm these initial findings, a phase 2A open label trial of EPI-743 for children with genetically-confirmed Leigh syndrome was conducted and herein we report the results. METHODS A single arm clinical trial was performed in children with genetically defined Leigh syndrome. Subjects were treated for 6 months with EPI-743 three times daily and all were eligible for a treatment extension phase. The primary objective of the trial was to arrest disease progression as assessed by neuromuscular and quality of life metrics. Results were compared to the reported natural history of the disease. RESULTS Ten consecutive children, ages 1-13 years, were enrolled; they possessed seven different genetic defects. All children exhibited reversal of disease progression regardless of genetic determinant or disease severity. The primary endpoints--Newcastle Pediatric Mitochondrial Disease Scale, the Gross Motor Function Measure, and PedsQL Neuromuscular Module--demonstrated statistically significant improvement (p<0.05). In addition, all children had an improvement of one class on the Movement Disorder-Childhood Rating Scale. No significant drug-related adverse events were recorded. CONCLUSIONS In comparison to the natural history of Leigh syndrome, EPI-743 improves clinical outcomes in children with genetically confirmed Leigh syndrome.

Waist circumference correlates with liver fibrosis in children with non alcoholic steatohepatitis

Objective: Waist circumference is widely accepted as a risk factor for cardiovascular disease and metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) is a feature of the metabolic syndrome. A contribution of metabolic syndrome, and especially of waist circumference, to liver fibrosis in children with NAFLD is strongly suspected. Design: Cross-sectional study. Setting: Department of Hepatogastroenterology and Nutrition, Paediatric Hospital “Bambino Gesù”, Rome, Italy. Patients: 197 consecutive Caucasian children with NAFLD (136 males and 61 females) aged 3–19 years. Main outcome measures: Multivariable logistic regression models were used to examine the contribution of gender, age, body mass index (BMI) and metabolic syndrome components (waist circumference, high-density lipoprotein (HDL)-cholesterol, triglycerides, blood pressure and glucose) to the odds of liver fibrosis as detected by liver biopsy. Results: 92% of the children had BMI ⩾85th percentile and 84% had a waist ⩾90th percentile for gender and age. Ten per cent of the children had metabolic syndrome and 67% had liver fibrosis, mostly of low degree. At multivariable analysis, waist was the only metabolic syndrome component to be associated with liver fibrosis. This was seen both when the components of the metabolic syndrome were coded as dichotomous (odds ratio (OR) = 2.40; 95% confidence interval (CI), 1.04 to 5.54) and continuous (OR = 2.07; 95% CI, 1.43 to 2.98 for a 5 cm increase). In the latter case, age was also associated with the outcome (OR = 0.70; 95% CI, 0.55 to 0.89 for a 1 year increase). Conclusions: Abdominal rather than generalised obesity contributes to liver fibrosis in children with NAFLD. Waist is also the only component of the metabolic syndrome to be associated with fibrosis in these children. Therefore, the presence of abdominal obesity is an additional criterion for the selection of children and adolescents who should undergo extensive investigation, including liver biopsy.

Endotoxin and plasminogen activator inhibitor-1 serum levels associated with nonalcoholic steatohepatitis in children.

Objectives: Recent evidence supports a role for endotoxemia in the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH). We investigated the association between serum levels of endotoxin, proinflammatory molecules, and histology in children with NAFLD. Patients and Methods: A total of 40 children, mean age of 11.9 ± 2.8 years (27 male and 13 female), with biopsy-proven NAFLD were consecutively enrolled. Anthropometrics, blood pressure, and parameters of the metabolic syndrome were collected. Serum levels of endotoxin, plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were measured by an enzyme-linked immunosorbent assay and compared with circulating levels of the same soluble factors in 9 age- and sex-matched normal weight controls (age 11.4 ± 1.7 years; 6 boys and 3 girls). Results: Children with NAFLD had markedly higher serum concentrations of endotoxin (P < 0.01), PAI-1 (P < 0.001), TNF-α, and IL-6 (P < 0.05) than control subjects. Endotoxin (P = 0.002), PAI-1, (P < 0.001), IL-6 (P = 0.002), TNF-α (P = 0.02), and body mass index (P = 0.03) were significantly associated with a NAFLD activity score ≥5 at the univariate analysis. At the stepwise regression analysis, endotoxin (P < 0.0001) and PAI-1 (P = 0.009) were the most significant predictors for NAFLD activity score. Conclusions: Our findings demonstrate that, apart from TNF-α and IL-6, endotoxin and PAI-1 may represent good markers of NASH. They also reinforce the hypothesis that elevated levels of endotoxin may contribute to the progression from NAFLD to NASH.