Lysosomal acid lipase activity and liver fibrosis in the clinical continuum of non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Recent evidence showed a reduced activity of the lysosomal acid lipase (LAL) in patients with non-alcoholic fatty liver disease (NAFLD) and cryptogenic cirrhosis (CC). However, the relationship between LAL activity and liver fibrosis has never been investigated. METHODS: Cross-sectional study including 575 outpatients referred for the management of cardio-metabolic and liver disease. The absence of liver fibrosis was defined by a FIB-4 < 1.30 and NAFLD fibrosis score (NFS) <-1.455. LAL activity was measured with dried blood spot technique. RESULTS: Overall, 515 patients had a diagnosis of NAFLD (454 NAFL and 61 biopsy-proven NASH) and 60 of CC. The value of LAL activity progressively decreased from healthy subjects to NAFL/NASH patients to CC (P < .001). LAL activity was reduced by 10% in patients with NAFL, by 20% in NASH and by 50% in CC. The prevalence of CC decreased across the tertiles of LAL activity: 22.2% in the lowest, 4.6% in the intermediate and 0.5% in the highest tertile. In NAFLD patients, 69.9% had a FIB4 < 1.30, and 43.1% a NFS <-1.455. Multivariate logistic regression analysis showed that Log (LAL activity) was associated with FIB-4 < 1.30 (Odds ratio [OR] 2.19 95% confidence interval [CI] 1.33-3.62, P = .002) and NFS < -1.455 (OR 2.43, 95% CI 1.51-3.91, P < .001) after adjustment for confounding factors. CONCLUSIONS: We found a progressive reduction of LAL activity according to liver disease severity. LAL activity was inversely associated with markers of liver fibrosis in patients with NAFLD.

Frataxin silencing alters microtubule stability in motor neurons: implications for Friedreich's ataxia.

To elucidate the pathogenesis of axonopathy in Friedreich's Ataxia (FRDA), a neurodegenerative disease characterized by axonal retraction, we analyzed the microtubule (MT) dynamics in an in vitro frataxin-silenced neuronal model (shFxn). A typical feature of MTs is their "dynamic instability", in which they undergo phases of growth (polymerization) and shrinkage (depolymerization). MTs play a fundamental role in the physiology of neurons and every perturbation of their dynamicity is highly detrimental for neuronal functions. The aim of this study is to determine whether MTs are S-glutathionylated in shFxn and if the glutathionylation triggers MT dysfunction. We hypothesize that oxidative stress, determined by high GSSG levels, induces axonal retraction by interfering with MT dynamics. We propose a mechanism of the axonopathy in FRDA where GSSG overload and MT de-polymerization are strictly interconnected. Indeed, using a frataxin-silenced neuronal model we show a significant reduction of neurites extension, a shift of tubulin toward the unpolymerized fraction and a consistent increase of glutathione bound to the cytoskeleton. The live cell imaging approach further reveals a significant decrease in MT growth lifetime due to frataxin silencing, which is consistent with the MT destabilization. The in vitro antioxidant treatments trigger the axonal re-growth and the increase in stable MTs in shFxn, thus contributing to identify new neuronal targets of oxidation in this disease and providing a novel approach for antioxidant therapies.

A Relative Deficiency of Lysosomal Acid Lypase Activity Characterizes Non-Alcoholic Fatty Liver Disease.

Lysosomal acid lipase (LAL) is a key enzyme in lipid metabolism. Initial reports have suggested a role for a relative acquired LAL deficiency in non-alcoholic fatty liver disease (NAFLD)-however, it is still unclear whether this mechanism is specific for NAFLD. We aimed to determine LAL activity in a cohort of NAFLD subjects and in a control group of hepatitis C virus (HCV)-infected patients, investigating the role of liver cirrhosis. A total of 81 patients with a diagnosis of NAFLD, and 78 matched controls with HCV-related liver disease were enrolled. For each patient, LAL activity was determined on peripheral dried blood spots (DBS) and correlated with clinical and laboratory data. A subgroup analysis among cirrhotic patients was also performed. LAL activity is significantly reduced in NAFLD, compared to that in HCV patients. This finding is particularly evident in the pre-cirrhotic stage of disease. LAL activity is also correlated with platelet and white blood cell count, suggesting an analytic interference of portal-hypertension-induced pancytopenia on DBS-determined LAL activity. NAFLD is characterized by a specific deficit in LAL activity, suggesting a pathogenetic role of LAL. We propose that future studies on this topic should rely on tissue specific analyses, as peripheral blood tests are also influenced by confounding factors.

Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis.

Does Lysosomial Acid Lipase Reduction Play a Role in Adult Non-Alcoholic Fatty Liver Disease?

Lysosomal Acid Lipase (LAL) is a key enzyme involved in lipid metabolism, responsible for hydrolysing the cholesteryl esters and triglycerides. Wolman Disease represents the early onset phenotype of LAL deficiency rapidly leading to death. Cholesterol Ester Storage Disease is a late onset phenotype that occurs with fatty liver, elevated aminotransferase levels, hepatomegaly and dyslipidaemia, the latter characterized by elevated LDL-C and low HDL-C. The natural history and the clinical manifestations of the LAL deficiency in adults are not well defined, and the diagnosis is often incidental. LAL deficiency has been suggested as an under-recognized cause of dyslipidaemia and fatty liver. Therefore, LAL activity may be reduced also in non-obese patients presenting non-alcoholic fatty liver disease (NAFLD), unexplained persistently elevated liver transaminases or with elevation in LDL cholesterol. In these patients, it could be indicated to test LAL activity. So far, very few studies have been performed to assess LAL activity in representative samples of normal subjects or patients with NAFLD. Moreover, no large study has been carried out in adult subjects with NAFLD or cryptogenic cirrhosis.

Glutathione metabolism in cobalamin deficiency type C (cblC).

BACKGROUND: Methylmalonic aciduria with homocystinuria, cblC defect, is the most frequent disorder of vitamin B12 metabolism. CblC patients are commonly treated with a multidrug therapy to reduce metabolite accumulation and to increase deficient substrates. However the long-term outcome is often unsatisfactory especially in patients with early onset, with frequent progression of neurological and ocular impairment. Recent studies, have shown perturbation of cellular redox status in cblC. To evaluate the potential contribution of oxidative stress into the patophysiology of cblC defect, we have analyzed the in vivo glutathione metabolism in a large series of cblC deficient individuals. METHODS: Levels of different forms of glutathione were measured in lymphocytes obtained from 18 cblC patients and compared with age-matched controls. Furthermore, we also analyzed plasma cysteine and total homocysteine. RESULTS: We found an imbalance of glutathione metabolism in cblC patients with a significant decrease of total and reduced glutathione, along with a significant increase of different oxidized glutathione forms. CONCLUSIONS: These findings show a relevant in vivo disturbance of glutathione metabolism underlining the contribution of glutathione pool depletion to the redox imbalance in treated cblC patients. Our study may be helpful in addressing future research to better understanding the pathogenetic mechanism of the disease and in developing new therapeutic approaches, including the use of novel vitamin B12 derivatives.

Riboflavin transporter 3 involvement in infantile Brown-Vialetto-Van Laere disease: two novel mutations.

BACKGROUND: Brown-Vialetto-Van Laere (BVVL) syndrome is a rare disorder characterised by progressive pontobulbar palsy and sensorineural deafness. Causative mutations in genes encoding human riboflavin transporter 2 (hRFT2) and 3 (hRFT3) have been identified in BVVL patients. METHODS AND RESULTS: We report the clinical and molecular features of a severe BVVL patient in whom screening of SLC52A3/hRFT2 was negative. Sequence analysis identified two novel compound heterozygous mutations in SLC52A2/hRFT3, namely c.155C>T and c.1255G>A, leading to the amino acid changes p.S52F and p.G419S, respectively. Functional studies show that these defects impair the gene expression of the corresponding transporter, resulting in a significant reduction of riboflavin transport. CONCLUSIONS: These findings support the pathogenetic role of SLC52A2/hRFT3 in BVVL with important clinical and therapeutic implications.

Frataxin silencing inactivates mitochondrial Complex I in NSC34 motoneuronal cells and alters glutathione homeostasis.

Friedreich's ataxia (FRDA) is a hereditary neurodegenerative disease characterized by a reduced synthesis of the mitochondrial iron chaperon protein frataxin as a result of a large GAA triplet-repeat expansion within the first intron of the frataxin gene. Despite neurodegeneration being the prominent feature of this pathology involving both the central and the peripheral nervous system, information on the impact of frataxin deficiency in neurons is scant. Here, we describe a neuronal model displaying some major biochemical and morphological features of FRDA. By silencing the mouse NSC34 motor neurons for the frataxin gene with shRNA lentiviral vectors, we generated two cell lines with 40% and 70% residual amounts of frataxin, respectively. Frataxin-deficient cells showed a specific inhibition of mitochondrial Complex I (CI) activity already at 70% residual frataxin levels, whereas the glutathione imbalance progressively increased after silencing. These biochemical defects were associated with the inhibition of cell proliferation and morphological changes at the axonal compartment, both depending on the frataxin amount. Interestingly, at 70% residual frataxin levels, the in vivo treatment with the reduced glutathione revealed a partial rescue of cell proliferation. Thus, NSC34 frataxin silenced cells could be a suitable model to study the effect of frataxin deficiency in neurons and highlight glutathione as a potential beneficial therapeutic target for FRDA.

Glutathione imbalance in patients with X-linked adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder of X-linked inheritance caused by a mutation in the ABCD1 gene which determines an accumulation of long-chain fatty acids in plasma and tissues. Recent evidence shows that oxidative stress may be a hallmark in the pathogenesis of X-ALD and glutathione plays an important role in the defense against free radicals. In this study we have analyzed glutathione homeostasis in lymphocytes of 14 patients with X-ALD and evaluated the balance between oxidized and reduced forms of glutathione, in order to define the role of this crucial redox marker in this condition. METHODS: Lymphocytes, plasma and erythrocytes were obtained from the whole blood of 14 subjects with X-ALD and in 30 healthy subjects. Total, reduced and protein-bound glutathione levels were measured in lymphocytes by HPLC analysis. Erythrocyte free glutathione and antioxidant enzyme activities, plasma thiols and carbonyl content were determined by spectrophotometric assays. RESULTS: A significant decrease of total and reduced glutathione was found in lymphocytes of patients, associated to high levels of all oxidized glutathione forms. A decline of free glutathione was particularly significant in erythrocytes. The increased oxidative stress in X-ALD was additionally confirmed by the decrease of plasma thiols and the high level of carbonyls. CONCLUSION: Our results strongly support a role for oxidative stress in the pathophysiology of X-ALD and strengthen the importance of the balance among glutathione forms as a hallmark and a potential biomarker of the disease.

Glutathione: a redox signature in monitoring EPI-743 therapy in children with mitochondrial encephalomyopathies.

BACKGROUND: Genetically defined Leigh syndrome (LS) is a rare, fatal inherited neurodegenerative disorder that predominantly affects children. Although mitochondrial dysfunction has clearly been associated with oxidative stress, few studies have specifically examined Leigh syndrome patients' blood glutathione levels. In this study, we analyzed the balance between oxidized and reduced glutathione in lymphocytes of 10 patients with genetically confirmed LS and monitored the effects of glutathione status following 6 months of treatment with EPI-743, a novel redox therapeutic. METHODS: Lymphocytes were obtained from blood samples of 10 children with a genetically confirmed diagnosis of LS and in 20 healthy subjects. Total, reduced, oxidized and protein-bound glutathione levels were determined by HPLC analysis. Erythrocyte superoxide dismutase and glutathione peroxidase enzyme activities were measured by spectrophotometric assays. Plasma total thiols, carbonyl contents and malondialdehyde were assessed by spectrophotometric and fluorometric assays. RESULTS: A significant impairment of all glutathione forms was detected in patients, including a profound decrease of total and reduced glutathione (GSH) associated with high levels of all oxidized glutathione forms (GSSG+GS-Pro; OX). These findings negatively correlated with the glutathione peroxidase activity, which underwent a significant decrease in patients. After treatment with EPI-743, all patients showed a significant increase in reduced glutathione levels and 96% decrease of OX/GSH ratio. CONCLUSIONS: The data presented here strongly support glutathione as a "redox blood signature" in mitochondrial disorders and its use as a clinical trial endpoint in the development of mitochondrial disease therapies.

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients.

Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

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.

All glutathione forms are depleted in blood of obese and type 1 diabetic children.

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of type 1 diabetes (T1D), where an increase in reactive oxygen species may contribute to the initial destruction of ß-cells. Accumulating evidence also suggests a role for oxidative stress in obesity, where it may potentiate the development of complications. OBJECTIVE: To analyze the in vivo homeostasis of glutathione in children with T1D at onset and in children who are obese, to evaluate the systemic content of all glutathione forms (total, reduced, oxidized, and protein-bound glutathione) and the balance among them. Moreover, since glutathione bound to hemoglobin is a clinical marker of oxidative stress in human blood, we analyzed glutathionyl-hemoglobin in T1D and in obese children. SUBJECTS: Children with T1D at onset (n = 30) or obesity (n = 30) at the first observation, and 30 healthy subjects chosen from the children who attended the outpatient clinic for minor problems. METHODS: We assessed circulating levels of various glutathione forms by performing reverse-phase high performance liquid chromatography. Glutathionyl-hemoglobin analysis was carried out by cation-exchange chromatography. RESULTS: In children with T1D and in obese children, we found a significant decrease of all glutathione forms including, for the first time, the content of total glutathione and glutathionylated proteins. The comparison among forms shows no significant imbalance in T1D patients, whereas in obese children it seems to suggest an attempt to rebalance the glutathione system homeostasis. CONCLUSIONS: Our findings consistently show in vivo evidence of glutathione depletion upon early onset of T1D and in obese children, thus evidencing glutathione as an early marker in these two metabolic conditions.

Emodin prevents intrahepatic fat accumulation, inflammation and redox status imbalance during diet-induced hepatosteatosis in rats.

High-fat and/or high-carbohydrate diets may predispose to several metabolic disturbances including liver fatty infiltration (hepatosteatosis) or be associated with necro-inflammation and fibrosis (steatohepatitis). Several studies have emphasized the hepatoprotective effect of some natural agents. In this study, we investigated the potential therapeutic effects of the treatment with emodin, an anthraquinone derivative with anti-oxidant and anti-cancer abilities, in rats developing diet-induced hepatosteatosis and steatohepatitis. Sprague-Dawley rats were fed a standard diet (SD) for 15 weeks, or a high-fat/high-fructose diet (HFD/HF). After 5 weeks, emodin was added to the drinking water of some of the SD and HFD/HF rats. The experiment ended after an additional 10 weeks. Emodin-treated HFD/HF rats were protected from hepatosteatosis and metabolic derangements usually observed in HFD/HF animals. Furthermore, emodin exerted anti-inflammatory activity by inhibiting the HFD/HF-induced increase of tumor necrosis factor (TNF)-α. Emodin also affected the hepatocytes glutathione homeostasis and levels of the HFD/HF-induced increase of glutathionylated/phosphorylated phosphatase and tensin homolog (PTEN). In conclusion, we demonstrated that a natural agent such as emodin can prevent hepatosteatosis, preserving liver from pro-inflammatory and pro-oxidant damage caused by HFD/HF diet. These findings are promising, proposing emodin as a possible hindrance to progression of hepatosteatosis into steatohepatitis.

High Compliance to Mediterranean Diet Associates with Lower Platelet Activation and Liver Collagen Deposition in Patients with Nonalcoholic Fatty Liver Disease.

The Mediterranean diet (Med-Diet) is considered the most effective dietary patterns to obtain weight loss in NAFLD patients. Previous evidence suggested that Med-Diet adherence could reduce cardiovascular risk and have a beneficial effect on NAFLD severity. Aim of the study was to investigate the relationship between Med-Diet adherence, platelet activation (PA), and liver collagen deposition. The study was performed in 655 consecutive NAFLD outpatients from the PLINIO study, a prospective observational cohort study aimed to identify non-conventional predictors of liver fibrosis progression in NAFLD. PA was measured by the serum thromboxane B2 (TxB2), and liver collagen deposition by N-terminal propeptide of type III collagen (Pro-C3). Adherence to the Med-diet was investigated by a short nine-item validated dietary questionnaire. Patients with high Med-Diet adherence were older and had less metabolic syndrome and lower serum triglycerides, GGT, TxB2, and Pro-C3. At multivariate regression analyses, in the linear model, the Med-Diet score negatively correlated with both TxB2 (Beta = −0.106; p = 0.009) and Pro-C3 (Beta = −0.121; p = 0.002) and in the logistic model high adherence inversely correlated with higher TxB2 tertiles (II tertile: OR = 0.576, p = 0.044; III tertile: OR = 0.556, p = 0.026) and Pro-C3 tertile (III tertile: OR = 0.488, p = 0.013). Low consumption of red meat inversely correlated with higher TxB2 tertile (II tertile: OR = 0.448, p < 0.001, III tertile: OR = 0.567, p = 0.004). In conclusion, NAFLD patients with high adherence to the Med-Diet show lower PA and liver collagen deposition, suggesting a protective role of the Med-Diet against NAFLD progression and cardiovascular risk. In addition, the correlation between TxB2 and Pro-C3 suggests a link between NAFLD severity and cardiovascular risk.

Novel large-range mitochondrial DNA deletions and fatal multisystemic disorder with prominent hepatopathy.

Hepatic involvement in mitochondrial cytopathies rarely manifests in adulthood, but is a common feature in children. Multiple OXPHOS enzyme defects in children with liver involvement are often associated with dramatically reduced amounts of mtDNA. We investigated two novel large scale deletions in two infants with a multisystem disorder and prominent hepatopathy. Amount of mtDNA deletions and protein content were measured in different post-mortem tissues. The highest levels of deleted mtDNA were in liver, kidney, pancreas of both patients. Moreover, mtDNA deletions were detected in cultured skin fibroblasts in both patients and in blood of one during life. Biochemical analysis showed impairment of mainly complex I enzyme activity. Patients manifesting multisystem disorders in childhood may harbour rare mtDNA deletions in multiple tissues. For these patients, less invasive blood specimens or cultured fibroblasts can be used for molecular diagnosis. Our data further expand the array of deletions in the mitochondrial genomes in association with liver failure. Thus analysis of mtDNA should be considered in the diagnosis of childhood-onset hepatopathies.

Effect of a common missense variant in LIPA gene on fatty liver disease and lipid phenotype: New perspectives from a single-center observational study.

Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive disease characterized by hypoalphalipoproteinemia, mixed hyperlipemia, and fatty liver (FL) due to mutations in LIPAse A, lysosomal acid type (LIPA) gene. The rs1051338 single-nucleotide polymorphism (SNP) in LIPA gene, in vitro, could adversely affect the LAL activity (LAL-A). Nonalcoholic fatty liver disease (NAFLD) is often associated with metabolic syndrome, and the diagnosis requires the exclusion of excess of alcohol intake and other causes of hepatic disease. The aim of the study was to evaluate the impact of rs1051338 rare allele on lipid phenotype, severity of FL, and LAL-A in patients suffering from dyslipidemia associated with NAFLD. We selected 74 subjects with hypoalphalipoproteinemia or mixed hyperlipemia and evaluated transaminases, liver assessment with controlled attenuation parameter (CAP), LAL-A, rs1051338 SNP genotype. The presence of rare allele caused higher levels of triglycerides and hepatic transaminase and lower levels of high-density lipoprotein cholesterol (HDL-C). Multivariate analysis highlighted independent association between rare allele and FL severity in subjects with NAFLD. The rs1051338 SNP may modulate FL severity and atherogenic dyslipidemia in patients suffering from NAFLD.

Antioxidant activity of Hydroxytyrosol and Vitamin E reduces systemic inflammation in children with paediatric NAFLD.

BACKGROUND: The rise in paediatric non-alcoholic fatty liver disease (NAFLD) is particularly alarming. We recently reported that Hydroxytyrosol (HXT) and Vitamin E (VitE) may improve oxidative stress, insulin resistance, and steatosis in children with biopsy-proven NAFLD. AIM: Here, we investigated if HXT+VitE may reduce systemic inflammation in the above-mentioned patients. METHODS: This study analysed the plasma levels of IL (interleukin)-6, IL-1ß, IL-10, tumour necrosis factor (TNF)-α, 4­hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2'deoxyguanosine (8-OHdG) in children enrolled in the HXT+VitE trial (ClinicalTrials.gov, NCT02842567). RESULTS: Changes in markers of systemic inflammation were found in both placebo (Pla) and HXT+VitE. In particular, after four months, the levels of IL-1ß and TNF-α were reduced in both groups, while IL-6 decreased, and IL-10 increased significantly only in the group treated with HXT+VitE. Children treated with HXT+VitE showed a significant decrease of 4-HNE and 8-OHdG that correlated with the improvement of triglyceride levels. Noticeably, only the 8-OHdG decrease correlated with steatosis amelioration and with the increase of IL-10 levels. CONCLUSION: The treatment with HXT and VitE reduced the NAFLD-related systemic inflammation in children, mainly by an increase of IL-10 circulating levels that occurred in response to DNA damage recovery, ultimately improving steatosis and hypertriglyceridemia.

Reduced Lysosomal Acid Lipase Activity in Blood and Platelets Is Associated With Nonalcoholic Fatty Liver Disease.

OBJECTIVES: To investigate whether blood total lysosomal acid lipase activity (BT-LAL) levels are uniquely associated with the noncirrhotic and cirrhotic stages of nonalcoholic fatty liver disease (NAFLD) and with protection from NAFLD in metabolically/genetically predisposed subjects and a normal liver. To clarify which enzyme-carrying circulating cells are involved in reduced BT-LAL of NAFLD. METHODS: In a cross-sectional study, BT-LAL was measured by a fluorigenic method in patients with NAFLD (n = 118), alcoholic (n = 116), and hepatitis C virus-related disease (n = 49), in 103 controls with normal liver and in 58 liver transplant recipients. Intracellular platelet and leukocyte LAL was measured in 14 controls and 28 patients with NAFLD. RESULTS: Compared with controls, (i) BT-LAL and LAL in platelets, but not in leukocytes, were progressively reduced in noncirrhotic NAFLD and in nonalcoholic steatohepatitis-related cirrhosis; (ii) platelet and leukocyte counts did not differ in patients with noncirrhotic NAFLD; and (iii) BT-LAL did not differ in alcoholic and hepatitis C virus noncirrhotic patients. BT-LAL progressively increased in controls with metabolic syndrome features according to their PNPLA3 rs738409 steatosis-associated variant status (II vs IM vs MM), and their BT-LAL was higher than that of noncirrhotic NAFLD, only when carriers of the PNPLA3 unfavorable alleles were considered. Liver transplant recipients with de novo NAFLD compared with those without de novo NAFLD had lower BT-LAL. DISCUSSION: LAL in blood and platelets is progressively and uniquely reduced in NAFLD according to disease severity. High BT-LAL is associated with protection from NAFLD occurrence in subjects with metabolic and genetic predisposition. Low LAL in platelets and blood could play a pathogenetic role in NAFLD.

Assaying ATP synthesis in cultured cells: a valuable tool for the diagnosis of patients with mitochondrial disorders.

Mitochondrial ATP synthase plays a central role in cell function by synthesising most of the ATP in human tissues. In different cells, active regulation of mitochondrial ATP synthase in response to cellular energy demand has been demonstrated, as well as its alteration under several pathological conditions affecting oxidative phosphorylation (OXPHOS). Traditionally, detection of OXPHOS defects is based on the spectrophotometric measurement of respiratory chain complex activities in muscle biopsies. Considering the broad clinical spectrum of mitochondrial disorders, and the difficulty in arriving at a single diagnostic method, in this study we propose measurement of ATP synthesis in mitochondria from skin fibroblasts as an effective screening tool. In the light of our results this assessment emerges as a useful marker of impaired energy production in primary OXPHOS disorders of childhood and as a tool with the potential to drive further molecular genetic studies.