Increased cytokine levels induced by high phenylalanine concentrations in late diagnosis PKU patients compared to early diagnosis: Anti-inflammatory effect of L-carnitine.

Phenylketonuria (PKU) was the first genetic disease to have an effective therapy, which consists of phenylalanine intake restriction. However, there are patients who do not adhere to treatment and/or are not submitted to neonatal screening. PKU patients present L-carnitine (L-car) deficiency, compound that has demonstrated an antioxidant and anti-inflammatory role in metabolic diseases. This study evaluated the effect caused by exposure time to high Phe levels in PKU patients at early and late diagnosis, through pro- and anti-inflammatory cytokines, as well as the L-car effect in patients under treatment. It was observed that there was a decrease in phenylalanine levels in treated patients compared to patients at diagnosis, and an increase in L-car levels in the patients under treatment. Inverse correlation between Phe versus L-car and nitrate plus nitrite versus L-car in PKU patients was also showed. We found increased proinflammatory cytokines levels: interleukin (IL)-1ß, interferons (IFN)-gamma, IL-2, tumor necrosis factor (TNF)-alpha, IL-8 and IL-6 in the patients at late diagnosis compared to controls, and IL-8 in the patients at early diagnosis and treatment compared to controls. Increased IL-2, TNF-alpha, IL-6 levels in the patients at late diagnosis compared to early diagnosis were shown, and reduced IL-6 levels in the treated patients compared to patients at late diagnosis. Moreover, it verified a negative correlation between IFN-gamma and L-car in treated patients. Otherwise, it was observed that there were increased IL-4 levels in the patients at late diagnosis compared to early diagnosis, and reduction in treated patients compared to late diagnosed patients. In urine, there was an increase in 8-isoprostane levels in the patients at diagnosis compared to controls and a decrease in oxidized guanine species in the treated patients compared to the diagnosed patients. Our results demonstrate for the first time in literature that time exposure to high Phe concentrations generates a proinflammatory status, especially in PKU patients with late diagnosis. A pro-oxidant status was verified in not treated PKU patients. Our results demonstrate the importance of early diagnosis and prompt start of treatment, in addition to the importance of L-car supplementation, which can improve cellular defense against inflammation and oxidative damage in PKU patients.

Cytokine profile and cholesterol levels in patients with Niemann-Pick type C disease presenting neurological symptoms: in vivo effect of miglustat and in vitro effect of N-acetylcysteine and coenzyme Q10.

Niemann Pick type C is an inborn error of metabolism (IEM), classified as a lysosomal storage disease (LSD) caused by a dysfunction in NPC transport protein, that leads to intracellular accumulation of non-esterified cholesterol and other lipids. Clinical manifestations are ample, with visceral and neurological symptoms. Miglustat, a molecule that reversibly inhibits glucosylceramide synthase is used as treatment for this disorder. Studies demonstrated the influence of oxidative stress and inflammation in IEM, as well in animal model of NP-C disease. Nonetheless, literature lacks data on patients, so our work aimed to investigate if there is influence of chronic inflammation in the pathophysiology of NP-C disease, and the effect of miglustat, N-acetylcysteine (NAC) and Coenzyme Q10 (CoQ10). We evaluated the plasmatic cytokines in NPC patients at diagnosis and during the treatment with miglustat. Additionally, we performed an in vitro study with antioxidants NAC (1 mM and 2.5 mM) and CoQ10 (5 µM and 10 µM), where we could verify its effect on inflammatory parameters, as well as in cholesterol accumulation. Our results showed that NP-C patients have higher plasmatic levels of pro and anti-inflammatory cytokines (IL-6, IL-8, and IL-10) at diagnosis and the treatment with miglustat was able to restore it. In vitro study showed that treatment with antioxidants in higher concentrations significantly decrease cholesterol accumulation, and NAC at 2.5 mM normalized the level of pro-inflammatory cytokines. Although the mechanism is not completely clear, it can be related to restoration in lipid traffic and decrease in oxidative stress caused by antioxidants.

Protective effects of L-carnitine on behavioral alterations and neuroinflammation in striatum of glutaryl-COA dehydrogenase deficient mice.

Glutaric acidemia type 1 (GA1) is caused by glutaryl-CoA dehydrogenase deficiency that leads to a blockage in the metabolic route of the amino acids lysine and tryptophan and subsequent accumulation of glutaric acid (GA), 3-hydroxyglutaric acids and glutarylcarnitine (C5DC). Patients predominantly manifest neurological symptoms, associated with acute striatal degeneration, as well as progressive cortical and striatum injury whose pathogenesis is not yet fully established. Current treatment includes protein/lysine restriction and l-carnitine supplementation of (L-car). The aim of this work was to evaluate behavior parameters and pro-inflammatory factors (cytokines IL-1ß, TNF-α and cathepsin-D levels), as well as the anti-inflammatory cytokine IL10 in striatum of knockout mice (Gcdh-/-) and wild type (WT) mice submitted to a normal or a high Lys diet. The potential protective effects of L-car treatment on these parameters were also evaluated. Gcdh-/- mice showed behavioral changes, including lower motor activity (decreased number of crossings) and exploratory activity (reduced number of rearings). Also, Gcdh-/- mice had significantly higher concentrations of glutarylcarnitine (C5DC) in blood and cathepsin-D (CATD), interleukin IL-1ß and tumor factor necrosis alpha (TNF-α) in striatum than WT mice. Noteworthy, L-car treatment prevented most behavioral alterations, normalized CATD levels and attenuated IL-1ß levels in striatum of Gcdh-/- mice. Finally, IL-1ß was positively correlated with CATD and C5DC levels and L-car was negatively correlated with CATD. Our results demonstrate behavioral changes and a pro-inflammatory status in striatum of the animal model of GA1 and, most importantly, L-car showed important protective effects on these alterations.

DNA damage in homocystinuria: 8-oxo-, 8-dihydro-2'-deoxyguanosine levels in cystathionine-ß-synthase deficient patients and the in vitro protective effect of N-acetyl­L­cysteine

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine ß-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS­deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2'- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 µM and 200 µM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria.

Oxidative Stress in Homocystinuria Due to Cystathionine ß-Synthase Deficiency: Findings in Patients and in Animal Models.

Homocystinuria is an inborn error of amino acid metabolism caused by deficiency of cystathionine ß-synthase (CBS) activity, biochemically characterized by homocysteine (Hcy) and methionine (Met) accumulation in biological fluids and high urinary excretion of homocystine. Clinical manifestations include thinning and lengthening of long bones, osteoporosis, dislocation of the ocular lens, thromboembolism, and mental retardation. Although the pathophysiology of this disease is poorly known, the present review summarizes the available experimental findings obtained from patients and animal models indicating that oxidative stress may contribute to the pathogenesis of homocystinuria. In this scenario, several studies have shown that enzymatic and non-enzymatic antioxidant defenses are decreased in individuals affected by this disease. Furthermore, markers of lipid, protein, and DNA oxidative damage have been reported to be increased in blood, brain, liver, and skeletal muscle in animal models studied and in homocystinuric patients, probably as a result of increased free radical generation. On the other hand, in vitro and in vivo studies have shown that Hcy induces reactive species formation in brain, so that this major accumulating metabolite may underlie the oxidative damage observed in the animal model and human condition. Taken together, it may be presumed that the disruption of redox homeostasis may contribute to the tissue damage found in homocystinuria. Therefore, it is proposed that the use of appropriate antioxidants may represent a novel adjuvant therapy for patients affected by this disease.

DNA damage in Fabry patients: An investigation of oxidative damage and repair.

Fabry disease (FD) is a lysosomal storage disorder associated with loss of activity of the enzyme α-galactosidase A. In addition to accumulation of α-galactosidase A substrates, other mechanisms may be involved in FD pathophysiology, such as inflammation and oxidative stress. Higher levels of oxidative damage to proteins and lipids in Fabry patients were previously reported. However, DNA damage by oxidative species in FD has not yet been studied. We investigated basal DNA damage, oxidative DNA damage, DNA repair capacity, and reactive species generation in Fabry patients and controls. To measure oxidative damage to purines and pyrimidines, the alkaline version of the comet assay was used with two endonucleases, formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (EndoIII). To evaluate DNA repair, a challenge assay with hydrogen peroxide was performed. Patients presented significantly higher levels of basal DNA damage and oxidative damage to purines. Oxidative DNA damage was induced in both DNA bases by H2O2 in patients. Fabry patients presented efficient DNA repair in both assays (with and without endonucleases) as well as significantly higher levels of oxidative species (measured by dichlorofluorescein content). Even if DNA repair be induced in Fabry patients (as a consequence of continuous exposure to oxidative species), the repair is not sufficient to reduce DNA damage to control levels.