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Gene 533 (2014) 469–476

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Gene

journal homepage: www.elsevier.com/locate/gene

Review

L-carnitine supplementation as a potential antioxidant therapy for inherited neurometabolic disorders

Graziela S. Ribas a,b,CarmenR.Vargasa,b,c,⁎,MoacirWajnera,b,c a Federal University of Rio Grande do Sul, Brazil b Serviço de Genética Médica, HCPA, Ramiro Barcelos 2350, Porto Alegre, RS 90035-903, Brazil c Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, UFRGS, Ramiro Barcelos 2700, Porto Alegre, RS 90035-003, Brazil article info abstract

Article history: In recent years increasing evidence has emerged suggesting that is involved in the Accepted 9 October 2013 pathophysiology of a number of inherited metabolic disorders. However the clinical use of classical Available online 19 October 2013 antioxidants in these diseases has been poorly evaluated and so far no benefit has been demonstrated. L- is an endogenous substance that acts as a carrier for fatty acids across the inner mitochondrial Keywords: membrane necessary for subsequent beta-oxidation and ATP production. Besidesitsimportantroleinthe L-Carnitine of , L-carnitine is also a potent antioxidant (free ) and thus may protect Oxidative stress fi Antioxidants tissues from oxidative damage. This review addresses recent ndings obtained from patients with some Neurometabolic disorders inherited neurometabolic diseases showing that L-carnitine may be involved in the reduction of oxidative damage observed in these disorders. For some of these diseases, reduced concentrations of L-carnitine may occur due to the combination of this compound to the accumulating toxic , especially organic acids, or as a result of restricted diets. Thus, L-carnitine supplementation may be useful not only to prevent tissue deficiency of this element, but also to avoid oxidative damage secondary to increased production of reactive species in these diseases. Considering the ability of L-carnitine to easily cross the blood–brain barrier, L-carnitine supplementation may also be beneficial in preventing neurological damage derived from oxidative injury. However further studies are required to better explore this potential. © 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction...... 470 2. Neuroprotective effects of L-carnitine...... 470 3. Antioxidant properties of L-carnitine...... 471 4. L-carnitine deficiencyininheriteddiseases...... 472 5. Use of L-carnitineasatherapeuticagentinthetreatmentofinheritedneurometabolicdisorders...... 472 6. Potential utilization of L-carnitineasapossibleantioxidantinthetreatmentofinheritedneurometabolicdiseases...... 472 7. Concludingremarks...... 474 Conflictofinterest...... 474 References...... 474

Abbreviations: LC, L-Carnitine; ALC, acetyl-L-carnitine; ATP, ; ROS, reactive species; H2O2, ; GPx, peroxidase; CAT, ; SOD, dismutase; HNE, 4-hydroxy-2-nonenal; GSH, reduced glutathione; HSP, heat shock protein; PI3K, phosphoinositol-3 kinase; CNS, central nervous system; PLC, propionyl-L-carnitine; MCAD, medium-chain acyl-coenzyme A dehydrogenase; LCAD, long-chain acyl-CoA dehydrogenase; VLCAD, very-long-chain acyl-CoA dehydrogenase; LCHAD, long-chain 3-hydroxyacyl-CoA dehydrogenase; CPT, carnitine palmitoyltransferase; PKU, phenylketonuria; TBARS, thiobarbituric acid reactive species; TAR, total antioxidant reactivity; MSUD, maple syrup urine disease; BCKAD, branched-chain α-keto acid dehydrogenase complex; BCAA, branched-chain amino acids; MDA, malondialdehyde; HD, Huntington's disease. ⁎ Corresponding author at: Serviço de Genética Médica, HCPA, Rua Ramiro Barcelos, 2350, CEP 90.035-903, Porto Alegre, RS, Brazil. Tel.: +55 51 21018011; fax: +55 51 21018010. E-mail address: [email protected] (C.R. Vargas).

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.10.017 470 G.S. Ribas et al. / Gene 533 (2014) 469–476

1. Introduction and removal of organic acids and xenobiotics from the mitochondria. Short-chain acylcarnitines, produced in the mitochondrial matrix by L-Carnitine (LC, β-hydroxy-y-trimethylaminobutyric acid) is a water the action of carnitine acetyltransferase, can diffuse across cellular mem- soluble molecule important in mammalian metabolism, especially for branes and be eliminated in the urine. This action helps to protect cells the normal mitochondrial oxidation of fatty acids. LC exists in the from the toxic effects caused by the accumulation of organic acids in as free LC, acetyl-L-carnitine (ALC) and other carnitine esters. the organism (Chapela et al., 2009). About 25% of carnitine in the body is obtained by endogenous biosynthesis from methionine and protein bound lysine in the liver 2. Neuroprotective effects of L-carnitine and kidney, but most LC is supplied by the , particularly from red and milk (Walter, 1996). Furthermore, over 99% of body carnitine In addition to its role in energy metabolism, LC may exert is intracellular. High tissue concentrations are observed in the liver cytoprotective effects. In the last years, numerous neuroprotective, (800–1500 mmol/kg), but also in skeletal muscle (2–3 mmol/kg). neuromodulatory, and neurotrophic properties have been dem- Circulating carnitine accounts for only about 0.5% of body carnitine, onstrated for this molecule. Despite the low level of β-oxidation and plasma levels can vary between 40 and 60 μmol/L (Stanley, 2004). in the brain, under metabolically compromised conditions the The uptake of LC into most tissues involves carrier-mediated transport blood levels of free acetyl-CoA and ketosis may become important systems, which maintain high tissue/plasma concentration ratios for brain functioning (Virmani and Binienda, 2004). In this particularly, (Pochini et al., 2013). Carnitine cannot be metabolized and is fatty acids are also needed for incorporation into brain structural lipids. excreted as free carnitine, in the urine, but also as acylcarnitine LC is actively transported through the blood–brain barrier by the organic (Stanley, 2004). cation transporter OCTN2 and accumulates in neural cells especially as LC performs important cellular functions in the mitochondrial acetyl-L-carnitine (ALC) (Mroczkowska et al., 1997; Shug et al., 1982). and peroxisomal metabolism. Cellular energy metabolism is largely Although the actions of LC in the brain are not yet fully established, it sustained by mitochondrial β-oxidation of fatty acids, especially is known that ALC can mediate the transfer of acetyl groups for when carbohydrate stores are depleted after fasting or prolonged acetylcholine synthesis in (Nałecz and Nałecz, 1996)andalso exercise. LC acts as a mediator of long chain fatty acid transport influence signal transduction pathways and gene expression (Binienda into the mitochondria, thus facilitating β-oxidation cycle and ATP and Ali, 2001). ALC was shown to be able to prevent decay and production. Therefore, any deficiency in carnitine availability or in accelerate regeneration of neurons in various and studies the carnitine-dependent mitochondrial transport system results in (Manfridi et al., 1992). Palmitoylcarnitine, other ester of carnitine, the curtailment of fatty acid oxidation (Stanley, 2004; Walter, 1996). can also stimulate the expression of GAP-43 (named also B-50, Besides, LC improves recycling of CoA by shuttling the short- neuromodulin, F1, pp45) (Nałecz et al., 2004), a protein involved chain acyl groups from the inside of the mitochondria to the , in neural development, neuroplasticity, and neurotransmission thus raising the levels of mitochondrial free CoA (Evangeliou and (Masliah and Terry, 1993; Nałecz et al., 2004)(Fig. 2). Vlassopoulos, 2003). Consequently, a reduced availability of carnitine Recent studies have reported that LC may protect cells against induces an increase in the acetyl Coa/CoASH ratio, and this change may oxidative damage in important neurodegenerative disorders, such as interfere in other ways in the intermediary metabolism, particularly in Parkinson's and Alzheimer's diseases (Abdul and Butterfield, 2007; , , catabolism and ketone body Beal, 2003). Underlying deleterious process in is production (Fig. 1). the increased metabolic stress due to mitochondrial dysfunction and In addition to its intrinsic interaction with bioenergetic processes, LC formation of (ROS) (Abdul and Butterfield, is also involved in the transesterification and excretion of acyl-CoA esters 2007; Hinerfeld et al., 2004). In this context, it is well established that

DIET

Transfers acetyl groups to acetylcholine synthesis EXOGENOUS SOURCE Stimulates Acelerates METHIONINE expression BRAIN regeneration LYSINE CARNITINE of GAP-43 of neurons ENDOGENOUS BIOSYNTHESIS Protects cells against oxidative injury

LIVER AND CIRCULATING SKELETAL Influences pathways of CARNITINE MUSCLE neuronal survival (0,5%)

Increase of free CoA availability Conjugation β-oxidation-oxidation of with organic fatty acids MITOCHONDRIA acids Difusion of shot-chain Acylcarnitines ATP production acylcarnitines to outside Upregulation formation of mitochondria Scavange of antioxidant ROS Excretion in urine

Fig. 1. Carnitine biosynthesis, distribution and main functions in the cells under physiological conditions. G.S. Ribas et al. / Gene 533 (2014) 469–476 471

THERAPY WITH LOW PROTEIN DIET

LOW FREE CARNITINE AVAILABILITY

Pathological Free CoA/Acyl-CoA deficiency disbalance

MITOCHONDRIA Accumulation of substrates and/or Low levels metabolites of β-oxidation Inhibition of oxidative Energy phosphorilation Conjugation with deficiency carnitine

Increased ROS formation

Reduction of antioxidants

Fig. 2. Possible roles of L-carnitine in neurometabolic disorders in which therapy is based on protein restriction. The red arrows indicate the benefits provided by supplementation with L-carnitine in these conditions. oxidative stress is also involved in the pathophysiology of some cytotoxicity, protein oxidation, peroxidation, and apoptosis in a inherited neurometabolic disorders, in which the accumulation of dose-dependent manner. Additionally, ALC and LC induced elevated toxic substrates in these pathological conditions may lead to mito- cellular GSH and heat shock protein (HSP) levels and activation of chondrial dysfunction and increased ROS generation (Wajner et al., phosphoinositol-3 kinase (PI3K), PKG, and ERK1/2 pathways, which 2004). Furthermore, the dietary treatment to which patients with play essential roles in neuronal survival (Abdul and Butterfield, these disorders are subjected may result in nutritional deficiencies 2007). that may contribute to the alterations in the antioxidant status In addition, recent studies have shown that ALC improves (Wajner et al., 2004). So, in this review we present recent data learning capacity in aging animals (Ando et al., 2001), improves from the literature showing a potential antioxidant role of LC in the the symptoms of Alzheimer's disease (Pettegrew et al., 2000), treatment of inherited neurometabolic disorders, which possibly attenuates the neurological damage following brain ischemia and represents a new approach for the use of this substance in the reperfusion (Calvani and Arrigoni-Martelli, 1999; Xie et al., 2006) treatment of these and other disorders. The antioxidant properties and improves cognitive functions in severe hepatic encephalopathy of LC are discussed below in more details. (Malaguarnera et al., 2011). It was reported that the increased levels of oxidized glutathione and protein nitration in patients 3. Antioxidant properties of L-carnitine with multiple sclerosis were decreased after ALC administration (Calabrese et al., 2003). These effects seem to be related to the Recent in vivo and in vitro studies reported that LC could effectively antioxidant properties and prevention of energy loss exerted by protect various cells against oxidative injury. In the work published by this substance. Similarly, the damage to the CNS provoked by Gülçin (2006), thus, LC was found to be an effective antioxidant agent methamphetamine was attenuated in vitro and in vivo by prior in different in vitro assays, especially due to its capacity to scavenge treatment of the animals or cultured cells with LC (Virmani et al., hydrogen peroxide and superoxide radical and also chelate transition 2002, 2003). metal ions. The protective effect of LC against H2O2-induced toxicity LC treatment also was shown to prevent oxidative injury in renal was confirmed by in vitro studies using SH-SY5Y neuroblastoma cells and models, in vascular dysfunction and in (Jing et al., 2011) and human hepatocyte HL7702 cell line (Li et al., diabetes mellitus (Rajasekar and Anuradha, 2007; Uysal et al., 2005). 2012). LC may also protect the endogenous antioxidant defense system, Zambrano et al. (2013) showed that LC improved the oxidative stress including GPx, CAT and SOD activities (Augustyniak and Skrzydlewska, in renal cortex of hypertensive rats through a specific modulation 2010; Binienda and Ali, 2001; Li et al., 2012) from peroxidative damage of NF-κB, Nrf2, and PPARα transcription factors leading to a lower and is very effective in normalizing age-associated alterations of oxidative production of superoxide radical. Similarly, in another study treat- status (Rosca et al., 2009; Sethumadhavan and Chinnakannu, 2006a,b; ment with LC provoked an upregulation of antioxidant enzymes, an Thangasamy et al., 2009). increase in the plasma total antioxidant capacity and a reduction of Furthermore, it was demonstrated that LC prevents in vivo behav- and superoxide radical production in the heart ioral, morphological, and neurochemical alterations in rats treated of spontaneously hypertensive rats (Miguel-Carrasco et al., 2010). with (an excitotoxin and free radical precursor) or 3- Furthermore, Ye et al. (2010) demonstrated that pretreatment with nitropropionic acid, and also reduces reactive oxygen species formation, LC inhibited H2O2-induced cell loss, intracellular reactive oxygen lipid peroxidation and mitochondrial dysfunction in synaptosomal species generation and lipid peroxidation in human proximal tubule fractions from these animals (Silva-Adaya et al., 2008). Furthermore, epithelial cell line (HK-2), implying that it may be a promising in vitro pretreatment of primary cortical neuronal cultures with ALC candidate for the treatment of oxidative and mitochondrial damages and LC significantly attenuated 4-hydroxy-2-nonenal (HNE)-induced in renal diseases (Ye et al., 2010). 472 G.S. Ribas et al. / Gene 533 (2014) 469–476

Propionyl-L-carnitine (PLC) has also been described as a potential 1994; Mayatepek et al., 1991; Ohtani et al., 1988; Sitta et al., 2009; antioxidant in several pathological conditions. It was reported that Weigel et al., 2008). PLC can protect human endothelial cells by decreasing lipid per- oxidation and oxidase activity and stimulating the gene 5. Use of L-carnitine as a therapeutic agent in the treatment of expression of some oxidative markers, such as heme oxygenase-1 and inherited neurometabolic disorders endothelial nitric oxide synthase (Calò et al., 2006). Other studies have demonstrated that PLC can exert cardioprotective effects which LC therapy has been advocated in a large number of inherited are mainly associated with antioxidant mechanisms and improvement neurometabolic disorders, not only by restoring its normal concentrations of the energy production by the cells (Broderick, 2008; Felix et al., in conditions associated with deficiency states, but also by reducing the 2001; Ferrari et al., 1991; Packer et al., 1991; Reznick et al., 1992; acyl CoA toxic accumulation. Both oral and intravenous preparations of Shug et al., 1991). In the ischemic injury, PLC enhanced mitochondrial LC are available for therapy (Chalmers et al., 1984)(Fig. 2). function, as shown by improved ATP replenishment and creatine Numerous studies have documented biochemical or clinical phosphate myocardial levels during the reperfusion phase (Di Lisa ameliorations to carnitine therapy in several organic acidemias, et al., 1989; Ferrari et al., 1991; Shug et al., 1991). and carnitine constitutes part of the standard treatment of these disorders in all major metabolic centers. Supplementation with 4. L-carnitine deficiency in inherited diseases carnitine is essential in these conditions in an attempt to facilitate the elimination of toxic acyl groups and presumably to replenish Organic acidemias are inborn errors of metabolism caused by the decreased intracellular and extracellular stores of free carnitine, deficient activity of specific enzymes of the catabolism of amino as well as to release CoASH for other essential oxidative pathways. acids, carbohydrates or lipids and characterized by mitochondrial Doses of LC can vary from 50 to 300mg/kg/day depending on the patient's accumulation of carboxylic (organic) acids and their acylated age and the nutritional and metabolic status (Burlina and Walter, 2002; metabolites (Wajner et al., 2004). The clinical symptoms are caused Evangeliou and Vlassopoulos, 2003). Beneficial effects of LC have been by the accumulation of acyl CoA compounds that disrupt inter- described for patients with propionic acidemia, methylmalonic acidemia, mediary metabolism. The subsequent hydrolysis of the acyl CoA isovaleric acidemia, 3-hydroxy-3-methylglutaric and glutaric type I compound to its free acid results in acidosis that can be - acidemias (Bykov, 2004; Dasouki et al., 1987; Davies et al., 1991; threatening. The major clinical manifestations of these diseases involve Hoffmann et al., 1996; Kölker et al., 2011; Roe et al., 1984; Wolff et al., the central nervous system (CNS), including lethargy, intermittent 1986). Although not widely used, LC has also been advocated in disorders coma, hypotonia/hypertonia, convulsions and delayed psychomotor oftheureacyclewhereitmayprotectthebrainfromsomeofthetoxic development. The accumulation of organic acids leads to a decreased effects of hyperammonemia (Mori et al., 1990). availability of free carnitine, due to its combination with these com- In relation to the defects of β-oxidation, the role of carnitine is less pounds, and increased urinary excretion of acylcarnitines (Reuter and clear. Despite the deficiency of free carnitine in medium-chain acyl- Evans, 2012). CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase It has been largely demonstrated that the accumulated metabolites (LCAD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) in organic acidemias may induce a disturbance of energy and deficiencies, some studies have shown that carnitine may favor the homeostasis (Wajner and Goodman, 2011). In organic acidemias formation of long and medium chain carnitine esters, which can be and in fatty acid oxidation defects, as observed in medium-chain toxic for the mitochondria (Primassin et al., 2008). On the other hand, acyl-coenzyme A dehydrogenase (MCAD), long-chain acyl-CoA Lee et al. (2005) reported that a short-term LC supplementation dehydrogenase (LCAD), very-long-chain acyl-CoA dehydrogenase improved the exercise tolerance, prevented the plasma carnitine con- (VLCAD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) centration reduction, and resulted in greater increases in excretion of deficiencies, it has been proposed that the compounds accumulating acylcarnitines in MCAD-deficient patients. So, more studies are in the liver are toxic causing liver dysfunction (Spiekerkoetter and necessary to better clarify the exact role exerted by LC treatment in Wood, 2010; Wajner and Goodman, 2011). They can also be released this group of disorders. in the circulation and reach other tissues, including the brain. Secondary LC deficiency usually is observed in organic acidemias and fatty acid 6. Potential utilization of L-carnitine as a possible antioxidant in the oxidation defects due to the combination of short, medium and long treatment of inherited neurometabolic diseases chain fatty acids and organic acids with LC (Table 1). Primary deficiency of LC is less common and occurs due to a reduced There is an increasing body of evidence in the literature, obtained activity of the enzymes involved in the transfer either of LC across both from in vitro and in vivo studies, showing that oxidative stress cellular membranes or of long chain fatty acids into mitochondria. can contribute to the pathophysiology of many inherited neurometabolic They include a defect of LC uptake across plasma membranes (carnitine disorders. Brain is highly susceptible to attack by reactive species for transport defect), and three disorders affecting the transfer of long chain several reasons, including its high oxygen consumption and low levels fatty acids from the cytoplasm into mitochondria namely carnitine of antioxidant defenses (Halliwell, 2006). The toxic metabolites that palmitoyltransferase (CPT) I deficiency, CPT II deficiency and carnitine/ are accumulated in some inherited neurometabolic disorders, such as acylcarnitine translocase deficiency. The clinical-laboratory presentation organic acids, amino acids and acylcarnitines can disturb mitochondrial of these disorders is heterogeneous and includes non-ketotic hy- homeostasis, disrupting cellular energy production leading to an poglycemia, hepatic encephalopathy, myopathy with myoglobinuria, increased ROS generation (Brusqueetal.,2002;ReisdeAssisetal., or cardiomyopathy (Mandel et al., 1988; Mongini et al., 1992; Rufini 2004; Scaini et al., 2012; Sgaravatti et al., 2003; Silva et al., 1999, 2000; et al., 1993). The major finding in the brain is swelling of astrocyte Ullrich et al., 1999; Viegas et al., 2011; Wajner and Coelho, 1997). cytoplasm as well as expanded mitochondria in nerve cells and myelin Therefore, disbalance in the oxidant/antioxidant status may provoke sheath splitting in the white matter (Kimura and Amemiya, 1990). LC harmful effects in the central nervous system. supplementation rapidly mitigates the signs and symptoms of this Phenylketonuria (PKU) is an inborn error of phenylalanine deficiency state and may prevent sudden death (Breningstall, 1990). metabolism clinically characterized by neurological symptoms, Finally, LC concentrations may also be reduced in other genetic such as mental retardation, developmental delay, epilepsy, and disorders including mitochondrial myopathies, disorders of the behavioral difficulties (Scriver and Kaufman, 2001). Phenylalanine cycle and, more recently, in patients with phenylketonuria accumulation can induce oxidative damage to biomolecules and (PKU) under protein-restriction therapy (Campos and Arenas, reduced antioxidant defenses both in animal tissues and in the G.S. Ribas et al. / Gene 533 (2014) 469–476 473

Table 1 Protective effects of L-carnitine treatment in various inherited neurometabolic diseases.

Study Experimental design Evaluated tissues Observed results

Sitta et al. (2009) PKU patients with good adherence Serum Decrease of L-carnitine levels to the treatment with low protein diet Increased TBARS Reduced TAR Correlation between carnitine and oxidative stress parameters PKU patients with poor adherence to the Serum Increased TBARS treatment with low protein diet Reduced TAR Sitta et al. (2011) PKU patients treated only with protein restricted Erythrocytes and plasma Increased TBARS diet and supplemented with a special formula of amino acids Reduced sulfhydryl content Increased carbonyl formation Reduced SOD and GSH-Px activity PKU patients treated for 6 months with a special Erythrocytes and plasma Normal TBARS formula containing L-carnitine (98 mg/day) and Normal sulfhydryl content (31.5 mcg/day) and low protein diet Normal GSH-Px activity Negative correlation between free L-carnitine levels and TBARS Positive correlation between GSH-Px activity and selenium levels Mescka et al. (2011) MSUD chemically-induced Wistar rats Cerebral cortex Reduced catalase activity Reduced GPx activity Increased TBARS formation Increased protein carbonyl content Reduced protein sulfhydryl content MSUD chemically-induced Wistar rats treated Cerebral cortex - Normal catalase activity with L-carnitine administrated intraperitoneally Normal GPx activity (100 mg/kg body weight) Normal TBARS Normal protein carbonylation Normal sulfhydryl oxidation Mescka et al. (2013) MSUD patients treated only with BCAA-restricted diet Plasma Increased malondialdehyde concentrations Increased protein carbonyl content MSUD patients treated with BCAA-restricted diet Plasma Reduced malondialdehyde in relation to and L-carnitine patients not supplemented with carnitine Negative correlation between free L-carnitine concentrations and malondialdehyde levels Ribas et al. (2010a,b) Untreated patients with methylmalonic and propionic acidemias Plasma Increased malondialdehyde concentrations Increased protein carbonyl formation Reduced protein sulfhydryl content Patients with methylmalonic and propionic acidemias treated Plasma Reduced malondialdehyde levels in relation to with oral L-carnitine (100 mg/kg/day) and low protein diet untreated patients Normal protein carbonyl formation Negative correlations between malondialdehyde and free and total L-carnitine concentrations Ribas et al. (2012) Untreated patients with methylmalonic and propionic acidemias Urine Increased isoprostanes concentrations Increased di-tyrosine concentrations Decreased antioxidant capacity Patients with methylmalonic and propionic acidemias treated Urine Normal isoprostanes and di-tyrosine concentrations with oral L-carnitine (100 mg/kg/day) and low protein diet Ribas et al. (2010a,b) Leukocytes from healthy individuals incubated in vitro with Leukocytes Increased DNA damage for all tested concentrations propionic acid (2, 3 and 5 mM) and methylmalonic acid of propionic and methylmalonic acids (0.5, 2, or 5 mM) Leukocytes from healthy individuals incubated in vitro with Leukocytes L-Carnitine prevented DNA damage in dose-dependent manner 5 mM propionic acid or 5 mM methylmalonic acid plus L-carnitine (30, 60, 90, 120 or 150 μM) Vamos et al. (2010) N171-82Q transgenic mouse (model of Huntington's disease) Brain Increased duration of survival in the treated group untreated and treated with L-carnitine intraperitoneally compared to the untreated animals administrated(250 mg/bodyweight kg) L-Carnitine treatment significantly reduced the numbers of cortical huntingtin aggregates in relation to untreated group Higher number of surviving striatal neurons in the carnitine treated group relative to the vehicle-treated group

affected patients (Fernandes et al., 2010; Ribas et al., 2011; Sanayama the supplementation of this therapy (Sitta et al., 2011). It was also et al., 2011). Recently, Sitta et al. (2009) verified a significant decrease verified a significant inverse correlation between lipid peroxidation of serum L-carnitine levels in PKU patients who strictly adhered to the and LC blood levels, indicating that LC is possibly involved in the treatment based on a low protein diet (confirmed by their lower levels prevention of this damage (Sitta et al., 2011). of phenylalanine in relation to patients who did not comply with the Oxidative stress was also reported in maple syrup urine disease diet). These patients presented increased lipid peroxidation, measured (MSUD), a neurometabolic inherited disorder caused by deficient by thiobarbituric acid reactive species (TBARS) formation, and decreased activity of the branched-chain α-keto acid dehydrogenase complex total antioxidant reactivity (TAR), both parameters were correlated with (BCKAD) (Barschak et al., 2006). The blockage of this pathway leads to low concentrations of LC. It was also found that treatment with a tissue accumulation of the branched-chain amino acids (BCAA) leucine, special formula containing LC and selenium was able to correct isoleucine and valine and their respective keto-acids which can induce lipid peroxidation and protein oxidative damage, measured by oxidative damage to the cells (Bridi et al., 2005). Changes in the sulfhydryl oxidation, which were increased in PKU patients before antioxidant status were observed in MSUD affected patients even after 474 G.S. Ribas et al. / Gene 533 (2014) 469–476 the institution of dietary treatment, suggesting that antioxidant sup- motor activity as compared with the control transgenic group. These plementation may be useful as adjuvant therapy. In this context, results suggest that LC may exert a neuroprotective effect probably by Mescka et al. (2011) showed that the intraperitoneal injection of LC decreasing the oxidative damage. (100 mg/kg) reduced TBARS and protein oxidation (measured by Taken together these findings suggest that L-carnitine supple- protein carbonyl formation and sulfhydryl oxidation) and increased mentation may be useful in the therapy of inherited neurometabolic catalase and GPx activities in cerebral cortex from chemically-induced diseases, not only by improving the energy status on the cells and MSUD rats. More recently, these authors showed that LC treatment avoid the accumulation of some toxic metabolites, but also by (50 mg/kg/day) reduced plasma malondialdehyde concentrations, but preventing the oxidative damage that occur in these disorders. not carbonyl formation, in patients with MSUD under treatment with protein restricted diet (Mescka et al., 2013). 7. Concluding remarks In various neurometabolic inherited pathologies, energy production impairment due to mitochondrial dysfunction leads to increased In recent years, numerous studies have shown that oxidative stress electron leakage and a higher formation of activated oxygen species. It may contribute in the pathogenesis of inherited neurometabolic diseases. has been demonstrated that several organic acids such as meth- There are increasing evidences showing beneficial effects of some ylmalonic acid, isovaleric acid, glutaric acid, 3-hydroxyglutaric acid antioxidants, such as N-, , folic acid, and 4-hydroxybutyric acid, which are produced inside the mitochondria and on this process in animal tissues, however little is and accumulate in the tissues of patients with inherited organic known about the effect of this supplementation in the affected patients. acidemias, can disrupt the activities of respiratory chain complexes, LC has demonstrated to exert antioxidant effects in vitro and in vivo. augmenting the reactive species formation and decreasing tissue There are a great number of studies, including clinical trials, showing antioxidant defenses (Bridi et al., 2005; de Oliveira Marques et al., 2003; antioxidant properties for this substance in several pathologies such Fighera et al., 1999, 2003; Fontella et al., 2000; Funchal et al., 2006; as diabetes, hypertension, renal and liver diseases and also in neu- Latini et al., 2002; Pettenuzzo et al., 2003; Solano et al., 2008). In some rodegenerative conditions. LC supplementation has been considered of these diseases (e.g., in propionic and methylmalonic acidemias), safe and with a low risk of adverse effects, which makes it a promising antioxidant treatment with ascorbic acid and alpha was able candidate for the prevention and treatment of oxidative alterations in to prevent learning/memory deficits and convulsions provoked by many diseases. in vivo chronic or acute administration of the specific organic acids in Several inherited neurometabolic disorders are associated with rats (Fighera et al., 1999, 2003; Pettenuzzo et al., 2002, 2003). These increased ROS production and decreased levels of antioxidants. It is findings reinforce the involvement of oxidative stress in the neurological important to emphasize that most of these disorders are clinically impairment in these disorders. characterized by neurological dysfunction and, since LC is capable of It is important to emphasize that, although many of these organic crossing the blood brain barrier, it could possibly contribute in the acids are produced predominantly in liver, they can be transported prevention of oxidative neuronal damage. across the blood–brain barrier and penetrate into the brain, particularly In this review we presented data from literature showing that LC is because the blood–brain barrier is less selective at early stages of probably involved in the reduction of oxidative damages in some development, and exert their neurotoxic properties. So, the use of inherited neurometabolic disorders. These findings may represent a antioxidants that can cross the blood–brain barrier, such as LC, may be new perspective for the use of LC as a possible antioxidant treatment beneficial in the prevention of the neurological damage caused by adjunct. For this purpose, further translational and clinical studies will these metabolites in case they elicit oxidative stress (Wajner et al., be necessary to fully explore this potential. 2004). In line with this hypothesis, Ribas et al. (2010a, 2012) showed that Conflict of interest patients with disorders of propionate metabolism (propionic and methylmalonic acidemias) under treatment with LC (100 mg/kg/day) The authors declare that there is no conflict of interest. and low protein diet presented lower levels of protein and lipid oxidative damage (measured by plasma MDA and carbonyl formation References and by urinary di-tyrosine and isoprostane measurements) in relation fi to untreated patients with these diseases. Besides, plasma MDA Abdul, H.M., eld, D.A., 2007. Involvement of PI3K/PKG/ERK1/2 signaling pathways in cortical neurons to trigger protection by cotreatment of acetyl-L-carnitine and concentrations were negatively correlated with total and free carnitine alpha-lipoic acid against HNE-mediated oxidative stress and neurotoxicity: levels, suggesting the contribution of LC on lipid peroxidation. In implications for Alzheimer's disease. Free Radic. Biol. Med. 42 (3), 371–384. addition to its antioxidant action, a reduction of the intracellular toxic Ando, S., et al., 2001. Enhancement of learning capacity and cholinergic synaptic function by carnitine in aging rats. J. Neurosci. Res. 66 (2), 266–271. concentrations of propionyl-CoA, propionic and methylmalonic acids Augustyniak, A., Skrzydlewska, E., 2010. 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