Cell Mol Neurobiol (2014) 34:157–165 DOI 10.1007/s10571-013-0002-0
REVIEW PAPER
Neurological Damage in MSUD: The Role of Oxidative Stress Angela Sitta • Graziela S. Ribas • Caroline P. Mescka Alethe´a G. Barschak • Moacir Wajner • Carmen R. Vargas
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Received: 19 March 2013 / Accepted: 26 October 2013 / Published online: 13 November 2013 Springer Science+Business Media New York 2013
Abstract Maple syrup urine disease (MSUD) is a metabolic disease caused by a deficiency in the branched-chain a-keto acid dehydrogenase complex, leading to the accumulation of branched-chain keto acids and their corresponding branched-chain amino acids (BCAA) in patients. Treatment involves protein-restricted diet and the supplementation with a specific formula containing essential amino acids (except BCAA) and micronutrients, in order to avoid the appearance of neurological symptoms. Although the accumulation of toxic metabolites is associated to appearance of symptoms, the mechanisms underlying the brain damage in MSUD remain unclear, and new evidence has emerged indicating that oxidative stress contributes to this damage. In this context, this review addresses some of the recent findings obtained from cells lines, animal studies, and from patients indicating that oxidative stress is an important A. Sitta G. S. Ribas M. Wajner C. R. Vargas (&) Servic¸o de Gene´tica Me´dica, Hospital de Clı´nicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre, RS CEP 90035-903, Brazil e-mail: [email protected] A. Sitta e-mail: [email protected] C. P. Mescka M. Wajner C. R. Vargas Programa de Po´s-Graduac¸a˜o em Cieˆncias Biolo´gicas: Bioquı´mica, UFRGS, Rua Ramiro Barcelos, 2600, Porto Alegre, RS CEP 90035-003, Brazil A. G. Barschak Departamento de Cieˆncias Ba´sicas da Sau´de, UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS CEP 90050-170, Brazil C. R. Vargas Programa de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, UFRGS, Av. Ipiranga, 2752, Porto Alegre, RS CEP 90610-000, Brazil
determinant of the pathophysiology of MSUD. Recent works have shown that the metabolites accumulated in the disease induce morphological alterations in C6 glioma cells through nitrogen reactive species generation. In addition, several works demonstrated that the levels of important antioxidants decrease in animal models and also in MSUD patients (what have been attributed to protein-restricted diets). Also, markers of lipid, protein, and DNA oxidative damage have been reported in MSUD, probably secondary to the high production of free radicals. Considering these findings, it is well-established that oxidative stress contributes to brain damage in MSUD, and this review offers new perspectives for the prevention of the neurological damage in MSUD, which may include the use of appropriate antioxidants as a novel adjuvant therapy for patients. Keywords MSUD Oxidative stress Free radicals Antioxidants
Maple Syrup Urine Disease Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is an inherited aminoacidopathy caused by mutations in the mitochondrial branched-chain a-keto acid dehydrogenase (BCKDH) complex (E.C.1.2.4.4). This enzyme complex catalyses an irreversible oxidative decarboxylation of branched- chain a-keto acids (BCKA) a-ketoisocaproate (KIC), a-keto-b-methylvalerate (KMV), and a-ketoisovalerate (KIV), and is involved in the metabolism of branched-chain amino acids (BCAA) leucine (Leu), isoleucine (Ileu), and valine (Val) (Chuang and Shih 2001; Harris et al. 2004). MSUD is regarded as one of the most significant and serious genetic disorders of amino acid metabolism, since
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deficient levels of the BCKDH complex cause toxic accumulation of BCAA and their related metabolites in biological fluids, thereby resulting in numerous and serious effects. The major clinical features presented by MSUD patients include convulsions, ketoacidosis, apnea, hypoglycemia, coma, ataxia, psychomotor delay, and mental retardation, as well as hypomyelination/demyelination, as evidenced by magnetic resonance imaging studies of the central nervous system (CNS). The disease affects approximately one in 185,000 newborns worldwide (Chuang and Shih 2001). Therapy for MSUD is based on a natural proteinrestricted diet with low BCAA content, supplemented with BCAA-free amino acid mixture enriched with trace elements, vitamins, and minerals, as well as the aggressive intervention during acute metabolic decompensation. Although the benefits of dietary treatment for MSUD are undeniable, natural protein restriction may increase the risk of nutritional deficiencies. Some of the nutritional deficiencies may result in a low total antioxidant status that can predispose and/or contribute to oxidative stress (Lombeck et al. 1978; Barschak et al. 2007, 2008a). Leu and/or a-ketoisocaproate acid are the main neurotoxic metabolites in MSUD, and the high concentrations of these metabolites are associated with the appearance of neurological symptoms (Chuang and Shih 2001; Snyderman et al. 1964). Furthermore, several studies have been
demonstrated that the metabolites accumulating in MSUD cause impairment of energy metabolism by inhibiting the electron transport chain (Sgaravatti et al. 2003), compromising the citric acid cycle (Ribeiro et al. 2008) and creatine kinase (E.C.2.7.3.2) activity in rat brain (Pilla et al. 2003). Other investigators demonstrated that the BCAA and/or BCKA that accumulate in MSUD provoke neuronal apoptosis (Jouvet et al. 2000), as well as convulsions (Coitinho et al. 2001), impairment of neurotransmitter synthesis and function (Zielke et al. 1997; Tavares et al. 2000), myelin alteration (Treacy et al. 1992; Tribble and Shapira 1983; Taketomi et al. 1983), and reduced uptake of essential amino acids by the brain (Arau´jo et al. 2001). Oxidative stress has been also related to the pathophysiology of MSUD. However, the mechanisms underlying neurological dysfunction in MSUD are still poorly understood. Figure 1 illustrates some mechanisms that may contribute to the neurological damage observed in MSUD.
Fig. 1 Possible mechanisms of neurotoxicity induced by leucine and a-keto-isocaproate (KIC) accumulation in MSUD. BCKDH deficiency leads to accumulation of KIC, which inhibits pyruvate dehydrogenase (PDH) and a-ketoglutarate dehydrogenase (aKGDH) leading to a reduced Krebs cycle flux and ATP depletion. Inhibition of PDH leads to lactate accumulation through lactate dehydrogenase (LDH) reaction. KIC also inhibits mitochondrial respiratory chain
contributing to an increased ROS formation. Leucine, the precursor of KIC, reduces the activity of creatine kinase (CK) and inhibits the antioxidant enzymes CAT and GPx, thereby contributing to a reduction in energy production and increased oxidative stress. Leucine also reduces the entrance of the large neutral amino acids (LNAA) tyrosine and tryptophan into the brain, leading to a reduced formation of the neurotransmitters dopamine and serotonin
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Free Radicals, Oxidative Stress and the Antioxidant System Free radicals and reactive species are any molecules capable of independent existence that contains one or more unpaired electrons. The occurrence of an unpaired electron makes them very reactive and unstable (Halliwell and
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Gutteridge 2007). These species can be formed by several mechanisms, both in physiological or in pathological conditions (Cadenas and Sies 1998). There are many types of free radicals in living systems, but the reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), the superoxide ion (O•2 ), and the hydroxyl radical (OH•-), are the most studied. In fact, the molecular oxygen (O2) is a free radical presenting two unpaired electrons (Halliwell and Gutteridge 2007; Gilbert 1981). The autoxidation of reduced respiratory chain components with O2 causes the production of the free radical superoxide (O•2 ), which is converted to H2O2 by the enzyme superoxide dismutase (SOD; E.C.1.15.1.1). H2O2, in the presence of ions of the transition metals iron or copper, generates the hydroxyl radical (OH•), the most dangerous free radical found in vivo (Chance et al. 1979). Besides ROS, other reactive species, such as nitric oxide (NO•) and the peroxynitrite (ONOO-), which are the most prominent reactive nitrogen species (RNS), are also produced in the cells (Halliwell and Gutteridge 2007). Reactive species are necessary for normal cell function, serving as signaling molecules for important physiological roles. They are continuously produced, but when produced at high concentrations or when the antioxidant defenses are deficient, they may damage molecules (e.g., proteins, lipids, carbohydrates, and DNA), leading to cell injury and death (McCord 2000; Fridovich 1999). To handle the continuous production of reactive species, organisms posses an antioxidant defense system. The importance of antioxidants in our metabolism and their influence on health and disease has been recognized during the last decades, since they act decreasing the chance of an uncontrolled rising of cellular reactive species concentration and preventing damage of cellular structures (Seifried et al. 2007). Antioxidants are exogenous or endogenous substances capable of reducing or neutralizing the formation of free radicals. The cell can protect against oxidative insults through nonenzymatic and enzymatic antioxidants. Examples of nonenzymatic antioxidant defenses are reduced glutathione (GSH), ascorbic acid, uric acid, melatonin, the cofactors selenium and coenzyme Q10, plasma proteins, a-tocopherol, b-carotene, bilirubin, and ubiquinol (Halliwell and Gutteridge 2007). Enzymatic antioxidant defenses are also important for cellular free radical detoxification. Examples of these are the isoenzymes of SOD containing copper and zinc (Cu, Zn-SOD) or manganese (Mn-SOD), catalase (CAT; E.C.1.11.1.6), glutathione peroxidase (GPx; E.C.1.11.1.9), glutathione reductase (GRd; E.C.1.8.1.7), and dehydroascorbate reductase (E.C.1.8.5.1) (Halliwell and Gutteridge 2007). In healthy individuals, production of free radicals/reactive species is approximately balanced with antioxidant defense systems, although the free radical scavenging has a
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notable reduction with the age (Halliwell and Gutteridge 2007). However, under conditions where free radical production is more prominent, the antioxidant defense response is not sufficient to maintain redox balance. This disturbance in the pro-oxidant–antioxidant balance in favor of the former is defined as oxidative stress, resulting in potential oxidative damage to the cell, and may represent a fundamental mechanism of human disease (Halliwell and Gutteridge 2007; Sies 1985).
Oxidative Stress and Neurological Damage The human brain is a complex organ with remarkable energy demands. Although it represents only 2 % of the total body weight, it accounts for 20 % of all oxygen consumption, reflecting its high rate of metabolic activity (Castro et al. 2010). This is an important characteristic that makes the brain prone to suffer oxidative damage. Other reasons are the high Ca2? traffic across neuronal membranes, the presence of excitotoxic amino acids, the fact that many transmitters are autoxidisable molecules, and the high content of polyunsaturated fatty, for example. As in other tissues, oxidative stress in the brain can damage neurons by several mechanisms, including increased lipid peroxidation (for example, hydroxynonenal, a product of lipid peroxidation, is highly neurotoxic), oxidative damage to DNA and proteins, and induction of apoptosis or necrosis (Halliwell 2001; Adibhatla and Hatcher 2010). The crucial role of ROS-inducing oxidative stress has been extensively demonstrated in many neurological disorders, including bipolar disorder and schizophrenia, and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis (Halliwell 2006). Oxidative stress is also believed to be a key event in contributing to CNS injuries such as stroke, traumatic brain injury, and spinal cord injury (Jenner and Olanow 1996; Liu et al. 1999; Perry et al. 2003; Andersen 2004; Halliwell 2006). Recently, an increased number of studies have been demonstrated that oxidative stress is present in many inborn errors of metabolism, including organic acidurias, aminoacidopathies, mitochondrial fatty acid oxidation disorders, and peroxisomal diseases (Wajner et al. 2004; Streck et al. 2003; Stefanello et al. 2005; Sirtori et al. 2005; Sitta et al. 2006, 2009; Barschak et al. 2006; Vargas et al. 2004; Deon et al. 2006). Although the cause of increased oxidative stress in these diseases is poorly understood, it may be presumed that the accumulation of toxic metabolites or their precursors could lead to an increased free radical production. Furthermore, dietary treatment with protein restriction to which the patients are submitted could cause nutritional deficiencies resulting in a reduction of
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antioxidants and contributing to the oxidative damage (Wajner et al. 2004). These studies suggest that oxidative stress may contribute, at least in part, for the neurological symptoms present by patients affected by inborn errors of metabolism. In this context, several in vitro and in vivo studies have been done testing the involvement of reactive species and antioxidant defenses in MSUD to better understand the mechanisms related to neurological damage in the disease. Here we briefly summarize the known literature data obtained from human and animal studies, suggesting that oxidative stress may represent a relevant pathomechanism of brain damage in MSUD.
MSUD and Oxidative Stress: Studies Using Cell Lines Glial cells play a vital role in the homeostatic regulation of CNS. Astrocytes, the most abundant glial cell type in the brain, are involved in neurotransmitter uptake, neuronal metabolic support, pH regulation, and protection against toxic episodes. In addition, glial cells preserve neuronal survival through inactivation of ROS. Because of its similarities with primary glial cells in culture, the C6 rat glioma cell line has provided a useful model to study glial cell properties, glial growth factors, and sensitivity of glial cells to various substances and conditions (Blanc et al. 1998; Takuma et al. 2004). Considering that the function of glial cells is altered in some neurodegenerative diseases, studies were realized in order to evaluate if this effect also occurs in MSUD, a disease presenting predominantly neurological symptoms. De Lima et al. (2007) investigated the effects of the BCAA accumulated in MSUD on C6 glioma cell morphology and cytoskeletal reorganization. The BCAA, in levels similar to those found in tissues from MSUD patients, induced marked morphological alterations. Among the BCAA, Val induced the most marked effect on the cell shape. On the other hand, immunocytochemistry staining with antiGFAP showed rearrangement of the cytoskeletal network, as well as altered organization of GFAP filaments after exposing C6 cells to BCAA. In addition, the authors observed that exposure to BCAA resulted in a marked reduction of GSH levels, and significantly increased nitric oxide (NO) production. Also, the morphological features caused by the BCAA on C6 cells were prevented by the use of the antioxidants GSH and Nx-nitro-L-arginine methyl ester (L-NAME). Similarly, in another study, Funchal et al. (2006a) showed that C6 glial cells exposed to the BCKA, at doses similar to those found in MSUD, changed their usual rounded morphology to a fusiform or process-bearing cell appearance. On the other hand, the exposure to high concentrations of these acids (10.0 mM) and/or for extended periods of time (24 h)
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elicited cell death. In order to evaluate whether oxidative stress was involved in the BCKA-induced morphological alterations and cell death, various parameters of oxidative stress in C6 cell homogenates, as well as the protective effects of antioxidants were analyzed. It was observed that cell exposure to low doses of all BCKA (1.0 mM) resulted in a marked reduction of nonenzymatic antioxidant defenses, as determined by total antioxidant reactivity (TAR) and GSH measurements. In addition, KIC provoked the reduction of the activity of SOD and GPx, whereas KMV caused a diminution only in SOD activity. Furthermore, NO production was observed to be increased by all BCKA. Finally, it was observed that the morphological features caused by BCKA on C6 cells were prevented by the use of the antioxidants GSH, alpha-tocopherol (trolox), and L-NAME. Taking together, these results strongly suggest that oxidative stress might be contributing to morphological alteration and death observed in C6 glioma cells, as well as in cytoskeletal reorganization elicited by BCAA and BCKA. It is presumed that these findings could explain, at least in part, the neuropathophysiology of MSUD. Another study from the same research group was performed in order to evaluate the effect of BCKA on S100B release from glial cells (Funchal et al. 2007). S100B is a calcium-binding protein expressed and secreted by astrocytes. Extracellular S100B stimulates glial cell proliferation, survive of neurons and extension of neurites in cell culture and is used as a parameter of glial cell death in pathological situations for brain injury (Donato 2001). In fact, elevation of peripheral (cerebrospinal fluid or serum) S100B concentrations has been observed in several common psychiatric disorders (Donato 2003). Funchal et al. showed that KIC and KIV, but not KMV, significantly enhanced S100B liberation from glial cells after 6 h of exposure. Furthermore, the stimulatory effect of the KIC and KIV on S100B release was prevented by coincubation with the energetic substrate creatine and with the L-NAME, a NO synthase inhibitor, indicating that energy deficit and NO were probably involved in this effect. In contrast, other antioxidants such as glutathione (GSH) and trolox (soluble vitamin E) were not able to prevent KIC- and KIV-induced increase of S100B liberation, suggesting that the alteration of S100B release caused by these BCKA is not mediated by oxidation of sulfydryl as well as by lipid peroxyl radicals. In the same way, another study (Funchal et al. 2006b) demonstrated that BCKA inhibited creatine kinase (CK) activity from C6 glioma cells and that this inhibition was totally prevented by preincubation with creatine and with L-NAME. CK has a central role in energy metabolism, especially for high energy consuming tissues, such as brain (Bessman and Carpenter 1985). A decrease in CK is found in neurodegenerative diseases and is correlated with
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neurodegeneration (Aksenov et al. 1997). So, the authors concluded that the inhibition of CK by the BCKA may contribute to the cerebral findings in MSUD. Considering that L-NAME prevented the inhibitory effect of the BCKA on CK activity from C6 glioma cells, NO is probably involved in those effects.
Oxidative Stress in MSUD: The Contribution from Animal Studies It is well-established that animal models provide a useful tool to explore the mechanisms of disease as well as to test potential therapies that could not justifiably be explored in humans without preclinical experimental data. In fact, studies investigating the role of oxidative stress in MSUD started with animal models. Fontella et al. (2002) verified that metabolites accumulated in MSUD stimulate lipid peroxidation in vitro in rat brain. Rat brain homogenates were incubated in the presence of BCAA, BCKA, or the hydroxy derivatives (L-2-hydroxycaproic acid, L-2-hydroxy3-methylvaleric acid, and L-2-hydroxyisovaleric acid), separately. They observed that all amino acids, keto acids, and hydroxy acids accumulated in MSUD stimulated to a variable degree the in vitro parameters of lipid peroxidation tested, named chemiluminescence and thiobarbituric acidreactive substances (TBARS). It is important to emphasize that lipid peroxidation may damage cell structures by altering the integrity, fluidity, and permeability of biomembranes, and by generating potentially toxic products being, therefore, associated to a crescent number of pathological conditions, including neurological diseases (Greenberg et al. 2008; Adibhatla and Hatcher 2010). Reinforcing these findings, Bridi et al. (2003) investigated the in vitro effect of BCAA on the lipid peroxidation, as well as on antioxidant parameters, in cerebral cortex of rats. Lleucine significantly increased chemiluminescence and TBARS measurements and markedly decreased the total radical-trapping antioxidant potential (TRAP) and the TAR values. L-isoleucine increased chemiluminescence and decreased TRAP measurements, but TAR and TBARS levels were not altered by this amino acid. Finally, L-valine diminished the TRAP measurement. The results of this study indicate a stimulation of lipid peroxidation and a reduction of brain capacity to efficiently modulate the damage associated to an increased production of reactive species by the BCAA. So, it can be speculate that oxidative stress was induced in vitro by BCAA, especially by Leu which altered all parameters investigated. Since Leu is probably the main metabolite causing oxidative stress in MSUD, another study evaluated the mechanisms involved in Leu-induced oxidative damage in cerebral cortex of young rats in vitro (Bridi et al. 2005a).
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The authors observed that the increase of TBARS induced by Leu was significantly attenuated when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus Trolox, DTT, GSH, and SOD, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of L-NAME and CAT did not affect TBARS values. It was also observed a dose-dependent inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and GPx, as well as a significant reduction in the membrane-protein thiol content from mitochondrial Leu-enriched preparations. Taken together, the results of this work indicate that a disbalance between reactive species formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage. Bridi et al. (2005b) demonstrated that besides BCAA, the BCKA accumulated in MSUD also stimulate in vitro lipid peroxidation and reduce antioxidant defenses in cerebral cortex of rats. This rat cerebral tissue was incubated in the presence of KIC, KMV, or KIV at concentrations similar to those found in MSUD patients. The major effects observed in this study were with KIC, which significantly increased chemiluminescence and TBARS measurements, decreased TRAP and TAR values, and markedly inhibited GPx activity. Taken all these findings together, it can be presumed that KIC and its precursor Leu provoke the most significant effects on oxidative stress parameters studied, which is in agreement with previous studies demonstrating that these metabolites are the most toxic in the MSUD (Chuang and Shih 2001). In accordance with in vitro findings, in the last years some in vivo studies demonstrated that metabolites accumulated in MSUD induce a pro-oxidative process, probably contributing to the neurological damage in the disease, and that the administration of antioxidants may exerts neuroprotection. Mescka et al. (2011) conducted a study using a chemically-induced acute model of MSUD (Bridi et al. 2006). A BCAA pool was injected subcutaneously in 14-day-old rats, and it was observed a markedly reduction in CAT and GPx activities in cerebral cortex homogenates. Additionally, TBARS levels and carbonyl and sulfhydryl content (parameters of protein oxidation) were also altered in brain by the BCAA. It was also demonstrated that the treatment with L-carnitine was capable to prevent all these alterations, showing a neuroprotective effect of this substance. In fact, some works have shown antiperoxidative effects of L-carnitine (Fariello and Calabrese 1988; Bertelli et al. 1994; Lowitt et al. 1995) and its capacity to prevent the formation of ROS, besides to regulate NO, cellular respiration, as well as the activity of antioxidant enzymes (Brown 1999; Kremser et al. 1995). Antioxidants showed also to prevent memory impairments in an animal model of MSUD (Scaini et al. 2012a).
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Wistar rats were submitted to acute and chronic administrations of a BCAA pool. These administrations induced long-term memory impairment in the inhibitory avoidance and CMIA (continuous multiple trials stepdown inhibitory avoidance) tasks. The coadministration of the antioxidants N-acetylcysteine (NAC) and deferoxamine (DFX) prevented inhibitory avoidance alterations, suggesting that cognition impairment after BCAA administration may be caused by oxidative brain damage. Another study demonstrated the occurrence of DNA damage (determined by the alkaline comet assay) in MSUD-induced animals (Scaini et al. 2012b). It is important to emphasize that DNA is a particularly important target for oxidation generating several classes of products (single- and double-strand breaks), inter/intra-strand crosslinks, DNA-proteins cross-links, and sugar fragmentation products. These products may elicit mutations, microsatellite instability, loss of heterozygosity, chromosomal aberrations, cytotoxicity, and neoplasic growth (Cooke et al. 2006). The acute administration of BCAA increased the DNA damage frequency and damage index in the hippocampus, whereas the chronic administration of BCAA increased the DNA damage frequency and damage index both in hippocampus and striatum. In addition, the antioxidant treatment with NAC and DFX was able to prevent DNA damage in these brain structures. NAC is known to reduce oxidative stress by improving thiol redox status and to scavenge superoxide and hydroxyl radicals and H2O2. However, when administered alone, NAC has pro-oxidant effects probably by its interaction with iron. So, the coadministration of DFX, a powerful iron chelator, has been used to improve NAC response (Aruoma et al. 1989; Ritter et al. 2004). NAC and DFX treatment also prevented the increase of brain acetylcholinesterase (AChE) activity induced in vivo by chronic administration of BCAA in rats (Scaini et al. 2012c). AChE is associated with brain development, learning, memory, and neuronal damage, and its activation leads to fast acetylcholine degradation and a subsequent downregulation of acetylcholine receptors, causing undesirable effects on cognitive functions (Soreq and Seidman 2001). So, the authors conclude that antioxidant treatment increases the efficiency of cholinergic transmission in MSUD by preventing the hydrolysis of acetylcholine, and consequently improving cognitive functions, such as learning and memory.
Oxidative Stress in MSUD Patients Contributing with the findings obtained from the cell culture and animal studies, in the last years, some works have demonstrated important alterations in oxidative stress
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biomarkers in MSUD-affected patients. One of these biomarkers is malondialdehyde (MDA), an end product of lipid peroxidation by reactive species, whose formation can be easily assessed by the measurement of TBARS. Barschak et al. (2006) demonstrated that TBARS were increased in plasma samples from five untreated patients with the classic form of MSUD, indicating an increased oxidative damage to lipids in these patients. Furthermore, the authors also demonstrated that TAR measurement was reduced in these samples, suggesting a poor quality of the tissue antioxidants in modulating the damage provoked by an increased ROS formation. Similar results were found by these researchers (Barschak et al. 2008b) in plasma from ten MSUD patients treated with a protein-restricted diet (low BCAA), and supplemented with a semisynthetic formula of essential amino acids, vitamins, and minerals. These patients presented an increased lipid peroxidation, as well as reduced antioxidant defenses, even during the treatment. Despite the high levels of Leu, Ileu, and Val presented by these patients, no significant correlations were observed between the concentrations of these amino acids and the oxidative stress parameters investigated. As occurs in phenylketonuria and in some organic acidemias (Sitta et al. 2011; Ribas et al. 2010), it is recognized that the low protein dietetic treatment to which the MSUD patients are submitted can contribute to alter antioxidant defenses, since strict protein ingest may reduce the absorption of vitamins and minerals which are essential to this defense system. One of these important elements is selenium, which is mainly present in foods like grains, cereals, and meat. Selenium exerts a fundamental antioxidant role, since it is the cofactor of the GPx enzyme. Some studies have demonstrated that selenium levels are reduced in plasma, whole blood and hair from patients with MSUD (Lombeck et al. 1978; Borglund et al. 1989; Barschak et al. 2007). Barschak et al. (2007) showed that this deficiency occurs in MSUD patients at diagnosis, but is more prominent in MSUD patients during the dietetic treatment. Treated MSUD patients also presented decreased erythrocyte GPx activity (Borglund et al. 1989; Lombeck et al. 1978; Barschak et al. 2007) that may be related, at least in part, to the reduction of this element, although a direct correlation between selenium concentrations and GPx activity in these patients had not been observed. On the other hand, Borglund et al. (1989) showed that selenium supplementation was able to increase tenfold the activity of GPx in a MSUD patient. These findings suggest that selenium supplementation could represent an important adjuvant therapy for MSUD. Also in this context, Mescka et al. (2012) demonstrated that MSUD patients treated with low protein diet present Lcarnitine deficiency. L-Carnitine is a small quaternary amine highly polar and water soluble, which may be
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biosynthesized by humans (25 %) or obtained from dietary sources (75 %), mainly from meats, milk, and nuts (Agarwal and Said 2004; Derin et al. 2004; Gulcin 2006). Usually it is present in plasma as free carnitine, which is responsible by transporting long-chain fatty acids across the inner mitochondrial membrane for utilization in metabolism through b-oxidation (Matalliotakis et al. 2000; Tastekin et al. 2007). Furthermore, L-carnitine has antioxidant properties, and it may protect cells from toxic oxygen metabolites (Derin et al. 2004). Thereby, in this work, it was investigated the effect of BCAA-restricted diet, supplemented or not with L-carnitine, on lipid and protein oxidation in MSUD patients. Significant increases of MDA and protein carbonyl content were observed in plasma from MSUD patients treated only with BCAA-restricted diet. However, patients under BCAA-restricted diet and supplemented with L-carnitine (50 mg/kg/day) presented a marked reduction of MDA content in relation to the carnitine-untreated MSUD patients. In addition, free L-carnitine concentrations were negatively correlated with MDA levels. These data show that L-carnitine may exert an antioxidant role, protecting cells against the lipid peroxidation, and this effect could represent an additional therapeutic approach to the patients affected by MSUD. In another work, Barschak et al. (2009) evaluated the concentrations of amino acids, branched-chain a-keto acids, and a-hydroxy acids in plasma samples from treated MSUD patients, and the authors observed that tryptophan and methionine were significantly reduced in relation to control values. This reduction may represent a mechanism of neurological dysfunction in MSUD, since the deficit of these amino acids in the brain can lead to an impaired protein and neurotransmitter synthesis (Arau´jo et al. 2001). Tryptophan and methionine can act as antioxidants through different mechanisms (Keithahn and Lerchl 2005; Watanabe et al. 2002; Levine et al. 1996; Stadtman 2004) and this role may be especially important in an organ like the brain, which is highly susceptible to the attack by reactive species. Besides, in the study published by Barschak et al. (2009), tryptophan and methionine concentrations presented a strongly inverse correlation with TBARS levels, suggesting that the low content of these amino acids in plasma could possibly be involved in the lipid peroxidation observed. The role of oxidative stress in the development of the clinical and biochemical manifestations of MSUD is not yet fully understood. Barschak et al. (2008a, b) showed that MSUD-treated patients present important biochemical alterations, such as reduced levels of glucose, cholesterol, creatinine, and albumin, as well as a high activity of the enzymes aspartate aminotransferase and lactate dehydrogenase. Associated to these findings, the authors observed increased levels of TBARS and a positive correlation
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between TBARS and plasma concentrations of triglycerides (Barschak et al. 2008a). Although all these data strongly suggest the occurrence of oxidative stress in patients with MSUD, and that Leu together with KIC can be considered the main neurotoxic agents in this disease, the studies in patients with MSUD have not shown a clear association between these metabolites and the markers of oxidative stress. So, it is possible that other factors may be related to oxidative stress in MSUD which needs to be better investigated.
Conclusions Oxidative stress has been associated to a crescent number of pathological conditions, including neurological diseases, since brain is an organ vulnerable to oxidative damage by reactive species. MSUD is a neurological disease, but the mechanisms underlying the neurotoxicity of this disorder remain unclear. However, in this review we described many in vitro and in vivo evidence in cell lines, animal models, and patients showing the occurrence of oxidative stress in MSUD, probably contributing to the neurological sequelae present in most patients. It is presumed that metabolites accumulated in MSUD induce excessive production of reactive species and/or deplete the tissue antioxidant capacity, leading to damage to biomolecules (lipids, proteins, and DNA). In addition, protein-restricted diet to which MSUD patients are submitted may lead to a decrease in the antioxidants intake (selenium and L-carnitine, for example), contributing to the oxidative damage verified in the disease. It is important to emphasize that the results from the presently available studies must be interpreted cautiously and confirmed by other in vivo studies, but it is possible to suggest that in the future antioxidants could be used as an adjuvant therapy for MSUD patients. Conflict of interest interest.
The authors declare that there is no conflict of
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REVIEW PAPER
Neurological Damage in MSUD: The Role of Oxidative Stress Angela Sitta • Graziela S. Ribas • Caroline P. Mescka Alethe´a G. Barschak • Moacir Wajner • Carmen R. Vargas
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Received: 19 March 2013 / Accepted: 26 October 2013 / Published online: 13 November 2013 Springer Science+Business Media New York 2013
Abstract Maple syrup urine disease (MSUD) is a metabolic disease caused by a deficiency in the branched-chain a-keto acid dehydrogenase complex, leading to the accumulation of branched-chain keto acids and their corresponding branched-chain amino acids (BCAA) in patients. Treatment involves protein-restricted diet and the supplementation with a specific formula containing essential amino acids (except BCAA) and micronutrients, in order to avoid the appearance of neurological symptoms. Although the accumulation of toxic metabolites is associated to appearance of symptoms, the mechanisms underlying the brain damage in MSUD remain unclear, and new evidence has emerged indicating that oxidative stress contributes to this damage. In this context, this review addresses some of the recent findings obtained from cells lines, animal studies, and from patients indicating that oxidative stress is an important A. Sitta G. S. Ribas M. Wajner C. R. Vargas (&) Servic¸o de Gene´tica Me´dica, Hospital de Clı´nicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre, RS CEP 90035-903, Brazil e-mail: [email protected] A. Sitta e-mail: [email protected] C. P. Mescka M. Wajner C. R. Vargas Programa de Po´s-Graduac¸a˜o em Cieˆncias Biolo´gicas: Bioquı´mica, UFRGS, Rua Ramiro Barcelos, 2600, Porto Alegre, RS CEP 90035-003, Brazil A. G. Barschak Departamento de Cieˆncias Ba´sicas da Sau´de, UFCSPA, Rua Sarmento Leite, 245, Porto Alegre, RS CEP 90050-170, Brazil C. R. Vargas Programa de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, UFRGS, Av. Ipiranga, 2752, Porto Alegre, RS CEP 90610-000, Brazil
determinant of the pathophysiology of MSUD. Recent works have shown that the metabolites accumulated in the disease induce morphological alterations in C6 glioma cells through nitrogen reactive species generation. In addition, several works demonstrated that the levels of important antioxidants decrease in animal models and also in MSUD patients (what have been attributed to protein-restricted diets). Also, markers of lipid, protein, and DNA oxidative damage have been reported in MSUD, probably secondary to the high production of free radicals. Considering these findings, it is well-established that oxidative stress contributes to brain damage in MSUD, and this review offers new perspectives for the prevention of the neurological damage in MSUD, which may include the use of appropriate antioxidants as a novel adjuvant therapy for patients. Keywords MSUD Oxidative stress Free radicals Antioxidants
Maple Syrup Urine Disease Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is an inherited aminoacidopathy caused by mutations in the mitochondrial branched-chain a-keto acid dehydrogenase (BCKDH) complex (E.C.1.2.4.4). This enzyme complex catalyses an irreversible oxidative decarboxylation of branched- chain a-keto acids (BCKA) a-ketoisocaproate (KIC), a-keto-b-methylvalerate (KMV), and a-ketoisovalerate (KIV), and is involved in the metabolism of branched-chain amino acids (BCAA) leucine (Leu), isoleucine (Ileu), and valine (Val) (Chuang and Shih 2001; Harris et al. 2004). MSUD is regarded as one of the most significant and serious genetic disorders of amino acid metabolism, since
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deficient levels of the BCKDH complex cause toxic accumulation of BCAA and their related metabolites in biological fluids, thereby resulting in numerous and serious effects. The major clinical features presented by MSUD patients include convulsions, ketoacidosis, apnea, hypoglycemia, coma, ataxia, psychomotor delay, and mental retardation, as well as hypomyelination/demyelination, as evidenced by magnetic resonance imaging studies of the central nervous system (CNS). The disease affects approximately one in 185,000 newborns worldwide (Chuang and Shih 2001). Therapy for MSUD is based on a natural proteinrestricted diet with low BCAA content, supplemented with BCAA-free amino acid mixture enriched with trace elements, vitamins, and minerals, as well as the aggressive intervention during acute metabolic decompensation. Although the benefits of dietary treatment for MSUD are undeniable, natural protein restriction may increase the risk of nutritional deficiencies. Some of the nutritional deficiencies may result in a low total antioxidant status that can predispose and/or contribute to oxidative stress (Lombeck et al. 1978; Barschak et al. 2007, 2008a). Leu and/or a-ketoisocaproate acid are the main neurotoxic metabolites in MSUD, and the high concentrations of these metabolites are associated with the appearance of neurological symptoms (Chuang and Shih 2001; Snyderman et al. 1964). Furthermore, several studies have been
demonstrated that the metabolites accumulating in MSUD cause impairment of energy metabolism by inhibiting the electron transport chain (Sgaravatti et al. 2003), compromising the citric acid cycle (Ribeiro et al. 2008) and creatine kinase (E.C.2.7.3.2) activity in rat brain (Pilla et al. 2003). Other investigators demonstrated that the BCAA and/or BCKA that accumulate in MSUD provoke neuronal apoptosis (Jouvet et al. 2000), as well as convulsions (Coitinho et al. 2001), impairment of neurotransmitter synthesis and function (Zielke et al. 1997; Tavares et al. 2000), myelin alteration (Treacy et al. 1992; Tribble and Shapira 1983; Taketomi et al. 1983), and reduced uptake of essential amino acids by the brain (Arau´jo et al. 2001). Oxidative stress has been also related to the pathophysiology of MSUD. However, the mechanisms underlying neurological dysfunction in MSUD are still poorly understood. Figure 1 illustrates some mechanisms that may contribute to the neurological damage observed in MSUD.
Fig. 1 Possible mechanisms of neurotoxicity induced by leucine and a-keto-isocaproate (KIC) accumulation in MSUD. BCKDH deficiency leads to accumulation of KIC, which inhibits pyruvate dehydrogenase (PDH) and a-ketoglutarate dehydrogenase (aKGDH) leading to a reduced Krebs cycle flux and ATP depletion. Inhibition of PDH leads to lactate accumulation through lactate dehydrogenase (LDH) reaction. KIC also inhibits mitochondrial respiratory chain
contributing to an increased ROS formation. Leucine, the precursor of KIC, reduces the activity of creatine kinase (CK) and inhibits the antioxidant enzymes CAT and GPx, thereby contributing to a reduction in energy production and increased oxidative stress. Leucine also reduces the entrance of the large neutral amino acids (LNAA) tyrosine and tryptophan into the brain, leading to a reduced formation of the neurotransmitters dopamine and serotonin
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Free Radicals, Oxidative Stress and the Antioxidant System Free radicals and reactive species are any molecules capable of independent existence that contains one or more unpaired electrons. The occurrence of an unpaired electron makes them very reactive and unstable (Halliwell and
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Gutteridge 2007). These species can be formed by several mechanisms, both in physiological or in pathological conditions (Cadenas and Sies 1998). There are many types of free radicals in living systems, but the reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), the superoxide ion (O•2 ), and the hydroxyl radical (OH•-), are the most studied. In fact, the molecular oxygen (O2) is a free radical presenting two unpaired electrons (Halliwell and Gutteridge 2007; Gilbert 1981). The autoxidation of reduced respiratory chain components with O2 causes the production of the free radical superoxide (O•2 ), which is converted to H2O2 by the enzyme superoxide dismutase (SOD; E.C.1.15.1.1). H2O2, in the presence of ions of the transition metals iron or copper, generates the hydroxyl radical (OH•), the most dangerous free radical found in vivo (Chance et al. 1979). Besides ROS, other reactive species, such as nitric oxide (NO•) and the peroxynitrite (ONOO-), which are the most prominent reactive nitrogen species (RNS), are also produced in the cells (Halliwell and Gutteridge 2007). Reactive species are necessary for normal cell function, serving as signaling molecules for important physiological roles. They are continuously produced, but when produced at high concentrations or when the antioxidant defenses are deficient, they may damage molecules (e.g., proteins, lipids, carbohydrates, and DNA), leading to cell injury and death (McCord 2000; Fridovich 1999). To handle the continuous production of reactive species, organisms posses an antioxidant defense system. The importance of antioxidants in our metabolism and their influence on health and disease has been recognized during the last decades, since they act decreasing the chance of an uncontrolled rising of cellular reactive species concentration and preventing damage of cellular structures (Seifried et al. 2007). Antioxidants are exogenous or endogenous substances capable of reducing or neutralizing the formation of free radicals. The cell can protect against oxidative insults through nonenzymatic and enzymatic antioxidants. Examples of nonenzymatic antioxidant defenses are reduced glutathione (GSH), ascorbic acid, uric acid, melatonin, the cofactors selenium and coenzyme Q10, plasma proteins, a-tocopherol, b-carotene, bilirubin, and ubiquinol (Halliwell and Gutteridge 2007). Enzymatic antioxidant defenses are also important for cellular free radical detoxification. Examples of these are the isoenzymes of SOD containing copper and zinc (Cu, Zn-SOD) or manganese (Mn-SOD), catalase (CAT; E.C.1.11.1.6), glutathione peroxidase (GPx; E.C.1.11.1.9), glutathione reductase (GRd; E.C.1.8.1.7), and dehydroascorbate reductase (E.C.1.8.5.1) (Halliwell and Gutteridge 2007). In healthy individuals, production of free radicals/reactive species is approximately balanced with antioxidant defense systems, although the free radical scavenging has a
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notable reduction with the age (Halliwell and Gutteridge 2007). However, under conditions where free radical production is more prominent, the antioxidant defense response is not sufficient to maintain redox balance. This disturbance in the pro-oxidant–antioxidant balance in favor of the former is defined as oxidative stress, resulting in potential oxidative damage to the cell, and may represent a fundamental mechanism of human disease (Halliwell and Gutteridge 2007; Sies 1985).
Oxidative Stress and Neurological Damage The human brain is a complex organ with remarkable energy demands. Although it represents only 2 % of the total body weight, it accounts for 20 % of all oxygen consumption, reflecting its high rate of metabolic activity (Castro et al. 2010). This is an important characteristic that makes the brain prone to suffer oxidative damage. Other reasons are the high Ca2? traffic across neuronal membranes, the presence of excitotoxic amino acids, the fact that many transmitters are autoxidisable molecules, and the high content of polyunsaturated fatty, for example. As in other tissues, oxidative stress in the brain can damage neurons by several mechanisms, including increased lipid peroxidation (for example, hydroxynonenal, a product of lipid peroxidation, is highly neurotoxic), oxidative damage to DNA and proteins, and induction of apoptosis or necrosis (Halliwell 2001; Adibhatla and Hatcher 2010). The crucial role of ROS-inducing oxidative stress has been extensively demonstrated in many neurological disorders, including bipolar disorder and schizophrenia, and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis (Halliwell 2006). Oxidative stress is also believed to be a key event in contributing to CNS injuries such as stroke, traumatic brain injury, and spinal cord injury (Jenner and Olanow 1996; Liu et al. 1999; Perry et al. 2003; Andersen 2004; Halliwell 2006). Recently, an increased number of studies have been demonstrated that oxidative stress is present in many inborn errors of metabolism, including organic acidurias, aminoacidopathies, mitochondrial fatty acid oxidation disorders, and peroxisomal diseases (Wajner et al. 2004; Streck et al. 2003; Stefanello et al. 2005; Sirtori et al. 2005; Sitta et al. 2006, 2009; Barschak et al. 2006; Vargas et al. 2004; Deon et al. 2006). Although the cause of increased oxidative stress in these diseases is poorly understood, it may be presumed that the accumulation of toxic metabolites or their precursors could lead to an increased free radical production. Furthermore, dietary treatment with protein restriction to which the patients are submitted could cause nutritional deficiencies resulting in a reduction of
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antioxidants and contributing to the oxidative damage (Wajner et al. 2004). These studies suggest that oxidative stress may contribute, at least in part, for the neurological symptoms present by patients affected by inborn errors of metabolism. In this context, several in vitro and in vivo studies have been done testing the involvement of reactive species and antioxidant defenses in MSUD to better understand the mechanisms related to neurological damage in the disease. Here we briefly summarize the known literature data obtained from human and animal studies, suggesting that oxidative stress may represent a relevant pathomechanism of brain damage in MSUD.
MSUD and Oxidative Stress: Studies Using Cell Lines Glial cells play a vital role in the homeostatic regulation of CNS. Astrocytes, the most abundant glial cell type in the brain, are involved in neurotransmitter uptake, neuronal metabolic support, pH regulation, and protection against toxic episodes. In addition, glial cells preserve neuronal survival through inactivation of ROS. Because of its similarities with primary glial cells in culture, the C6 rat glioma cell line has provided a useful model to study glial cell properties, glial growth factors, and sensitivity of glial cells to various substances and conditions (Blanc et al. 1998; Takuma et al. 2004). Considering that the function of glial cells is altered in some neurodegenerative diseases, studies were realized in order to evaluate if this effect also occurs in MSUD, a disease presenting predominantly neurological symptoms. De Lima et al. (2007) investigated the effects of the BCAA accumulated in MSUD on C6 glioma cell morphology and cytoskeletal reorganization. The BCAA, in levels similar to those found in tissues from MSUD patients, induced marked morphological alterations. Among the BCAA, Val induced the most marked effect on the cell shape. On the other hand, immunocytochemistry staining with antiGFAP showed rearrangement of the cytoskeletal network, as well as altered organization of GFAP filaments after exposing C6 cells to BCAA. In addition, the authors observed that exposure to BCAA resulted in a marked reduction of GSH levels, and significantly increased nitric oxide (NO) production. Also, the morphological features caused by the BCAA on C6 cells were prevented by the use of the antioxidants GSH and Nx-nitro-L-arginine methyl ester (L-NAME). Similarly, in another study, Funchal et al. (2006a) showed that C6 glial cells exposed to the BCKA, at doses similar to those found in MSUD, changed their usual rounded morphology to a fusiform or process-bearing cell appearance. On the other hand, the exposure to high concentrations of these acids (10.0 mM) and/or for extended periods of time (24 h)
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elicited cell death. In order to evaluate whether oxidative stress was involved in the BCKA-induced morphological alterations and cell death, various parameters of oxidative stress in C6 cell homogenates, as well as the protective effects of antioxidants were analyzed. It was observed that cell exposure to low doses of all BCKA (1.0 mM) resulted in a marked reduction of nonenzymatic antioxidant defenses, as determined by total antioxidant reactivity (TAR) and GSH measurements. In addition, KIC provoked the reduction of the activity of SOD and GPx, whereas KMV caused a diminution only in SOD activity. Furthermore, NO production was observed to be increased by all BCKA. Finally, it was observed that the morphological features caused by BCKA on C6 cells were prevented by the use of the antioxidants GSH, alpha-tocopherol (trolox), and L-NAME. Taking together, these results strongly suggest that oxidative stress might be contributing to morphological alteration and death observed in C6 glioma cells, as well as in cytoskeletal reorganization elicited by BCAA and BCKA. It is presumed that these findings could explain, at least in part, the neuropathophysiology of MSUD. Another study from the same research group was performed in order to evaluate the effect of BCKA on S100B release from glial cells (Funchal et al. 2007). S100B is a calcium-binding protein expressed and secreted by astrocytes. Extracellular S100B stimulates glial cell proliferation, survive of neurons and extension of neurites in cell culture and is used as a parameter of glial cell death in pathological situations for brain injury (Donato 2001). In fact, elevation of peripheral (cerebrospinal fluid or serum) S100B concentrations has been observed in several common psychiatric disorders (Donato 2003). Funchal et al. showed that KIC and KIV, but not KMV, significantly enhanced S100B liberation from glial cells after 6 h of exposure. Furthermore, the stimulatory effect of the KIC and KIV on S100B release was prevented by coincubation with the energetic substrate creatine and with the L-NAME, a NO synthase inhibitor, indicating that energy deficit and NO were probably involved in this effect. In contrast, other antioxidants such as glutathione (GSH) and trolox (soluble vitamin E) were not able to prevent KIC- and KIV-induced increase of S100B liberation, suggesting that the alteration of S100B release caused by these BCKA is not mediated by oxidation of sulfydryl as well as by lipid peroxyl radicals. In the same way, another study (Funchal et al. 2006b) demonstrated that BCKA inhibited creatine kinase (CK) activity from C6 glioma cells and that this inhibition was totally prevented by preincubation with creatine and with L-NAME. CK has a central role in energy metabolism, especially for high energy consuming tissues, such as brain (Bessman and Carpenter 1985). A decrease in CK is found in neurodegenerative diseases and is correlated with
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neurodegeneration (Aksenov et al. 1997). So, the authors concluded that the inhibition of CK by the BCKA may contribute to the cerebral findings in MSUD. Considering that L-NAME prevented the inhibitory effect of the BCKA on CK activity from C6 glioma cells, NO is probably involved in those effects.
Oxidative Stress in MSUD: The Contribution from Animal Studies It is well-established that animal models provide a useful tool to explore the mechanisms of disease as well as to test potential therapies that could not justifiably be explored in humans without preclinical experimental data. In fact, studies investigating the role of oxidative stress in MSUD started with animal models. Fontella et al. (2002) verified that metabolites accumulated in MSUD stimulate lipid peroxidation in vitro in rat brain. Rat brain homogenates were incubated in the presence of BCAA, BCKA, or the hydroxy derivatives (L-2-hydroxycaproic acid, L-2-hydroxy3-methylvaleric acid, and L-2-hydroxyisovaleric acid), separately. They observed that all amino acids, keto acids, and hydroxy acids accumulated in MSUD stimulated to a variable degree the in vitro parameters of lipid peroxidation tested, named chemiluminescence and thiobarbituric acidreactive substances (TBARS). It is important to emphasize that lipid peroxidation may damage cell structures by altering the integrity, fluidity, and permeability of biomembranes, and by generating potentially toxic products being, therefore, associated to a crescent number of pathological conditions, including neurological diseases (Greenberg et al. 2008; Adibhatla and Hatcher 2010). Reinforcing these findings, Bridi et al. (2003) investigated the in vitro effect of BCAA on the lipid peroxidation, as well as on antioxidant parameters, in cerebral cortex of rats. Lleucine significantly increased chemiluminescence and TBARS measurements and markedly decreased the total radical-trapping antioxidant potential (TRAP) and the TAR values. L-isoleucine increased chemiluminescence and decreased TRAP measurements, but TAR and TBARS levels were not altered by this amino acid. Finally, L-valine diminished the TRAP measurement. The results of this study indicate a stimulation of lipid peroxidation and a reduction of brain capacity to efficiently modulate the damage associated to an increased production of reactive species by the BCAA. So, it can be speculate that oxidative stress was induced in vitro by BCAA, especially by Leu which altered all parameters investigated. Since Leu is probably the main metabolite causing oxidative stress in MSUD, another study evaluated the mechanisms involved in Leu-induced oxidative damage in cerebral cortex of young rats in vitro (Bridi et al. 2005a).
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The authors observed that the increase of TBARS induced by Leu was significantly attenuated when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus Trolox, DTT, GSH, and SOD, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of L-NAME and CAT did not affect TBARS values. It was also observed a dose-dependent inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and GPx, as well as a significant reduction in the membrane-protein thiol content from mitochondrial Leu-enriched preparations. Taken together, the results of this work indicate that a disbalance between reactive species formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage. Bridi et al. (2005b) demonstrated that besides BCAA, the BCKA accumulated in MSUD also stimulate in vitro lipid peroxidation and reduce antioxidant defenses in cerebral cortex of rats. This rat cerebral tissue was incubated in the presence of KIC, KMV, or KIV at concentrations similar to those found in MSUD patients. The major effects observed in this study were with KIC, which significantly increased chemiluminescence and TBARS measurements, decreased TRAP and TAR values, and markedly inhibited GPx activity. Taken all these findings together, it can be presumed that KIC and its precursor Leu provoke the most significant effects on oxidative stress parameters studied, which is in agreement with previous studies demonstrating that these metabolites are the most toxic in the MSUD (Chuang and Shih 2001). In accordance with in vitro findings, in the last years some in vivo studies demonstrated that metabolites accumulated in MSUD induce a pro-oxidative process, probably contributing to the neurological damage in the disease, and that the administration of antioxidants may exerts neuroprotection. Mescka et al. (2011) conducted a study using a chemically-induced acute model of MSUD (Bridi et al. 2006). A BCAA pool was injected subcutaneously in 14-day-old rats, and it was observed a markedly reduction in CAT and GPx activities in cerebral cortex homogenates. Additionally, TBARS levels and carbonyl and sulfhydryl content (parameters of protein oxidation) were also altered in brain by the BCAA. It was also demonstrated that the treatment with L-carnitine was capable to prevent all these alterations, showing a neuroprotective effect of this substance. In fact, some works have shown antiperoxidative effects of L-carnitine (Fariello and Calabrese 1988; Bertelli et al. 1994; Lowitt et al. 1995) and its capacity to prevent the formation of ROS, besides to regulate NO, cellular respiration, as well as the activity of antioxidant enzymes (Brown 1999; Kremser et al. 1995). Antioxidants showed also to prevent memory impairments in an animal model of MSUD (Scaini et al. 2012a).
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Wistar rats were submitted to acute and chronic administrations of a BCAA pool. These administrations induced long-term memory impairment in the inhibitory avoidance and CMIA (continuous multiple trials stepdown inhibitory avoidance) tasks. The coadministration of the antioxidants N-acetylcysteine (NAC) and deferoxamine (DFX) prevented inhibitory avoidance alterations, suggesting that cognition impairment after BCAA administration may be caused by oxidative brain damage. Another study demonstrated the occurrence of DNA damage (determined by the alkaline comet assay) in MSUD-induced animals (Scaini et al. 2012b). It is important to emphasize that DNA is a particularly important target for oxidation generating several classes of products (single- and double-strand breaks), inter/intra-strand crosslinks, DNA-proteins cross-links, and sugar fragmentation products. These products may elicit mutations, microsatellite instability, loss of heterozygosity, chromosomal aberrations, cytotoxicity, and neoplasic growth (Cooke et al. 2006). The acute administration of BCAA increased the DNA damage frequency and damage index in the hippocampus, whereas the chronic administration of BCAA increased the DNA damage frequency and damage index both in hippocampus and striatum. In addition, the antioxidant treatment with NAC and DFX was able to prevent DNA damage in these brain structures. NAC is known to reduce oxidative stress by improving thiol redox status and to scavenge superoxide and hydroxyl radicals and H2O2. However, when administered alone, NAC has pro-oxidant effects probably by its interaction with iron. So, the coadministration of DFX, a powerful iron chelator, has been used to improve NAC response (Aruoma et al. 1989; Ritter et al. 2004). NAC and DFX treatment also prevented the increase of brain acetylcholinesterase (AChE) activity induced in vivo by chronic administration of BCAA in rats (Scaini et al. 2012c). AChE is associated with brain development, learning, memory, and neuronal damage, and its activation leads to fast acetylcholine degradation and a subsequent downregulation of acetylcholine receptors, causing undesirable effects on cognitive functions (Soreq and Seidman 2001). So, the authors conclude that antioxidant treatment increases the efficiency of cholinergic transmission in MSUD by preventing the hydrolysis of acetylcholine, and consequently improving cognitive functions, such as learning and memory.
Oxidative Stress in MSUD Patients Contributing with the findings obtained from the cell culture and animal studies, in the last years, some works have demonstrated important alterations in oxidative stress
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biomarkers in MSUD-affected patients. One of these biomarkers is malondialdehyde (MDA), an end product of lipid peroxidation by reactive species, whose formation can be easily assessed by the measurement of TBARS. Barschak et al. (2006) demonstrated that TBARS were increased in plasma samples from five untreated patients with the classic form of MSUD, indicating an increased oxidative damage to lipids in these patients. Furthermore, the authors also demonstrated that TAR measurement was reduced in these samples, suggesting a poor quality of the tissue antioxidants in modulating the damage provoked by an increased ROS formation. Similar results were found by these researchers (Barschak et al. 2008b) in plasma from ten MSUD patients treated with a protein-restricted diet (low BCAA), and supplemented with a semisynthetic formula of essential amino acids, vitamins, and minerals. These patients presented an increased lipid peroxidation, as well as reduced antioxidant defenses, even during the treatment. Despite the high levels of Leu, Ileu, and Val presented by these patients, no significant correlations were observed between the concentrations of these amino acids and the oxidative stress parameters investigated. As occurs in phenylketonuria and in some organic acidemias (Sitta et al. 2011; Ribas et al. 2010), it is recognized that the low protein dietetic treatment to which the MSUD patients are submitted can contribute to alter antioxidant defenses, since strict protein ingest may reduce the absorption of vitamins and minerals which are essential to this defense system. One of these important elements is selenium, which is mainly present in foods like grains, cereals, and meat. Selenium exerts a fundamental antioxidant role, since it is the cofactor of the GPx enzyme. Some studies have demonstrated that selenium levels are reduced in plasma, whole blood and hair from patients with MSUD (Lombeck et al. 1978; Borglund et al. 1989; Barschak et al. 2007). Barschak et al. (2007) showed that this deficiency occurs in MSUD patients at diagnosis, but is more prominent in MSUD patients during the dietetic treatment. Treated MSUD patients also presented decreased erythrocyte GPx activity (Borglund et al. 1989; Lombeck et al. 1978; Barschak et al. 2007) that may be related, at least in part, to the reduction of this element, although a direct correlation between selenium concentrations and GPx activity in these patients had not been observed. On the other hand, Borglund et al. (1989) showed that selenium supplementation was able to increase tenfold the activity of GPx in a MSUD patient. These findings suggest that selenium supplementation could represent an important adjuvant therapy for MSUD. Also in this context, Mescka et al. (2012) demonstrated that MSUD patients treated with low protein diet present Lcarnitine deficiency. L-Carnitine is a small quaternary amine highly polar and water soluble, which may be
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biosynthesized by humans (25 %) or obtained from dietary sources (75 %), mainly from meats, milk, and nuts (Agarwal and Said 2004; Derin et al. 2004; Gulcin 2006). Usually it is present in plasma as free carnitine, which is responsible by transporting long-chain fatty acids across the inner mitochondrial membrane for utilization in metabolism through b-oxidation (Matalliotakis et al. 2000; Tastekin et al. 2007). Furthermore, L-carnitine has antioxidant properties, and it may protect cells from toxic oxygen metabolites (Derin et al. 2004). Thereby, in this work, it was investigated the effect of BCAA-restricted diet, supplemented or not with L-carnitine, on lipid and protein oxidation in MSUD patients. Significant increases of MDA and protein carbonyl content were observed in plasma from MSUD patients treated only with BCAA-restricted diet. However, patients under BCAA-restricted diet and supplemented with L-carnitine (50 mg/kg/day) presented a marked reduction of MDA content in relation to the carnitine-untreated MSUD patients. In addition, free L-carnitine concentrations were negatively correlated with MDA levels. These data show that L-carnitine may exert an antioxidant role, protecting cells against the lipid peroxidation, and this effect could represent an additional therapeutic approach to the patients affected by MSUD. In another work, Barschak et al. (2009) evaluated the concentrations of amino acids, branched-chain a-keto acids, and a-hydroxy acids in plasma samples from treated MSUD patients, and the authors observed that tryptophan and methionine were significantly reduced in relation to control values. This reduction may represent a mechanism of neurological dysfunction in MSUD, since the deficit of these amino acids in the brain can lead to an impaired protein and neurotransmitter synthesis (Arau´jo et al. 2001). Tryptophan and methionine can act as antioxidants through different mechanisms (Keithahn and Lerchl 2005; Watanabe et al. 2002; Levine et al. 1996; Stadtman 2004) and this role may be especially important in an organ like the brain, which is highly susceptible to the attack by reactive species. Besides, in the study published by Barschak et al. (2009), tryptophan and methionine concentrations presented a strongly inverse correlation with TBARS levels, suggesting that the low content of these amino acids in plasma could possibly be involved in the lipid peroxidation observed. The role of oxidative stress in the development of the clinical and biochemical manifestations of MSUD is not yet fully understood. Barschak et al. (2008a, b) showed that MSUD-treated patients present important biochemical alterations, such as reduced levels of glucose, cholesterol, creatinine, and albumin, as well as a high activity of the enzymes aspartate aminotransferase and lactate dehydrogenase. Associated to these findings, the authors observed increased levels of TBARS and a positive correlation
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between TBARS and plasma concentrations of triglycerides (Barschak et al. 2008a). Although all these data strongly suggest the occurrence of oxidative stress in patients with MSUD, and that Leu together with KIC can be considered the main neurotoxic agents in this disease, the studies in patients with MSUD have not shown a clear association between these metabolites and the markers of oxidative stress. So, it is possible that other factors may be related to oxidative stress in MSUD which needs to be better investigated.
Conclusions Oxidative stress has been associated to a crescent number of pathological conditions, including neurological diseases, since brain is an organ vulnerable to oxidative damage by reactive species. MSUD is a neurological disease, but the mechanisms underlying the neurotoxicity of this disorder remain unclear. However, in this review we described many in vitro and in vivo evidence in cell lines, animal models, and patients showing the occurrence of oxidative stress in MSUD, probably contributing to the neurological sequelae present in most patients. It is presumed that metabolites accumulated in MSUD induce excessive production of reactive species and/or deplete the tissue antioxidant capacity, leading to damage to biomolecules (lipids, proteins, and DNA). In addition, protein-restricted diet to which MSUD patients are submitted may lead to a decrease in the antioxidants intake (selenium and L-carnitine, for example), contributing to the oxidative damage verified in the disease. It is important to emphasize that the results from the presently available studies must be interpreted cautiously and confirmed by other in vivo studies, but it is possible to suggest that in the future antioxidants could be used as an adjuvant therapy for MSUD patients. Conflict of interest interest.
The authors declare that there is no conflict of
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