Preliminary results of PBA-loaded nanoparticles development and the effect on oxidative stress and neuroinflammation in rats submitted to a chemically induced chronic model of MSUD.

Maple syrup urine disease (MSUD) is a genetic disorder that leads the accumulation of branched-chain amino acids (BCAA) leucine (Leu), isoleucine, valine and metabolites. The symptomatology includes psychomotor delay and mental retardation. MSUD therapy comprises a lifelong protein strict diet with low BCAA levels and is well established that high concentrations of Leu and/or its ketoacid are associated with neurological symptoms. Recently, it was demonstrated that the phenylbutyrate (PBA) have the ability to decrease BCAA concentrations. This work aimed the development of lipid-based nanoparticles loaded with PBA, capable of targeting to the central nervous system in order to verify its action mechanisms on oxidative stress and cell death in brain of rats subjected to a MSUD chronic model. PBA-loaded nanoparticles treatment was effective in significantly decreasing BCAA concentration in plasma and Leu in the cerebral cortex of MSUD animals. Furthermore, PBA modulate the activity of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase enzymes, as well as preventing the oxidative damage to lipid membranes and proteins. PBA was also able to decrease the glial fibrillary acidic protein concentrations and partially decreased the reactive species production and caspase-3 activity in MSUD rats. Taken together, the data indicate that the PBA-loaded nanoparticles could be an efficient adjuvant in the MSUD therapy, protecting against oxidative brain damage and neuroinflammation.

Melatonin ameliorates oxidative stress and DNA damage of rats subjected to a chemically induced chronic model of Maple Syrup Urine Disease.

Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched α-ketoacid dehydrogenase complex (BCKDC) activity. Branched-chain amino acids (BCAA) accumulation is, at least in part, responsible for neurological disturbances characteristic of this metabolic disorder. Experimental studies demonstrated that high levels of BCAA induce brain oxidative stress. Considering that many antioxidants are obtained from the diet, the dietary restriction in MSUD patients probably produce deficiency of vitamins and micronutrients involved in antioxidant defenses. Supplementation with synthetic melatonin has been used to prevention and treatment of pathological conditions, including brain diseases. In this study, we aimed at investigating the potential neuroprotective effect of melatonin treatment in a MSUD experimental model. Infant rats (7 day old) received twice daily subcutaneous injections of a BCAA pool (0.21472 g/kg, 190 mmol/L leucine, 59 mmol/L isoleucine and 69 mmol/L valine in saline solution (15.8 µL/g per weight/injection) or saline alone, and supplemented with melatonin (10 mg/kg, intraperitoneal) for 21 days. Oxidative stress parameters, i.e. antioxidant enzyme activity, reactive species production and damage to lipids and proteins, were assessed in the cerebral cortex, hippocampus and striatum at twenty-eight days of age. In addition, the damage to blood cell DNA was evaluated. The chronic administration of BCAA pool in infant rats induced significant oxidative stress (p < 0.05) - such as oxidation of lipids and proteins, imbalance in antioxidant enzymes activities - damages in DNA (p < 0.05) and in brain structures (cerebral cortex, hippocampus and striatum). Notably, melatonin supplementation was able to ameliorate the oxidative (p < 0.05) and antioxidant (p < 0.05) parameters in the brain and blood of the rat model of MSUD. Our results show that melatonin could be a promising therapeutic agent for MSUD.

L-carnitine Prevents Oxidative Stress in the Brains of Rats Subjected to a Chemically Induced Chronic Model of MSUD.

Maple syrup urine disease (MSUD), or branched-chain α-keto aciduria, is an inherited disorder that is caused by a deficiency in branched-chain α-keto acid dehydrogenase complex (BCKAD) activity. Blockade of this pathway leads to the accumulation of the branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, and their respective ketoacids in tissues. The main clinical symptoms presented by MSUD patients include ketoacidosis, hypoglycemia, opisthotonos, poor feeding, apnea, ataxia, convulsions, coma, psychomotor delay, and mental retardation. Although increasing evidence indicates that oxidative stress is involved in the pathophysiology of this disease, the mechanisms of the brain damage caused by this disorder remain poorly understood. In the present study, we investigated the effect of BCAAs on some oxidative stress parameters and evaluated the efficacy of L-carnitine (L-car), an efficient antioxidant that may be involved in the reduction of oxidative damage observed in some inherited neurometabolic diseases, against these possible pro-oxidant effects of a chronic MSUD model in the cerebral cortex and cerebellum of rats. Our results showed that chronic BCAA administration was able to promote both lipid and protein oxidation, impair brain antioxidant defenses, and increase reactive species production, particularly in the cerebral cortex, and that L-car was able to prevent these effects. Taken together, the present data indicate that chronic BCAA administration significantly increased oxidative damage in the brains of rats subjected to a chronic model of MSUD and that L-car may be an efficient antioxidant in this disorder.

DNA damage in an animal model of maple syrup urine disease.

Maple syrup urine disease is an inborn error of metabolism caused by a severe deficiency of the branched chain alpha-ketoacid dehydrogenase complex. Neurological dysfunction is a common finding in patients with maple syrup urine disease. However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly understood. In this study, we investigated whether acute or chronic administration of a branched chain amino acid pool (leucine, isoleucine and valine) causes transient DNA damage, as determined by the alkaline comet assay, in the brain and blood of rats during development and whether antioxidant treatment prevented the alterations induced by branched chain amino acids. Our results showed that the acute administration of branched chain amino acids increased the DNA damage frequency and damage index in the hippocampus. However, the chronic administration of branched chain amino acids increased the DNA damage frequency and damage index in both the hippocampus and the striatum, and the antioxidant treatment was able to prevent DNA damage in the hippocampus and striatum. The present study demonstrated that metabolite accumulation in MSUD induces DNA damage in the hippocampus and striatum and that it may be implicated in the neuropathology observed in the affected patients. We demonstrated that the effect of antioxidant treatment (N-acetylcysteine plus deferoxamine) prevented DNA damage, suggesting the involvement of oxidative stress in DNA damage.

Antioxidant administration prevents memory impairment in an animal model of maple syrup urine disease.

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder resulting from deficiency of branched-chain α-keto acid dehydrogenase complex leading to branched chain amino acids (BCAA) leucine, isoleucine, and valine accumulation as well as their corresponding transaminated branched-chain α-keto acids. MSUD patients present neurological dysfunction and cognitive impairment. Here, we investigated whether acute and chronic administration of a BCAA pool causes impairment of acquisition and retention of avoidance memory in young rats. We have used two administration protocols. Acute administration consisted of three subcutaneous administrations of the BCAA pool (15.8 µL/g body weight at 1-h intervals) containing 190 mmol/L leucine, 59 mmol/L isoleucine, and 69 mmol/L valine or saline solution (0.85% NaCl; control group) in 30 days old Wistar rats. Chronic administration consisted of two subcutaneous administrations of BCAA pool for 21 days in 7 days old Wistar rats. N-acetylcysteine (NAC; 20 mg/kg) and deferoxamine (DFX; 20 mg/kg) co administration influence on behavioral parameters after chronic BCAA administration was also investigated. BCAA administration induced long-term memory impairment in the inhibitory avoidance and CMIA (continuous multiple-trials step-down inhibitory avoidance) tasks whereas with no alterations in CMIA retention memory. Inhibitory avoidance alterations were prevented by NAC and DFX. BCAA administration did not impair the neuropsychiatric state, muscle tone and strength, and autonomous function evaluated with the SHIRPA (SmithKline/Harwell/ImperialCollege/RoyalHospital/Phenotype Assessment) protocol. Taken together, our results indicate that alterations of motor activity or emotionality probably did not contribute to memory impairment after BCAA administration and NAC and DFX effects suggest that cognition impairment after BCAA administration may be caused by oxidative brain damage.

Evaluation of acetylcholinesterase in an animal model of maple syrup urine disease.

Maple syrup urine disease is an inherited metabolic disease predominantly characterized by neurological dysfunction. However, the mechanisms underlying the neuropathology of this disease are still not defined. Therefore, the aim of this study was to investigate the effect of acute and chronic administration of a branched-chain amino acids (BCAA) pool (leucine, isoleucine, and valine) on acetylcholinesterase (AChE) activity and gene expression in the brain and serum of rats and to assess if antioxidant treatment prevented the alterations induced by BCAA administration. Our results show that the acute administration of a BCAA pool in 10- and 30-day-old rats increases AChE activity in the cerebral cortex, striatum, hippocampus, and serum. Moreover, chronic administration of the BCAA pool also increases AChE activity in the structures studied, and antioxidant treatment prevents this increase. In addition, we show a significant decrease in the mRNA expression of AChE in the hippocampus following acute administration in 10- and 30-day-old rats. On the other hand, AChE expression increased significantly after chronic administration of the BCAA pool. Interestingly, the antioxidant treatment was able to prevent the increased AChE activity without altering AChE expression. In conclusion, the results from the present study demonstrate a marked increase in AChE activity in all brain structures following the administration of a BCAA pool. Moreover, the increased AChE activity is prevented by the coadministration of N-acetylcysteine and deferoxamine as antioxidants.

A chemically-induced acute model of maple syrup urine disease in rats for neurochemical studies.

We report a chemically-induced model of maple syrup urine disease (MSUD) in 10- and 30-day-old rats produced by subcutaneous administration of a branched-chain amino acids (BCAA) pool along with the analyses of plasma and brain amino acid levels by HPLC at 0-120 min after administration. We observed an increase of plasma leucine (Leu), isoleucine (Ile) and valine (Val) concentrations in both 10- and 30-day-old rats. These increases were accompanied by a concomitant reduction of plasma concentrations of methionine (Met), phenylalanine (Phe), tyrosine (Tyr), histidine (His), alanine (Ala), lysine (Lys), and ornithine (Orn) in 10-day-old rats and of Met, Phe, Tyr, tryptophan (Trp), and Orn in 30-day-old rats. These results are similar to those observed in MSUD patients during crises, when plasma levels of large neutral amino acids (LNAA) are also reduced when BCAA concentrations are increased. In the brain, increased concentrations of Leu, Ile and Val were achieved in 10-day-old rats at all times after injection. In contrast, no differences in cerebral concentrations of BCAA were observed in 30-day-old rats. In conclusion, the present MSUD model, using 10- rather than 30-day-old rats, has a similar amino acid profile to that of MSUD untreated patients and is suitable to investigate the mechanisms of brain damage characteristic of this disorder.