Effect of Leucine Administration to Female Rats During Pregnancy and Lactation on Oxidative Stress and Enzymes Activities of Phosphoryltransfer Network in Cerebral Cortex and Hippocampus of the Offspring

Maple Syrup Urine Disease is an inborn error of metabolism caused by severe deficiency in the activity of branched-chain α-keto acid dehydrogenase complex. Neurological disorder is common in patients with maple syrup urine disease. Although leucine is considered the main toxic metabolite, the mechanisms underlying the neuropathology of brain injury are poorly understood. In the present study, we evaluated the possible preventive effect of the co-administration of creatine plus pyruvate on the effects elicited by leucine administration to female Wistar rats during pregnancy and lactation on some oxidative stress parameters as well as the activities of some enzymes involved in the phosphoryltransfer network in the brain cortex and hippocampus of the offspring at 21 days of age. Leucine administration induced oxidative stress and altered the activities of pyruvate kinase, adenylate kinase, mitochondrial and cytosolic creatine kinase. Co-administration of creatine plus pyruvate was partially effective in the prevention of some alterations provoked by leucine administration on the oxidative stress but not in the enzymes of phosphoryltransfer network. These results suggest that non-treated maternal hyperleucinemia may be toxic to the brain of the offspring.     

Inhibition of the Mitochondrial Respiratory Chain by Phenylalanine in Rat Cerebral Cortex

Phenylketonuria (PKU) is biochemically characterized by the accumulation of phenylalanine (Phe) and its metabolites in tissues of affected children. Neurological damage is the clinical hallmark of PKU, and Phe is considered the main neurotoxic metabolite in this disorder. However, the mechanisms of neurotoxicity are poorly known. The main objective of the present work was to measure the activities of the mitochondrial respiratory chain complexes (RCC) and succinate dehydrogenase (SDH) in brain cortex of Wistar rats subjected to chemically induced hyperphenylalaninemia (HPA). We also investigated the in vitro effect of Phe on SDH and RCC activities in the cerebral cortex of 22-day-old rats. HPA was induced by subcutaneous administration of 2.4 mol/g body weight -methylphenylalanine, a phenylalanine hydroxylase inhibitor, once a day, plus 5.2 M/g body weight phenylalanine, twice a day, from the 6th-21st postnatal day. The results showed a reduction of SDH and complex I + III activity in brain cortex of rats subjected to HPA. We also verified that Phe inhibited the in vitro activity of complexes I + III, possibly by competition with NADH. Considering the importance of SDH and RCC for the maintenance of energy supply to brain, our results suggest that energy deficit may contribute to the Phe neurotoxicity in PKU.     

Role of Catalase and Superoxide Dismutase Activities on Oxidative Stress in the Brain of a Phenylketonuria Animal Model and the Effect of Lipoic Acid

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase which leads to accumulation of phenylalanine and its metabolites in tissues of patients with severe neurological involvement. Recently, many studies in animal models or patients have reported the role of oxidative stress in PKU. In the present work we studied the effect of lipoic acid against oxidative stress in rat brain provoked by an animal model of hyperphenylalaninemia (HPA), induced by repetitive injections of phenylalanine and α-methylphenylalanine (a phenylalanine hydroxylase inhibitor) for 7 days, on some oxidative stress parameters. Lipoic acid prevented alterations on catalase (CAT) and superoxide dismutase (SOD), and the oxidative damage of lipids, proteins, and DNA observed in HPA rats. In addition, lipoic acid diminished reactive species generation compared to HPA group which was positively correlated to SOD/CAT ratio. We also observed that in vitro Phe inhibited CAT activity while phenyllactic and phenylacetic acids stimulated superoxide dismutase activity. These results demonstrate the efficacy of lipoic acid to prevent oxidative stress induced by HPA model in rats. The possible benefits of lipoic acid administration to PKU patients should be considered.     

Homocysteine induces energy imbalance in rat skeletal muscle: Is creatine a protector?

Homocystinuria is a neurometabolic disease caused by a severe deficiency of cystathionine beta‐synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction. In this study, we investigated the effect of chronic hyperhomocysteinemia on the cell viability of the mitochondrion, as well as on some parameters of energy metabolism, such as glucose oxidation and activities of pyruvate kinase, citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, respiratory chain complexes and creatine kinase in gastrocnemius rat skeletal muscle. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injections of homocysteine (0.3–0.6 µmol/g body weight) and/or creatine (50 mg/kg body weight) from the 6th to the 28th days of age. The animals were decapitated 12 h after the last injection. Homocysteine decreased the cell viability of the mitochondrion and the activities of pyruvate kinase and creatine kinase. Succinate dehydrogenase was increased other evaluated parameters were not changed by this amino acid. Creatine, when combined with homocysteine, prevented or caused a synergistic effect on some changes provoked by this amino acid. Creatine per se or creatine plus homocysteine altered glucose oxidation. These findings provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function, more studies are needed to elucidate them. Although creatine prevents some alterations caused by homocysteine, it should be used with caution, mainly in healthy individuals because it could change the homeostasis of normal physiological functions. Copyright © 2012 John Wiley & Sons, Ltd.     

Maternal phenylketonuria. Review with emphasis on pathogenesis

Maternal phenylketonuria (PKU) refers to fetal damage from PKU in the pregnant woman. The progeny from such pregnancies are almost always microcephalic and mentally subnormal and have an increased frequency of congenital heart disease and low birth weight. Treatment with a phenylalanine-restricted diet, if begun before conception, seems to protect the fetus. The degree of protection is much less if dietary treatment is delayed until the pregnancy is in progress. The origin of fetal damage in maternal PKU is not known. Due to placental concentration of amino acids, the fetus is exposed to a higher concentration of phenylalanine than that in the mother, but it is not certain that phenylalanine is the toxic agent. Animal models made hyperphenylalaninemic by the administration of phenylalanine, often accompanied by a phenylalanine hydroxylase inhibitor, do not reproduce the full maternal PKU syndrome; but fetuses and newborns from these models have had reduced growth of the body and brain, and offspring later may show evidence of impaired learning ability.     

Alanine Prevents the Reduction of Pyruvate Kinase Activity in Brain Cortex of Rats Subjected to Chemically Induced Hyperphenylalaninemia

The mechanisms by which phenylalanine is toxic to the brain in phenylketonuria are not fully understood. Considering that brain glucose metabolism is reduced in these patients, our main objective was to determine pyruvate kinase activity in brain cortex of rats subjected to acute and chronic chemically induced hyperphenylalaninemia. The effect of alanine administration on the enzyme activity in the treated rats was also investigated. We also studied the in vitro effect of the two amino acids on pyruvate kinase activity in brain cortex of nontreated rats. The results indicated that phenylalanine inhibits pyruvate kinase in vitro and in vivo and that alanine prevents the inhibitory effect of phenylalanine on the enzyme activity. Considering the crucial role pyruvate kinase plays in glucose metabolism in brain, it is possible that inhibition of this enzyme activity may contribute to the brain damage characteristic of this disease.     

Gender differences in the expression of genes involved during cardiac development in offspring from dams on high fat diet

Previously we have demonstrated that maternal high fat diet (HF) during pregnancy increase cardiovascular risk in the offspring, and pharmacological intervention using statins in late pregnancy reduced these risk factors. However the effects of maternal HF-feeding and statin treatment during pregnancy on development of heart remain unknown. Hence we measured expression of genes involved in cell cycle progression (cyclin G1), ventricular remodelling brain natriuretic peptide (BNP), and environmental stress response small proline-rich protein 1A (SPRR 1A) in the offspring left ventricle (LV) from dams on HF with or without statin treatment. Female C57 mice were fed a HF diet (45 % kcal fat) 4 weeks prior to conception, during pregnancy and lactation. From the second half of the pregnancy and throughout lactation, half of the pregnant females on HF diet were given a water-soluble statin (Pravastatin) in their drinking water (HF + S). At weaning offspring were fed HF diet to adulthood (generating dam/offspring dietary groups HF/HF and HF + S/HF). These groups were compared with offspring from dams fed standard chow (C 21 % kcal fat) and fed C diet from weaning (C/C). LV mRNA levels for cyclin G1, BNP and SPRR 1A were measured by RT-PCR. Heart weights and BP in HF/HF offspring were higher versus C/C group. Maternal Pravastatin treatment reduced BP and heart weights in HF + S/HF female offspring to levels found in C/C group. LV cyclin G1 mRNA levels were lower in HF/HF versus both C/C and HF + S/HF offspring. BNP mRNA levels were elevated in HF/HF females but lower in males versus C/C. BNP gene expression in HF + S/HF offspring was similar to HF/HF. SPRR 1A mRNA levels were similar in all treatment groups. Statins given to HF-fed pregnant dams reduced cardiovascular risk in adult offspring, and this is accompanied by changes in expression of genes involved in adaptive remodelling in the offspring LV and that there is a gender difference.     

Alterations in Inflammatory Mediators, Oxidative Stress Parameters and Energetic Metabolism in the Brain of Sepsis Survivor Rats

Sepsis is characterized by biochemical alterations in the central nervous system at early times and cognitive impairment at late times after induction in sepsis animal model. In order to understand at least in part the mechanism of disease, we have evaluated the effects of sepsis on cytokine levels in the cerebrospinal fluid (CSF); oxidative parameters; the activity of the electron transport chain enzymes; and creatine kinase (CK) activity in the brain of sepsis survivor rats 10 days after cecal ligation and perforation (CLP). Male Wistar rats underwent CLP with "basic support" or sham-operated. Ten days after surgery, the animals were killed and prefrontal cortex, cortex, hippocampus, striatum, cerebellum, and CSF were obtained. It was found a decrease in the levels of TNF-α (P = 0.001), IL-1β (P = 0.008), IL-6 (P = 0.038), and IL-10 (P = 0.022) in the CSF; an increase in the TBARS only hippocampus (0.027); an up-regulation in the activity of complex II (P = 0.024), III (P = 0.018), and IV (P = 0.047) only in the prefrontal cortex; a decrease in the CK activity in the cerebellum (P = 0.001) and striatum (P = 0.0001), and an increase in the hippocampus (P = 0.0001) and cortex (P = 0.0001). Oxidative stress and mitochondrial alterations observed during early times in sepsis, persisted up to 10 days after surgery. The cytokines levels during the early times were found at high levels, decreasing to low levels after 10 days. In conclusion, these findings may contribute for a better comprehension of the cognitive damage in sepsis survivor rats.     

Proline Reduces Creatine Kinase Activity in the Brain Cortex of Rats

Type II hyperprolinemia is an inherited disorder caused by a deficiency of ¹-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurological symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of the present study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity of brain cortex of Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered twice a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the brain cortex of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in cerebral cortex of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase forthe maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.     

Large Neutral Amino Acid Supplementation Exerts Its Effect through Three Synergistic Mechanisms: Proof of Principle in Phenylketonuria Mice

Background

Phenylketonuria (PKU) was the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. However, despite this effect, neuropsychological outcome in PKU still remains suboptimal and the phenylalanine-restricted diet is very demanding. To improve neuropsychological outcome and relieve the dietary restrictions for PKU patients, supplementation of large neutral amino acids (LNAA) is suggested as alternative treatment strategy that might correct all brain biochemical disturbances caused by high blood phenylalanine, and thereby improve neurocognitive functioning.

Objective

As a proof-of-principle, this study aimed to investigate all hypothesized biochemical treatment objectives of LNAA supplementation (normalizing brain phenylalanine, non-phenylalanine LNAA, and monoaminergic neurotransmitter concentrations) in PKU mice.

Methods

C57Bl/6 Pah-enu2 (PKU) mice and wild-type mice received a LNAA supplemented diet, an isonitrogenic/isocaloric high-protein control diet, or normal chow. After six weeks of dietary treatment, blood and brain amino acid and monoaminergic neurotransmitter concentrations were assessed.

Results

In PKU mice, the investigated LNAA supplementation regimen significantly reduced blood and brain phenylalanine concentrations by 33% and 26%, respectively, compared to normal chow (p<0.01), while alleviating brain deficiencies of some but not all supplemented LNAA. Moreover, LNAA supplementation in PKU mice significantly increased brain serotonin and norepinephrine concentrations from 35% to 71% and from 57% to 86% of wild-type concentrations (p<0.01), respectively, but not brain dopamine concentrations (p = 0.307).

Conclusions

This study shows that LNAA supplementation without dietary phenylalanine restriction in PKU mice improves brain biochemistry through all three hypothesized biochemical mechanisms. Thereby, these data provide proof-of-concept for LNAA supplementation as a valuable alternative dietary treatment strategy in PKU. Based on these results, LNAA treatment should be further optimized for clinical application with regard to the composition and dose of the LNAA supplement, taking into account all three working mechanisms of LNAA treatment.     

Cerebral biochemical abnormalities in experimental maternal phenylketonuria: gangliosides and sialoglycoproteins

The present study sought a biochemical explanation for retarded brain development in the heterozygous offspring of the phenylketonuric (PKU) mother. Two rat models of simulated maternal PKU, one induced by p-chlorophenylalanine and phenylalanine and the other by phenylacetate, were employed in this investigation. Maternal PKU had no influence on cerebral concentrations of DNA, protein, and cholesterol, which were normal in the 2 d old pup. However, there was a noticeable disruption of the normal ganglioside pattern and a significant reduction of sialoglycoproteins. Concomitant with a delayed drop in the gangliosides Q1b and D3, was a slower rise in M1 and D1a. At least 66% of sialoglycoproteins located on SDS-PAGE gel chromatograms, by radioactivity incorporated in vivo from radiolabeled N-acetylmannosamine and by (3H) sialic acid released by Neuraminidase from periodate-(3H)borohydride labeled glycoproteins, have mobilities of the cell adhesion molecules N-CAM and D-CAM. Whether the reduction of the sialoglycoproteins induced by maternal PKU is mainly in these cell adhesion molecules requires further investigation. Interference with the function of gangliosides and certain sialoglycoproteins during cerebral development may contribute to the brain dysfunction observed in the offspring of PKU mothers not on diet control during pregnancy.     

Effect of thyroid hormones on the activity of pyruvate kinase and lactate dehydrogenase in mature rat brain

The enzymatic activities of two "key" enzymes of the glycolytic pathway, pyruvate kinase and lactic dehydrogenase, were studied in seven areas of the brain in male adult rats in states of pharmacologically induced hyper and hypothyroidism. The brain areas were: anterior cortex, adenohypophysis, hypothalamus, amygdaline nucleus, septum, hippocampus and cerebellum. In T3 treated animals, pyruvate kinase activity showed significant increase in all the areas studied while lactic dehydrogenase activity decreased. In propyl-thiouracil treated animals these enzyme activities showed no significant variations from those in animals of the control group.     

The role of tubulin in the mitochondrial metabolism and arrangement in muscle cells

Tubulin, a well-known component of the microtubule in the cytoskeleton, has an important role in the transport and positioning of mitochondria in a cell type dependent manner. This review describes different functional interactions of tubulin with cellular protein complexes and its functional interaction with the mitochondrial outer membrane. Tubulin is present in oxidative as well as glycolytic type muscle cells, but the kinetics of the in vivo regulation of mitochondrial respiration in these muscle types is drastically different. The interaction between VDAC and tubulin is probably influenced by such factors as isoformic patterns of VDAC and tubulin, post-translational modifications of tubulin and phosphorylation of VDAC. Important factor of the selective permeability of VDAC is the mitochondrial creatine kinase pathway which is present in oxidative cells, but is inactive or missing in glycolytic muscle and cancer cells. As the tubulin-VDAC interaction reduces the permeability of the channel by adenine nucleotides, energy transfer can then take place effectively only through the mitochondrial creatine kinase/phosphocreatine pathway. Therefore, closure of VDAC by tubulin may be one of the reasons of apoptosis in cells without the creatine kinase pathway. An important question in tubulin regulated interactions is whether other proteins are interacting with tubulin. The functional interaction may be direct, through other proteins like plectins, or influenced by simultaneous interaction of other complexes with VDAC.     

Differential Effects of Intrauterine Growth Restriction on the Regional Neurochemical Profile of the Developing Rat Brain

Intrauterine growth restricted (IUGR) infants are at increased risk for neurodevelopmental deficits that suggest the hippocampus and cerebral cortex may be particularly vulnerable. Evaluate regional neurochemical profiles in IUGR and normally grown (NG) 7-day old rat pups using in vivo ¹H magnetic resonance (MR) spectroscopy at 9.4 T. IUGR was induced via bilateral uterine artery ligation at gestational day 19 in pregnant Sprague–Dawley dams. MR spectra were obtained from the cerebral cortex, hippocampus and striatum at P7 in IUGR (N = 12) and NG (N = 13) rats. In the cortex, IUGR resulted in lower concentrations of phosphocreatine, glutathione, taurine, total choline, total creatine (P < 0.01) and [glutamate]/[glutamine] ratio (P < 0.05). Lower taurine concentrations were observed in the hippocampus (P < 0.01) and striatum (P < 0.05). IUGR differentially affects the neurochemical profile of the P7 rat brain regions. Persistent neurochemical changes may lead to cortex-based long-term neurodevelopmental deficits in human IUGR infants.     

Canine erythrocyte pyruvate kinase. I. Properties of the normal enzyme

We have compared the kinetic, immunological, and electrophoretic properties of human and canine erythrocyte pyruvate kinase. Both enzymes are allosteric and subject to positive and negative regulation. The allosteric properties of the canine enzyme are more pronounced than those of the human enzyme; however, the properties of both enzymes are consistent with a regulatory function in the glycolytic pathway of their respective erythrocytes. Antiserum against the human enzyme gives precipitin lines of partial identity between the human and canine enzymes on immunodiffusion. The anti-human serum inactivates the enzymatic activity of both enzymes, although it is more effective against the human enzyme than the canine. The two enzymes have slightly different mobilities on starch gel electrophoresis. While we have demonstrated differences between erythrocyte pyruvate kinase from dogs and that from humans, we conclude that the enzymes are sufficiently similar in properties and function to allow use of the dog as a model for human erythrocyte pyruvate kinase deficiency.     

Experimental Evidence that Phenylalanine Provokes Oxidative Stress in Hippocampus and Cerebral Cortex of Developing Rats

High levels of phenylalanine (Phe) are the biochemical hallmark of phenylketonuria (PKU), a neurometabolic disorder clinically characterized by severe mental retardation and other brain abnormalities, including cortical atrophy and microcephaly. Considering that the pathomechanisms leading to brain damage and particularly the marked cognitive impairment in this disease are poorly understood, in the present study we investigated the in vitro effect of Phe, at similar concentrations as to those found in brain of PKU patients, on important parameters of oxidative stress in the hippocampus and cerebral cortex of developing rats. We found that Phe induced in vitro lipid peroxidation (increase of TBA-RS values) and protein oxidative damage (sulfhydryl oxidation) in both cerebral structures. Furthermore, these effects were probably mediated by reactive oxygen species, since the lipid oxidative damage was totally prevented by the free radical scavengers α-tocopherol and melatonin, but not by L-NAME, a potent inhibitor of nitric oxide synthase. Accordingly, Phe did not induce nitric oxide synthesis, but significantly decreased the levels of reduced glutathione (GSH), the major brain antioxidant defense, in hippocampus and cerebral cortex supernatants. Phe also reduced the thiol groups of a commercial GSH solution in a cell-free medium. We also found that the major metabolites of Phe catabolism, phenylpyruvate, phenyllactate and phenylacetate also increased TBA-RS levels in cerebral cortex, but to a lesser degree. The data indicate that Phe elicits oxidative stress in the hippocampus, a structure mainly involved with learning/memory, and also in the cerebral cortex, which is severely damaged in PKU patients. It is therefore presumed that this pathomechanism may be involved at least in part in the severe cognitive deficit and in the characteristic cortical atrophy associated with dysmyelination and leukodystrophy observed in this disorder.     

Combination of Omega-3 Fatty Acids,Lithium, and Aripiprazole Reduces Oxidative Stress in Brain of Mice with Mania

Manic episode in bipolar disorder (BD) was evaluated in the present study with supplementation of omega-3 fatty acids in combination with aripiprazole and lithium on methylphenidate (MPD)-induced manic mice model. Administration of MPD 5 mg/kg bw intraperitoneally (i.p.) caused increase in oxidative stress in mice brain. To retract this effect, supplementation of omega-3 fatty acids 1.5 ml/kg (p.o.), aripiprazole 1.5 mg/kg bw (i.p.), and lithium 50 mg/kg bw (p.o) were given to mice. Omega-3 fatty acids alone and in combination with aripiprazole- and lithium-treated groups significantly reduced the levels of superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation products (thiobarbituric acid reactive substances) in the brain. MPD treatment significantly decreased the reduced glutathione (GSH) level and glutathione peroxidase (GPx) activity, and they were restored by supplementation of omega-3 fatty acids with aripiprazole and lithium. There is no remarkable difference in the effect of creatine kinase (CK) activity between MPD-induced manic model and the treatment groups. Therefore, our results demonstrate that oxidative stress imbalance and mild insignificant CK alterations induced by administration of MPD can be restored back to normal physiological levels through omega-3 fatty acids combined with lithium and aripiprazole that attributes to effective prevention against mania in adult male Swiss albino mice.