Mechanism of the aerobic oxidation of acetoacetate and 2- methylacetoacetate catalyzed by Mb: implications for ketogenic disorders

Acetoacetato (AA) e 2-metilacetoacetato (MAA) são compostos β-cetoácidos acumulados em diversas desordens metabólicas como no diabetes e na isoleucinemia, respectivamente. Examinamos o mecanismo de oxidação aeróbica de AA e MAA iniciada por intermediários reativos de mioglobina de coração de cavalo (Mb) gerados pela adição de H2O2. Uma rota quimioluminescente que envolve um intermediário dioxetânico cuja termólise gera espécies α-dicarbonílicas (metilglioxal e biacetilo) foi proposta e estudada. Emissão de luz ultra fraca acompanha a reação, e sua intensidade aumenta linearmente pelo aumento da concentração tanto de Mb (10-500 µM) quando AA (10-100 mM). Estudos de consumo de oxigênio mostraram que MAA é, como esperado, quase uma ordem de grandeza mais reativo que AA. Estudos de EPR com captação de spin, utilizando MNP, possibilitaram detectar adutos de MAA atribuíveis a um radical centrado no Cα (aN = 1.55 mT) e ao radical acetila (aN = 0.83 mT). O sinal do radical acetila é totalmente suprimido por sorbato, um conhecido e eficiente supressor de espécies tripletes, o que é consistente com uma rota reacional envolvendo um intermediário dioxetânico. Clivagem-α da ligação carbonila-carbonila do produto biacetilo triplete produziria, de fato, radicais acetila. Além disso, utilizando AA como substrato para Mb/H2O2, um sinal de EPR atribuível ao aduto MNP-AA• (aN = 1.46 mT e aH = 0.34 mT) foi observado e confirmado por efeito isotópico. O consumo de oxigênio e o rendimento de compostos α-dicarbonílicos foram dose-dependentes à concentração de AA ou MAA (1-50 mM) bem como à concentração de H2O2 adicionado às misturas de reação contendo Mb (até 1:10 quando medido o consumo de oxigênio, e até 1:25 quando medido o rendimento de compostos α-dicarbonílicos) e tert-butilhidroperóxido (até 1:200). Os perfis de pH (5,8-7,8) para consumo de oxigênio e rendimento de compostos α-dicarbonílicos mostraram maiores rendimentos para baixos valores de pH, indicativo de ferrilMb formada no ciclo peroxidático da proteína. Avaliando os níveis de lesão de Mb, os β-cetoácidos diminuíram o nível de desorganização protéica na estrutura secundária e terciária elicitada por H2O2. Ainda, houve maior preservação da estrutura primária da proteína, sendo que MAA protegeu mais em comparação a AA, embora quando utilizado este último composto, foi mostrado que há acetilação dose-dependente de Mb. Acetoacetato aumentou a velocidade de descoramento da hemeproteína, provavelmente por ataque de espécies tripletes geradas no sistema. Músculos de rato, plantar e sóleo, expostos ex vivo a concentrações citotóxicas de glicose oxidase (GOX, gera H2O2 em fluxo), foram protegidas pelos ésteres etílicos AAE e MAAE. Foi detectado biacetilo no meio intracelular em músculos expostos a MAAE e GOX. A concentração deste composto α-dicarbonílico é claramente relacionada à abundância de Mb em cada um dos tipos de músculos estudados. Em resumo, Mb tratada com metabólitos β-cetoácidos (AA e MAA) gera radicais centrados em carbono e produtos α-dicarbonílicos altamente reativos no estado triplete. Experimentos realizados com tecido muscular ex vivo sugerem que esta reação possivelmente ocorra in vivo. Levantamos a hipótese de que a geração de espécies carbonílicas reativas e seus adutos em condições de desbalanço metabólico possam contribuir para a compreensão das bases moleculares de desordens cetogênicas.Acetoacetate (AA) and 2-methylacetoacetate (MAA) are β-ketoacids accumulated in several metabolic disorders such as diabetes and isoleucinemia, respectively. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by the reactive enzyme intermediates formed by the reaction of muscle horse myoglobin (Mb) with H2O2. A chemiluminescent route involving a dioxetane intermediate whose thermolysis yields triplet α-dicarbonyl species (methylglyoxal and diacetyl) is envisaged. Accordingly, the ultraweak light emission that accompanies the reaction increases linearly by raising the concentration of both Mb (10-500 µM) and AA (10- 100 mM). Oxygen uptake studies revealed that MAA is, expectedly, almost one order of magnitude more reactive than AA. EPR spin-trapping studies with MNP detected spin adducts from MAA attributable to an α-carbon-centered radical (aN = 1.55 mT) and to an acetyl radical (aN = 0.83 mT). As the acetyl radical signal is totally suppressed by sorbate, a well-known efficient triplet species quencher, the dioxetane hypothesis seems to be reliable. The α-cleavage of the carbonyl-carbonyl bond of a putative excited triplet diacetyl product would, in fact, leads to an acetyl radical. Furthermore, using AA as substrate for Mb/H2O2, an EPR signal assignable to a MNP-AA• adduct (aN = 1.46 mT and aH = 0.34 mT) was observed and confirmed by isotope effect. Oxygen consumption and α-dicarbonyl yield were also dependent on AA or MAA concentrations (1-50 mM) as well as on the concentration of peroxide added to the Mb-containing reaction mixtures: H2O2 (up to 1:10 when measuring oxygen uptake and up to 1:25 when measuring the α-dicarbonyl yield) and t-butOOH (up to 1:200). The pH profiles (5.8-7.8) of oxygen consumption and α-dicarbonyl yield show higher reaction rates at lower pHs, indicative of a ferrylMb intermediate. Evaluating Mb lesion, both β-ketoacids reduced disorganization of the secondary and tertiary protein structure elicited by H2O2. Therefore, Mb primary structure was more preserved, and MAA was more protective than AA. Moreover using the later compound, it was shown that Mb acetylation is dose-dependent. Acetoacetate increased the rate of the hemeprotein bleaching, probably due to the attack of triplet products generated in the system. Plantaris and soleous rat muscles exposed to damaging concentrations of glucose oxidase (GOX, generates H2O2 in flux), was cytoprotected by AAE and MAAE. Intracellular diacetyl was detected in muscle samples exposed to MAAE and GOX. The α-dicarbonyl concentration is clearly related to the Mb abundance in the muscle types. In summary, Mb treated with peroxides reacts with β-ketoacid metabolites (AA and MAA), yielding carbon-centered radicals and highly reactive α-dicarbonyl products in the triplet state. Experiments carried out ex vivo with muscle tissue showed that this reaction possibly occurs in vivo. A new route for generation and accumulation of carbonyl reactive species and adducts is here proposed to occur in unbalanced metabolic situations, such as is the case of ketogenic disorders

Additional file 1: of Role of cytochrome c in α-synuclein radical formation: implications of α-synuclein in neuronal death in Maneb- and paraquat-induced model of Parkinson’s disease

This contains Figure S1, S2, Scheme S1. Figure S1 contains heat maps showing changes in gene expression as mentioned in Fig. 5c. Figure S2 shows Western blots for α-synuclein, caspase-9, and cleaved caspase-3. Scheme S1 shows an outline of immuno-spin trapping method in which anti-DMPO antibody is used to detect protein radicals. (PDF 361 kb

Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.

none6siCeruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe(2+) oxidation in plasma of mammals. In addition to its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA, and desferal, whereas heparin and bathocuproine have no effect. Catalase or superoxide dismutase additions do not interfere with the CPH-oxidation yield, demonstrating that oxygen-derived free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for reactive oxygen species detection and quantification.mixedGanini D.; Canistro D.; Jang J.; Stadler K.; Mason R.P.; Kadiiska M.B.Ganini D.; Canistro D.; Jang J.; Stadler K.; Mason R.P.; Kadiiska M.B

Myoglobin-H2O2 catalyzes the oxidation of beta-ketoacids to alpha-dicarbonyls: Mechanism and implications in ketosis

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are accumulated in metabolic disorders such as diabetes and isoleucinemia. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by myoglobin (Mb)/H2O2. We propose a chemiluminescent route involving a dioxetanone intermediate whose thermolysis yields triplet alpha-dicarbonyl species (methylglyoxal and diacetyl). the observed ultraweak chemiluminescence increased linearly on raising the concentration of either Mb (10-500 mu M) or AA (10-100 mM). Oxygen uptake studies revealed that MAA is almost a 100-fold more reactive than AA. EPR spin-trapping studies with MNP/MAA revealed the intermediacy of an alpha-carbon-centered radical and acetyl radical. the latter radical, probably derived from triplet diacetyl, is totally suppressed by sorbate, a well-known quencher of triplet carbonyls. Furthermore, an EPR signal assignable to MNP-AA(center dot) adduct was observed and confirmed by isotope effects. Oxygen consumption and a-dicarbonyl yield were shown to be dependent on AA or MAA concentrations (1-50 mM) and on H2O2 or tert-butOOH added to the Mb-containing reaction mixtures. That ferrylMb is involved in a peroxidase cycle acting on the substrates is suggested by the reaction pH profiles and immunospin-trapping experiments. the generation of radicals and triplet dicarbonyl products by Mb/H2O2/beta-ketoacids may contribute to the adverse health effects of ketogenic unbalance. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Ciencia e Tecnologia (INCT) RedoxomaNational Institute of Environmental Health Sciences (NIEHS)Universidade Federal de São Paulo, Dept Ciencias Exatas & Terra, Inst Ciencias Ambientais Quim & Farmaceut, BR-00972270 Diadema, SP, BrazilUniv São Paulo, Inst Quim, Dept Bioquim, BR-01498 São Paulo, BrazilNatl Inst Environm Hlth Sci, Free Rad Metab Grp, Lab Toxicol & Pharmacol, NIH, Res Triangle Pk, NC 27709 USAUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, Inst Ciencias Ambientais Quim & Farmaceut, BR-00972270 Diadema, SP, BrazilWeb of Scienc

Free Radical Metabolism by Cytochrome P450-2E1 and NADPH Oxidase Activation Forms Protein Radicals and Tyrosine Nitration in Obesity-Associated Non-alcoholic Fatty Liver Disease

NIEHS, Res Triangle Pk, NC USAUniversidade Federal de São Paulo, UNIFESP, São Paulo, BrazilUniv Illinois, Chicago, IL USAUniversidade Federal de São Paulo, UNIFESP, São Paulo, BrazilWeb of Scienc

Astaxanthin Supplementation Delays Physical Exhaustion and Prevents Redox Imbalances in Plasma and Soleus Muscles of Wistar Rats

Astaxanthin (ASTA) is a pinkish-orange carotenoid commonly found in marine organisms, especially salmon. ASTA is a powerful antioxidant and suggested to provide benefits for human health, including the inhibition of LDL oxidation, UV-photoprotection, and prophylaxis of bacterial stomach ulcers. Exercise is associated to overproduction of free radicals in muscles and plasma, with pivotal participation of iron ions and glutathione (GSH). Thus, ASTA was studied here as an auxiliary supplement to improve antioxidant defenses in soleus muscles and plasma against oxidative damage induced by exhaustive exercise. Long-term 1 mg ASTA/kg body weight (BW) supplementation in Wistar rats (for 45 days) significantly delayed time to exhaustion by 29% in a swimming test. ASTA supplementation increased scavenging/iron-chelating capacities (TEAC/FRAP) and limited exercise-induced iron overload and its related pro-oxidant effects in plasma of exercising animals. On the other hand, ASTA induced significant mitochondrial Mn-dependent superoxide dismutase and cytosolic glutathione peroxidase antioxidant responses in soleus muscles that, in turn, increased GSH content during exercise, limited oxidative stress, and delayed exhaustion. We also provided significant discussion about a putative “mitochondrial-targeted” action of ASTA based on previous publications and on the positive results found in the highly mitochondrial populated (oxidative-type) soleus muscles here

The peroxidase activity of mitochondrial superoxide dismutase

Maria Montesori svoju koncepciju odgoja zasniva na spoznajama o razvojnim fazama dječje osjetljivosti. Zadovoljavanje tih razvojnih faza polazi od dviju pretpostavki, da djeca imaju prirođene snage za samorazvoj i da se prirođene snage aktiviraju ako su djeca okružena povoljnom sredinom (Matijević, 2001). Zadaća Montessori odgojitelja je da osigura odgovarajuću sredinu i materijale koji će poticati samoaktivnost djece. On nije poučavatelj ni predavač već osoba koja daje inicijalne upute te usmjerava i potiče aktivnosti pojedinog djeteta. Svako dijete se razvija svojim tempom i ima određene prirođene unutarnje potrebe koje se pojavljuju u različito vrijeme, zbog toga Maria Montessori zagovara individualizirani odgoj. Materijali i prostor su važan dio samoodgoja djeteta. Oni trebaju dati potporu djetetovu samorazvoju. Svi su materijali u Montessori prostoru uredno složeni, a od svake vrste materijala postoji samo jedan komplet. Dok se jedno dijete koristi nekim materijalom, drugo dijete koje poželi isti materijal treba pričekati. To je važno sredstvo u socijalizaciji jer privikava na uljudnu međuljudsku komunikaciju. Montessori pedagogija polazi od ideje slobode, odnosno slobodnog odgoja. Dijete samo inicira i upravlja aktivnostima kojima se hoće baviti prema svojim potrebama i interesima. Preko slobodnog izbora dijete uči donositi odluke, ono uči što je to kreativnost. Odgojitelj pokazuje djetetu kako da likovne aktivnosti izvede sam. Ono samo bira vrstu likovnog materijala i temu svog rada, dok mu odgojitelj, bez govorenja, pokazuje kako se koristi tim materijalom. Trajanje likovne aktivnosti ovisi o djetetu i zadovoljavanju njegovih potreba.Maria Montessori based her basic idea of education on insights of development stages of children's sensitivities. Fulfilment of this development stages is based on two hypothesis, children have inborn forces for self-development and this inborn forces are activated if children are surrounded with suitable environment (Matijević, 2001). Montessori educators have a task to prepare suitable environment and materials that will encourage children's self-activity. They are not a teacher or lecturer, but a person that gives initiative instructions and a person that directs and encourages activites of every single child. Every child develops differently, in its own pace, and every child has its own internal needs which appear in different time. Therefore, Maria Montessori advocates individual education. Materials and environment are very important part of child's self-education. They need to give support for child's self-development. All materials in Montessori environment are neatly arranged and there's only one set of every sort of material. While one child is using some material and some other child is interested in using the same material, it has to wait until the first child is done with the activity. This is very important for child's social development because it teaches children how to be polite while communicating with others. Montessori pedagogy is based on freedom. Therefore, child initiates and manages activities which are result of its needs and interests. While having freedom to choose its own activities, child is learning to make its own decisions, it is learning what creativity is. Educator is showing the children how to do art activities all by themselves. Child chooses what type of art material it wants to use and it chooses a topic of its piece, while educator, without speaking, shows the child how to use this material. Duration of this activity depends on the child and on satisfaction of its needs

Redox Status and Neuro Inflammation Indexes in Cerebellum and Motor Cortex of Wistar Rats Supplemented with Natural Sources of Omega-3 Fatty Acids and Astaxanthin: Fish Oil, Krill Oil, and Algal Biomass

Health authorities worldwide have consistently recommended the regular consumption of marine fishes and seafood to preserve memory, sustain cognitive functions, and prevent neurodegenerative processes in humans. Shrimp, crabs, lobster, and salmon are of particular interest in the human diet due to their substantial provision of omega-3 fatty acids (n-3/PUFAs) and the antioxidant carotenoid astaxanthin (ASTA). However, the optimal ratio between these nutraceuticals in natural sources is apparently the key factor for maximum protection against most neuro-motor disorders. Therefore, we aimed here to investigate the effects of a long-term supplementation with (n-3)/PUFAs-rich fish oil, ASTA-rich algal biomass, the combination of them, or krill oil (a natural combination of both nutrients) on baseline redox balance and neuro-inflammation indexes in cerebellum and motor cortex of Wistar rats. Significant changes in redox metabolism were only observed upon ASTA supplementation, which reinforce its antioxidant properties with a putative mitochondrial-centered action in rat brain. Krill oil imposed mild astrocyte activation in motor cortex of Wistar rats, although no redox or inflammatory index was concomitantly altered. In summary, there is no experimental evidence that krill oil, fish oil, oralgal biomass (minor variation), drastically change the baseline oxidative conditions or the neuro-inflammatory scenario in neuromotor-associated rat brain regions

Effects of acute creatine supplementation on iron homeostasis and uric acid-based antioxidant capacity of plasma after wingate test

<p>Abstract</p> <p>Background</p> <p>Dietary creatine has been largely used as an ergogenic aid to improve strength and athletic performance, especially in short-term and high energy-demanding anaerobic exercise. Recent findings have also suggested a possible antioxidant role for creatine in muscle tissues during exercise. Here we evaluate the effects of a 1-week regimen of 20 g/day creatine supplementation on the plasma antioxidant capacity, free and heme iron content, and uric acid and lipid peroxidation levels of young subjects (23.1 ± 5.8 years old) immediately before and 5 and 60 min after the exhaustive Wingate test.</p> <p>Results</p> <p>Maximum anaerobic power was improved by acute creatine supplementation (10.5 %), but it was accompanied by a 2.4-fold increase in pro-oxidant free iron ions in the plasma. However, potential iron-driven oxidative insult was adequately counterbalanced by proportional increases in antioxidant ferric-reducing activity in plasma (FRAP), leading to unaltered lipid peroxidation levels. Interestingly, the FRAP index, found to be highly dependent on uric acid levels in the placebo group, also had an additional contribution from other circulating metabolites in creatine-fed subjects.</p> <p>Conclusions</p> <p>Our data suggest that acute creatine supplementation improved the anaerobic performance of athletes and limited short-term oxidative insults, since creatine-induced iron overload was efficiently circumvented by acquired FRAP capacity attributed to: overproduction of uric acid in energy-depleted muscles (as an end-product of purine metabolism and a powerful iron chelating agent) and inherent antioxidant activity of creatine.</p