Free Radical Biology & Medicine - COnnecting REpositories .Original Contribution Myoglobin-H 2O 2...
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Free Radical Biology & Medicine 51 (2011) 733743
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Free Radical Biology & Medicine
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Myoglobin-H2O2 catalyzes the oxidation of -ketoacids to -dicarbonyls:Mechanism and implications in ketosis
Douglas Ganini a, Marcelo Christoff a, Marilyn Ehrenshaft b, Maria B. Kadiiska b,Ronald P. Mason b, Etelvino J.H. Bechara a,c,a Departamento de Bioqumica, Instituto de Qumica, Universidade de So Paulo, So Paulo, SP, Brazilb Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USAc Departamento de Cincias Exatas e da Terra, Instituto de Cincias Ambientais, Qumicas e Farmacuticas, Universidade Federal de So Paulo, Diadema, SP, Brazil
Abbreviations: AA, acetoacetate; AGEs, advancedcatalase; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; ELsorbent assay; EPR, electron paramagnetic resonancexylenol orange; GSH, reduced glutathione; HPLC, high praphy; HRP, horseradish peroxidase; MAA, 2-methylMNP, methylnitrosopropane; NK, nuclear factor RAGE, AGE receptor; SOD, superoxide dismutase; tert-Bide; TNF-, tumor necrosis factor . Corresponding author at: Departamento de Cincias
Cincias Ambientais, Qumicas e Farmacuticas, UniversiProf. Artur Riedel, 275, 09972270 Diadema, SP, Brazil.
E-mail address: email@example.com (E.J.H. Becha
0891-5849 2011 Elsevier Inc.doi:10.1016/j.freeradbiomed.2011.05.002
Open access under the Else
a b s t r a c ta r t i c l e i n f o
Article history:Received 1 March 2011Revised 14 April 2011Accepted 2 May 2011Available online 8 May 2011
Keywords:MyoglobinAcetoacetate2-MethylacetoacetateFree radicalsTriplet carbonylsMethylglyoxalDiacetylKetosis
Acetoacetate (AA) and 2-methylacetoacetate (MAA) are accumulated in metabolic disorders such as diabetesand isoleucinemia. Here we examine the mechanism of AA andMAA aerobic oxidation initiated by myoglobin(Mb)/H2O2. We propose a chemiluminescent route involving a dioxetanone intermediate whose thermolysisyields triplet -dicarbonyl species (methylglyoxal and diacetyl). The observed ultraweak chemiluminescenceincreased linearly on raising the concentration of either Mb (10500 M) or AA (10100 mM). Oxygen uptakestudies revealed that MAA is almost a 100-fold more reactive than AA. EPR spin-trapping studies with MNP/MAA revealed the intermediacy of an-carbon-centered radical and acetyl radical. The latter radical, probablyderived from triplet diacetyl, is totally suppressed by sorbate, a well-known quencher of triplet carbonyls.Furthermore, an EPR signal assignable to MNP-AA adduct was observed and confirmed by isotope effects.Oxygen consumption and -dicarbonyl yield were shown to be dependent on AA or MAA concentrations (150 mM) and on H2O2 or tert-butOOH added to the Mb-containing reaction mixtures. That ferrylMb is involvedin 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/-ketoacidsmay contribute to the adverse health effects of ketogenic unbalance.
glycated end products; CAT,ISA, enzyme-linked immuno-; FOX1, ferrous oxidation oferformance liquid chromatog-acetoacetate; Mb, myoglobin;; OPD, o-phenylenediamine;
utOOH, tert-butyl hydroperox-
Exatas e da Terra, Instituto dedade Federal de So Paulo, RuaFax: +55 11 4043 6428.ra).
vier OA license.
2011 Elsevier Inc.Open access under the Elsevier OA license.
Acetoacetate (AA) is a carbohydrate, protein, and lipid catabo-lite that is normally found in human plasma at concentrations below0.5 mM, but can reach 610 mM during ketosis, a characteristic ofdiabetesmellitus types 1 and 2 , strenuous physical exercise , andthe Atkins diet . Another -ketoacid, 2-methylacetoacetate (MAA)
accumulates in isoleucinemic patients, whose 2-methylacetoacetyl-CoA thiolase is defective , leading to massive urinary excretion ofMAA, triglylglycine, and 2-methyl-3-hydroxybutyrate . Isoleucine-mic patients are characterized by recurrent ketoacidosis, lethargy,vomiting, and usually manifest mental retardation, convulsions, andcoma .
AA is reportedly toxic to cells in culture due to generation offree radicals and activation of pro-oxidant signaling pathways. Forexample, exposure of monocyte cultures to AA (23 mM) results inhigher secretion of tumor necrosis factor- (TNF-) and depletionof reducedglutathione (GSH) [7,8], and inhigher interleukin-6 secretionwhen in the presence of phorbol 12-myristate 13-acetate . Mitogen-activated protein kinase (MAPK) increases in AA (0.520 mM)-treatedprimary rat hepatocyte cultures , and endothelial cells exposed toAA (520 mM) display higher levels of malondialdehyde and undergogrowth inhibition . Paradoxically, AA was also shown to exhibitantioxidantproperties, for example, inneurons stressedbyglutamate orhypoxia . In addition, AA increases the contractile performance ofstunned myocardium by acting as a -adrenergic inotropic sub-stance due to its utilization as carbon fuel  and NAD(P)+-linkedsubstrate .
Scheme 2. Proposed reaction mechanism for the chemiluminescent oxidation ofAA by Mb/H2O2.
734 D. Ganini et al. / Free Radical Biology & Medicine 51 (2011) 733743
It has long been known that myoglobin catalyzes the chemi-luminescent oxidation of AA to formate and triplet methylglyoxal,probably via a dioxetane intermediate . Myeloperoxidase and, more recently, peroxynitrite  were also shown to triggerthe oxidation of -ketoacids, in the latter case demonstrated toyield methylglyoxal and diacetyl from AA and MAA as substrates,respectively (Scheme 1). Noteworthy in this regard is that clinicalstudies with diabetic patients show a high correlation betweenplasma levels of AA and methylglyoxal, but not glucose .
Reactive carbonyl species such asmethylglyoxal, an-oxoaldehyde,can aggregate proteins and generate protein and DNA adducts ,reportedly leading to activation of pro-oxidant signaling cascades ,cellular malfunction, and aging [21,22]. Various pathological states,such as diabetes type I and II, cardiovascular diseases, inflammation, andchronic renal failure are shown to be related to accumulation of meth-ylglyoxal and carbonyl protein adducts, collectively named advancedglycated end products (AGEs) . The progression of diabeticmicro- and macrovascular manifestations such as retinopathy andchronic kidney failure is shown in different animal models and hu-mans to be correlated to indexes of AGEs such as carboxyethyl--amine-lysine (CML), pentosidine, glyoxal-derived lysine dimer, andmethylglyoxal-derived lysine dimer . Recently, a receptor forAGEs, RAGE, was characterized and described to be directly asso-ciated with the initiation of microvascular diseases . SolubleAGEs, such as the well-characterized CML, induce receptor expres-sion and lead to the activation of signaling cascades through MAPKand NF .
Here, we aim to clarify the mechanism of the Mb/peroxide-catalyzed oxidation of AA and MAA through free radicals to meth-ylglyoxal and diacetyl, respectively, in normally aerated buffer(Scheme 2). This may shed light on the molecular bases of the clin-ical complications of ketoacidosis and rhabdomyolysis. Acting as aperoxidase in the presence of H2O2, the very unstable Compound Iform of Mb is stabilized by electron transfer from the hememoiety tothe Tyr103 residue, giving rise to Mb Compound II . One ormore of these species is expected to oxidize AA or MAA yielding aresonance-stabilized enoyl radical (AA or MAA) (Scheme 2). Reac-tion of these radicals with dioxygen would form the correspondingalkylperoxyl radicals that can propagate the chain reaction to the -hydroperoxides AAOOH or MAAOOH. The hydroperoxides mightthen undergo cyclization to dioxetane intermediates followed bytheir thermolysis to methylglyoxal or diacetyl products in the tripletstate. Both ground state [19,20] and excited state  -dicarbonylproducts are very reactive toward proteins, DNA, and a wide spec-trum of biomolecules. Myoglobin is therefore expected to be dam-aged by theAAandMAA radical intermediates and dicarbonyl products.In this regard, we emphasize here that the reaction of Mb with lipidperoxides in kidneys of rats intramuscularly injected with glycerol
Scheme 1. Chemical structures of acetoacetate (AA) and 2-methylacetoacetate (MAA)anions and of their oxidation products: methylglyoxal and diacetyl, respectively.
seems to contribute to the acute kidney failure caused by rhabdomy-olysis .
Materials and methods
Ethyl acetoacetate and ethyl 3-methylacetoacetate were fromAldrich Co. (Saint Louis, MO, USA). Glyoxal (40%), methylglyoxal,diacetyl, quinoxalinol, 2-methylquinoxaline, 2,3-dimethyquinoxaline,xylenol orange, horse myoglobin (Mb), sorbic acid, sodium pyruvate,glucose oxidase (from Aspergillus niger), o-phenylenediamine (OPD),butanone, methylnitrosopropane (MNP), catalase (CAT), Cu,Zn-super-oxide dismutase (SOD) (from bovine erythrocytes), and 70% tert-butylhydroperoxide were from Sigma Co. (St. Louis, MO, USA). Hydrogenperoxide (30%), methanol, acetonitrile, phosphoric acid (85%), aceticacid, HPLC grade solvents, and salts used for buffer preparation ofthe highest purity available were from Merck Co. (Darmstadt,Germany). DMPO was f