Evidence That 3-Hydroxy-3-Methylglutaric Acid Promotes Lipid and Protein Oxidative Damage and Reduces the Nonenzymatic Antioxidant Defenses in Rat Cerebral Cortex Guilhian Leipnitz, 1 Bianca Seminotti, 1 Josue ´ Haubrich, 1 Manuela B. Dalcin, 1 Karina B. Dalcin, 1 Alexandre Solano, 1 Giorgia de Bortoli, 1 Rafael B. Rosa, 1 Alexandre U. Amaral, 1 Carlos S. Dutra-Filho, 1 Alexandra Latini, 2 and Moacir Wajner 1-4 * 1 Departamento de Bioquı ´mica, Instituto de Cie ˆncias Ba ´sicas da Sau ´de, Universidade Federal de Rio Grande do Sul, Porto Alegre, RS, Brazil 2 Departamento de Bioquı ´mica, Centro de Cie ˆncias Biolo ´gicas, Universidade Federal de Santa Catarina, Floriano ´polis, SC, Brazil 3 Hospital de Clı ´nicas, Servic ¸o de Gene ´tica Me ´dica, Porto Alegre, RS, Brazil 4 Universidade Luterana do Brasil, Canoas, Brazil In the present work we investigated the in vitro effect of 3-hydroxy-3-methylglutarate (HMG) that accumu- lates in 3-hydroxy-3-methylglutaryl-CoA lyase defi- ciency (HMGLD) on important parameters of oxidative stress in rat cerebral cortex. It was observed that HMG induced lipid peroxidation by significantly increasing chemiluminescence and levels of thiobarbituric acid-re- active substances (TBA-RS). This effect was prevented by the antioxidants a-tocopherol, melatonin, N-acetyl- cysteine, and superoxide dismutase plus catalase, suggesting that free radicals were involved in the lipid oxidative damage. On the other hand, HMG did not change TBA-RS levels in intact or disrupted mitochon- drial preparations, indicating that generation of oxi- dants by this organic acid was dependent on cytosolic mechanisms. HMG also induced protein oxidative damage in cortical supernatants, which was reflected by increased carbonyl content and sulfhydryl oxidation. Furthermore, HMG significantly reduced the nonenzy- matic antioxidant defenses total-radical trapping anti- oxidant potential, total antioxidant reactivity, and reduced glutathione (GSH) levels in rat cerebral cortex. HMG-induced GSH reduction was totally blocked by melatonin pretreatment. We also verified that the decrease of GSH levels provoked by HMG in cortical supernatants was not due to a direct oxidative effect of this organic acid, because exposition of commercial GSH and purified membrane protein-bound thiol groups to HMG in the absence of cortical supernatants did not decrease the reduced sulfhydryl groups. Finally, the activities of the main antioxidant enzymes were not altered by HMG exposure. Our data indicate that oxidative stress elicited in vitro by HMG may pos- sibly contribute at least in part to the pathophysiology of the brain injury in HMGLD. V V C 2007 Wiley-Liss, Inc. Key words: 3-hydroxy-3-methylglutaric acid; 3-hydroxy- 3-methylglutaric aciduria; oxidative stress 3-Hydroxy-3-methylglutaric aciduria (HMGA; Mckusick 246450) is an autossomal recessive neurometa- bolic disorder of leucine catabolism and of ketogenesis caused by 3-hydroxy-3-methylglutaryl-CoA lyase defi- ciency (HMGLD; Faull et al., 1976; Wysocki et al., 1976). This mitochondrial and peroxissomal enzyme cat- alyzes the cleavage of 3-hydroxy-3-methylglutaril-CoA to acetoacetate acid and acetyl-CoA. The disorder is biochemically characterized by predominant tissue accu- mulation and high urinary excretion of large quantities of 3-hydroxy-3-methylglutarate (HMG), in addition to 3-methylglutaconate, 3-methylglutarate, 3-hydroxyiso- valerate, and 3-methylcrotonylglycine (Bonafe ´ et al., 2000; Sweetman et al., 2001). The average concentra- tion of urinary HMG in HMGA patients is about 1,300 mmol/mol of creatinine, but it can rise to 11,000 mmol/ Contract grant sponsor: FAPERGS; Contract grant sponsor: PRONEX II; Contract grant sponsor: CNPq-Brazil. *Correspondence to: Moacir Wajner, Departamento de Bioquı ´mica, Instituto de Cie ˆncias Ba ´sicas da Sau ´de, Universidade Federal de Rio Grande do Sul. Rua Ramiro Barcelos No. 2600 Anexo, CEP 90035- 003, Porto Alegre, RS, Brasil. E-mail: mwajner@ufrgs.br Received 18 May 2007; Revised 19 July 2007; Accepted 26 July 2007 Published online 16 October 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21527 Journal of Neuroscience Research 86:683–693 (2008) ' 2007 Wiley-Liss, Inc.