FreeRadicalBiology& Medicine, Vol. 12, pp. 471-478, 1992 0891-5849/92 $5.00 + .00 Printed in the USA. All rights reserved. Copyright © 1992 Pergamon Press Ltd. Original Contribution ARE DIOXETANES CHEMILUMINESCENT INTERMEDIATES IN LIPOPEROXIDATION? PAOLO DI MASCIO,*t LuIZ H. CATALANI,* and ETELVINO J. H. BECHARA* *Instituto de Quimica, Universidade de Silo Paulo, C.P. 20780, 01498, S~io Paulo, SP, Brazil; and tInstitut f'tir Physiologische Chemie I, Universitlit Dtisseldorf, Moorenstr. 5, D-4000 DUsseldorf, Germany (Received 25 July 1991; Revised 18 December 1991; Accepted 27 January 1992) Abstract--Ultraweak chemiluminescence arising from lipoperoxidation has been attributed by several authors to the radiative deactivation of singlet oxygen and triplet carbonyl products. The latter emitters have been suggested to come from annihilation of RO" and ROO" radicals as well as from the thermolysis of dioxetane intermediates formed by (2 + 2) cycloaddition of JO2 to polyunsaturated fatty acids. This article questions possible dioxetane intermediacy in lipoperoxidation, as the literature clearly states that addition of tO2 to alpha-hydrogen-containing alyphatic olefins yields only the corresponding allylic hydroperoxides. These compounds may undergo dark thermal or Lewis acid-assisted decomposition to the same product obtained from dioxe- tane cleavage. Here, reexamining the chemiluminescence properties of dioxygenated tetramethylethylene and linoleic acid and comparing them with those of tetraethyldioxetane, a hindered dioxetane, we corroborate the literature information that only steric hindrance leads to dioxetane formation upon singlet oxygen addition to electron-poor olefins, albeit in very low yields. Proton nuclear magnetic resonance (tH-NMR) analysis, quenching by dioxygen and energy transfer studies to 9,10-dibro- moanthracene, as well as gas chromatography (GC) analysis oftriphenylphosphine-treated and untreated photo- and chemically dioxygenated olefins support our final conclusion that dioxetane formation during lipoperoxidation can be safely excluded on the basis of the data presently available. Keywords--Singlet molecular oxygen, Linoleic acid, Triplet ketones, Dioxetanes, Chemiluminescence, Free radicals INTRODUCTION Lipid peroxidation has been pointed out as a key chemical event of the oxidative stress associated with several inborn and acquired disorders. Disruption of organelle and cell membranes together with calcium homeostasis alterations are the main supramolecular events linked to lipid peroxidation.l Not clarified yet, however, is whether the phenomenon constitutes an early, triggering step of the clinical manifestations of the diseases or a terminal, consequent process. The low-level chemiluminescence which accompa- nies the peroxidation of polyunsaturated fatty acids (PUFA) has been used as a tool in kinetic and mecha- nistic studies of biological samples to estimate the ex- tent of the reactions and even to indicate tissue dam- age promoted by pro-oxidants injected in experimen- tal animals. 2 Triplet carbonyls and singlet oxygen Address correspondence to Prof. Dr. Etelvino J. H. Bechara, In- stituto de Quimica, Departamento de Bioquimica, Universidade de S~lo Paulo, C.P. 20780, 01498, S~loPaulo, SP, Brazil. formed in the annihilation of intermediate alkylper- oxyl radicals (ROO") have been identified as the che- miluminescence emitters. 3 The disproportionation of alkoxyl radicals (RO") is also a putative source of trip- let ketones. 4 Several authors have suggested parallel formation of electronically excited carbonyls in lipoperoxida- tion via the thermal cleavage of dioxetane interme- diates supposedly formed by (2 + 2) cycloaddition of singlet oxygen (IO2) to PUFA. 2'5-7 However, it has long been known that 1,2-addition of 10 2 to alkenes yields dioxetanes only in the case of electron-rich ole- fins lacking alpha-hydrogens or rigid olefins) We thus decided to reexamine the reaction of ~O2 with appro- priate olefins--tetramethylethylene (TME), tetraeth- ylethylene (TEE), and linoleic acid (LA)--with the aim of verifying the possible involvement of dioxe- tane intermediates in the chemiluminescence asso- ciated with lipoperoxidation. Singlet oxygen was generated both photochemically and by the ther- molysis of 1,4-dimethylnaphthalene-l,4-endoperox- ide (DMNO2). 9 471