532 RADIATION RESEARCH 145, 532-541 (1996) 0033-7587/96 $5.00 1996 by Radiation Research Society. All rights of reproduction in any form reserved. Catalytic Metals, Ascorbate and Free Radicals: Combinations to Avoid 1 Garry R. Buettner and Beth Anne Jurkiewicz ESR Facility and Radiation Research Laboratory, EMRB 68, The University of Iowa, Iowa City, IA 52242-1101 Buettner, G. R. and Jurkiewicz, B. A. Catalytic Metals, Ascorbate and Free Radicals: Combinations to Avoid. Radiat. Res. 145, 532-541 (1996) Trace levels of transition metals can participate in the metal- catalyzed Haber-Weiss reaction (superoxide-driven Fenton reac-tion), as well as catalyze the oxidization of ascorbate. Generally ascorbate is thought of as an excellent reducing agent; it is able to serve as a donor antioxidant in free radical-mediated oxidative processes. However, as a reducing agent it is also able to reduce redox-active metals such as copper and iron, thereby increasing the pro-oxidant chemistry of these metals. Thus ascorbate can serve both as a pro- oxidant and antioxidant. In general, at low ascorbate concentrations, ascorbate is prone to be a pro-oxidant, and at high concentrations, it will tend to be an antioxidant. Hence, there is a crossover effect. We propose that the “position” of this crossover effect is a function of the catalytic metal concentration. In this presentation, we discuss: (1) the role of catalytic metals in free radical-mediated oxidations; (2) ascorbate as both a pro-oxidant and as an antioxidant; (3) catalytic metal catalysis of ascorbate oxidation;(4) Use of ascorbate to determine adventitious catalytic metal concentrations; (5) use of ascorbate radical as a marker of oxidative stress; and 6) use of ascorbate and iron as free radical pro-oxidants in photodynamic therapy of cancer. 1996 by Radiation Research Society INTRODUCTION A major turning point in the research and understanding of free radicals in biology and medicine occurred in 1968-1969 with the discovery that erythrocuprein functions enzymatically as a superoxide dismutase (1). However, per- haps just as important was the discovery that an enzyme, xanthine oxidase, produces superoxide (2-4). Soon after these discoveries Beauchamp and Fridovich made the very important observation that hydroxyl radical appeared to be produced in the superoxide-generating system of xan- thine-xanthine oxidase (5). They proposed that the mecha- nism of hydroxyl radical production was that proffered by Haber, Weiss, and Willstätter (6-8), i.e. 1 Presented at the Fenton Centennial Symposium at the 42 nd Annual Meeting of the Radiation Research Society, San Jose, CA, April 1995 O 2 •− /HO 2 • + H 2 O 2 → O 2 + H 2 O + HO • . However, studies of kinetics of this reaction found it to be very slow with measured rate constants of <0.0001 (9), 0.13 (10), and 2.3 M –1 s –1 at neutral pH values (11). Thus, on a biological time scale, it is a very slow reaction at the cellular concentrations of these two reactants and thus can be considered to be a negligible process. So the ques- tion becamehow can HO • be formed in a superoxide- generating system?Iron, or more generally catalytic transition metals! It was soon realized that the addition of iron to a system generating superoxide enhances the peroxidation of mem- brane lipids (12). Additional work demonstrated that super- oxide could reduce ADP–Fe(III) to ADP–Fe(II) and that this iron facilitated the apparent production of HO • (13). The role that iron plays in free radical oxidations was not appreciated until the Pinawa meeting 2 when it was presented that adventitious levels of iron in buffer solu- tions were on the order of 1 µM, and that this level of iron would change the results observed in superoxide-generat- ing systems (14). However, just as important, it was demonstrated that chelating agents will alter the reactivity of iron in superoxide-generating systems. It was shown that EDTA 3 enhances the reactivity of iron toward O 2 •− while DETAPAC 4 drastically slows the O 2 •− reaction with iron (14). Currently, chelating agents are being widely researched and used as tools to study free radical oxida- tions and as potential clinical agents to address human health problems. 2 International Conference on Singlet Oxygen and Related Species in Chemistry and Biology, 21-26 August 1977, Pinawa, Manitoba Canada. The symposium papers were published in Photochem. Photobiol. 28 (4, 5) (1978). 3 Abbreviations: Asc •- , ascorbate free radical; Asc 2- , ascorbate dianion; AscH - , ascorbate monanion; AscH 2 , ascorbic acid; DETAPAC, diethylenetriaminepentaacetic acid; DTPA=DETAPAC; EDTA, ethylenediaminetetraacetic acid; POBN, α-[4-pyridyl 1-oxide]-N-tert-butyl nitrone; TBARS, thiobarbituric acid reactive substances; TO • , toco- pheroxyl free radical; TOH, tocopherol. 4 It was at the Pinawa meeting that the acronym DETAPAC was introduced. This was a simple name we used to label the container of our stock solution of this chelating agent. DTPA is the more accepted abbre- viation, but nearly all researchers refer to it verbally as DETAPAC.