Original Contribution THE EFFECT OF COPPER SUPPLEMENTATION ON RED BLOOD CELL OXIDIZABILITY AND PLASMA ANTIOXIDANTS IN MIDDLE-AGED HEALTHY VOLUNTEERS EDMOND ROCK,* ANDRZEJ MAZUR,* JACQUELINE M. O’CONNOR, ² MAXINE P. BONHAM, ² YVES RAYSSIGUIER,* and JOHN J. STRAIN ² *Unite ´ Maladies Me ´taboliques et Micronutriments, INRA-CRNH Saint Gene `s Champanelle, France ² NICHE, University of Ulster, Coleraine, Northern Ireland (Received 6 April 1999; Revised 4 November 1999; Accepted 4 November 1999) Abstract—A multicenter European study (FoodCue) was undertaken to provide data on the significance of increased dietary copper as a pro-oxidant or antioxidant in vivo. The present work describes the effect of Cu supplementation on (2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH)-induced red blood cell oxidation in middle-aged people. Double-blinded copper supplementation was achieved in 26 healthy volunteers (50 –70 years) with pills containing 3 mg CuSO 4 , 3 mg Cu glycine chelate (CuG) and 6 mg CuG. Each 6 week supplementation period was preceded and followed by 6 weeks of washout (WO) on placebo. The results show significant increases in time necessary to achieve 50% hemolysis (LT 50 ) after 3CuSO 4 and 6CuG compared with values after WO periods. Cu supplementation did not increase the levels of (Cu,Zn)SOD activity in red blood cells. Resistance to hemolysis was significantly and positively correlated (r = .30, p  .01) with - and -carotene content in the plasma. Together, these data suggest that intake of copper as high as 7 mg/d has no pro-oxidant activity and may rather result in protection of red blood cells against oxidation. The decreased oxidizability of red blood cells did not result from increased (Cu,Zn)SOD activity and may occur through other mechanisms such as changes in membrane antioxidant content. © 2000 Elsevier Science Inc. Keywords—Copper supplementation, Human, Red blood cell oxidizability, Antioxidants, Carotene, Free radicals INTRODUCTION Copper (Cu) is an essential nutrient and is necessary to maintain a number of physiological functions [1]. Al- though overt Cu deficiency is not believed to be a sig- nificant nutritional problem for populations, the current recommendation of 1.5 to 3 mg Cu/d [2] is not com- monly attained and may lead to suboptimal Cu status in the population. When overt Cu deficiency occurs, symp- toms include neutropenia, cardiac disorders, osteoporo- sis, and anemia [3]. Anemia is classically observed dur- ing severe Cu deficiency in laboratory animals. Many studies, including those from our laboratory [4 – 6], have established that red blood cells (RBC) from Cu-deficient rats become more susceptible to in vitro–induced oxida- tive stress. Such a pro-oxidant effect of Cu is attributed to a decline of Cu-Zn SOD and extracellular SOD activ- ities primarily involved in the removal of superoxide anion, a highly toxic reactive oxygen species. This ac- tivity was shown to be very sensitive to Cu intake, particularly in animals and humans consuming low Cu- containing diets [7]. On the other hand, Cu ions are well known to partic- ipate in radical reactions such as the conversion of su- peroxide to hydrogen peroxide and hydroxyl radicals. Moreover, unstable Cu(III) form may be produced by interaction of hydrogen peroxide or ascorbate with cu- pric ion (CuII) [8]. It is assumed that such reactions are involved, at least in part, during Cu-induced oxidation of low-density lipoprotein (LDL) in vitro [9]. Pro-oxidant activity of excess Cu intake has been shown in rats with increased spin trap adduct in the bile, lipid peroxides in the mitochondria, and 8-hydroxydeoxyguanosine in the liver and kidney (reviewed in [10]). Antioxidant nutri- ents (vitamin E, selenium) protect against Cu toxicity, findings that are consistent with oxidative damage in- Address correspondence to: Dr. Edmond Rock, Unite ´ Maladies Me ´ taboliques et Micronutriments, INRA-CRNH, 63 122 Saint Gene `s Champanelle, France; Tel: +33 473- 62-41-69; Fax: +33 473-62-46- 38; E-Mail: rock@clermont.inra.fr. Free Radical Biology & Medicine, Vol. 28, No. 3, pp. 324 –329,2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter PII S0891-5849(99)00241-5 324