Original Contribution
In Vivo Oxidative Modification of Erythrocyte Membrane Proteins in Copper Deficiency

https://doi.org/10.1016/S0891-5849(96)00430-3Get rights and content

Abstract

Oxidative stress has been postulated to contribute to the pathology associated with dietary copper deficiency. In vivo, erythrocytes are probable targets of oxidative damage because they are exposed to high concentrations of oxygen and contain heme iron that can autoxidize, which results in the formation of superoxide anions. Activity of the important antioxidant enzyme, copper, zinc superoxide dismutase, decreases markedly in erythrocytes during copper deficiency. The effect of dietary copper deficiency on indicators of oxidative stress was examined in erythrocyte membranes of rats maintained on a purified copper-deficient diet for 35 days after weaning. Erythrocytes were separated into young and old populations on a Percoll gradient prior to membrane isolation and quantification of lipid peroxides and protein carbonyls. Protein carbonyls, determined by Western blot immunoassay, were detected predominantly in both the alpha and beta chains of spectrin. Alpha and beta subunits of spectrin in erythrocyte membranes from copper-deficient rats contained higher amounts of carbonyls than controls, regardless of the population of erythrocytes studied. This study suggests that spectrin may be a specific target for oxidative damage when erythrocyte copper, zinc superoxide dismutase activity is reduced by copper deficiency. Copyright © 1997 Elsevier Science Inc.

Introduction

It is well-known that copper is an essential nutrient for man and animals and that its deficiency in diets can have serious pathological consequences.1, 2, 3, 4 However, the molecular mechanisms that produce the pathology associated with copper deficiency are not completely understood. Copper deficiency can reduce the activities of Cu, Zn-superoxide dismutase (SOD), ceruloplasmin, and catalase,5, 6, 7 all of which are enzymatic components of the antioxidant defense system. Thus, increased susceptibility to oxidative damage may be one of the biological consequences that contributes significantly to the pathology of copper deficiency. Evidence that oxidative damage can occur during copper deficiency is contained in reports of increased lipid hydroperoxide content in liver mitochondria and microsomes from copper-deficient rats[8] and increased susceptibility of tissues from copper-deficient rats to lipid peroxidation.9, 10 Increased production of breath ethane is further evidence that lipid peroxidation is increased in copper-deficient rats.[11] In fact, there are enough reports of oxidative damage or increased susceptibility to oxidative damage during copper deficiency to suggest that copper could be characterized as an antioxidant nutrient.[12]

Because they are exposed to high concentrations of oxygen and to superoxide anions that are constantly generated from the autoxidation of oxyhemoglobin,13, 14 erythrocytes may be targets for oxidative damage during copper deficiency. The intracellular concentration of superoxide anion is normally controlled by superoxide dismutase, an enzyme whose erythrocyte activity is reduced by copper deficiency.15, 16 Concentration of superoxide anion could thus increase and oxidatively modify cell components. A report of increased malondialdehyde content in erythrocytes from copper-deficient rats supports the contention that erythrocytes are oxidatively damaged during copper deficiency.[17]

Although most studies have confined their examination of oxidant damage during copper deficiency to lipid components, damage also may occur to protein components of the erythrocyte membrane. It is known that erythrocyte membrane proteins can be oxidatively crosslinked by malondialdehyde,18, 19, 20 phenylhydrazine,[20] and o-phenanthroline/CuSO4.[21] These findings suggest that erythrocyte membrane proteins are susceptible to oxidative modification and may be damaged in vivo if intracellular conditions favor the production or accumulation of oxidants. Although enzymatic defenses against oxidants are compromised by copper deficiency, it has not been demonstrated that oxidative damage to erythrocyte membrane proteins occurs. Thus, the objective of the present study was to determine if the oxidative stress produced by copper deficiency can damage proteins associated with the erythrocyte membrane.

Section snippets

Animals and Diets

Male, weanling Sprague–Dawley rats (SASCO, Inc., Omaha, NE)2 were housed in individual stainless steel cages in quarters maintained at 22°C and 50% humidity with a 12-h light/dark cycle. Upon arrival at our facility, the rats were fed a purified diet

Results

Rats consuming copper-deficient diet containing 0.5 μg Cu/g for five weeks had lower liver copper concentrations, hemoglobin concentrations, hematocrits, and ceruloplasmin oxidase activity than their counterparts consuming copper-adequate diet containing 5.9 μg Cu/g (Table 1). Four rats consuming the copper-deficient diet died before the experiment was completed. Reduced liver copper concentrations, anemia, and reduced ceruloplasmin activities are characteristic of low-copper status33, 34, 35

Discussion

Because of their physiological role as oxygen carriers, erythrocytes are exposed to continuous oxidative stress and require effective defensive mechanisms to prevent or reduce oxidative damage from endogenously generated reactive oxygen intermediates. Cu, Zn-SOD, which regulates the intercellular concentration of O2&̇minus;, is an important enzymatic component of the erythrocyte oxidant defense system. Loss of CuZnSOD activity could reduce the dismutation of O2&̇minus; and increase the

Acknowledgements

This work was supported in part by a Sigma Xi Grant-in-Aid of Research to Thomas P. LaBerge. We gratefully acknowledge the technical assistance of Eric L. Risovi, Thomas J. Matzke, and Anne C. Thomas.

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