Evaluation of erythrocyte copper chaperone for superoxide dismutase (CCS) as a biomarker for marginal copper deficiency.

Abstract

Copper is an essential dietary trace metal responsible for the functioning of at least 12 mammalian enzymes. Despite current increases in cases of copper deficient humans, a sensitive, reliable, and easily evaluated copper deficiency biomarker has yet to be identified. Multiple studies using rodent models were used to evaluate currently accepted blood biomarker proteins through Western blotting and activity assays. In the first study, male postweanling Sprague Dawley rats were fed copper deficient (CuD) or copper adequate (CuA) diets and deionized water and sampled after one, two, and four weeks. A group fed an iron deficient diet (FeD) was also included and sampled after two weeks. Lastly, after the four weeks a repletion study was performed for two weeks with samples taken after a total of six weeks of study. Superoxide dismutase (Sod1) and ceruloplasmin (Cp) were influenced by CuD and FeD diets, while the copper chaperone for superoxide dismutase (CCS) and the ratio of CCS/Sod1 were influenced only by CuD diet and after just one week. Blood cuproenzymes from CuD repleted rats normalized after two weeks on the CuA diet. Next, for two weeks male postweanling Sprague Dawley rats were fed CuA or CuD diets and compared with a group of marginally copper deficient (CuM) rats whose water was copper supplemented. Although differences between the CuD and CuM groups versus the CuA groups were identified, only CCS showed statistically different results between all groups, allowing for the identification of marginally copper deficient individuals. Further, there was high negative statistical correlation between liver copper and erythrocyte CCS. For all mouse trials, Swiss Webster mice were fed either CuD or CuA diets for five weeks. In one experiment, postweanling mice were housed in solid bottom cages and thus had access to their feces. In another postweanling mice were housed in stainless steal mesh bottom cages. CuD mice from solid bottom cages were found to be less deficient, likely due to coprophagia. These animals showed no reduction in plasma Cp or Sod1 expression, but did have higher CCS expression. Lastly, adult Swiss Webster mice were house in stainless steal mesh bottom cages and then evaluated for copper deficiency. CuD adult mice had no changes in Cp activity, liver copper levels, hemoglobin quantities, or Sod1 and Cp expression, but they did have a significantly elevated expression of CCS. These rodent model results indicated that CCS maybe an excellent biomarker for marginal copper deficiency in human diagnosis due to CCS’s reliability and sensitivity to variable copper status.