Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with a putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats (Rattus novegicus), albino mice (Mus musculus), Cp -/-mice, and adult humans (Homo sapiens) to investigate the copper-iron interaction. Dietary copper-deficient (CuD) rats and mice were produced using a modified AIN-76A diet. Results confirmed that o-dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp -/-mice contained no detectable Cp diamine oxidase activity. Importantly, no ferroxidase activity was detectable for CuD rats, mice, or Cp -/-mice compared to robust activity for copper-adequate (CuA) rodent controls using western membrane assay. Immunoblot protocols detected major reductions (60-90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT-PCR. Data are consistent with apo-Cp being less stable than holo-Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in Cp is a sensitive biomarker for copper deficiency.

Ceruloplasmin (CP) is a multicopper oxidase and the most abundant copper binding protein in mammalian plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with CP's putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats, albino mice, Cp −/− mice, and adult humans to investigate the copper‐iron interaction and further characterize CP as a biomarker. Dietary copper‐deficient (CuD) rats and mice were produced using a modified AIN‐76A diet. Results confirmed that o‐dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp −/− mice contained no detectable Cp diamine oxidase activity. Importantly, following western blotting no detectable ferroxidase activity was observed for CuD rats, mice, or Cp −/− mice compared to robust activity for copper‐adequate (CuA) rodent controls. Immunoblot protocols detected major reductions (60–90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT‐PCR. Data support that apo‐Cp is less stable than holo‐Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in CP activity is a sensitive biomarker for copper deficiency. Supported by NIH HD‐39708 and USDA NIFA 2006‐35200‐17378.