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Copper transport and accumulation were studied in virgin and lactating C57BL/6 mice, with and without expression of ceruloplasmin (Cp), to assess the importance of Cp to these processes. One hour after i.p. injection of tracer (64)Cu, liver and kidney accounted for 80% of the radioactivity, and mammary gland 1%, while in lactating Cp+/+ mice 2-4 days post partum, uptake by mammary gland was 9-fold higher and that of liver and other organs was decreased, with (64)Cu rapidly appearing in milk. Parallel studies in Cp-/- mice (siblings from same colony) gave virtually identical results. However, their milk contained less (64)Cu, and actual copper contents determined by furnace atomic absorption were less than half those for milk from normal dams. Liver copper concentrations of pups born to Cp-/- dams also were half those of pups from wild type dams. Copper in pup brains was unaffected; but iron concentrations were reduced. We conclude that absence of Cp, while not affecting entry of exchangeable copper from the blood into the mammary gland, does have a significant effect on the availability of this metal to the newborn through the milk and in the form of stores accumulating in gestation.

In the blood plasma of humans and rats, ceruloplasmin is the major copper-binding protein and ferroxidase, accounting for 70% of the copper present in the plasma, with the rest binding primarily to albumin and a macroglobulin. Systematic studies with fresh plasma were carried out to compare what occurs in the mouse. C57BL6 mice had half as much copper and pPD (p-phenylene diamine) oxidase activity as humans and rats, 20-40% as much ferroxidase activity as humans (determined using three different assays) and less inhibition by azide. Plasma from ceruloplasmin knockout mice had no pPD oxidase activity, but retained >50% ferroxidase activity (which was not as affected by azide). Modelling of mouse ceruloplasmin against the known X-ray structure of human ceruloplasmin indicated subtle but potentially significant changes in the pPD- and azide-binding sites. Purification and in-gel assays after native PAGE confirmed that mouse ceruloplasmin had ferroxidase activity but revealed an additional ferroxidase in ceruloplasmin knockout mouse plasma, which is also seen in size-exclusion chromatography. In the wild-type mouse, the 'ceruloplasmin' peak contained approximately 55% of the total copper, but ceruloplasmin knockout plasma exposed a major additional peak (180 kDa) which co-eluted with ferroxidase activity. Two other ferroxidases (700 and 2000 Da) were also detected in mouse and human plasma. Mammalian blood thus contains copper components and ferroxidases not reported previously.

To establish for the first time how mice might differ from rats and humans in terms of copper transport, excretion, and copper binding proteins, plasma and organ cytosols from adult female C57CL6 mice were fractionated and analyzed by directly coupled size exclusion HPLC-ICP-MS, before and after i.p. injection of large doses of (65)Cu. Plasma from untreated mice had different proportions of Cu associated with transcuprein/macroglobulin, ceruloplasmin and albumin than in humans and rats, and two previously undetected copper peaks (Mr 700 k and 15 k) were observed. Cytosols had Cu peaks seen previously in rat liver (Mr > 1,000 k, 45 k and 11 k) plus one of 110 kDa. (65)Cu (141 microg) administered over 14 h, initially loaded plasma albumin and mainly entered liver and kidney (especially 28 kDa and 11 kDa components). Components of other organs were less (but still significantly) enriched. (63)Cu/(65)Cu ratios returned almost to normal by 14 days, indicating a robust system for excreting excess copper. We conclude that there are significant differences but also strong similarities in copper metabolism between mice, rats and humans; that the liver is able to buffer enormous changes in copper status; and that a large number of mammalian copper proteins remain to be identified.