Copper (Cu), as an essential metal, plays a crucial role in biochemical reactions and in physiological regulations. Cu in plasma is mostly bound to proteins; about 65-90% of Cu is tightly binds with caeruloplasmin and the rest of Cu is loosely binds with albumin and transcuprein. A small but significant relatively "free" fraction, probably complexed with amino acids, is present at around 5% of the total concentration. We developed and validated a new method for direct measurement of free Cu in serum by ultrafiltration with AMICON®Ultra 100K device and determination with AAS. Also, we checked that there is no trace of albumin in the ultrafiltrates and we demonstrated the ultrafiltration of a known concentration of Cu added in artificial serum without albumin and, on the contrary, the retention of the Cu in artificial serum with albumin. The ultrafiltration procedure and the instrumental determination showed a good repeatability and a very low limit of detection (1μg/L). The method was applied to 30 healthy subjects, the mean value of the total Cu (994.8μg/L) is included in the normal range for healthy people and the values of free Cu (23.6μg/L) corresponding to 2.37% of the Cu total. The determination of free Cu by this simple and cheap method may be useful to measure the most bioavailable Cu fraction possibly implicated in neuro-degenerative and oxidative-stress related diseases.
Keywords: Albumin; Free-copper; Speciation; Ultrafiltration.
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