Direct determination of free Zn concentration in samples of biological interest.

The speciation of essential metal ions in biological fluids, such as blood plasma and serum, is of fundamental importance to understand the homeostasis of these elements. The activity of metal ions such as Zn in extracellular media is thought to affect their interaction with membrane-bound transporters, and thus is critical for their cellular uptake. Previous approaches to determine "free" Zn (i.e. the hexa-aquo ion) are based on separation by either chromatography or ultrafiltration, or on metallochromic dyes. However, both types of approach are prone to affect the relevant equilibria. These drawbacks can be circumvented with the electroanalytical technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping), since it can measure free zinc concentration without perturbing the sample speciation. Here, a Bovine Serum Albumin (BSA) + Zn synthetic mixture and Fetal Bovine Serum (FBS) are analyzed as proof of concept. Adsorption of BSA on the surface of the Hanging Mercury Drop Electrode (HMDE), despite the advantage of its renewal, is so intense that it blocks appropriate attainment of the required equilibrium, and only estimations of [Zn] can be derived. In contrast, a rotating disc electrode with a thin mercury film deposited on it (TMF-RDE) is advantageous because of its small volume and enhanced mass transfer. Protein adsorption can be prevented by covering the TMF-RDE with Nafion. A free Zn concentration [Zn] = 2.7 nmol L was found at pH 7.0, total Zn 20 μμmol L and BSA 600 μμmol L. A sample of FBS with fixed pH 7.2 (MOPS 0.08 mol L) yielded [Zn] = 0.25 nmol L. This methodology opens the way to free metal concentration determinations in biological fluids.