Flow cytometry detection of zinc ion bioavailability using the fluorescent probe Zinpyr-1: Relationship with the total intracellular zinc and metallothioneins

Zinc is an essential trace element for the biological function of more than 300 enzymes and for codifing, via zinc-fingers, many proteins involved in genomic stability and antioxidant response. The development of methods able to evaluate the intracellular availability of zinc ions is a matter of interest both in biological and nutritional field of research. In fact, the simple assessment of plasma zinc is not an optimal marker of the zinc status because of the existence of many homeostatic mechanisms which serve to maintain stable circulating levels even in the presence of a marginal deficiency. The intracellular zinc ion availability is, in turn, strictly related to the function of proteins with high affinity for zinc called metallothioneins (MT). The release of zinc from MT in response to oxidative stress is pivotal to activate the antioxidant response and DNA-repair mechanisms. A flow cytometry method consisting into the assessment of the free zinc levels with the zinc-specific probe, Zinpyr-1 (ZP-1), before and after induction with NO-donor, has been developed in order to evaluate this phenomenon. The validity of this method was verified by modulating "in vitro" the total intracellular concentration of zinc and the expression of MT. Preliminary applications of this method in peripheral blood mononuclear cells from young donors, demonstrate that in relatively few time and with a simple experimental tool, it is possible to get informations on the intracellular zinc ion availability and on the levels of free zinc that can be released by MT.