Parameters Influencing Zinc in Experimental Systems in Vivo and in Vitro

Abstract

Abstract:In recent years, the role of zinc in biological systems has been a subject of intense research. Despite wide increase in our knowledge and understanding of zinc homeostasis, numerous questions remain to be answered, encouraging further research. In particular, the quantification of intracellular zinc ions and fluctuation, as well as the function of zinc in signaling processes are being intensely investigated. The determination of free intracellular zinc ions is difficult and error-prone, as concentrations are extremely low (in the pico-to nanomolar range), but techniques exist involving fluorescent probes and sensors. In spite of zinc deficiency being accepted as a global problem, causing death and disease worldwide, to date there are no markers to reliably assess a person's zinc status. This review summarizes the difficulties and major pitfalls when working with zinc in in vitro and in vivo research. Additionally, it specifies important aspects for zinc substitution and supplementation, including the bioavailability of zinc and its intestinal absorption. In particular, it is intended to help researchers with yet minor experience working with zinc efficiently set up experiments and avoid commonly occurring mistakes, starting with the choice and preparation of reagents and instrumentation, and concluding with possibilities for measuring the status of zinc in humans.

A Guide to Human Zinc Absorption: General Overview and Recent Advances of In Vitro Intestinal Models

“…Speciation of zinc in cell culture medium or buffer severely affects its availability and cellular uptake in in vitro experiments [220,287]. A particular problem in this context is fetal calf serum (FCS), which proves to be an unpredictable factor due to its variability [288] and contains about 60% albumin in its protein fraction [289].…”

Metals Are Main Actors in the Biological World

“…It is clear from this work that metals in biology are a proverbial "double-edged sword", i.e., they are vital for the activity of a number of critical enzymes and cell processes, yet their dyshomeostasis causes pathological effects that damage both the organism in general and cells in particular. Such an aspect is very clear also in the paper by Ollig et al [6], that, in addition to describing the role of zinc in living organisms, deals with the methodological problems and pitfalls related to measuring intracellular free zinc levels, elucidating the technical problems correlated to the in vivo investigation of the metallomics world.…”

A practical guide to the preparation and use of metal ion‐buffered systems for physiological research

“…27 In addition, it has been reported that even membraneimpermeable high-affinity ligands applied extracellularly may alter intracellular trace metal homeostasis, presumably by establishing a steep gradient for efflux of ions like Zn 2+ . 28,29 For these and other reasons, the concentration of strong ligands should be kept as low as possible when metal ion-buffered systems are used in living tissues or cells. Provided that steps are taken to reduce the initial amount of contaminating metal ions present, 1 mmol/L of a PAPC ligand is often sufficient for effective buffering and reduction in background Zn 2+ contamination below the threshold for detection with indicators like FluoZin-3 (but see section 3.4).…”