Introduction

One of the major advances in the understanding of the cellular biology of Zn(II) has been the development of techniques to visualize and quantify the dynamic intracellular pools of Zn(II) (1,2). Labile Zn(II) is bound weakly to proteins and other macromolecules as well as to low molecular weight thiols such as glutathione (3,4). Study of these chelatable or labile pools of Zn(II) has thrown new light on mechanisms underlying processes as diverse as cell secretion, apoptosis, neuro-transmission, Ras-mediated signaling pathways, and zinc transport pathways (3,4,5,6,7). A relatively neglected area to date, however, has been the detection and measurement of labile Zn(II) in extracellular fluids, including the major fluids of the body and in vitro culture secretions or other cell-conditioned media.

There are several reasons why it may be important to measure the concentration of labile extracellular Zn(II). It has been estimated that 23% of the world's population are Zn(II) deficient and at heightened risk of infection, growth retardation, and other manifestations of Zn(II) deficiency (8,9). Meta-analysis of trials conducted in Vietnam, Bangladesh, and Indonesia have found that dietary zinc supplementation reduces the incidence of pneumonia by 41% and diarrhea by 18% (9). Zn(II) deficiency also complicates a variety of chronic clinical conditions, such as sickle cell anemia, diabetes, rheumatoid arthritis, and asthma (10,11,12). Traditionally, serum or plasma Zn(II) concentrations, as measured by processes such as atomic absorption spectrometry (AAS), have been used as markers of zinc deficiency. However, one limitation in using such measures is that a significant proportion of the total serum or plasma Zn(II) is tightly incorporated in the metalloprotein a-2-macroglobulin; the function of this Zn(II) is not known but it is poorly exchangeable and does not appear to be a determinant of Zn(II) nutriture (10,13). In fact, the bulk of the 2–3 g of Zn(II) in the body comprises structural and catalytic Zn(II) contained within several hundred Zn(II) metalloenzymes and other intra- and extracellular Zn(II) metalloproteins. These fixed pools of Zn(II) are largely unaffected, even in severe Zn(II) deficiency (3,10). For example, Zn(II) deprivation in rats resulted in a progressive fall in plasma Zn(II) levels to about 40% of initial levels but did not decline further despite prolonged deprivation (14).

The most relevant plasma Zn(II) pool is thought to be the labile Zn(II) bound to albumin and en route for uptake by the various organ systems. Although the speciation of Zn(II) between different plasma pools has been estimated by fractionation studies coupled with a total Zn(II) measure such as by A AS, a laboratory test that would largely measure the labile Zn(II) content in extracellular fluids, against a background of fixed Zn(II) in metalloproteins, would be an important adjunct in assessing Zn(II) deficiency.

There is also a need to measure labile Zn(II) in culture media because many cellular processes are affected by Zn(II) and a number of cytokines and growth factors contain, or are dependent upon, Zn(II) (10,15,16). The optimal levels of extracellular Zn(II) for in vitro cultures remain poorly defined. In addition, some cell types release or actively secrete Zn(II) (17,18). A technique to measure labile Zn(II) in extracellular fluids would enable quantification of Zn(II) release from cells as well as provide a means to monitor available Zn(II) concentrations in a variety of buffers and media.

Our studies over the past decade have focused on the measurement and visualization of labile Zn(II) in subcellular compartments, whole cells, and tissues using the UV-excitable fluorophore zinquin ethyl ester ((Figure 1)A) (12)(18,19,20). Zinquin is sensitive to nM concentrations of free Zn(II), highly specific for Zn(II) among other metal cations, and preferentially reactive with the most labile (free or thermodynamically exchangeable) Zn(II) pools. Zinquin can also react with another category of Zn(II), which is Zn(II) partially complexed by a biomolecule such that binding sites remain available to zinquin (21). For this reason, the Zn(II) detected by zinquin includes both exchangeable Zn(II) and more tightly bound but accessible Zn(II). For the purposes of this manuscript, we use the term labile Zn(II) to include all zinquin-detectable Zn(II) that is capable of being quenched by the chelator EGTA. Here we have used zinquin in a rapid and sensitive fluorometric assay for the measurement of labile Zn(II) in minute volumes of plasma and other extracellular fluids.