Free Zn Concentrations Research Articles

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release

Aberrant Zn(2+) homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn(2+) modulates cardiac ryanodine receptor gating and Ca(2+) dynamics in isolated cardiomyocytes. We reveal that Zn(2+) is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn(2+) concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca(2+). At concentrations of free Zn(2+) >1 nm, Zn(2+) is the main activating ligand, and the dependence on Ca(2+) is removed. Zn(2+) is therefore a higher affinity activator of RyR2 than Ca(2+). Millimolar levels of free Zn(2+) were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn(2+) modulates both the frequency and amplitude of Ca(2+) waves in a concentration-dependent manner and that physiological levels of Zn(2+) elicit Ca(2+) release in the absence of activating levels of cytosolic Ca(2+). This highlights a new role for intracellular Zn(2+) in shaping Ca(2+) dynamics in cardiomyocytes through modulation of RyR2 gating.

Development of an In Situ Ion-Exchange Technique for the Determination of Free Cd, Co, Ni, and Zn Concentrations in Freshwaters

It is now well established that the bioavailability of metals toward aquatic organisms varies as a function of the free metal concentration. The ion-exchange technique (IET), which consists of equilibrating a calibrated cation-exchange resin with the water sample, is one of the few existing speciation methods that provide sufficient sensitivity and specificity to measure free metals in natural waters. In the present study, we developed an in situ IET (field-IET) in which the resin was directly equilibrated on site using dialysis cassettes. The field-IET was tested in six Canadian Shield lakes and in the Athabasca River (AB, Canada) for Cd2+, Co2+ (only in the Athabasca River), Ni2+, and Zn2+. Measurements were compared with those obtained on samples collected from the same sites with in situ diffusion samplers and analyzed in the laboratory (lab-IET). IET results were also compared with predictions from the Windermere Humic Aqueous Model [WHAM VII; the Co and Ni carbonato complex formation constants were updated according to the US National Institute of Science and Technology (NIST compilation)]. Good agreement was obtained between the field-IET and the lab-IET for Co2+ and Ni2+ concentrations, but field-IET Cd2+ and Zn2+ concentrations were systematically higher than the lab-IET results (factors of ~1.5× and ~2.4×, respectively). Uncertainties in the field-IET resin calibration and a significant Zn contamination could explain these results. WHAM VII predicted the free metal concentrations reasonably well, except for Ni2+ concentrations in the lakes, probably due to inappropriate formation constants for complexation of Ni with dissolved organic matter. This study showed that the field-IET, with its relative simplicity and its low detection limits, could be a useful method for the determination of free metal concentrations in acidic to neutral freshwaters.

The biochemical effects of extracellular Zn(2+) and other metal ions are severely affected by their speciation in cell culture media.

Investigations of physiological and toxicological effects of metal ions are frequently based on in vitro cell culture systems, in which cells are incubated with these ions in specialized culture media, instead of their physiological environment. This allows for targeted examination on the cellular or even molecular level. However, it disregards one important aspect, the different metal ion speciation under these conditions. This study explores the role of culture conditions in investigations with zinc ions (Zn(2+)). Their concentration is buffered by several orders of magnitude by fetal calf serum. Due to the complexity of serum and its many zinc-binding components, zinc speciation in culture media cannot be completely predicted. Still, the primary effect is due to the main Zn(2+)-binding protein albumin. Buffering reduces the free Zn(2+) concentration, thereby diminishing its biological effects, such as cytotoxicity and the impact on protein phosphorylation. This is not limited to Zn(2+), but is also observed with Ag(+), Cu(2+), Pb(2+), Cd(2+), Hg(2+), and Ni(2+). Usually, the serum content of culture media, and thereby their metal buffering capacity, is only a fraction of that in the physiological cellular environment. This leads to systematic over-estimation of the effects of extracellular metal ions when standard cell culture conditions are used as model systems for assessing potential in vivo effects.

Hypoxia lowers SLC30A8/ZnT8 expression and free cytosolic Zn2+ in pancreatic beta cells.

Aims/hypothesisHypoxic damage complicates islet isolation for transplantation and may contribute to beta cell failure in type 2 diabetes. Polymorphisms in the SLC30A8 gene, encoding the secretory granule zinc transporter 8 (ZnT8), influence type 2 diabetes risk, conceivably by modulating cytosolic Zn2+ levels. We have therefore explored the role of ZnT8 and cytosolic Zn2+ in the response to hypoxia of pancreatic islet cells.MethodsHuman, mouse or rat islets were isolated and exposed to varying O2 tensions. Cytosolic free zinc was measured using the adenovirally expressed recombinant targeted zinc probe eCALWY4. Gene expression was measured using quantitative (q)RT-PCR, western (immuno-) blotting or immunocytochemistry. Beta cells were identified by insulin immunoreactivity.ResultsDeprivation of O2 (1% vs 5% or 21%) for 24 h lowered free cytosolic Zn2+ concentrations by ~40% (p < 0.05) and ~30% (p < 0.05) in mouse and human islet cells, respectively. Hypoxia similarly decreased SLC30A8 mRNA expression in islets, and immunoreactivity in beta cells. Implicating lowered ZnT8 levels in the hypoxia-induced fall in cytosolic Zn2+, genetic ablation of Slc30a8 from mouse islets lowered cytosolic Zn2+ by ~40% (p < 0.05) and decreased the induction of metallothionein (Mt1, Mt2) genes. Cell survival in the face of hypoxia was enhanced in small islets of older (>12 weeks) Slc30a8 null mice vs controls, but not younger animals.Conclusions/interpretationThe response of pancreatic beta cells to hypoxia is characterised by decreased SLC30A8 expression and lowered cytosolic Zn2+ concentrations. The dependence on ZnT8 of hypoxia-induced changes in cell survival may contribute to the actions of SLC30A8 variants on diabetes risk in humans.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3266-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

The impact of electrodic adsorption on Zn, Cd and Pb speciation measurements with AGNES

The systems assayed in this work (Cd with iodide, Cd with polyacrylic acid, Pb or Zn with xylenol orange, etc.) as well as others previously studied in our lab (Pb/Cd/Zn with humic acids, Pb with pyridinedicarboxylic acid, Pb with organic extracts from river waters, etc.), exhibit induced adsorption of the metallic electroactive species on the mercury electrode. This fact, however, does not hinder the possibility of a correct determination of the free metal concentration in the solution with AGNES (Absence of Gradients and Nernstian Equilibrium Stripping). The equilibrium state (the target situation aimed at the end of the deposition stage of AGNES) is not disturbed by induced adsorption, while different strategies (e.g. the use of charge as stripping analytical signal) can avoid a possible kinetic interference on the quantification stage. The impact of adsorption on the blanks proves to be very small and, if needed, it could be further minimized by choosing a sufficiently high gain or variants of AGNES (such as AGNELSV or AGNES–SCP) that do not require the explicit subtraction of the blank. Some surfactants in solution, even without complexing the metal, might block the electrode surface, so that refined methodologies have to be used in such systems. In the case of the dispersant accompanying Nanotek ZnO nanoparticles, the complete blocking of the electrode can be reached in around 100s, and, thus, electrodes with renovating drops are key for dealing with such kind of difficulties. The free Zn concentration in equilibrium with these nanoparticles determined by AGNES (with minimization of the deposition times and suppression of any stirring) is consistent with the one expected according to its primary particle size.

Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology

Zinc plays a central role in all living cells as a cofactor for enzymes and as a structural element enabling the adequate folding of proteins. In eukaryotic cells, metals are highly compartmentalized and chelated. Although essential to characterize the mechanisms of Zn(2+) homeostasis, the measurement of free metal concentrations in living cells has proved challenging and the dynamics are difficult to determine. Our work combines the use of genetically encoded Förster resonance energy transfer (FRET) sensors and a novel microfluidic technology, the RootChip, to monitor the dynamics of cytosolic Zn(2+) concentrations in Arabidopsis root cells. Our experiments provide estimates of cytosolic free Zn(2+) concentrations in Arabidopsis root cells grown under sufficient (0.4nM) and excess (2nM) Zn(2+) supply. In addition, monitoring the dynamics of cytosolic [Zn(2+) ] in response to external supply suggests the involvement of high- and low-affinity uptake systems as well as release from internal stores. In this study, we demonstrate that the combination of genetically encoded FRET sensors and microfluidics provides an attractive tool to monitor the dynamics of cellular metal ion concentrations over a wide concentration range in root cells.

Availability of Zinc and the Ligands Citrate and Histidine to Wheat: Does Uptake of Entire Complexes Play a Role?

Organic ligands in soils affect the availability of trace metals such as Zn to plants. This study investigated the effects of two of these ligands, citrate and histidine, on Zn uptake by wheat under hydroponic conditions. Uptake of (65)Zn in the presence of these ligands was compared to uptake in the presence of EDTA at the same free Zn concentration (Zn(2+) ~ 50 nM). In the presence of citrate Zn root uptake was enhanced ~3.5 times and in the presence of histidine, by a factor of ~9, compared to the EDTA treatments. Citrate uptake was slightly reduced in the treatment containing ligands and Zn compared to the treatment containing the same ligand concentration but no Zn. In addition, a higher uptake of Zn than of citrate was observed. This suggests that the enhanced Zn uptake was primarily due to increased supply of Zn(2+) by diffusion and dissociation of Zn-citrate complexes at the root surface. Histidine uptake was much higher than citrate uptake and not influenced by the presence of Zn. As histidine forms stronger complexes with Zn than citrate, the results suggest that the enhancement of Zn uptake in the presence of histidine was in part due to the uptake of undissociated Zn-histidine complexes.

Determination of the Complexing Capacity of Wine for Zn Using the Absence of Gradients and Nernstian Equilibrium Stripping Technique

The complexing capacity of synthetic (0.011 M tartrate in 13.5% ethanol) and real wine (Raimat Abadia) in titrations with added total Zn concentrations up to 0.03 M has been determined following the free Zn concentrations with AGNES (absence of gradients and Nernstian equilibrium stripping) technique. A correction to find the preconcentration factor or gain (Y(1)) really applied at each one of the ionic strengths reached due to Zn additions along the titration has been applied. The standard implementation of AGNES to real wine led to the observation of two anomalous behaviors: (a) an increasingly negative current in the deposition stage (labeled as "HER" effect) and (b) a minimum in the currents of the stripping stage plot (labeled as the "dip" effect). A practical strategy to apply AGNES avoiding the dip effect has been developed to quantify properly free Zn concentrations. The van den Berg-Ružic-Lee linearization method (assuming the existence of just 1:1 complexes) has been adapted to consider the dilution effect and the ionic strength changes. Aggregated stability constants and total ligand concentrations have been calculated from synthetic and wine titration data. The found complexing capacity in the studied wine (c(T,L) = 0.0179 ± 0.0007 M) indicates the contribution of ligands other than tartrate (which is confirmed to be the main one).

Application of Zinpyr-1 for the investigation of zinc signals in Escherichia coli

Changes of the pico- to nanomolar concentration of free intracellular Zn(2+) are part of the signal transduction in mammalian cells. These zinc signals regulate the enzymatic activity of target proteins such as protein tyrosine phosphatases. For Escherichia coli, previous studies have reported diverging concentrations from femto- to picomolar, raising the question if Zn(2+) could also have a function in bacterial signaling. This manuscript explores the use of the low molecular weight fluorescent probe Zinpyr-1 in E. coli. The probe detects free Zn(2+) in these bacteria. Comparable to mammalian cells, other metal ions, especially Hg(2+) and Cd(2+), interfere with the detection of Zn(2+). Moreover, experiments in E. coli were particularly prone to artifacts based on cellular autofluorescence, necessitating corrections that are not required in mammalian cells. Based on measurements in lysates of E. coli and the mammalian cell line Jurkat, similar values between 0.1 and 0.2nM free Zn(2+) were found. For E. coli, this corresponds to less than one free zinc ion per cell. Moreover, phosphatase inhibition by Zn(2+) was only observed in Jurkat, but not E. coli. This excludes a function for zinc signals as a regulator of bacterial phosphatases. Still, changes in the free Zn(2+) concentration were observed in response to elevated extracellular Zn(2+) and pH, or to addition of the detergent NP-40, suggesting that other processes could be controlled by the free intracellular Zn(2+) concentration.

Influence of essential elements on cadmium uptake and toxicity in a unicellular green alga: The protective effect of trace zinc and cobalt concentrations

Within the biotic ligand model (BLM) construct, major cations are considered to be simple competitors for metal binding to uptake sites and may offer some protection against metal-induced toxicity, but the influence of essential trace elements and cell preconditioning to different micronutrient concentrations on metal uptake and toxicity is considered negligible. To test these underlying assumptions, we monitored Cd uptake and toxicity in a green alga (Chlamydomonas reinhardtii) after long-term exposures (60 h) to a range of environmentally realistic free Zn(2+) , Co(2+) , Fe(3+) , Mn(2+) , Ca(2+) , and Cu(2+) concentrations buffered with nitrilotriacetic acid. A 200-fold increase in free [Mn(2+) ] as well as a 100-fold increase in free [Fe(3+) ] did not affect Cd uptake or toxicity, whereas a 50-fold increase in free [Ca(2+) ] effectively offered some protection, as predicted by the BLM. However, a 10-fold increase in free [Cu(2+) ] significantly enhanced Cd toxicity by a factor of approximately 2, whereas a 100-fold increase in free [Zn(2+) ] and [Co(2+) ] from 10(-11) to 10(-9) M significantly decreased Cd uptake and toxicity by more than twofold. These effects did not change with prior algal acclimation to different essential micronutrient concentrations. Low essential trace metal concentrations may strongly affect the uptake and toxicity of Cd in freshwater algae and should be taken into consideration in future developments of the BLM.

Substituent-dependent fluorescent sensors for zinc ions based on carboxamidoquinoline

A series of carboxamidoquinoline-based fluorescent sensors (the AQZ family) were synthesized and characterized. The AQZ family members were highly soluble in water and showed good selectivity for Zn(2+)via enhanced fluorescence in aqueous buffer solution. Fluorescence signals could be tuned from dual-wavelength ratiometric changes to changes in the intensity of a single wavelength upon binding Zn(2+) through the introduction of different substituents onto the quinoline ring. Concentrations of free Zn(2+) of 10(-5)-10(-6) M could be detected using the sensors. Changes of substituents and their positions on the quinoline ring influenced the sensitivity for Zn(2+), but had little effect on Zn(2+) affinities.

The use of permeation liquid membranes for free zinc measurements in aqueous solution

Environmental context The free Zn ion concentration in environmental aqueous systems is an important factor in determining Zn deficiency or toxicity to organisms as this species is directly bioavailable. The permeation liquid membrane technique, a tool to measure either free or bioavailable metal concentrations in solution depending on its setup, was evaluated for the first time for Zn speciation in simplified plant nutrient solutions. The technique is low-cost and applicable to a broad range of aqueous samples. Abstract The bioavailability of Zn in environmental water phases strongly depends on its speciation. One important species in studies on Zn deficiency or toxicity to organisms is the free ion. The permeation liquid membrane (PLM) technique is a tool to measure free metal concentrations with a short analysis time of 1 h and at low cost. However, so far it has only been validated for Cd, Cu, Ni and Pb. In this study we tested the effect of carrier concentrations and pH on Zn transport across the organic PLM membrane and the ability of the technique to measure free Zn in synthetic plant nutrient solution. We found that Zn membrane transport is dependent on the concentration of the carrier molecule lauric acid (LA), whereas variations in the concentration of the other carrier molecule, the crown ether Kryptofix 22DD, showed no effect, suggesting that Zn is not transported by the ‘classical’ PLM transport mechanism by binding to the crown ether. Zn preconcentration increased with increasing pH and decreased with increasing ligand concentrations. Using 0.05 M LA, Zn membrane transport is expected to be rate limiting (permeability criterion &lt;&lt;1) and the free Zn concentration can be measured. Under these conditions, PLM measurements agreed well with speciation calculations and with Donnan membrane technique (DMT) measurements in the presence of ligands forming negatively charged Zn complexes (ethylenediaminetetraacetate or citrate). In the presence of L-histidine higher free Zn concentrations than calculated were measured by PLM and DMT, suggesting that positively charged complexes contributed to cross-membrane transport in both methods.

Does the Zinc Neuroprotective Effectiveness of Mean Prevention of Intracellular Zinc Accumulation in Water in INDIA

Zinc (Zn) plays an important role in the activity of all cells, including neurons. Both the neurotoxic and neuroprotective effects of zinc have been well established, but the exact mechanism of its dual abilities still remains unclear. The same effect may also be found in other cells, but it should be remembered that lower sensitivity to hypoxia prolongs in time the cytotoxic effect of Zn+2 excess. It seems that the apparent dualism depends primarily on the energetic condition of the cell, and also on the efficacy of ion pumps, genetically conditioned mechanisms regulating Zn cell efflux and Zn sequestration inside the cell and also on the concentration of extracellular free Zn.

Tissue zinc levels in a child with hypercalprotectinaemia and hyperzincaemia: A case report and a review of the literature

Background. A girl suffering from a rare syndrome of unknown aetiology, termed hypercalprotectinaemia, was evaluated for tissue zinc status, because calprotectin is a protein which chelates Zn at multiple binding-sites, which might have affected the distribution of Zn in her body. Methods. Measurement of serum, urine, hair and nail zinc (Zn) concentration, complemented with measurement of total Zn in ultrafiltrates of plasma. Results. Her serum Zn concentration was 105–133 μmol/L. Zn levels in her hair (102 μg/g), nail (90 μg/g) and urine (3–12 μmol/L; 20–80 μg/dL) were all at the lower end of the reference intervals described in the sparse literature. Zn concentrations in ultrafiltrates of plasma were below the detection limit (<100 nmol/L). Thus, the elevated serum Zn did not translate into a similarly increased level of Zn in any of the tissues tested, nor in free Zn concentrations. Instead it appeared to be a result of Zn being chelated to binder proteins, most probably calprotectin. Conclusion. Her grossly elevated serum calprotectin concentration is probably able to raise circulating total Zn concentrations without raising ionized concentrations, but this Zn remains confined to the circulating blood as well as to excreted body fluids, particularly faeces.

Bioavailability pathways underlying zinc-induced avoidance behavior and reproduction toxicity in Lumbricus rubellus earthworms

We investigated possible bioavailability pathways underlying zinc-induced avoidance behavior and sublethal reproduction impairment in Lumbricus rubellus. Clay-loam (pH 7.3) and sandy soil (three pH values of 4.3–6.0) were amended with zinc sulfate at six soil concentrations of total Zn ranging from 0.1 to 36 mmol/kg dw. Estimated and measured concentrations of free and exchangeable Zn ranged 10 −4 to 7.1 mmol/l. Avoidance behavior responses were fast and could be directly predicted from the activity of free zinc ions without a modifying pH effect. The repellent effect is thus likely mediated by a direct action of Zn 2+ ions on epidermal chemosensitive receptors. Body zinc uptake, however, was determined by proton competition with free Zn 2+ sorption. Excess accumulation of body Zn was a good predictor of reproduction decline, which is indicative of internal zinc poisoning. The results indicated that zinc affects earthworms via both direct and indirect mechanisms of external and internal exposure.

Redox Regulation of Intracellular Zinc: Molecular Signaling in the Life and Death of Neurons

Zn(2+) has emerged as a major regulator of neuronal physiology, as well as an important signaling agent in neural injury. The intracellular concentration of this metal is tightly regulated through the actions of Zn(2+) transporters and the thiol-rich metal binding protein metallothionein, closely linking the redox status of the cell to cellular availability of Zn(2+). Accordingly, oxidative and nitrosative stress during ischemic injury leads to an accumulation of neuronal free Zn(2+) and the activation of several downstream cell death processes. While this Zn(2+) rise is an established signaling event in neuronal cell death, recent evidence suggests that a transient, sublethal accumulation of free Zn(2+) can also play a critical role in neuroprotective pathways activated during ischemic preconditioning. Thus, redox-sensitive proteins, like metallothioneins, may play a critical role in determining neuronal cell fate by regulating the localization and concentration of intracellular free Zn(2+).

Chemical composition and Zn bioavailability of the soil solution extracted from Zn amended variable charge soils

Abstract A study on variable charge soils (volcanic Italian and podzolic Scottish soils) was performed to investigate the influence of soil properties on the chemical composition of soil solution. Zinc speciation, bioavailability and toxicity in the soil solution were examined. The soils were spiked with increasing amounts of Zn (0, 100, 200, 400 and 1000 mg/kg) and the soil solutions were extracted using rhizon soil moisture samplers. The pH, total organic carbon (TOC), base cations, anions, total Zn and free Zn 2+ in soil solution were analysed. A rapid bioassay with the luminescent bacterium Escherichia coli HB101 pUCD607 was performed to assess Zn toxicity. The influence of soil type and Zn treatments on the chemical composition of soil solution and on Zn toxicity was considered and discussed. Different trends of total and free Zn concentrations, base cations desorption and luminescence of E. coli HB101 pUCD607 were observed. The soil solution extracted from the volcanic soils had very low total and free Zn concentrations and showed specific Zn 2+/Ca 2+ exchange. The soil solution from the podzolic soil had much higher total and free Zn concentrations and showed no evidence of specific Zn 2+/Ca 2+ exchange. In comparison with the subalkaline volcanic soils, the acidic podzol showed enhanced levels of toxic free Zn 2+ and consequently stronger effects on E. coli viability.

Application of the new electroanalytical technique AGNES for the determination of free Zn concentration in river water

Absence of gradients and Nernstian equilibrium stripping (AGNES) is a recently developed electroanalytical technique specifically designed for the direct determination of free concentrations of metal ions. AGNES is applied here to the determination of free Zn concentration in a river water sample. The method has been validated with synthetic solutions of low ionic strengths containing Zn and 2,6-pyridinedicarboxylic acid and then applied to synthetic river waters and to a natural sample collected from Besòs River in Montcada i Reixac (Catalonia, North-Eastern Spain). In the river sample, an average free Zn concentration of 12.8(4) nM was obtained, while the total dissolved Zn concentration was 0.51(8) microM. To control and maintain pH and pCO(2) constant during AGNES measurements, a novel device for N(2)/CO(2) mixed purging has been developed.

Design, synthesis and biological application of chemical probes for bio-imaging

Biological imaging has revealed many new biological findings, among them GFP and other fluorescent proteins and small molecule based fluorescent probes have been widely used, especially in the past two decades. In this tutorial review the design concept and application of chemical probes are described-these are FRET based probes, Zn(2+) probes and MRI probes. Fluorescence resonance energy transfer (FRET) has been used extensively for the design principle for fluorescent probes. One of the most significant advantages of probes with FRET modulation is that these can enable ratiometric measurement in living cells, which reduces the artefacts from microscopic imaging systems. The design strategy for the development of a small molecular FRET probe is described, in terms of avoiding close contact between donor fluorophore and acceptor fluorophore in aqueous solution. Furthermore, a strategy to design a FRET probe with modulating overlap integral of donor and acceptor is introduced. Numerous tools for Zn(2+) sensing in living cells have become available in the last decade. Among them, fluorescence imaging using fluorescent probes has been the most popular approach. Some of these probes can be used to visualize Zn(2+) in living cells. Some of the biological functions of Zn(2+) were clarified using these probes, especially in neuronal cells, which contain a high concentration of free Zn(2+). Real-time imaging of enzyme activities in vivo offers valuable information in understanding living systems and in developing medicine for various types of diseases. Magnetic resonance imaging (MRI) is expected to be one of the most promising in vivo imaging techniques in the scientific and medical fields. One of the most promising nuclide for MRI is (19)F. (19)F atoms are concentrated in the form of solid salts mostly in bones and teeth in our bodies. The MRI signal of the intrinsic (19)F is hardly detectable. A novel design strategy for (19)F MRI probes in detecting protease activity is described.

Determination of Free Cd, Cu and Zn Concentrations in Lake Waters by In Situ Diffusion Followed by Column Equilibration Ion-exchange

Combining in situ diffusion and column ion-exchange equilibration, we measured free metal ion concentrations (Cd, Cu and Zn) in water samples collected from the epilimnion of 14 lakes in the Rouyn-Noranda area (600 km north-west of Montreal, QC, Canada). Lakes were selected to represent a wide range of physico-chemical characteristics (hardness, pH, dissolved organic matter—DOM, degree of metal contamination), to determine the influence of these parameters on metal speciation. Total dissolved metal concentrations, as determined within the diffusion cells, varied over one to two orders of magnitude: [Cd] 0.19–2.9 nM; [Cu] 36–190 nM; [Zn] 7–2,800 nM. The proportion of total dissolved metal present as free Cd2+ and Zn2+ was relatively constant for the 14 selected lakes, despite the wide pH (4.5–8) and DOM (3–23 mg C/L) ranges, probably reflecting the inverse relationship observed between pH and DOM; this proportion did, however, vary with DOM and pH for Cu. Our experimental free metal ion concentrations were compared with those calculated with the thermodynamic models WHAM (Windermere Humic Aqueous Model VI) and ECOSAT 4.7 (incorporating the NICA-Donnan model). Measured and calculated values were in reasonable agreement for both Cd and Zn although measured values were generally slightly higher, i.e. less than one order of magnitude. For several lakes, measured free Cu concentrations were, however, much higher than the calculated values, suggesting that these models overestimate Cu complexation. The gap between measured and calculated free metal ion concentration becomes more important as the total metal concentration decreases and as pH increases.