Intracellular zinc during cell activation and zinc deficiency

IntroductionZinc is an essential trace element having manifold functions within living cells. Zinc deficiency but also zinc excess impairs cell-specific functions whereas a balanced zinc level is required for an adequate cell behavior.Material and methodsThis study deals with the impact of cellular priming due to stimulation with interleukin (IL)-1, IL-2, IL-4, IL-6 or the chemokine CXCL12a and its subsequent influence on the intracellular free zinc concentration. Since cellular priming and activation is essential for proper immunological reactions, and across that highly cell-type specific, we investigated T cells, B cells, and peripheral blood mononuclear cells (PBMCs). Additionally, alterations of the intracellular zinc content was investigated by inducing zinc deficiency using the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) with subsequent re-supplementation of zinc, hence generating an intracellular zinc flux. Evaluation of zinc staining with FluoZin3-AM, Zinpyr-1 and Zinquin was done by flow cytometry or by fluorescence microscopy.ResultsOur results indicate that cellular priming for different periods of time (10 minutes/one hour) causes decreased intracellular free zinc concentrations in the FluoZin3-AM staining and increased zinc concentrations stained with Zinpyr-1. Furthermore, zinc supplementation after induced zinc deficiency leads to a fast and excessive rise of the intracellular free zinc levels in most cellular compartments.ConclusionOur study emphasizes the importance of zinc homeostasis and zinc distribution during cellular priming and for certain signaling cascades especially in T and B cells. Moreover, we demonstrated that zinc re-supplementation of zinc deficient cells results in significantly elevated intracellular free zinc concentrations compared to untreated controls. Hence, this underlines the need of a balanced zinc homeostasis for proper immune cell function.     

Dietary phytate,zinc and hidden zinc deficiency

Epidemiological data suggest at least one in five humans are at risk of zinc deficiency. This is in large part because the phytate in cereals and legumes has not been removed during food preparation. Phytate, a potent indigestible ligand for zinc prevents it's absorption. Without knowledge of the frequency of consumption of foods rich in phytate, and foods rich in bioavailable zinc, the recognition of zinc deficiency early in the illness may be difficult. Plasma zinc is insensitive to early zinc deficiency. Serum ferritin concentration ≤ 20 μg/L is a potential indirect biomarker. Early effects of zinc deficiency are chemical, functional and may be “hidden”. The clinical problem is illustrated by 2 studies that involved US Mexican-American children, and US premenopausal women. The children were consuming home diets that included traditional foods high in phytate. The premenopausal women were not eating red meat on a regular basis, and their consumption of phytate was mainly from bran breakfast cereals. In both studies the presence of zinc deficiency was proven by functional responses to controlled zinc treatment. In the children lean-mass, reasoning, and immunity were significantly affected. In the women memory, reasoning, and eye-hand coordination were significantly affected. A screening self-administered food frequency questionnaire for office might help caregiver's identify patients at risk of zinc deficiency.     

Serum zinc levels and zinc binding capacity in thalassemia

Recently, it has been reported that serum zinc binding capacity (ZnBC) is a very important criterion to evaluate body zinc (Zn) status. It has been shown that chronic Zn deficiency occur in the patients with thalassemia major (TM). Zn deficiency in TM may cause hyperzincuria, high ferritin levels, hepatic iron load, hepatic dysfunction. This study was undertaken to determine serum Zn levels and ZnBC in different thalassemia forms and sickle cell disease (SCD). The study has been carried out on 30 Thalassemia Major (TM), 34 Thalassemia Intermedia (TI), 31 Thalassemia Trait (TT) and 10 SCD. As control group,13 healthy children and 20 adults were included. Serum Zn and ZnBC were determined by atomic absorption, then saturation index (SI%: serum Zn/ZnBC x 100) was calculated. Serum Zn levels in all patients were lower than control (p < 0.01). Serum ZnBC was at a normal level in patients with TT and TI but it was found to be lower in TM and SCD than control (p < 0.01). While serum Zn levels decrease and ZnBC increase in nutritionaL Zn deficiency, serum Zn levels decrease but ZnBC doesn't increase in patients with thalassemia.     

Kinetics of zinc status and zinc deficiency in Berardinelli-Seip syndrome

Berardinelli-Seip syndrome (BSS) is a very rare disorder characterized by near-complete absence of adipose tissue from birth or early infancy, hypoleptinemia, hypertriglyceridemia, insulin resistance, diabetes mellitus, and other clinical signals. It is caused by mutations in AGPAT2 or Gng3lg. We evaluated 10 BSS patients and 10 healthy subjects. A single dose of 382.43 μmol zinc was administered intravenously before and after 3 months of oral zinc supplementation. Blood samples were collected from the contralateral arm at 0, 30, 60, 90, and 120 min after zinc injection. Plasma and serum were obtained to measure hematological and biochemical parameters. Urine was collected to measure creatinine, protein, and zinc. Basal serum zinc levels were similar in controls and BSS patients. However, serum zinc profiles were significant reduced in BSS patients in comparison with controls. The change in total-body zinc clearance was more significant in BSS patients, indicating that these patients had suboptimum zinc deficiency.     

Nuclear zinc in liver of cadmium and zinc treated rats

X-ray microanalysis was performed to detect quantitatively, the variation of the nuclear zinc in the liver cells of rats. The nuclear zinc concentration showed statistical decrease and increase in response to cadmium and zinc treatments, respectively. The results suggest that the liver responds differently to cadmium and zinc treatments. The difference in response to either treatment may reflect different mechanisms of zinc transport and metabolism in the liver. The difference in binding affinity of metallothionein (MT) may suggest the involvement of Mt in the metabolism and transport of zinc, an effect, which may be modified by treatment.     

Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals

Zinc(II) ions are essential for all forms of life. In humans, they have catalytic and structural functions in an estimated 3,000 zinc proteins. In addition, they interact with proteins transiently when they regulate proteins or when proteins regulate cellular zinc re-distribution. As yet, these types of zinc proteins have been explored poorly. Therefore the number of zinc/protein interactions is potentially larger than that given by the above estimate. Confronted with such a wide range of functions, which affect virtually all aspects of cellular physiology, investigators have begun to elucidate the molecular mechanisms of cellular homeostatic control of zinc, especially the functions of transporter, sensor, and trafficking proteins, such as metallothioneins, in providing the correct amounts of zinc ions for the synthesis of zinc metalloproteins. The sulfur-containing amino acid cysteine in proteins has an important role in the cellular mobility of zinc ions. Sulfur-coordination environments provide sufficiently strong interactions with zinc ions; they can undergo fast ligand-exchange; and they can serve as molecular redox switches for zinc binding and release. For the cellular functions of zinc, the free zinc ion concentrations (zinc potentials, pZn = −log[Zn²⁺]) and the zinc buffering capacity are critically important parameters that need to be defined quantitatively. In the cytoplasm, free zinc ions are kept at picomolar concentrations as a minute fraction of the few hundred micromolar concentrations of total cellular zinc. However, zinc ion concentrations can fluctuate under various conditions. Zinc ions released intracellularly from the zinc/thiolate clusters of metallothioneins or secreted from specialized organelles are potent effectors of proteins and are considered zinc signals. The cellular zinc buffering capacity determines the threshold between physiological and pathophysiological actions of zinc ions. When drugs, toxins, other transition metal ions or reactive compounds compromise zinc buffering, large zinc ion fluctuations can injure cells through effects on redox biology and interactions of zinc ions with proteins that are normally not targeted.

Saccharomyces cerevisiae vacuole in zinc storage and intracellular zinc distribution

Previous studies of the yeast Saccharomyces cerevisiae indicated that the vacuole is a major site of zinc storage in the cell. However, these studies did not address the absolute level of zinc that was stored in the vacuole nor did they examine the abundances of stored zinc in other compartments of the cell. In this report, we describe an analysis of the cellular distribution of zinc by use of both an organellar fractionation method and an electron probe X-ray microanalysis. With these methods, we determined that zinc levels in the vacuole vary with zinc status and can rise to almost 100 mM zinc (i.e., 7 x 10(8) atoms of vacuolar zinc per cell). Moreover, this zinc can be mobilized effectively to supply the needs of as many as eight generations of progeny cells under zinc starvation conditions. While the Zrc1 and Cot1 zinc transporters are essential for zinc uptake into the vacuole under steady-state growth conditions, additional transporters help mediate zinc uptake into the vacuole during "zinc shock," when zinc-limited cells are resupplied with zinc. In addition, we found that other compartments of the cell do not provide significant stores of zinc. In particular, zinc accumulation in mitochondria is low and is homeostatically regulated independently of vacuolar zinc storage. Finally, we observed a strong correlation between zinc status and the levels of magnesium and phosphorus accumulated in cells. Our results implicate zinc as a major determinant of the ability of the cell to store these other important nutrients.     

Dietary zinc intake,supplemental zinc intake and serum zinc levels and the prevalence of kidney stones in adults

ObjectiveThe association between zinc intake and the risk of kidney stones remains controversial. We examined the associations between dietary zinc intake, supplemental zinc intake and serum zinc levels and the prevalence of kidney stones in adults.MethodsAdult participants from the 2007–2016 NHANES were included. Restricted cubic splines were adopted to assess the dose-response relationships.ResultsDietary zinc intake was linearly associated with the prevalence of kidney stones (P_{for non-linearity} = 0.50), and the odds ratios (95% confidence intervals) of kidney stones were 0.75 (0.51–1.04) for 10 mg/day, 0.65 (0.39-0.97) for 20 mg/day, 0.53 (0.30-0.94) for 30 mg/day and 0.45 (0.22-0.95) for 40 mg/day. The linear relationship was also observed among women and overweight/obese individuals. No association was found between supplemental zinc intake and the prevalence of kidney stones. A non-linear relationship was found between serum zinc levels and the prevalence of kidney stones (P_{for non-linearity} = 0.02), and the odds ratios (95% confidence intervals) of kidney stones were 0.52 (0.33-0.82) for 70 ug/dL, 0.43 (0.24-0.77) for 90 ug/dL, 0.56 (0.32-0.98) for 110 ug/dL and 0.77 (0.37–1.62) for 130 ug/dL. The non-linear relationship was also observed among men and overweight/obese individuals.ConclusionsDietary zinc intake and serum zinc levels were inversely associated with the prevalence of kidney stones in adults, and there may be effect modification by participant sex and body mass index. The present analysis is limited in its ability to establish causality.     

Intracellular free zinc and zinc buffering in human red blood cells

Summary The effect of vasopressin on voltage-sensitive Ca²⁺ currents in the rat insulinoma cell line RINm5F has been investigated in patch-clamp whole-cell and single-channel current recording experiments. In the whole-cell recording configuration the dominant inward current in the presence of tetrodotoxin was noninactivating and had a high voltage threshold. This current was much enhanced when external Ca²⁺ was replaced by Ba²⁺ and was blocked by 1 m nifedipine. It can therefore be classified as an L-current. Vasopressin enhanced the L-current without changing the voltage threshold of activation or the voltage at which the peak current was observed. Vasopressin effects were seen at concentrations as low as 0.01nm, and the maximal effect was observed at about 1nm. In higher concentrations the vasopressin effects were weaker, with effects at 50nm of about the same magnitude as at 0.01nm. In single-channel current recording experiments carried out with the cell-attached configuration there were no effects on single L-channel currents when vasopressin was added to the bath solution, but in experiments in which vasopressin (5nm) was infused into the patch pipette a marked increase in the apparent channel open state probability was observed. We conclude that vasopressin, a peptide that is known to markedly enhance glucose-evoked insulin secretion, stimulates opening of the voltage-sensitive Ca²⁺ channels in insulin-secreting cells.     

Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease

Many bacteriophage and prophage genomes encode an HNH endonuclease (HNHE) next to their cohesive end site and terminase genes. The HNH catalytic domain contains the conserved catalytic residues His-Asn-His and a zinc-binding site [CxxC]₂. An additional zinc ribbon (ZR) domain with one to two zinc-binding sites ([CxxxxC], [CxxxxH], [CxxxC], [HxxxH], [CxxC] or [CxxH]) is frequently found at the N-terminus or C-terminus of the HNHE or a ZR domain protein (ZRP) located adjacent to the HNHE. We expressed and purified 10 such HNHEs and characterized their cleavage sites. These HNHEs are site-specific and strand-specific nicking endonucleases (NEase or nickase) with 3- to 7-bp specificities. A minimal HNH nicking domain of 76 amino acid residues was identified from Bacillus phage γ HNHE and subsequently fused to a zinc finger protein to generate a chimeric NEase with a new specificity (12–13 bp). The identification of a large pool of previously unknown natural NEases and engineered NEases provides more ‘tools’ for DNA manipulation and molecular diagnostics. The small modular HNH nicking domain can be used to generate rare NEases applicable to targeted genome editing. In addition, the engineered ZF nickase is useful for evaluation of off-target sites in vitro before performing cell-based gene modification.     

Zinc availability from zinc lipoate and zinc sulfate in growing rats

The purpose of this study was to investigate the effect of zinc lipoate and zinc sulfate on zinc availability in growing rats. 6 . 6 male albino rats were fed purified diets based on corn starch, egg albumen, sucrose, soy bean oil and cellulose over a 4-week period (diet Ia: 10 mg Zn/kg as zinc sulfate, diet Ib: 10 mg Zn/kg as zinc lipoate, diet IIa: 10 mg Zn/kg as zinc sulfate +0.4% phytic acid, diet IIb: 10 mg Zn/kg as zinc lipoate +0.4% phytic acid, diet IIIa: 20 mg Zn/kg as zinc sulfate + 0.4% phytic acid, diet IIIb: 20 mg Zn/kg as zinc lipoate + 0.4% phytic acid). Zinc lipoate and zinc sulfate both proved to be highly available zinc sources. When 0.4% phytic acid were present in the diets, apparent zinc absorption was generally depressed but was higher from zinc lipoate in tendency than from zinc sulfate. Comparable results were evident for femur zinc, plasma zinc and metallothionein concentrations in liver tissues. This indicates that zinc lipoate could be a valuable zinc source under conditions of low zinc availability. Nevertheless the absence or presence of phytic acid was a more important factor influencing zinc availability than the type of zinc source investigated.     

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2′-deoxycytidine (AZA) increased intracellular (after 24 and 48 h) and total cellular zinc levels (after 48 h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48 h. MT mRNA was significantly enhanced after 24 h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells.     

Effect of phosphorus and zinc nutrition on soybean seed phytic Acid and zinc

The relationships between nutrient P and Zn levels and the phytic acid, P, and Zn concentrations in soybean (Glycine max L. Merr. cv `Williams 79') seed were studied. Phytic acid increased linearly from 4.2 to 19.2 milligrams per gram as nutrient P treatment was varied from 2.0 to 50 milligrams per liter and Zn was held constant at 0.05 milligrams per liter. Leaf P concentration during seed development was found to be closely related to the concentrations of seed P and phytic acid. Leaf and seed Zn concentrations both responded positively to increasing nutrient Zn treatment. The effects of P treatment on plant and seed P and phytic acid were largely independent of the effects of Zn treatment on leaf and seed Zn. Phytic acid to Zn molar ratios ranging from 3.6 to 33.8 were observed.

The effects of nutrient P treatments on the concentrations of phytic acid, seed P, and leaf P were also studied in the P-sensitive (gene np) cultivars `Harosoy' and `Clark' and their respective P-tolerant (gene Np) near-isogenic lines L66-704 and L63-1677. In general, the positive relationships observed among nutrient P, leaf P, seed P, and phytic acid concentrations were similar to those observed in the studies with Williams 79. When fertilized with low or moderate nutrient P (2.5 and 25.0 milligrams P per liter, respectively) no significant differences in any parameter were observed between Harosoy or Clark and their respective P-tolerant isolines. When fertilized with high nutrient P (100 milligrams P per liter), Harosoy seed had a significantly higher concentration of phytic acid (30 milligrams per gram) than did seed of its P-tolerant near-isogenic line L66-704 (24.2 milligrams per gram phytic acid), whereas no significant difference was observed between Clark and its P-tolerant near-isogenic line L63-1677 (22.8 and 21.6 milligrams per gram, respectively). Variation in the phytic acid concentrations in the mature seed of the cultivars and isolines more closely paralleled leaf P concentrations observed during seed development (49 days after flowering), than those observed at the onset of seed development (14 days after flowering). Electrophoresis and ion-exchange chromatography revealed that partially phosphorylated intermediates do not appear when phytic acid accumulation is greatly reduced by limiting the nutrient P or when accumulation is greatly accelerated by excess P.

     

Association between plasma zinc concentration and zinc kinetic parameters in premenopausal women

The objective of this study was to measure relationships between plasma zinc (Zn) concentrations and Zn kinetic parameters and to measure relationships of Zn status with taste acuity, food frequency, and hair Zn in humans. The subjects were 33 premenopausal women not taking oral contraceptives and dietary supplements containing iron and Zn. Main outcomes were plasma Zn concentrations, Zn kinetic parameters based on the three-compartment mammillary model using 67Zn as a tracer, electrical taste detection thresholds, and food frequencies. Lower plasma Zn was significantly (P < 0.01) associated with smaller sizes of the central and the lesser peripheral Zn pools, faster disappearance of tracer from plasma, and higher transfer rate constants from the lesser peripheral pool to the central pool and from the central pool to the greater peripheral pool. The break points in the plasma Zn-Zn kinetics relationship were found between 9.94 and 11.5 micromol/l plasma Zn. Smaller size of the lesser peripheral pool was associated with lower frequency of beef consumption and higher frequency of bran breakfast cereal consumption. Hypozincemic women with plasma Zn <10.7 micromol/l or 700 ng/ml had decreased thresholds of electrical stimulation for gustatory nerves. Our results based on Zn kinetics support the conventional cutoff value of plasma Zn (10.7 micromol/l or 700 ng/ml) between normal and low Zn status.     

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl₂, which attenuated the abnormal increase in extracellular glutamate induced with high K⁺ in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K⁺ was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺.     

Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasis

Our knowledge of the molecular mechanisms of intracellular homeostatic control of zinc ions is now firmly grounded on experimental findings gleaned from the study of zinc proteomes and metallomes, zinc transporters, and insights from the use of computational approaches. A cell's repertoire of zinc homeostatic molecules includes cytosolic zinc-binding proteins, transporters localized to cytoplasmic and organellar membranes, and sensors of cytoplasmic free zinc ions. Under steady state conditions, a primary function of cytosolic zinc-binding proteins is to buffer the relatively large zinc content found in most cells to a cytosolic zinc(ii) ion concentration in the picomolar range. Under non-steady state conditions, zinc-binding proteins and transporters act in concert to modulate transient changes in cytosolic zinc ion concentration in a process that is called zinc muffling. For example, if a cell is challenged by an influx of zinc ions, muffling reactions will dampen the resulting rise in cytosolic zinc ion concentration and eventually restore the cytosolic zinc ion concentration to its original value by shuttling zinc ions into subcellular stores or by removing zinc ions from the cell. In addition, muffling reactions provide a potential means to control changes in cytosolic zinc ion concentrations for purposes of cell signalling in what would otherwise be considered a buffered environment not conducive for signalling. Such intracellular zinc ion signals are known to derive from redox modifications of zinc-thiolate coordination environments, release from subcellular zinc stores, and zinc ion influx via channels. Recently, it has been discovered that metallothionein binds its seven zinc ions with different affinities. This property makes metallothionein particularly well positioned to participate in zinc buffering and muffling reactions. In addition, it is well established that metallothionein is a source of zinc ions under conditions of redox signalling. We suggest that the biological functions of transient changes in cytosolic zinc ion concentrations (presumptive zinc signals) complement those of calcium ions in both spatial and temporal dimensions.     

Synthesis and characterization of nanocrystalline zinc sulfide via zinc thiobenzoate-lutidine single-source precursor

ZnS nanocrystals were prepared both in the form of mesoporous powder and thin films by one step thermal decomposition technique from a single-source procure (SSP) [Zn(SOCPh)₂Lut₂·H₂O]. The final product was characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), N₂ adsorption-desorption isotherm, UV-Vis absorption spectroscopy and photoluminescence (PL) study. Structural analyses of the prepared ZnS revealed the formation of cubic crystallites with diameters around 5 and 10 nm for the thin films and powder materials, respectively. On the other hand, the powder form showed mesoporous nature (type IV isotherm) with an average pore diameter of 37.9 Å and BET specific surface area of 51.73 m²/g.