Role of metallothionein in regulating the abundance of histochemically reactive zinc in rat tissues

The objectives of this study were (i) to investigate the modulating effects of zinc nutrition on histochemically reactive zinc in the rat intestine and liver and (ii) to assess the relationship between histochemically reactive zinc and metallothionein-bound zinc in these tissues under varying zinc nutrition. Male Wistar rats were fed a zinc-deficient (3 mg zinc/kg), adequate-zinc (30 mg zinc/kg, ad libitum or pair-fed), or zinc-supplemented (155 mg zinc/kg) diet for 2 or 6 weeks. Plasma N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide-reactive zinc reflected dietary zinc intake. Abundance of the intestine histochemically reactive zinc was correlated with dietary zinc intake after 2 weeks of dietary treatment. Dietary zinc intake had no effect on the abundance of the intestine histochemically reactive zinc after 6 weeks of dietary treatment and the hepatic histochemically reactive zinc after both 2 and 6 weeks of dietary treatment. This lack of effect of dietary zinc intake on the abundance of histochemically reactive zinc was associated with a higher level of metallothionein. The molecular-mass distribution profile revealed that N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide-reactive zinc and metallothionein-bound zinc represented two different, but interrelated, pools of zinc. Overall, these results suggested that the abundance of histochemically reactive zinc was homeostatically regulated, which was partially achieved through the regulation of metallothionein levels in rats.     

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.     

Bioavailability of two organic forms of zinc in comparison to zinc sulphate for weaning pigs fed a diet composed mainly of wheat,barley and soybean meal

This study was performed to compare the bioavailability of two organic zinc compounds, a zinc glycinate complex and a zinc amino acid chelate with that of zinc sulphate in growing pigs fed a basal diet composed mainly of wheat, barley and soybean meal. The experiment included 96 pigs with an average body weight of 8 kg, allotted to ten groups of nine to ten pigs each. The first group received the basal diet, containing 42 mg of native zinc per kg, without zinc supplementation over a period of five weeks. The other nine groups received the basal diet supplemented with 15, 30 or 50 mg of zinc/kg as zinc sulphate, zinc glycinate or the zinc amino acid chelate. Pigs fed the unsupplemented diet had a lower growth performance (body weight gain, feed conversion ratio) than the other nine groups. Supplementation of 15 mg zinc/kg diet (irrespective of zinc form) was sufficient to yield optimum growth performance. Plasma zinc concentration and activity of alkaline phosphatase were rising with increasing zinc supplementation levels up toa maximum reached at a supplementary level of 30 or 50 mg/kg diet for activity of alkaline phosphatase and plasma zinc concentration, respectively. The response of those parameters to zinc supplementation did, however, not differ between thethree zinc compounds considered. The apparent digestibility of zinc from the diet was also not different for the three zinc compounds. In conclusion, these findings show that the bioavailability of the two organic zinc compounds did not differ from that of zinc sulphate in growing pigs fed a diet with wheat, barley and soybean meal as major components.     

Bioavailability of two organic forms of zinc in comparison to zinc sulphate for weaning pigs fed a diet composed mainly of wheat, barley and soybean meal

This study was performed to compare the bioavailability of two organic zinc compounds, a zinc glycinate complex and a zinc amino acid chelate with that of zinc sulphate in growing pigs fed a basal diet composed mainly of wheat, barley and soybean meal. The experiment included 96 pigs with an average body weight of 8 kg, allotted to ten groups of nine to ten pigs each. The first group received the basal diet, containing 42 mg of native zinc per kg, without zinc supplementation over a period of five weeks. The other nine groups received the basal diet supplemented with 15, 30 or 50 mg of zinc/kg as zinc sulphate, zinc glycinate or the zinc amino acid chelate. Pigs fed the unsupplemented diet had a lower growth performance (body weight gain, feed conversion ratio) than the other nine groups. Supplementation of 15 mg zinc/kg diet (irrespective of zinc form) was sufficient to yield optimum growth performance. Plasma zinc concentration and activity of alkaline phosphatase were rising with increasing zinc supplementation levels up to a maximum reached at a supplementary level of 30 or 50 mg/kg diet for activity of alkaline phosphatase and plasma zinc concentration, respectively. The response of those parameters to zinc supplementation did, however, not differ between the three zinc compounds considered. The apparent digestibility of zinc from the diet was also not different for the three zinc compounds. In conclusion, these findings show that the bioavailability of the two organic zinc compounds did not differ from that of zinc sulphate in growing pigs fed a diet with wheat, barley and soybean meal as major components.     

The influence of zinc status on the kinetics of zinc uptake into cultured endothelial cells

To better understand cellular zinc homeostasis and characterize the zinc transport process, a mammalian cell culture model was utilized to investigate the influence of zinc status on the kinetics of zinc uptake. Culturing conditions were optimized to induce moderate zinc deficiency and zinc excess while still sustaining the general health of the cells. Cells were grown in (1) control medium of 10% fetal bovine serum (FBS) in minimum essential medium (MEM; 5.0 micromol zinc/L), (2) low zinc medium (10% dialyzed FBS in MEM; 1.5 micromol zinc/L), or (3) zinc back medium (10% dialyzed FBS in MEM with zinc added as ZnCl(2); 5.0 micromol zinc/L). Bovine pulmonary artery endothelial cells (BPAEC), porcine aortic endothelial cells (PAEC), and porcine venous endothelial cells (PVEC) were evaluated as to their responsiveness to our zinc-deficient conditions. Zinc uptake was faster (P < 0.001) in all three cell types when they were grown in low zinc medium compared with controls; the increases were 32% in PAEC, 37% in PVEC, and 66% in BPAEC. Further kinetic analysis with BPAEC demonstrated a 31% increase (P < 0.05) in the maximum rate of zinc uptake (Jmax) grown in low zinc medium compared with controls, but no difference (P > 0.05) between the low zinc group and the control group in the concentration at which uptake was half-maximal (K). Zinc uptake into BPAEC grown in excess zinc conditions was not different (P > 0.05) unless the medium contained greater than 50 micromol zinc/L. In conclusion, BPAEC increased their ability for zinc uptake in response to moderate zinc deficiency, but did not change their kinetics of zinc uptake during moderate zinc excess.     

The dynamic nature of zinc availability from foods in vivo

This paper presents a review of data from animal experiments demonstrating that the relative availability of zinc from foods is affected by an interaction between the source and amount of zinc consumed. Zinc availability from foods was determined by whole body counting after feeding 65Zn-labeled meals containing varying amounts of zinc. Relative to availability from zinc chloride, zinc availability from foods such as chicken, milk, and peanut butter was greater when determined using 98 rather than 16 micrograms zinc in the meal. In rats fed the higher dose of zinc, there were greater differences in zinc availability among the 15 foods studied, and zinc availability was greater from some foods than from zinc chloride. After an in vitro enzymatic digestion, neither zinc solubility nor the partitioning of zinc between low and high molecular weight substances was useful for predicting zinc availability in vivo. These data indicate that zinc availability from food is not a constant proportion of availability from a zinc salt. In view of the dynamic nature of zinc availability in vivo, the inability to accurately simulate different physiological responses to varying quantities of certain foods may limit the usefulness of in vitro methods.     

Molecular distribution of zinc in biliary and pancreatic secretions

Rat bile and pancreatic fluid were examined for the presence of low molecular weight zinc complexes. Fluids were collected separately by cannulation, and zinc distribution in collected samples was analyzed by gel filtration on Sephadex G-50. Most of the zinc in bile was associated with low molecular weight zinc complexes; only a small amount of zinc was present in the high molecular weight fraction. In contrast, pancreatic secretions did not contain low molecular weight zinc complexes, but there were considerable amounts of zinc bound to high molecular weight compounds. The addition of zinc to bile resulted in an increased amount of zinc in the low molecular weight fraction, while the addition of zinc to pancreatic fluid resulted primarily in an increase in zinc bound to the high molecular weight components. Like pancreatic fluid, homogenates of pancreatic tissue had no low molecular weight zinc complex. In rats whose bile and pancreatic fluid were removed and not returned into the intestine, the amount of zinc bound to low molecular weight complexes in intestinal homogenates was reduced. This alteration of the molecular distribution of zinc in intestinal homogenates by removal of bile and pancreatic fluid suggests the potential importance of low molecular weight zinc complexes for zinc homeostasis.     

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.     

The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signaling

Free zinc ions are potent effectors of proteins. Their tightly controlled fluctuations ("zinc signals") in the picomolar range of concentrations modulate cellular signaling pathways. Sulfur (cysteine) donors generate redox-active coordination environments in proteins for the redox-inert zinc ion and make it possible for redox signals to induce zinc signals. Amplitudes of zinc signals are determined by the cellular zinc buffering capacity, which itself is redox-sensitive. In part by interfering with zinc and redox buffering, reactive species, drugs, toxins, and metal ions can elicit zinc signals that initiate physiological and pathobiochemical changes or lead to cellular injury when free zinc ions are sustained at higher concentrations. These interactions establish redox-inert zinc as an important factor in redox signaling. At the center of zinc/redox signaling are the zinc/thiolate clusters of metallothionein. They can transduce zinc and redox signals and thereby attenuate or amplify these signals.     

A study of zinc distribution in human serum.

The partition of zinc in human serum between two major zinc-binding proteins, albumin and alpha2-macroglobulin, was studied in 28 control subjects and in 156 hospitalized patients. Albumin-bound zinc was both the major and the more dynamic of the serum zinc components. Over a wide range of values the concentrations of albumin-bound zinc and total serum zinc were highly correlated (r=0.91) with each other, as were concentrations of albumin and albumin-bound zinc (r=0.69). alpha2-Macroglobulin-bound zinc was not strongly correlated either with total serum zinc or with the serum concentration of alpha2-macroglobulin. Twenty-four hour urinary excretion of zinc was not correlated with any of the serum zinc parameters. To a large extent it appears that total serum zinc concentration reflects serum albumin concentration.     

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.     

Metals on the move: zinc ions in cellular regulation and in the coordination dynamics of zinc proteins

Homeostatic control maintains essential transition metal ions at characteristic cellular concentrations to support their physiological functions and to avoid adverse effects. Zinc is especially widely used as a catalytic or structural cofactor in about 3000 human zinc proteins. In addition, the homeostatic control of zinc in eukaryotic cells permits functions of zinc(II) ions in regulation and in paracrine and intracrine signaling. Zinc ions are released from proteins through ligand-centered reactions in zinc/thiolate coordination environments, and from stores in cellular organelles, where zinc transporters participate in zinc loading and release. Muffling reactions allow zinc ions to serve as signaling ions (second messengers) in the cytosol that is buffered to picomolar zinc ion concentrations at steady-state. Muffling includes zinc ion binding to metallothioneins, cellular translocations of metallothioneins, delivery of zinc ions to transporter proteins, and zinc ion fluxes through cellular membranes with the result of removing the additional zinc ions from the cytosol and restoring the steady-state. Targets of regulatory zinc ions are proteins with sites for transient zinc binding, such as membrane receptors, enzymes, protein–protein interactions, and sensor proteins that control gene expression. The generation, transmission, targets, and termination of zinc ion signals involve proteins that use coordination dynamics in the inner and outer ligand spheres to control metal ion association and dissociation. These new findings establish critically important functions of zinc ions and zinc metalloproteins in cellular control.     

Kinetics of zinc desorption from soils

Summary The kinetics of zinc desorption by DTPA were investigated in several soils. The rate of desorption of soil zinc and adsorbed zinc was rapid initially and gradually declined with time. The desorption reaction can be described by a two constant rate equation, C=At^B. The rate of zinc desorption in soil containing freshly adsorbed zinc was considerably higher than soil zinc, possibly due to the higher solubility and zinc concentration on the surfaces of soil particulate matter. The amount of zinc desorbed by DTPA, however, continued to decrease with increasing aging time. Elevated temperature further enhanced zinc aging and reduced zinc extractability. Recrystallization of adsorbed zinc in soil which subsequently increased the bonding strength of adsorbed zinc, could be responsible for the reduction of zinc desorption by DTPA.Contribution from the Department of Agronomy and Range Science, University of California, Davis, Ca.     

Effects of arachidonic acid and cyclo (his-pro) on zinc transport across small intestine and muscle tissues

Previously we have shown that arachidonic acid (AA) plus zinc or cyclo (his-pro) (CHP) plus zinc improve clinical signs of diabetes in streptozotocin-induced diabetic rats. Since streptozotocin destroys pancreatic beta-cells, we hypothesize that the effect of either AA or CHP, plus zinc on glucose metabolism is via mobilization of intracellular zinc which in turn stimulates glucose uptake by peripheral tissues. We now report the relationship between zinc and AA and between zinc and CHP in controlling zinc influx and efflux across hindlimb muscle cells isolated from three-month old rats. Although CHP increased muscle zinc influx in a dose-dependent manner, AA was not effective. However, AA was more effective in stimulating zinc efflux than CHP. We have previously demonstrated that AA stimulates intestinal zinc uptake and absorption, and now present evidence that CHP also influences intestinal zinc transport. These results suggest that both AA and CHP affect glucose uptake in muscle cells via stimulating intestinal zinc absorption and muscle cell zinc flux.     

Appraisal of the methodology and applications for measurement of the zinc content of blood components as indicators of zinc status

Methods for the measurement of the zinc content of blood components (plasma, erythrocytes, platelets, mononuclear leukocytes, and polymorphonuclear neutrophils) as indicators of the zinc status of animals have been reviewed and evaluated. The values of plasma or serum zinc concentrations as indicators of zinc status in experimental animals or humans is questionable. Consequently, the zinc content of blood cellular components has been suggested as better indicators of zinc status. Methodological problems, such as incomplete cellular separation or zinc contamination, occur in some of the procedures applied to the quantitation of blood cellular component zinc. Specialized microprobe procedures involving, for example, proton-induced X-ray fluorescence, scanning ion technology, mass spectrometery, or laser microprobe mass analysis might be used to measure the absolute zinc level in individual cells subjected to minimal handling. Then, more conventional methods can be tested against the true standard. Nevertheless, there appears to be species differences in regard to the response of blood cellular zinc concentrations to dietary zinc deficiency. Blood cellular component zinc is conserved during severe zinc deficiency in the rat. In contrast, the zinc content of blood cellular components in humans may reflect the whole body zinc status.     

Zinc Supplementation or Regulation of its Homeostasis: Advantages and Threats

To accomplish its multifunctional biological roles, zinc requires precise homeostatic mechanisms. There are efficient mechanisms that regulate zinc absorption from the alimentary tract and its excretion by the kidney depending on the organism demands. The regulatory mechanisms of cellular zinc inflow, distribution, and zinc outflow are so efficient that symptoms of zinc deficiency are rare, and symptoms connected with its massive accumulation are even more rare. The efficiency of homeostatic mechanisms that prevent zinc deficiency or excessive zinc accumulation in the organism is genetically conditioned. It seems that an essential element of zinc homeostasis is the efficiency of zinc transmembrane exchange mechanisms. Intracellular free zinc concentration is higher than in extracellular space. Physiologically, the active outflow of zinc ions from the cell depends on the increase of its concentration in extracellular space. The ion pumps activity depends on the efficiency by which the cell manages energy. Considering the fact that zinc deficiency accelerates apoptosis and that excessive zinc accumulation inside cells results in a toxic effect that forces its death brings about several questions: Is intensification and acceleration of changes in zinc metabolism with age meaningful? Is there a real zinc deficiency occurring with age or in connection with the aforementioned pathological processes, or is it just a case of tissue and cell redistribution? When discussing factors that influence zinc homeostasis, can we consider zinc supplementation or regulation of zinc balance in the area of its redistribution? To clarify these aspects, an essential element will also be the clear understanding of the nomenclature used to describe changes in zinc balance. Zinc homeostasis can be different in different age groups and depends on sex, thus zinc dyshomeostasisrefers to changes in its metabolism that deviate from the normal rates for a particular age group and sex. This concept is very ample and implies that zinc deficiency may result from a low-zinc diet, poor absorption, excessive loss of zinc, zinc redistribution in intra- and extracellular compartments, or a combination of these factors that is inadequate for the given age and sex group. Such factor or factors need to be considered for preventing particular homeostasis disorders (or dyshomeostasis). Regulation of zinc metabolism by influencing reversal of redistribution processes ought to be the main point of pharmacologic and nonpharmacologic actions to reestablish zinc homeostasis. Supplementation and chelation are of marginal importance and can be used to correct long-term dietary zinc deficiency or zinc poisoning or in some cases in therapeutic interventions. In view of its biological importance, the problem posed by the influence of zinc metabolism requires further investigation. To date, one cannot consider, for example, routine zinc supplementation in old age, because changes of metabolism with age are not necessarily a cause of zinc deficiency. Supplementation is warranted only in cases in which deficiency has been established unambiguously. An essential element is to prevent sudden changes in zinc metabolism, which lead to dyshomeostasis in the terms defined here. The primary prophylaxes, regular physical activity, efficient treatment of chronic diseases, are all elements of such prevention.     

Investigation of excretion and absorption of different zinc salts in puppies.

The effects of dietary zinc on zinc absorption and excretion were evaluated with six 12-week-old beagles. The dogs were fed a commercial dry food later supplemented with 2 and 4mg/kg of body weight per day of either zinc sulphate, zinc acetate or zinc oxide. The concentrations of zinc in the urine and faeces of all treatment groups were increased depending on the type of zinc salts and the dosage in the diet (P < 0.05). The apparent absorption of zinc salts ranged from 0.20 to 0.36. The zinc salt supplements at both dosages did not affect the digestibility of crude protein, fat and fibre. The zinc concentration in plasma varied with the type of zinc salt and with the dosage. The mean concentrations ranged from 61+/- 1.46 microg to 73 +/- 1.57 microg/dl in the unsupplemented groups, whereas it was 115 +/- 2.33 microg/dl in the group supplemented with zinc acetate at the highest dosage.     

Contribution of zinc fraction to available zinc extracted,by some chemical methods,from rice growing soils of North-Western Himalayas

A laboratory and greenhouse investigation was undertaken to study the distribution and contribution of zinc fractions to available zinc in submerged rice. Most of the total zinc was present as Al- and Fe-oxide bound (52.8%) and residual zinc (27.8%). The exchangeable (non-specifically and specifically absorbed), organically bound and Mn-oxide bound zinc fractions averaged 0.7, 1.1, 6.3 and 4.9 per cent of the total zinc, respectively. 0.1 M HCl, EDTA-(NH₄)₂CO₃ and dithizone extractants showed significant correlation with per cent yield, Zn concentration and zinc uptake by grain and the critical limits were 3.0, 1.9 and 1.0 µg^–1, respectively. Organically bound zinc exhibited significant correlation with per cent yield and zinc uptake by grain whereas specifically absorbed zinc correlated with Zn concentration in grain. Mn-oxide boudn zinc and Al- and Fe-oxide bound zinc fractions were also correlated with zinc concentration and zinc uptake by grain.