Copper absorption in young men fed adequate and low zinc diets

Copper absorption was measured at two levels of dietary zinc in six healthy young men who were confined to a metabolic unit for a 75 d study of zinc utilization. A diet of conventional foods was fed, providing either 16.5 or 5.5 mg zinc and 1.3 mg copper daily. Copper absorption was determined by feeding⁶⁵Cu, a stable isotope of copper, once during the 16.5 mg Zn diet and near the beginning and end of the 5.5 mg Zn diet. Apparent copper absorption averaged 48.1% when the 16.5 mg Zn diet was fed. This was significantly higher than the averages of 37.2 and 38.5% when the 5.5 mg Zn diet was fed. Absorption also differed significantly among subjects. Fecal copper did not differ between diets or among subjects. All subjects were in positive copper balance at both levels of dietary zinc. These results suggest that a dietary zinc intake slightly above the Recommended Dietary Allowance of 15 mg/d does not increase fecal copper loss and does not interfere with copper absorption.     

Interaction between copper and zinc in metal accumulation in rats with particular reference to the synthesis of induced-metallothionein

The effectiveness of Zn at moderating the pro-oxidant effects of Cu was evaluated in two rat models that differed in the route and mode of administration. The endpoints investigated included measurement of the concentrations of Cu, Zn, metallothionein and glutathione concentrations, as well as SOD and catalase activity, in liver, kidneys and intestine. In a sub-chronic animal model, the hepatic accumulation of Cu was achieved by administration of dietary Cu (1.8 g/kg solid diet) for 30 days after which oral Zn (6g/kg solid diet) was given. Cu treatment induced an increase in the hepatic and intestinal concentration of Cu of 66 and 455%, respectively, that was not associated with synthesis of metallothionein synthesis, but rather appeared to be related to the higher activity of SOD. Subsequent administration with Zn after dietary Cu induced an increase in the hepatic and intestinal metallothionein content of more twice and reduced the Cu content to control values. Thus, Zn could act as both a competitor for absorption on the luminal side of the intestinal epithelium inducing the synthesis of metallothionein. In the second animal model, we studied the effects of interaction between Cu and Zn administered by i.p. injection at the dose of 3 and 10mg/kg, respectively; Zn was administered subsequent to Cu overload. In this case, when Zn was administered, Cu was already deposited in tissues and thus there is no competition between two metals at the level of membrane transport. In this experimental model treatment with Cu alone induced liver metallothionein synthesis, and the subsequent treatment with Zn did not decrease the hepatic content of Cu. One explanation for these observations is that Zn induces the synthesis of metallothionein, which binds Cu for which it has a higher affinity. Moreover, after treatment with Zn, SOD activity in the liver decreases of almost 30% with respect to treatment with alone Cu, suggesting that Zn has a protective effect.     

日本沼虾MT基因克隆、组织差异性表达及与饲料铜、锌含量的相关性

为了更全面理解日本沼虾(Macrobrachium nipponense)的铜/锌营养生理作用,研究利用RACE技术从日本沼虾肝胰腺中克隆获得一金属硫蛋白基因cDNA全长(mn-MT),并对该基因分子特征、组织表达谱和饲料铜/锌水平对其表达的影响进行分析。结果显示:(1)mn-MT cDNA全长665 bp,含编码59个氨基酸的180 bp的开放阅读框,预测该多肽的理论分子量为6.085 kD,等电点为7.73。该蛋白中半胱氨酸含量最高(30.5%),其次是赖氨酸(16.95%)和丝氨酸(10.17%)。相似性分析显示mn-MT氨基酸序列与美洲海螯虾、斑节对虾和中华绒螯蟹MT的相似性分别达到78%、75%和75%。(2)qRT-PCR分析显示, mn-MT mRNA在肝胰腺、血细胞、鳃、胃、卵巢、肠和肌肉中都有表达,其中肝胰腺中表达量最高。(3)用4组铜添加量分别为0、20、40及160 mg/kg的饲料和3组锌添加水平分别为0、35和210 mg/kg的饲料饲喂初重为(0.1010.002) g日本沼虾56d后,分析各组虾肝胰腺的mn-MT mRNA表达。mn-MT mRNA表达随饲料铜水平的提高而升高,到40 mg/kg组达到最高(P0.05),而后开始下降;饲料中高锌(210 mg/kg)显著提高mn-MT表达(P0.05), 0和35 mg/kg组间差异不显著(P0.05)。结果表明饲料中铜/锌均可影响mn-MT表达,且呈现不同的剂量依赖效应。       

Levels of copper and zinc in diets for growing and finishing pigs can be reduced without detrimental effects on production and mineral status

One hundred and sixty pigs were used to evaluate dietary copper (Cu) and zinc (Zn) supplementation on performance, fecal mineral levels, body mineral status and carcass and meat quality. Diets differed in mineral form (MF) (Cu and Zn in the form of proteinate amino acid chelate (organic) or sulfate (inorganic)) and inclusion level (IL) (27 mg/kg of total Cu and 65 mg/kg of total Zn ('low') or 156 mg/kg of total Cu and 170 mg/kg of total Zn ('high')) according to a 2 × 2 factorial arrangement of treatments. Pigs were used from 25 to 107 kg body weight (BW) and fed their respective diets ad libitum. Blood and fecal samples were collected on days 14 and 77 of the experiment. Blood was analyzed for concentration of Cu and Zn, hemoglobin (Hb), Cu content of red blood cells (RBC Cu) and alkaline phosphatase (ALP) and feces for Cu and Zn concentration. Hot carcass weight (HCW) and backfat depth were measured at slaughter and indices of meat quality were assessed on a section of longissimus thoracis. Liver, kidney and bone samples were collected immediately after slaughter and liver and kidney were tested for Cu and Zn content, while bone was only tested for Zn. Over the entire experimental period (25 to 107 kg BW) no significant treatment differences in average daily gain (ADG) or average daily feed intake (ADFI) occurred; however, feed conversion ratio (FCR) was improved by the inclusion of proteinate amino acid chelate (P = 0.012). Copper and Zn concentrations in feces were in direct proportion to the IL in the diet. Blood mineral levels were within normal physiological ranges in all treatments and tissue Cu and Zn concentrations increased with dietary IL (P < 0.05). Results indicate that Cu and Zn fecal concentrations were reduced by approximately 6-fold for Cu and by 2.5-fold for Zn by feeding 27 mg/kg Cu and 65 mg/kg Zn, in either the proteinate amino acid chelate or the sulfate form, compared with a diet containing 156 mg/kg Cu and 170 mg/kg Zn. This decrease in total dietary Cu and Zn did not reduce performance or mineral status of pigs.     

Dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats

The hypothesis was tested that dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats, which in turn causes a decreased copper absorption. Male rats were fed adequate-copper (5 mg Cu/kg) diets containing either fructose or glucose (709.4 g monosaccharide/kg) for a period of 5 wk. Fructose vs glucose significantly lowered copper concentrations in plasma and the liver, but did not alter hepatic copper mass. Fructose feeding resulted in a significantly lesser intestinal solubility of copper as based on either a smaller soluble fraction of copper in the liquid phase of small intestinal contents or a lower copper concentration in the liquid phase. The latter fructose effect can be explained by the observed fructose-induced increase in volume of liquid phase of intestinal digesta. After administration of a restricted amount of diet extrinsically labeled with⁶⁴Cu, rats fed fructose also had significantly lower soluble⁶⁴Cu fraction in the digesta of the small intestine. Although this study shows that fructose lowered intestinal copper solubility, only a slight reduction of apparent copper absorption was observed. It is suggested that the fructose-induced lowering of copper status in part counteracted the fructose effect on copper absorption at the level of the intestinal lumen.     

The effects of cadmium and copper on the renal uptake and metallothionein binding of gold in the rat and hamster

Rats and hamsters, (pre)-treated with copper and cadmium, were used to investigate whether species-differences in renal metallothionein synthesis in response to gold were determined by changes in the kidney concentrations of other metals. The effects of both dietary copper limitation and excess on the renal metabolism of gold also were studied in the rat. In this species, all of the pre-treatments affected the renal concentrations of total and metallothionein-bound copper, but none of them altered either the kidney uptake or thionein-binding of gold. Incorporation of zinc into the metallothionein, which accompanied the binding of gold in this fraction of the kidney, however, was influenced slightly by the pretreatments. In hamsters, pretreatment with cadmium, which increased the concentrations of total and thionein-bound zinc in the kidneys, also did not affect the renal uptake of gold, although it increased significantly the binding of gold to the metallothionein fraction of the renal cytosol. This increased binding of gold also was accompanied by further increases in the zinc and copper contents of the metallothionein; the contents of total and thionein-bound cadmium, however, remained essentially unchanged. Concentrations of copper and zinc in the hamster kidney were not affected significantly by subcutaneous administration of copper alone (five daily doses, each of 3.2 mg Cu/kg body wt.), but were increased when gold was given during the copper-treatment. The concentrations of gold, copper and zinc in the renal metallothionein fraction also were increased under these conditions. From these results it seems that kidney metallothionein synthesis in response to gold may be related to the changes in either the concentration or distribution of zinc, rather than copper.     

Effect of acute stress on the absorption and distribution of zinc and on Zn-metallothionein production in the liver of the chick.

A study has been made of the effects of chloroform inhalation, Escherichia coli endotoxin injection and hydrocortisone injection on the absorption of a single intragastric dose of 65Zn by the chick. Injection of hydrocortisone increased the absorption of the 65Zn by 30-55% in both Zn-deficient and Zn-supplemented chicks. The influence of chloroform and endotoxin was less consistent; the former treatment only increased 65Zn absorption and endotoxin was less consistent; the former treatment only increased 65Zn absorption in Zn-supplemented chicks fed ad libitum whereas endotoxin only increased that in Zn-supplemented chicks on a restricted food intake. Injection of endotoxin increased the hepatic uptake of the absorbed 65Zn in both Zn-deficient and Zn-supplemented chicks, whereas hydrocortisone had a similar effect in the Zn-supplemented birds only. Chloroform inhalation increased hepatic 65Zn uptake in Zn-deficient chicks only. The increase in hepatic Zn concentrations in the stressed chicks was mainly associated with a protein in the cytosol identified as metallothionein. Both endotoxin and hydrocortisone decreased total plasma Zn concentrations in Zn-supplemented and Zn-deficient chicks; chloroform decreased plasma 65Zn content only.     

Rainbow trout metallothionein

Two forms of hepatic metallothionein were isolated and purified from rainbow trout injected intraperitoneally with cadmium chloride. Both forms showed similarities with mammalian metallothioneins, had a high cystein content (30 mol%), and were void of aromatic amino acids and histidine. The molecular weight was estimated to be about 6000 dalton for the apothioneins, and the thiol groups of the cysteine residues complexed with the heavy metals (Cd, Cu, Zn) in a SH/Me⁺⁺ ratio of about 2.4. The amount of copper in metallothionein from rainbow trout was very high, greater than the amount of cadmium and zinc after injections of 3 mg cadmium/kg body weight. The total metal content of cadmium, copper and zinc in metallothionein 1 and 2 were about 7 and 8 atoms per molecule respectively.     

Copper uptake in wild type and copper metallothionein-deficient Saccharomyces cerevisiae. Kinetics and mechanism

The mechanism of copper uptake in Saccharomyces cerevisiae has been investigated using a combination of 64Cu2+ and atomic absorption spectrophotometry. A wild type copper-resistant CUP 1R-containing strain and a strain carrying a deletion of the CUP1 locus (yeast copper metallothionein) exhibited quantitatively similar saturable energy-dependent 64Cu2+ uptake when cultures were pregrown in copper-free media (medium [Cu] approximately 15 nM). The kinetic constants for uptake by the wild type strain were Vmax = 0.21 nmol of copper/min/mg of protein and Km = 4.4 microM. This accumulation of 64Cu2+ represented net uptake as confirmed by atomic absorption spectrophotometry. This uptake was not seen in glucose-starved cells, but was supported in glycerol- and ethanol-grown ones. Uptake was inhibited by both N3- and dinitrophenol and was barely detectable in cultures at 4 degrees C. When present at 50 microM, Zn2+ and Ni2+ inhibited by 50% indicating that this uptake process was relatively selective for Cu2+. 64Cu2+ accumulation was qualitatively and quantitatively different in cultures either grown in or preincubated with cold Cu2+. Either treatment resulted in the appearance of a fast phase (t 1/2 approximately 1 min) of 64Cu2+ accumulation which represented isotopic exchange since it did not lead to an increase in the mass of cell-associated copper; also, it was not energy-dependent. Exchange of 64Cu2+ into this pool was not inhibited by Zn2+. Pretreatment with Cu2+ caused a change in the rate of net accumulation as well; a 3-h incubation of cells in 5 microM medium Cu2+ caused a 1.6-fold increase in the velocity of energy-dependent uptake. Prior addition of cycloheximide abolished this Cu2(+)-dependent increase and, in fact, inhibited the 64Cu2+ uptake velocity by greater than 85%. The exchangeable pool was also absent in cycloheximide, Cu2(+)-treated cells suggesting that exchangeable Cu2+ derived from the copper taken up initially by the energy-dependent process. The thionein deletion mutant was similar to wild type in response to medium Cu2+ and cycloheximide indicating that copper metallothionein is not directly involved in Cu2+ uptake (as distinct from retention) in yeast.     

Effects of pretreatment with cadmium on the discriminative uptake of subsequent cadmium, copper or zinc by the liver

1. Effects of pretreatment with cadmium (Cd) on the uptake by the liver of subsequent Cd, copper (Cu) and zinc (Zn) were examined at two different time intervals to elucidate the biological discrimination mechanism among metals of similar chemical properties. 2. Pretreatment with 0.3 mg Cd/kg body wt 6 hr but not 24 hr before a subsequent dose of 0.8 mg metal/kg body wt enhanced the disappearance rate from plasma and accumulation rate in the liver of Cu (and Zn) but not of Cd. 3. Synthesis of metallothionein was induced with different time-courses depending on the time interval between the pretreatment and subsequent treatment, which coincided with the accumulation curves for Cu (and Zn) but not for Cd. 4. Although uptake of Cd was not enhanced by any pretreatment, metallothionein synthesis was enhanced depending on the timing of pretreatment.     

Copper metabolism in analbuminaemic rats fed a high-copper diet

Copper metabolism in male Nagase analbuminaemic (NA) rats was compared with that in male Sprague Dawley (SD) rats fed purified diets containing either 5 or 100 mg Cu/kg diet. Dietary copper loading increased hepatic and kidney copper concentrations in both strains to the same extent, but baseline values were higher in the NA rats. There was no strain difference in true and apparent copper absorption nor in faecal endogenous and urinary copper excretion. NA rats had higher levels of radioactivity in kidneys at 2 hr after intraperitoneal administration of ⁶⁴Cu. As based on the distribution of added ⁶⁴Cu, about 70% of plasma copper appeared to be in the non-protein compartment in the NA rats, whereas in SD rats, it was only about 1%. It is concluded that the NA rats are able to maintain a relatively normal metabolism of copper, even after dietary copper challenge. In the NA rats, zinc concentrations in kidneys, liver and urinary zinc excretion were elevated when compared with SD rats. The high-copper diet did not affect tissue zinc concentrations and apparent zinc absorption in both strains of rats.     

Zinc attenuates forskolin-stimulated electrolyte secretion without involvement of the enteric nervous system in small intestinal epithelium from weaned piglets

In a previous study, we found that secretagogue-stimulated electrolyte secretion was attenuated by dietary and serosal zinc in piglet small intestinal epithelium in Ussing chambers. Several studies show that the enteric nervous system (ENS) is involved in regulation of electrolyte and/or fluid transport in intestinal epithelium from many species. The aim of the present study is to examine the mechanisms behind the attenuating effect of zinc on electrolyte secretion and to study whether the ENS is involved in this effect of zinc in vitro. Twenty-four piglets (six litters of four piglets) were allocated randomly to one of two dietary treatments consisting of a basic diet supplemented with 100 mg zinc/kg (Zn(100)) or 2500 mg zinc/kg (Zn(2500)), as ZnO. All the piglets were killed at 5-6 days after weaning and in vitro experiments with small intestinal epithelium in Ussing chambers were carried out. Furthermore, zinc, copper, alkaline phosphatase (AP) and metallothionein (MT) in mucosa, liver, and plasma were measured. These measurements showed that zinc status was increased in the Zn(2500) compared to the Zn(100) fed piglets. The in vitro studies did not confirm previous findings of attenuating effects of dietary zinc and zinc in vitro on the 5-HT induced secretion. But it showed that the addition of zinc at the serosal side attenuated the forskolin (FSK) (cAMP-dependent) induced ion secretion in epithelium from piglets fed with Zn(100) diet. Blocking the ENS with lidocaine or hexamethonium apparently slightly reduced this effect of zinc in vitro, but did not remove the effect of zinc. Consequently, it is suggested that zinc attenuates the cAMP dependent ion secretion mainly due to an effect on epithelial cells rather than affecting the mucosal neuronal pathway.     

Isolation of an intestinal metallothionein induced by parenteral zinc

An intestinal zinc-binding protein, induced by parenteral zinc administration, has been isolated and characterized. Based upon its elution behavior in two chromatographic systems, Zn²⁺/protein ratio of 5.0–5.6 gram atoms/mole, a Zn²⁺/SH ratio of about 3.0, paucity of both aromatic amino acids and absorbance at 280 nm, abundance of cysteic acid residues (28–31%), and low molecular weight (6,000–7,000 daltons), the protein meets the criteria for classification as a metallothionein and is more properly named zinc-thionein. Orally administered ⁶⁵Zn was found to bind to intestinal zinc-thionein and thus this intracellular protein may function as a component of the mechanism responsible for mammalian zinc homeostasis at the level of intestinal absorption.     

Reducing effect of ingesting tannic acid on the absorption of iron, but not of zinc, copper and manganese by rats

Interest in the beneficial effects of polyphenols, including tannic acid (TA), is increasing, although, these compounds also have adverse effects; for example, on the absorption of iron (Fe), and possibly other trace minerals. We examined the effect of a graded dose of TA on the absorption of Fe and compared with that of zinc (Zn), copper (Cu) and manganese (Mn) in rats. We also investigated the effect of TA on cecal fermentation which plays a role in absorption. In Experiment 1, to set the optimum dose of Fe, male Sprague-Dawley rats (weighing 70-90 g) after acclimatization were fed with different levels of dietary Fe (5, 10, 20, 30 and 35 mg/kg). We observed that the hematocrit (Ht), serum Fe concentration and transferrin saturation (%) were each reduced in those rats fed less than 20 mg/kg Fe in a dose-dependent manner. In Experiment 2, the rats were fed with test diets containing the minimum required level of Fe, 30 mg/kg diet, with (5, 10, 15 and 20 g/kg diet) or without TA for a period of three weeks. Feeding a diet containing more than 10 g TA/kg diet, but not 5 g TA/kg diet, reduced the hemoglobin concentration (Hb), Ht and serum Fe concentration due to decreased Fe absorption. In contrast, the Zn, Cu and Mn absorption was not affected by TA feeding. It is also demonstrated that liver Fe, but not the Zn, Cu and Mn contents, were lower in the TA groups than in the TA-free control group. Feeding TA slightly decreased the pH value of the cecal contents with an increase in the major short-chain fatty acid pool. About 15% of the ingested TA were recovered in the feces of each TA-fed group. Our results demonstrate that more than 10 g TA/kg diet induced anemia by reducing the Fe absorption, although there was no effect on the absorption of other important trace minerals. Our findings suggest that the usual intake of polyphenols is relatively safe, but that a high intake by supplementation or by dietary habit of tannin affects only the Fe level.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

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.     

Copper supplementation reverses dietary iron overload-induced pathologies in mice

Dietary iron overload in rodents impairs growth and causes cardiac hypertrophy, serum and tissue copper depletion, depression of serum ceruloplasmin (Cp) activity and anemia. Notably, increasing dietary copper content to ~25-fold above requirements prevents the development of these physiological perturbations. Whether copper supplementation can reverse these high-iron-related abnormalities has, however, not been established. The current investigation was thus undertaken to test the hypothesis that supplemental copper will mitigate negative outcomes associated with dietary iron loading. Weanling mice were thus fed AIN-93G-based diets with high (>100-fold in excess) or adequate (~80 ppm) iron content. To establish the optimal experimental conditions, we first defined the time course of iron loading, and assessed the impact of supplemental copper (provided in drinking water) on the development of high-iron-related pathologies. Copper supplementation (20 mg/L) for the last 3 weeks of a 7-week high-iron feeding period reversed the anemia, normalized serum copper levels and Cp activity, and restored tissue copper concentrations. Growth rates, cardiac copper concentrations and heart size, however, were only partially normalized by copper supplementation. Furthermore, high dietary iron intake reduced intestinal ⁶⁴Cu absorption (~60%) from a transport solution provided to mice by oral, intragastric gavage. Copper supplementation of iron-loaded mice enhanced intestinal ⁶⁴Cu transport, thus allowing sufficient assimilation of dietary copper to correct many of the noted high-iron-related physiological perturbations. We therefore conclude that high- iron intake increases the requirement for dietary copper (to overcome the inhibition of intestinal copper absorption).     

Induction and characterization of metallothionein in different organs ofOstrea edulis L.

In this study, the induction of metallothionein (MT) in one species of oysterOstrea edulis exposed to copper (Cu) and zinc (Zn) was examined. The quantity of total protein (mg protein/mg dry wt) in each sample and the measurement of MT were determined by the Lowry and silver (Ag)-saturation methods. Our results show that the gonads are the organs producing the greatest quantity of MT in the controls and in the groups induced by Cu and Zn, followed by the mantle, gut, and by muscle and plasma. Competition by Zn with respect to Cu for the production of MT has been established.