Inhibition of intestinal copper absorption by divalent cations and low-molecular-weight ligands in the rat

l-histidine (His) has been shown to enhance the inhibitory effect of zinc on intestinal copper absorption. This study was aimed at examining whether this effect of His was also extended to the interactions of other divalent cations: ferrous iron, tin, and cobalt, using an in vivo perfusion system in rats. Copper absorption and intestinal content of this element significantly decreased in the presence of 2 mM His and ferrous iron. Iron accumulation was greater when His was present than when omitted. A fivefold excess of tin inhibited copper absorption only when His was present. Citrate, at the same concentration as His, had no effect on copper absorption, but hepatic copper levels were increased, as compared to the absence of either His or citrate. Addition of 0.5 or 1.0 mM cobaltous salt plus His resulted in a sharp decrease in copper intestinal absorption, with an increase in intestinal tissue retention. These results confirm earlier findings with zinc and His, and suggest that a general phenomenon, either accelerating the removal of copper from the intestinal lumen or increasing, the retention of this element by the intestinal tissue, is a common feature of the interaction between cations of similar electronic configuration to copper and a high-affinity ligand, such as His.     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

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.     

Zinc uptake by isolated rat enterocytes: effect of low molecular weight ligands

The luminal phase of zinc intestinal absorption has not been well characterized. This study was intended to elucidate the possible role of low molecular weight (LMW) ligands in zinc intestinal transport in an isolated rat enterocyte system. Under these in vitro conditions, zinc uptake by the isolated enterocytes was rapid, leveling off within 1 min. Kinetic analysis revealed that both a mediated and diffusion component were involved in zinc uptake in the absence of LMW ligands by the cells. For the mediated component of zinc transport, the Kt and Vmax were 64.1 microM and 13.9 nmol/20 sec/mg protein, respectively. Zinc uptake was not affected by the addition of metabolic inhibitors. In the presence of histidine or cysteine (2:1 ligand:zinc molar ratio), zinc uptake was greatly reduced and occurred solely via mediated transport. Zinc uptake was also significantly decreased upon the addition of EDTA to the assay media. Other amino acids tested had no effect on zinc uptake by the cells. Albumin markedly reduced zinc uptake by the cells. Histidine and other potential LMW ligands were unable to facilitate albumin-inhibited zinc uptake. The results of this study suggest that the intestinal absorption of zinc may not be effected in the form of chelates with LMW ligands. Amino acids such as histidine and cysteine significantly reduce the uptake of the metal by isolated rat enterocytes, making questionable their putative role as necessary vehicles in the luminal phase of zinc absorption.     

Mechanism of copper transport from plasma to hepatocytes

The effects of plasma components on the kinetics of copper transport by rat hepatocytes were examined in an attempt to determine how copper is mobilized from plasma for uptake by the liver. Specific protein-facilitated transport was indicated by saturation kinetics, competition by related substrates, and similar kinetic parameters for uptake and efflux. For copper uptake, Km = 11 +/- 0.6 microM and Vmax = 2.7 +/- 0.6 nmol Cu/(min X mg protein). Zinc is a competitive inhibitor of copper uptake, and copper competes for zinc uptake. Copper efflux from preloaded cells is biphasic. The kinetic parameters for the initial rapid phase are similar to the parameters for uptake. Copper transport by hepatocytes is strictly passive. A variety of metabolic inhibitors have no effect on uptake and initial rates are solely dependent on extracellular-intracellular concentration gradients. Albumin markedly inhibits copper uptake by a substrate removal mechanism, and histidine facilitates albumin-inhibited copper uptake. The active species that delivers copper to hepatocytes under conditions of excess albumin and excess histidine is the His2Cu complex. Experiments with [3H]His2 64Cu showed that the transported species is free ionic copper. The kinetic parameters of copper transport by hepatocytes isolated from the brindled mouse model of Menkes' disease are normal. However, these cells show a decreased capacity to accumulate copper on prolonged incubation. An intracellular metabolic defect seems to be involved.     

The effect of various dietary zinc concentrations on the biological interactions of zinc,copper, and iron in rats

Three groups (14 rats each) were fed one of the following diets for 8 wks: a control purified basal diet containing 12 ppm zinc, 5 ppm copper, and 35 ppm iron; the basal diet with less than 2 ppm zinc; or the basal diet supplemented with 1000 ppm zinc. Rats fed the zinc-deficient diet had decreased weight gain, moderate polydipsia, and intermittent mild diarrhea. The zinc-supplemented rats had a cyclical pattern of food intake and weight loss from weeks 5 to 8. Tissue concentrations suggest that zinc and copper were not mutually antagonistic with chronic dietary imbalances. If tissue element concentrations reflected intestinal uptake, then competition and/or inhibition of intestinal uptake occurred between zinc and iron. The fluctuations in tissue element concentrations that occurred with increased duration of the study were at variance with previous studies of shorter time periods. The dietary proportions of zinc, copper, and iron appear to influence zinc, copper, and iron metabolism at the intestinal and cellular transport levels over a given period of time.     

Copper accumulation in the soluble and particulate fractions of renal cortex in the streptozotocin-diabetic rat

The accumulation and subcellular distribution of copper in the kidney of streptozotocin-diabetic rats were investigated. Male Sprague-Dawley rats received streptozotocin (50 mg/kg body wt on two consecutive days) intraperitoneally and were fed either commercial or purified diet. The concentrations of copper, zinc, iron, and manganese present in intact kidney, renal cortex, and renal medulla were compared at various times. Chow-fed diabetic rats had a renal copper concentration 2.6 times greater than age-matched controls after 2 weeks. The concentration of zinc was only 30% higher in diabetic kidney than in control tissue, whereas the iron and manganese concentrations were similar for both groups. The additional complement of renal copper was localized entirely in the cortex and was significantly reduced by oral treatment with penicillamine, a copper chelating agent. When diabetic rats were fed purified diet (15-20 ppm Cu), the quantity of copper accumulated in the renal cortex increased from 2.3 to 8.7-fold higher than in control tissue from 1 to 4 weeks, respectively, after injection with streptozotocin. Copper levels in. both the soluble and particulate (165, 000g pellet) fractions of diabetic renal cortex were similarly increased at each time. Gel filtration Chromatographic analysis of the cytosol showed that all of the copper accumulated in the soluble fraction was associated with metallothionein. The distribution of excess copper in the particulate fraction was determined by differential centrifugation. The additional quantity of metal was localized in the crude nuclear fraction of renal cortex in the diabetic rat. Further analysis revealed that the lysosomal fraction from 3-weeek diabetic rats had a copper level 16-fold higher than in the controls. The possibility that accumulation of excessive levels of copper in the streptozotocin-diabetic kidney may contribute to the development of diabetic nephropathy is discussed.     

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).     

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ～90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.     

Brain tissue accumulates 67copper by two ligand-dependent saturable processes. A high affinity, low capacity and a low affinity, high capacity process

We characterized the mechanism of copper accumulation by the brain, using rat hypothalamic tissue slices incubated with 67Cu as a model system. Two ligand-dependent saturable processes were discerned: a high affinity, low capacity process and a low affinity, high capacity process. Vo versus [S] for the high affinity process was a hyperbolic function having an apparent Km and Vmax of 6 microM copper and 23 pmol/min/mg protein, respectively. Vo versus [S] for the low affinity process was a sigmoidal function having an "apparent Km" (So5) and maximal velocity at saturating [S] of 40 microM copper and 425 pmol/min/mg protein, respectively. The two processes were similar in that each exhibited: (a) a requirement for complexing of copper for optimal 67Cu accumulation; (b) a broad ligand specificity with respect to amino acids (histidine, cysteine, threonine, glycine) and peptides (Gly-His-Lys, glutathione) and ineffectiveness of albumin in serving as a facilitatory ligand; (c) a requirement for thermic but not metabolic energy. In spite of these similarities, a 50- or 1000-fold molar excess of ligand (histidine) inhibited 67Cu accumulation by the low affinity process by 60 and 85%, respectively, whereas excess histidine facilitated 67Cu accumulation by the high affinity process by 1.6-4-fold. These results are consistent with 1) a carrier-mediated facilitated diffusion, analogous to that of neutral amino acids, as a means of transporting complexed copper into brain tissue, and 2) the existence of two distinct carrier sites interacting in a positive cooperative manner: a high and a low affinity site.     

Inhibition of zinc absorption by iron depends on their ratio

Previous studies upon zinc-iron interactions gave conflicting results that could come from differences in protocol design or in trace element status of subjects. The present work assessed the influence of zinc : iron ratio and iron deficiency upon zinc absorption. The digestive absorption of zinc sulphate (100 mol Zn/l) in presence of iron gluconate was studied in perfused jejunal loops (n = 6/group) of normal rats (range 0–1000 mol Fe/l) and iron deficient rats (200–750 mol Fe/l). In normal rats no significant iron inhibition on zinc absorption occurred at Fe:Zn ratio below 2:1. At higher ratios zinc uptake and net absorption decreased significantly (p<0.05). Between 2:1 and 5:1 a dose dependent inhibition of zinc absorption occurred and reached a plateau beyond this ratio. In iron deficient animals no changes in zinc uptake, mucosal retention and absorption compared to normal animals occurred at ratio 2:1. At higher ratios differences were observed at every zinc absorption step except for mucosal retention at 7.5:1 ratio.

Iron-zinc interactions depend on their ratio and on previous trace elements status of subjects. Due to the wide and unknown variations that were likely to occur between the subjects of previous human and experimental studies, these results could explain some of the discrepancies between their results.     

Effect of zinc on L-threonine transport across the jejunum of rabbit

Zinc is an essential trace element for life. Many metalloenzymes involved in the metabolism of carbohydrates, lipids, protein, and nucleic acids require zinc for their functions. The aim of this study was to characterize how zinc acts on the intestinal amino acid absorption in rabbit. Results obtained show that zinc inhibits both L-threonine accumulation in the jejunum tissue, and mucosal-to-serosal transepithelial flux of this amino acid in a dose-dependent way. The inhibition does not increase by a 10-min previous intestinal exposure of the mucosa to the heavy metal, and is not reversed by washing the intestinal tissue with saline solution or 10mM EDTA, but is appreciably reversed with 10mM dithioerythritol. Zinc seems not to modify amino acid diffusion across the intestinal epithelium. The inhibition of intestinal amino acid transport by zinc seems to be of a competitive type, and appears to be a result of impairment of the active transport that is altered by its binding to proteins (prevailing to thiol groups) of the brush-border membrane of enterocytes.     

Kinetics and mechanism of tolerance induction on acclimation ofVillorita cyprinoides (Hanley) to copper and zinc

The typical euryhaline clamVillorita cyprinoides (Hanley) was acclimated to copper and zinc at salinity 13 × 10⁻³ and < 1 × 10⁻³ (fresh water). Acclimation enhanced the lethal tolerance, as denoted by dose-survival curves, which was more pronounced after zinc acclimation. In fresh water copper acclimation sensitized the organisms. The copper accumulation trend was significantly changed consequent to metal acclimation, especially after zinc acclimation, indicating some tissue metal regulatory effect. Acclimation to copper equiped the organism to survive for longer periods with increased body burden of copper, while zinc acclimation supressed the uptake of the more toxic ion copper. The earlier report of increased uptake of zinc by this organism during combined exposure with copper is corelated in the present context. The role of metallothionein like protein in providing protection against metal toxicity, the environmental implication of acclimation phenomena are indicated     

Histidine Absorption across Apical Surfaces of Freshwater Rainbow Trout Intestine: Mechanistic Characterization and the Influence of Copper

The essential amino acid histidine performs critical roles in health and disease. These functions are generally attributed to the amino acid itself, but could also be mediated by a positive effect on trace element bioavailability. Mechanistic information regarding the absorption of histidine across the gastrointestinal tract is essential for understanding the interplay between amino acid and mineral nutrients and the implications of these interactions for nutrition and toxicology. Using intestinal brush-border membrane vesicles obtained from freshwater rainbow trout, absorption of histidine over the range 0.78–780 μm was found to be saturable, with a maximal transport rate (J _max) of 9.1 ± 0.8 nmol mg protein⁻¹ min⁻¹ and a K _m (histidine concentration required to reach 50% of this level) of 339 ± 68 μm. Histidine uptake was highly specific as 10-fold elevated levels of a variety of amino acids with putative shared transporters failed to significantly inhibit uptake. Elevated levels of d-histidine, however, impaired uptake of the natural l-isomer. The presence of “luminal” copper (8.3 μm) significantly increased both the J _max and K _m of histidine transport. This suggests that chelated copper–histidine species cross the brush-border epithelium through transport pathways distinct from those used by histidine alone.     

Tissue-and metal-specific effects of thiolacrylic acids in rats

Kidney copper increased 12- to 18-fold above the normal level in rats administered alpha-mercapto-beta-(2-furyl)acrylic acid (MFA). Kidney zinc increased twofold; plasma zinc increased more than 10-fold and liver zinc increased 30–50%. No other changes in copper, iron, and zinc concentrations were found in these tissues or in bone, brain, heart, lung, skeletal muscle, spleen, or testis. Related compounds produced similar effects, although MFA and its disulfide were the most potent of the compounds tested. These increases in tissue copper and zinc were largely complete after 2–5 d of daily administration of compound. Increased plasma zinc returned toward normal with a half-life of 1.0 d for the process, after dosing was ended; albumin was identified as the species binding the excess zinc in plasma. Kidney copper and zinc, which had increased in the ratio of 3 Cu/Zn, returned to normal levels after dosing was stopped with half-lives of 2.1–2.5 d. Consistent with the observations of highly tissuespecific effects of MFA, copper and zinc balances over 8 weeks of trials were found to be not greatly affected by administration of the compound. Thus, it was not established whether excess metal in affected organs derived from enhanced retention of dietary metal or redistribution from other tissues. Kidney copper and zinc and serum zinc increased even in zinc-deficient rats administered MFA.     

Distinct mechanisms of zinc uptake at the apical and basolateral membranes of caco-2 cells.

Zinc uptake mechanisms at the apical and basolateral membrane borders of caco-2 cells were examined. This human-derived cell line possesses many morphological and functional characteristics of absorptive small intestinal cells. By day 14, confluent and well-differentiated monolayers were formed when the cells were grown on porous polycarbonate filters. Labelled zinc was placed on the apical or basal side of the monolayer and its uptake by the cells, as well as its transport across the monolayer, were measured. Zinc uptake by the cells from the apical side was found to be a saturable process (Kt = 41 microM; Vmax = 0.3 nmols/cm2/10 min) with a diffusional term at higher concentrations (1.0 sec/cm). Apical uptake was not affected by metabolic inhibitors or potential zinc ligands. Zinc uptake from the basolateral side was concentration dependent (Kd = 1.3 sec/cm) and was partially inhibited (30%) by ouabain and vanadate, suggesting that the (Na-K)-ATPase on the basolateral membrane is involved in the serosal uptake of zinc by the cell. Transport of zinc across the monolayers from the apical or basolateral compartment was concentration dependent and was not affected by metabolic inhibitors. Zinc transport from the basolateral side was greater than 2-fold greater than apical transport. Hence, separate mechanisms can be distinguished with respect to zinc uptake at the apical and basolateral membranes of caco-2 cells.     

Regulation of copper absorption by copper availability in the Caco-2 cell intestinal model

Relatively little is known about the individual steps in intestinal copper absorption and whether or how they may be regulated. Polarized Caco-2 cell monolayers with tight junctions offer an already tested model in which to study intestinal metal transport. This model was used to examine potential effects of cellular copper availability on copper absorption. Uptake and transport were determined on application of (64)Cu(II) to the brush border. In the range of 0.2-2 micro M, uptake was dose dependent and was approximately 20% of dose/90 min. Overall transport of (64)Cu across the basolateral surface was approximately 0.3%. When cellular copper levels were depleted 40% by 18-h pretreatment with the specific copper chelator triethylenetetraamine, uptake and overall transport were markedly increased, going to 80 and 65% of dose, respectively. Cellular retention of (64)Cu fell fourfold, from 6 to 1.5%. Depletion of copper with the chelator was rapid and preceded initial changes in uptake and overall transport by 4 h. A lesser depletion of cellular copper (13%) failed to enhance copper uptake but doubled the rate of overall transport, as measured with (64)Cu and by atomic absorption. As previously reported, preexposure of the cells to excess copper (10 micro M, 18 h) also enhanced copper uptake ( approximately 3-fold). In contrast, ascorbate (10-1,000 micro M) failed to significantly alter uptake and transport of 1 micro M (64)Cu. Our findings are consistent with the concepts that, in the low physiological range, copper availability alters the absorption capacity of the intestine to support whole body homeostasis and that basolateral transport is more sensitively regulated than uptake.     

Copper-sodium linkage during intestinal absorption: inhibition by amiloride.

The possible association between copper and sodium small intestinal absorption in the rat was investigated in the presence or absence of the electrolyte transport inhibitors amiloride, acetazolamide, and furosemide, at pharmacologic concentrations, using an in situ perfusion procedure. Amiloride (1 mM) produced a significant decrease in copper, net water, and sodium absorption, in solutions with sodium. Copper tissue retention was not altered, but was much higher in the absence of sodium. Acetazolamide and furosemide (1 mM), in separate experiments, had no effect on copper removal from the lumen, but generally reduced sodium and water transport. The presence or absence of sodium in the perfusate influenced rates of copper uptake. These data are compatible with a more effective passage of copper across the enterocyte basolateral membrane in the presence of sodium than in its absence.     

Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity,low affinity,and histidine-facilitated pathways

Zinc is both a vital nutrient and an important toxicant to aquatic biota. In order to understand the interplay between nutrition and toxicity, it will be important to determine the mechanisms and the factors that regulate zinc uptake. The mechanism of apical intestinal Zn(II) uptake in freshwater rainbow trout and its potential modification by the complexing amino acid histidine was investigated using brush-border membrane vesicles (BBMVs). Following characterisation of the BBMV preparation, zinc uptake in the absence of histidine was both time- and concentration-dependent and consisted of two components. A saturable phase of uptake was described by an affinity constant of 57+/-17 microM and a transport capacity of 1867+/-296 nmol mg membrane protein(-1) min(-1). At higher zinc levels (>500 microM) a linear, diffusive component of uptake was evident. Zinc transport was also temperature-dependent, with Q10 values suggesting zinc uptake was a carrier-mediated process. Zinc uptake by vesicles in the presence of histidine was correlated to a mono-histidine species (Zn(His)+) at all Zn(II) concentrations examined.