Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes.

The process of hepatobiliary copper (Cu) secretion is still poorly understood: Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting P-type ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 microM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated Km of 8.6 microM and a Vmax of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT.

A plasma membrane NADH oxidase is involved in copper uptake by plasma membrane vesicles isolated from rat liver.

The accumulation of copper (Cu) by hepatocytes is initiated by the binding of Cu in either a CuHis2 complex or as a CuHisAlb ternary complex, followed by transfer of the metal alone across the cell membrane. In this paper, we provide evidence that the transfer involves reduction of cupric (Cu(II)) copper to cuprous (Cu(I)) copper and further we show that membrane-bound NADH oxidase can provide the electron required for the reduction. 64Cu uptake by rat liver plasma membrane vesicles is stimulated by the addition of NADH, but not NAD+. The stimulation increases the Vmax from 4.75 +/- 0.02 to 8.38 +/- 0.40 nmol Cu/mg protein per min (P < 0.05, mean +/- S.E., n = 3) without significantly altering the K0.5 (1.52 +/- 0.17 and 2.10 +/- 0.22 mumol/l; with n values of 1.30 +/- 0.01 and 1.43 +/- 0.10, respectively; analysing by the Hill equation). Correspondingly, addition of CuHis2 stimulated NADH-oxidase activity by a maximum of 7.4 +/- 2.1 nmol/mg protein per min (P < 0.01, mean +/- S.E., n = 5) at 5 mumol/l and a NADH concentration of 150 mumol/l. Ascorbic acid also stimulated copper uptake, and points to a reductive dissociation of copper prior to its movement into the cell. Our data indicate that membrane bound enzymes can provide an electron for the reduction of copper prior to uptake and suggest a physiological role for the plasma membrane NADH oxidase.

Metallothionein in Menkes' disease: induction in cultured muscle cells.

Menkes' disease is an inherited disturbance of copper metabolism. Addition of copper to the medium of cultured fibroblasts and lymphoblasts from patients with Menkes' disease results in an increased induction of metallothionein. We investigated the metallothionein induction in response to copper and zinc in muscle cells (myoblasts and myotubes). Metallothionein synthesis was analyzed by gel electrophoresis of labeled proteins and metallothionein synthesis in muscle cells was compared with the synthesis in fibroblasts. The induction by copper was higher both in muscle cells and in fibroblasts from the Menkes' patient compared to the control cells. Hybrid myotubes obtained by fusion of control myoblasts and Menkes' myoblasts render a system in which complementation can be studied. Metallothionein synthesis in hybrid myotubes occurred at a level intermediate between the synthesis in Menkes' and control myotubes. The abnormal accumulation of copper-induced metallothionein was only partially corrected by fusion with normal cells. Metallothionein induction by zinc was similar in Menkes' and control fibroblasts. Combination of copper and zinc yielded no differences in additional metallothionein synthesis for Menkes' cells and control fibroblasts. Therefore, metallothionein induction in Menkes' disease can primarily be accounted for by copper rather than by zinc.

Trace metal uptake in liver cells. 1. Influence of albumin in the medium on the uptake of copper by hepatoma cells.

Cultured rat hepatoma cells (HTC-cells) were used to study the transfer of copper from a well-defined medium to and across the cell membrane and particularly the role of albumin in this process. HTC-cells, maintained in a minimal salt-glucose medium, accumulated far more copper than when maintained in the same medium, but supplemented with albumin. In the latter case, the Cu uptake strongly depended on the molar Cu/albumin ratio. The results suggest a role of albumin in the uptake of trace metals. The results indicate the presence of two types of binding sites for copper on the cell membrane. The sites of the first type bind copper very strongly and are probably responsible for the uptake of copper under physiological conditions. Their number was estimated to be about 10(6) per cell. Those of the second type only bind copper when the molar Cu/albumin ratio exceeds a value of about 1, i.e., under extreme, unphysiological conditions. Furthermore, the results suggest a direct interaction of the Cu-albumin complex with these strong binding sites as a first step in Cu uptake processes.

Trace element uptake in liver cells. 2. Effect of different proteins in the medium on the uptake of copper and zinc by hepatoma cells.

Cultured rat hepatoma cells (HTC-cells) were used to study the uptake of copper and zinc from a minimal salt-glucose medium, supplemented with albumin from different species or with ovalbumin. Competitive equilibrium dialysis showed that at low molar ratios of metal/protein (less than 1) the affinity for copper of human and bovine albumin was about equal, but that of dog albumin or ovalbumin was much lower. Only a small difference in affinity for zinc could be detected between human albumin and ovalbumin. Supplementing the medium with the different proteins the rate of copper uptake in the cell at a given molar Cu/protein ratio increased as follows: human albumin congruent to bovine albumin less than dog albumin less than ovalbumin. When the molar Cu/protein ratio was increased, a discontinuity was seen with all three albumin species at a ratio of about 1. In contrast, the zinc uptake mimics that of Cu/ovalbumin, and no discontinuity was observed using different molar Zn/protein ratios. These results indicate that the rate of copper and zinc uptake depends strongly on its affinity for the protein: a low affinity leads to a high uptake. The results suggest further that at physiologic concentrations zinc is taken up by a mechanism different from that for copper.

Copper status in rats fed diets supplemented with either vitamin E, vitamin A, or beta-carotene.

Copper status was measured in rats fed copper-adequate, purified diets supplemented with either vitamin E (250 IU/kg), vitamin A (40,000 IU/kg), or beta-carotene (2 g/kg). It was hypothesized that the extra intake of the antioxidants would spare vitamin C resulting in a decreased copper status as shown previously after supplementation with vitamin C. A significant increase in plasma ascorbate concentration was observed after beta-carotene supplementation, but not after supplemental vitamin E or vitamin A. Extra intake of either beta-carotene or vitamin A slightly, but significantly, raised plasma copper concentrations. Beta-carotene also slightly raised liver copper concentration. Supplemental vitamin E had no effect on plasma and liver copper concentrations. It is concluded that the observed relatively small effects of supplemental vitamin A and beta-carotene on copper status in rats are not mediated by changes in plasma vitamin C concentration.

Ascorbic acid supplementation and copper status in rats.

The effect of a high concentration (1%, w/w) of ascorbic acid in a Cu-adequate (150 mumol/kg) purified diet was studied in rats. After 6 wk, ascorbic acid had significantly reduced Cu concentrations in muscle and bone. The estimated whole body content of Cu in rats fed ascorbic acid was reduced by 20%. Within 1 d after oral administration of 64Cu, the recovery of the dose in feces was increased in rats fed ascorbic acid, suggesting that the vitamin depresses intestinal absorption of Cu. After intraperitoneal (ip) administration of 64Cu, the rate of loss of the dose from the body was decreased in rats fed ascorbic acid. This study suggests that the ascorbic acid induces a decreased efficiency of intestinal Cu absorption, which in turn triggers mechanisms to preserve Cu in the body stores. This is supported by the observation that the feeding of a Cu-deficient diet (5 mumol/kg) had similar effects, although more pronounced.

Iron, copper, and zinc status in rats fed supplemental nickel.

Literature data concerning the effect of increasing dietary Ni concentrations on Fe, Cu, and Zn status in rats are sparse and, in part, controversial. Therefore, the effects of the addition of either 0, 3, 50, or 100 mg Ni/kg diet on Fe, Cu, and Zn status of rats were investigated in two separate experiments. Purified diets were used that were composed according to the established nutrient requirements of rats. Ni in kidney was increased with increasing Ni intakes. Dietary Ni did not significantly influence Fe concentrations in plasma, liver, kidney, femur, and spleen. Likewise, the addition of Ni to the diet did not alter Cu status. Zn concentrations in femur were significantly decreased after feeding the diets with 100 mg Ni/kg. However, Zn in plasma, liver, kidney, and spleen was not affected. It is concluded that variations in dietary Ni concentrations have no major impact on Fe, Cu, and Zn status in rats.

Impaired iron status in rats as induced by copper deficiency.

The hypothesis was tested that marginal copper deficiency affects iron status. Copper restriction (1 vs 5 mg Cu/kg diet) significantly lowered iron concentrations and transferrin saturation in plasma and reduced blood hemoglobin, hematocrit, and iron concentrations in tibia and femur, but raised iron concentrations in liver. Marginal copper deficiency did not affect feed intake and bodyweight gain.

Ascorbic acid feeding of rats reduces copper absorption, causing impaired copper status and depressed biliary copper excretion.

The feeding of diets enriched with ascorbic acid (10 g/kg) to rats has previously been shown to lower plasma and liver copper concentrations. The present studies corroborate this. We hypothesized that ascorbic acid initially reduces copper absorption, this effect being masked later by the stimulatory effect on copper absorption of the impaired copper status. We also hypothesized that the impaired copper status as induced by ascorbic acid feeding is followed by a diminished biliary excretion of copper in an attempt to preserve copper homeostasis. Our hypotheses are supported by the present studies. Ascorbic acid feeding initially reduced apparent copper absorption, and in the course of the experiment this effect tended to turn over into a stimulatory effect. Copper deficiency, as induced by feeding a diet containing 1 mg Cu/kg instead of 5 mg Cu/kg, systematically increased copper absorption. Biliary excretion of copper in rats given ascorbic acid was unaffected initially but became depressed after prolonged ascorbic acid feeding. A similar time course was seen for fecal endogenous copper excretion that was calculated as the difference between true and apparent copper absorption. Copper deficiency systematically reduced biliary copper excretion and fecal endogenous copper loss.

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 with 64Cu, rats fed fructose also had significantly lower soluble 64Cu 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.

Iron and zinc status in rats with diet-induced marginal deficiency of vitamin A and/or copper.

The hypothesis was tested that there are interactions of marginal copper and vitamin A deficiency regarding iron and zinc status. Copper restriction (1 vs 5 mg Cu/kg diet) significantly lowered copper concentrations in plasma and tissues of rats and reduced blood hemoglobin, hematocrit, and iron concentrations in tibia and femur, but raised iron concentrations in liver. Vitamin A restriction (0 vs 4000 IU vitamin A/kg diet) reduced plasma retinol concentrations and induced a fall of blood hemoglobin and hematocrit. Neither copper nor vitamin A restriction for up to 42 d affected feed intake and body wt gain. There were no interrelated effects of vitamin A and copper deficiency on iron status. Copper deficiency slightly depressed liver, spleen, and kidney zinc concentrations. Vitamin A deficiency lowered zinc concentrations in heart, but only when the diets were deficient in copper.

Iron status in rats fed a purified diet without vitamin A.

The effect of vitamin A deficiency on iron status was investigated in rats. After 28 d of feeding either low or high vitamin A diets (0 vs 4000 IU of vitamin A per kg feed), the final body weight was slightly but significantly lowered by the low vitamin A diet. Plasma retinol concentrations were decreased in rats fed diets low in vitamin A. Marginal vitamin A deficiency produced slightly, but significantly lower blood hemoglobin concentrations; it did not clearly affect hematocrit. The concentration of iron in liver was significantly higher when diets low in vitamin A were fed while significantly lower levels were observed in femur.

Absorption and retention studies of trace elements and minerals in rats using radiotracers and whole-body counting.

A description is given of a whole-body counting technique using radiotracers, permitting the determination of true absorption and endogenous excretion of trace elements and minerals in the rat in vivo. This non-invasive counting method involves oral and intraperitoneal administration of tracer doses of a radioisotope in a cross-over fashion and subsequent measurement of the whole-body retention in a whole-body counter. Thus, true absorption can be determined in one animal which contributes to the reduction of animal use. To study the variations in counting response due to radioisotope distribution, to size or shape of the animal body, the influence of the position of a point source and distribution over different phantoms to simulate various body sizes are experimentally evaluated for 64Cu, 65Zn, 59Fe and 28Mg. Results from 2 studies, with 64Cu and 28Mg, as an example for a trace element and a mineral respectively, are presented and illustrate that absorption as measured by apparent absorption does not necessarily reflect true absorption. True absorption as determined by the whole-body retention method using radioisotopes corrects for faecal losses of endogenous origin.

129I and 36Cl concentrations in lichens collected in 1990 from three regions around Chernobyl.

129I and 36Cl were measured by accelerator mass spectrometry in 11 lichen samples (Parmelia sulcata) collected in 1990 from three regions (Novozybkov, Bragin and Ovruc) near Chernobyl. Previously measured activities of 137Cs were highest in the samples from the Novozybkov region while the measured activities of 36Cl and 129I in this study were highest in the samples from the Bragin region. The regional distribution patterns of the 36Cl and 129I show a positive correlation suggesting that these volatile radionuclides were deposited in the same manner.

Influence of ascorbic acid supplementation on copper metabolism in rats.

An attempt was made to unravel further the mechanism by which high dietary concentrations of ascorbic acid influence copper metabolism. The addition of ascorbic acid to the diet of rats caused about a twofold increase in plasma ascorbate concentrations and reduced group mean plasma and tissue concentrations of Cu. The effect of 10 g ascorbic acid/kg diet was greater than that of 1 g/kg. Ascorbic acid feeding reduced blood haemoglobin concentrations and packed cell volume values. Dietary ascorbic acid caused a significant decrease in apparent Cu absorption from the intestine. Ascorbate, intravenously administered together with 64Cu, caused an increase in 64Cu in the liver. Ascorbate, at concentrations occurring in plasma after ascorbic acid feeding, promoted the uptake of 64Cu by isolated hepatocytes. Thus, ascorbate stimulated the efficiency of hepatic uptake of Cu. Ascorbate, intravenously administered together with 64Cu, stimulated accumulation of 64Cu in bile of rats with a bile duct cannula. In rats fed on ascorbic acid, intravenously administered 64Cu was recovered in bile at increased rates. Dietary ascorbic acid enhanced the recovery of intraperitoneally administered 64Cu in faeces. The ascorbate-induced stimulation of biliary 64Cu excretion may reflect an increased hepatic uptake of 64Cu and be caused by an increased specific activity of Cu in liver pools. It is suggested that dietary ascorbic acid reduces tissue Cu concentrations primarily by interfering with intestinal Cu absorption. Ascorbate increases the efficiency of hepatic uptake of Cu, but this effect may not be causatively related with the reduced tissue Cu concentrations after ascorbic acid feeding.

The accumulation of copper by microvillar vesicles isolated from human placenta.

Copper uptake from copper-dihistidine complexes by microvillar vesicles from human placenta was studied. Uptake occurred in two phases: a rapid initial binding followed by approximately linear uptake to equilibrium at approximately 5 min. The uptake showed temperature dependence, was saturable (apparent Vmax 10.5 +/- 1.6 nmol/(mg protein.4 min), apparent Km of 0.6 +/- 0.12 mumol/L) and decreased with increasing osmotic pressure, showing that the Cu uptake arose from accumulation within the vesicles and not from extravesicular binding or isotope exchange. Ceruloplasmin blocked uptake of 64Cu from 64Cu-dihistidine by the vesicles, with 50% inhibition achieved at a protein concentration of 5-10 mumol/L and a 64Cu-dihistidine concentration of 1.5 mumol/L. The effect was specific, because glucose oxidase, a noncopper protein, increased apparent uptake by binding copper and in turn being bound to the nitrocellulose membranes used to separate vesicles from incubation medium. Adding increasing concentrations of histidine also decreased uptake. The data presented indicate that the placenta can accumulate copper from copper-dihistidine, that ceruloplasmin can interfere with uptake and that this system will be very valuable in elucidating the first stage in transfer of copper across the placenta.

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.

High protein intake raises apparent but not true magnesium absorption in rats.

Earlier studies with rats have shown that greater protein intake raises apparent magnesium absorption (ingested magnesium minus fecal magnesium). We addressed the question of whether high protein intake affects true magnesium absorption. Rats were fed either a normal (175 g casein/kg) or a high (525 g casein/kg) protein diet. Extra protein was added at the expense of the glucose component of the diet; the diets were balanced for magnesium, calcium and phosphorus. The high protein diet enhanced apparent magnesium absorption. True magnesium absorption, as measured with the use of oral and intraperitoneal administration of tracer doses of 28Mg, was found to be unaffected by high protein intake. Endogenous magnesium excretion in feces was significantly depressed in rats fed the high protein diet, which was associated with elevated urinary magnesium excretion. High protein intake reduced the amount of magnesium in whole ileal digesta but raised the amount and concentration of magnesium in the liquid phase. We conclude that improvement of apparent magnesium absorption in rats fed high protein diets is due to depressed fecal excretion of endogenous magnesium.

Differences in hepatic processing of dietary and intravenously administered copper in rats.

The biliary pathway represents the major excretory route for copper (Cu). It has been suggested that glutathione (GSH) plays a role in this process. However, biliary secretion of endogenous Cu is unaffected in canalicular multispecific organic anion transporter (cmoat)/multi-drug resistance protein (mrp2)-deficient GY/TR- rats, which is a mutant rat strain expressing defective canalicular adenosine triphosphate (ATP)-dependent GSH-conjugate transport and which is unable to secrete GSH into bile. Secretion of Cu after iv Cu load is markedly impaired in GY/TR- rats when compared with normal Wistar (NW) rats. Administration, iv, of 65, 325, or 2300 nmol/100 g body wt CuSO4 dose-dependently increased Cu secretion in normal Wistar (NW) rats. Secretion rates in GY/TR rats were much lower and plateaued with higher loads at a level of about 35 nmol/h/100 g body wt. Clearance of an intravenous (iv) bolus of 64Cu (250 nmol/100 g body wt) was faster in GY/TR- rats than in controls, but secretion of 64Cu into bile was clearly reduced in the mutants. Specific activity of biliary Cu was similar in both groups. To investigate the removal of excess dietary Cu via bile, GY/TR and NW rats received water supplemented with Cu (CuSO4 8 mmol/L) for up to 12 weeks (Cu-fed) or tap water (controls). Cu feeding resulted in an increase of biliary Cu secretion from approximately 6 to approximately 30 nmol/h/100 g body wt within two weeks, both in NW and GY/TR- rats; Cu secretion also did not further increase during the course of the experiment. Hepatic Cu content was similar in NW and GY/TR- rats and progressively increased during Cu feeding. Our data indicate that biliary secretion of diet-derived Cu proceeds exclusively via a saturable Cu transporting system, which is distinct from cmoat/mrp2 and which is independent of biliary GSH. This transport may be mediated by the recently identified Cu-ATPase. In contrast, excess hepatic Cu after iv Cu load depends on cmoat/mrp2 activity for rapid removal. It is concluded that iv administered and dietary (endogenous) Cu is, in part, processed differently by rat liver, which might be related to differences in Cu redox state.