59Fe is retained from an elemental 59Fe powder supplement without effects on 65Zinc, 47Calcium and 67Copper in young pigs.

In vivo counting with the use of a germanium detector evaluated the retention of an elemental 59Fe powder supplement while measuring potential interactions with zinc, calcium and copper. Effects of dietary iron and zinc on in vivo retentions of 59Fe, 65Zn, 67Cu and 47Ca were studied in young pigs. In Experiment 1, 4-d-old piglets fed a cereal-based diet were randomly assigned to one of four treatment groups (2 x 2 factorial arrangement, n = 5 per group). Variables were dietary iron source (either elemental iron or FeSO4, each at 100 mg iron/kg diet) and the dosage form of radioactive iron (either elemental 59Fe powder or 59FeSO4). Experiment 2 (2 x 3 factorial arrangement) was performed using two levels of iron (100 and 200 mg/kg, as elemental iron) and three levels of zinc (25, 50 and 100 mg/kg). Piglets were also dosed with 47Ca, 65Zn and 67Cu; all radioisotopes were measured for 8 d. Apparent absorption of elemental 59Fe powder was 13 +/- 1%, whereas 59Fe sulfate was significantly (P < 0.05) higher at 26 +/- 1%. The FeSO4 diet decreased 65Zn retention in Experiment 1, in contrast to the elemental iron diet, which did not have this effect in either experiment. Apparent 65Zn absorption averaged 44 +/- 2, 35 +/- 1 and 27 +/- 2% for the three levels of zinc (25, 50 and 100 mg/kg), respectively. Retention of 47Ca was not affected by dietary iron or zinc; retention of 67Cu was not affected by dietary iron. The data demonstrate good bioavailability of elemental iron without effects on zinc, copper and calcium.

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.

Dietary raw versus retrograded resistant starch enhances apparent but not true magnesium absorption in rats.

Dietary raw (RS2) vs. retrograded resistant starch (RS3) raises apparent magnesium absorption in rats. The mechanism proposed is that RS2 enhances magnesium avaibility for absorption; it does this by increasing ileal solubility of magnesium due to a reduction in pH as a consequence of RS2 fermentation in the gut. The mechanism implies that dietary RS2 vs. RS3 would raise true magnesium absorption and stimulate reabsorption of endogenous magnesium, leading to a lower fecal excretion of endogenous magnesium. Dietary lactulose vs. glucose raises apparent magnesium absorption, and the mechanism proposed is similar to that for the stimulatory effect of RS2 vs. RS3. Thus, we measured in rats fed RS3, RS2, glucose or lactulose true magnesium absorption on the basis of the retention of the orally and intraperitoneally administered radiotracer 28Mg. Feeding rats RS2 instead of RS3 significantly enhanced apparent but not true magnesium absorption, because RS2 lowered fecal excretion of endogenous magnesium. When compared with dietary glucose, lactulose significantly raised both apparent and true magnesium absorption, but did not affect fecal excretion of endogenous magnesium. It is suggested that the proposed mechanism by which RS2 and lactulose would enhance magnesium absorption is disproved by the present data.

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.

Adenosine triphosphate-dependent copper transport in human liver.

BACKGROUND/AIM: The recent cloning and sequencing of the Wilson disease gene indicates that hepatic copper (Cu) transport is mediated by a P-type ATPase. The location of this Cu-transporting protein within the hepatocyte is not known; in view of its proposed function and current concepts of hepatic Cu transport, it may reside in intracellular membranes (endoplasmic reticulum (ER), lysosomes) and/or in the bile canalicular membrane. The objective of this study was to establish characteristics and localization of ATP-dependent Cu transport in human liver. METHODS: We have investigated Cu transport in vesicles of human liver plasma membranes showing a gradual increase in enrichment of canalicular domain markers: i.e. basolateral liver plasma membranes (blLPM), a mixed population of basolateral and canalicular (XLPM) and canalicular liver plasma membranes (cLPM). RESULTS: In the presence of ATP (4 mM) and an ATP-regenerating system, uptake of radiolabeled Cu (64Cu, 10 microM) into cLPM vesicles and, to a lesser extent, into blLPM and XLPM was clearly stimulated when compared to control AMP values. Initial uptake rates of ATP-dependent Cu transport were 5.6, 7.8 and 13.7 nmol.min-1.mg-1 protein for blLPM, XLPM and cLPM, respectively, and showed no relationship with marker enzyme activity of ER and lysosomes (glucose-6-phosphatase and acid-phosphatase, respectively). Leucine aminopeptidase activity, as a marker for the cLPM, significantly correlated with ATP-dependent uptake rates measured in different membrane preparations: r = 0.70 (n = 9, p < 0.05). Estimated K(m) and Vmax values of ATP-dependent Cu uptake were 49.5 microM and 36.9 nmol.min-1.mg-1 protein, respectively. CONCLUSION: This study provides biochemical evidence for the presence of an ATP-dependent Cu transport system in human liver (cCOP), mainly localized at the canalicular domain of the hepatocytic plasma membrane.

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.

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.

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.

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.

Dietary fructose v. glucose in rats raises urinary excretion, true absorption and ileal solubility of magnesium but decreases magnesium retention.

Apparent Mg absorption, that is Mg intake minus faecal excretion, was found to be greater in rats fed on diets containing fructose instead of glucose. This effect of fructose was accompanied by enhanced urinary excretion and diminished retention of Mg. True Mg absorption was then determined with the use of oral and intraperitoneal administration of tracer doses of 28 Mg. True Mg absorption was significantly greater in rats fed on fructose. There was no significant effect of fructose v. glucose on faecal excretion of endogenous Mg. It was hypothesized that fructose enhances the solubility of Mg in the ileal lumen and thereby facilitates its absorption. The distribution of Mg between the solid and liquid phases of the ileum was determined in rats fed on either glucose or fructose. Fructose reduced the amount of Mg in the solid phase but raised both the amount and the concentration of Mg in the liquid phase. We conclude that the dietary-fructose-induced stimulation of Mg absorption in rats is caused by a raised solubility of ileal Mg, but the mechanism by which fructose exerts this effect and why it was not associated with a decrease in faecal excretion of endogenous Mg remain unknown. Dietary fructose v. glucose did not systematically affect the apparent absorption of Ca and P.

Dietary ascorbic acid lowers the concentration of soluble copper in the small intestinal lumen of rats.

We tested the hypothesis that ascorbic acid in the diet of rats lowers the concentration of soluble Cu in the small intestine, causing a decrease in apparent Cu absorption. Male rats were fed on diets adequate in Cu (5 mg Cu/kg) without or with 10 g ascorbic acid/kg. The diet with ascorbic acid was fed for either 6 or 42 d. Ascorbic acid depressed tissue Cu concentrations after a feeding period of 42, but not after 6 d. Dietary ascorbic acid lowered apparent Cu absorption after 6, but not after 42 d. The lowering of tissue Cu concentrations after long-term ascorbic acid feeding may have increased the efficiency of Cu absorption, and thus counteracted the inhibitory effect of ascorbic acid. Dietary ascorbic acid caused a significant decrease in the Cu concentrations in the liquid phase of both the proximal and distal parts of the small intestinal lumen. This effect was due to both a decrease in the amount of Cu in the liquid digesta and an increase in the volume of the liquid phase; only the latter effect for the distal intestine was statistically significant. We conclude that ascorbic acid supplementation lowers Cu absorption by decreasing the concentration of soluble Cu in the small intestine.

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.

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.

Iron status in rats fed on diets containing marginal amounts of vitamin A.

Severe vitamin A deficiency in rats is known to cause anaemia associated with growth retardation and impaired water retention. However, study of the effect of marginal vitamin A intake is of more interest because such intake may mirror the situation in humans in many developing countries. Therefore, in two experiments, the effect of marginal vitamin A deficiency on Fe status was investigated in male rats. After 28 d of feeding either low- or high-vitamin A diets (0 or 120 v. 1200 retinol equivalents/kg feed), body weight and feed intake were not influenced by the level of vitamin A in the diet. Liver weight was lowered by vitamin A deficiency. Water intake was not influenced in rats fed on a low-vitamin A diet. Plasma retinol concentrations were decreased in rats fed on diets low in vitamin A. Marginal vitamin A deficiency produced slightly lower blood haemoglobin concentrations; it did not systematically affect packed cell volume. The concentration of Fe in liver was significantly higher when diets low in vitamin A were fed, but hepatic Fe mass was not affected. Significantly lower Fe levels were observed in femurs of rats with vitamin A deficiency. The effects on liver and femur Fe concentrations were seen with diets adequate in Fe but not with diets deficient in Fe. The efficiency of apparent Fe absorption was significantly increased by low intakes of vitamin A, provided that the dietary Fe concentration was adequate. It is speculated that depressed uptake of Fe by bone marrow is the primary feature of altered Fe status in rats with marginal vitamin A deficiency.

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.

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.

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.

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.

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.