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.

Serum adrenal steroid concentrations in premature infants.

To evaluate serum adrenal steroid concentrations in preterm infants, 17-hydroxyprogesterone (17-OHP), 17-hydroxypregnenolone, 11-deoxycortisol, cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, 18-hydroxycorticosterone, and aldosterone values were determined in 9 sick and 13 healthy premature infants. Serum steroid concentrations were compared to previously reported data from healthy full-term infants. 17-OHP, 11-deoxycortisol, and aldosterone values were higher in sick preterm infants than in healthy preterm infants. Compared to healthy full-term infants, the premature infants-had significantly higher 17-hydroxypregnenolone, 17-OHP, and DHEA sulfate concentrations. Cortisol values were not different between the sick and healthy preterm infants and were similar to full-term values. Aldosterone values were also similar between the premature and the full-term infants. The findings of elevated steroid precursors in preterm infants and low cortisol levels in stressed sick preterm infants may indicate a relative immaturity of adrenal enzyme activity and inadequate adrenal reserve for stress.

Plasma corticosteroid concentrations during spironolactone administration: evidence for adrenal biosynthetic blockade in man.

Spironolactone, a mineralocorticoid antagonist, may also inhibit aldosterone biosynthesis. In vitro studies suggest that spironolactone and its major metabolites inhibit adrenal 18- and 11 beta-hydroxylase activity. We examined various adrenal corticosteroids and their precursors, plasma renin activity, aldosterone excretion rate, and serum and urine electrolytes in normal subjects before and on days 5 and 10 of spironolactone administration (400 mg/day). Plasma corticosteroids were also examined 60 min after ACTH (Cortrosyn) 0.25-mg iv bolus. RIAs were performed after extensive chromatography; there was no interference of spironolactone and its metabolites in the assays. All studies were performed in supine subjects in metabolic balance on a constant 120-meq sodium intake. Plasma renin activity was increased (P less than 0.001) on both days 5 and 10 of spironolactone. Plasma aldosterone (PA) and the aldosterone excretion rate increased (P less than 0.01) on day 5 of spironolactone but decreased (P less than 0.01) from day 5 to 10. Both 11-deoxycorticosterone and 18-hydroxycorticosterone were increased from day 5 to 10. Corticosterone, progesterone, and dehydroepiandrosterone did not increase significantly during spironolactone administration. Incremental PA response to ACTH was less than control on day 10 of spironolactone, but other corticosteroid responses to ACTH were not different during control and days 5 or 10 of treatment. Reduction in PA and further elevation in its precursors during the second 5-day period of spironolactone therapy suggests inhibition of aldosterone biosynthesis during this phase of treatment in normal man. The disproportionate increments in 18-hydroxycorticosterone and 11-deoxycorticosterone suggest biosynthetic inhibition at the 18-dehydrogenase and 11 beta-hydroxylase sites.

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.

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.

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.

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.

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 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.

An integrated system for large scale cell culture.

The potential of tissue culture as a means of production of various viruses for use in vaccines, or various biomolecules such as the interferons, interleukins, various enzymes and growth factors, has been steadily increasing over the last few years. If the full potential for the production of these products is to be realized, significant technological advances in the area of large scale cell culture are required. Most cells producing the above mentioned molecules and products grow only when attached to a surface. Therefore, the ability to harness cell culture as a tool for large scale production of products is dependent on the availability of large surface areas that are biocompatible with cell attachment and growth and are presented in such a form that proper nutrient solutions are continually being offered to the cells. This presentation will describe a closed loop circumfusion system with an available surface area scaleble to 72 m2. This system is integrated with a computer based controller providing programmable monitoring, control, and documentation of the culture. The system has been extensively tested in both in-house and field trials. This testing has shown the system to be compatible with the typical production environment rigors while highlighting the economic potentials of production using this unique system.

Electron-microscopic localization of A2B5 cell surface antigen in monolayer cultures of murine cerebellum and retina.

Immuno-electron microscopy was performed on live, cultured, early postnatal cerebellar and retinal cells of the mouse to identify A2B5 antigen-bearing elements. In cerebellar cultures, granule cells, some immature oligodendroglia, and astroblasts express A2B5 antigen on their cell surfaces. The typical features of astroblasts include large cisternae of the endoplasmic reticulum and a mixed population of intermediate-sized filaments and microtubules. Immature oligodendroglia cells express the antigen on their cell bodies and on processes filled with cytoplasm. Cytoplasm-free membranous whorls, however, are devoid of A2B5 antigen, but not of 0 or NS-1 antigens. In retinal cultures, A2B5 antigen is observed on differentiating neurons with the exception of photoreceptor cells as identified by ribbon synapses.

Light and electron microscopic structure of Golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure).

After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguished by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pices of tissue left in a 1% K2Cr2O7 solution for 13 h before impregnation in a 0.5% AgNO3 solution for 2 h. Thick sections (30-50 micron) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.