Effects of residual and reapplied biosolids on performance and mineral status of grazing beef steers.

An experiment was designed to assess the mineral status of 60 Angus yearling beef steers grazing bahiagrass pastures fertilized with large amounts of biosolids from three sources: Baltimore, MD; Tampa, FL; and Largo, FL. Biosolids were classified as exceptional quality and thus had no regulatory restrictions on loading rate. They differed primarily in concentration of Mo (12 to 56 mg/kg of DM). Residual treatments (biosolids applied only the previous year) for Baltimore biosolids were applied at 22.4 and 44.8 t/ha, and Tampa biosolids were either 16.8 or 33.6 t/ ha. The reapplied treatments (applied in consecutive years) for both Baltimore and Tampa sludges were applied at 22.4, 44.8, 16.8 , and 33.6 t/ha, respectively. The two Largo biosolids treatments were either 56 or 112 t/ha and were applied only in the 2nd yr. Liver biopsies and blood samples were collected on d 1, 95, and 180. Liver and plasma were analyzed for minerals and blood was analyzed for hemoglobin, hematocrit, and superoxide dismutase of polymorphonuclear neutrophils. Experimental animals were generally adequate in macromineral status and Co, Fe, and Mn throughout the experiment. Copper deficiency was evident based on the clinical signs of hair coat discoloration, very low plasma Cu at d 95, and the continuous decline in liver Cu over 180 d. A sharp decline in plasma Cu was observed for all treatments from d 1 to 95, after which Cu concentrations rebounded to normal concentrations (> 0.65 microg/mL) by d 180. Liver Mo was well below concentrations indicating toxicity (> 5.0 mg/kg). The steep decline in liver Cu over the first 95 d reflects the dietary Cu deficiency and the possibility of high forage S (0.26 to 0.52%) interfering with Cu metabolism. Biosolids application to bahiagrass pastures was not detrimental to mineral status except for declining Cu stores; however, the controls likewise declined, but to a lesser degree.

Endothelial cell calcium mobilization to acetylcholine is attenuated in copper-deficient rats.

Dietary copper deficiency significantly attenuates nitric oxide (NO)-mediated vascular smooth muscle relaxation and vasodilation. There is evidence for both increased inactivation of the NO radical by superoxide anion, and oxidative damage to the endothelium where NO is produced. The current study was designed to examine the NO synthetic pathway in the endothelium during copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient (CuD, 0.4 mg Cu/kg diet) diet for four weeks. Cremasteric arterioles (approximately 100 microm diameter) were isolated and used for the experiments. Western blot analysis of the arteriole endothelial nitric oxide synthase (eNOS) concentration did not show a difference between dietary groups. Acetylcholine (Ach)-induced vasodilation was significantly reduced in the CuD group both before and after pretreatment with the eNOS substrate L-arginine. Endothelial intracellular calcium ([Ca2+]i) stimulated by 10(-6) M Ach was significantly inhibited in the arterioles from CuD rats. Coincident with the inhibition of [Ca2+]i and vasodilation was a depression of vascular Cu/Zn-SOD activity and an increase in plasma peroxynitrite activity. These data suggest that endothelial Ca2+ signaling and agonist-stimulated NO-mediated vascular dilation are likely reduced by increased oxidative damage in copper-deficient rats.

Effects of pasture applied biosolids on performance and mineral status of grazing beef heifers.

Angus x Hereford heifers (n = 50) were randomly assigned to bahiagrass pastures treated with biosolids varying in mineral content and evaluated for mineral status, with special attention to Cu. Biosolids and NH4NO3 were all applied at the rate of either 179 kg N/ha (X) or twice this (2X). Fertilizer was applied to .81-ha pastures for the following treatments: 1) Baltimore biosolids (1X = 179 kg N/ha); 2) Baltimore biosolids (2X = 358 kg N/ha); 3) Tampa biosolids (1X = 179 kg N/ha); 4) Tampa biosolids (2X = 358 kg N/ha); or 5) control NH4NO3 (1X = 179 kg N/ha) applied at two times. Pastures were divided into five blocks with each treatment represented once in each block. Copper loads varied from 8.8 to 42.2 kg/ha, and Mo loads varied from .27 to 1.11 kg/ha. Heifers (two per pasture) grazed their assigned pastures exclusively for 176 d. Liver biopsies were taken from all animals at d 1, 99, and 176, and blood samples on d 1, 50, 99, 135, and 176. Liver and plasma were analyzed for selected mineral contents, and blood was analyzed for hemoglobin and hematocrit. Experimental animals were generally low in mineral status when assigned to pastures and deficient in Se and P. By d 50, plasma Ca, Mg, Se, P, and Zn were adequate for all treatments. Plasma Cu declined (P < .03) for all treatments from d 50 to 176. Plasma Cu reflected depleted liver Cu storage, with the two Tampa and highest Baltimore treatment means lower in plasma Cu than the control at 176 d. Liver Fe concentrations were adequate for all treatments, and Mo concentrations (< 2.18 mg/kg) did not approach levels indicative of toxicity. Liver Cu declined (P < .05) with time for all treatments. By d 99, animals receiving the two Baltimore treatments and the lowest Tampa application rate had lower (P < .05) liver Cu than the control, and all treatments were lower at 176 d. The decline of animal Cu status (liver and plasma) reflects the low Cu status of bahiagrass and the possibility of high forage S (.30 to .47%) interfering with Cu metabolism. Forage Mo was low but was slightly higher in biosolids-treated pastures. High levels of biosolids applications to bahiagrass pastures were not detrimental to mineral status except Cu, which had a tendency to decline in plasma and for all biosolids treatments declined in liver.

Use of echinacea in medicine.

Echinacea, also known as the purple coneflower, is an herbal medicine that has been used for centuries, customarily as a treatment for the common cold, coughs, bronchitis, upper respiratory infections, and some inflammatory conditions. Research on echinacea, including clinical trials, is limited and largely in German. More information is needed before a definitive statement about the efficacy of echinacea can be made. Future work needs to clearly identify the species of echinacea and distinguish between the efficacy of the different plant parts (roots versus upper plant parts). Although many of the active compounds of echinacea have been identified, the mechanism of action is not known, nor is the bioavailability, relative potency, or synergistic effects of the active compounds known. Interpretation of existing literature suggests that echinacea should be used as a treatment for illness, not as a means for prevention of illness. The consensus of the studies reviewed in this article is that echinacea is indeed effective in reducing the duration and severity of symptoms, but that this effect is noted only with certain preparations of echinacea. Studies show that the plant and its active components affect the phagocytic immune system, but not the specifically acquired immune system.

Wine modifies the effects of alcohol on immune cells of mice.

Ethanol may be detrimental to immune cells due to the generation of free radicals during detoxification. If this is true, then alcoholic beverages that contain antioxidants, like red wine, should be protective against immune cell damage. We investigated this by giving mice either a red muscadine wine (Vitis rotundifolia), a cabernet sauvignon (Vitis vinifera), ethanol (all at 6% alcohol) or water in the water bottles as the sole fluid for 8 wk. Plasma antioxidant capacity was measured with alphaalpha-diphenyl-beta-picrylhydrazyl and was more than doubled in the mice that consumed wine compared to control mice that consumed water or ethanol. Cytochrome P450-2E1 levels and glutathione-S-transferase activity were modified in such a way as to be interpreted as protective. An immune response was elicited by an intraperitoneal injection of lipopolysaccharide. Later (24 h), natural killer cells and T-lymphocytes derived from the circulation were quantitated in the leukocyte fraction by flow cytometry. Ethanol consumption, as ethanol, significantly suppressed baseline cell numbers relative to the other groups. However, the mice that consumed the same amount of alcohol as wine had baseline cell numbers not different from the water-consuming controls. The lymphocyte response to lipopolysaccharide challenge was inhibited in the mice that consumed ethanol, but was normal in those that consumed the same amount of alcohol in the form of wine. We conclude that there are phytochemicals acting as antioxidants and impacting on the detoxification pathway in the wine that offset the detrimental effects of ethanol on immunity.

Altered copper status in adult men with cystic fibrosis.

OBJECTIVES: To measure indices of copper status in adult men with cystic fibrosis (CF). A previous study in children showed changes in copper homeostasis compared to controls. This study was designed to investigate whether this observation persisted into adulthood. METHODS: This was a case-control age-matched study using seven men with CF and six healthy men. Blood samples were drawn into metal free tubes and fractionated into plasma, polymorphonuclear cells, mononuclear cells and erythrocytes. Cell fractions were assayed for copper and CuZn-superoxide dismutase; plasma was assayed for ceruloplasmin. RESULTS: The men with cystic fibrosis had significantly greater plasma copper and ceruloplasmin activity, yet had significantly lower copper-zinc superoxide dismutase activity in mononuclear and polymorphonuclear cells. Furthermore, the mononuclear cells of the cystic fibrosis subjects had about 45% percent less copper-zinc superoxide dismutase protein. Cellular copper levels were not statistically different between the two groups. A significant correlation was found between lung function and copper-zinc superoxide dismutase activity in the polymorphonuclear cells. Iron status was normal. CONCLUSIONS: The results indicate that individuals with cystic fibrosis have altered copper distribution compared to control individuals. Some aspects are characteristic of an inflammatory response; however, other measures suggest that copper homeostasis may be abnormal. It is not known whether the deviation in copper homeostasis in these individuals is a result of poor copper absorption, inadequate dietary intake, a result of their chronic inflammation or a direct effect due to the defect in ion transport caused by the disease. However, this research suggests that the severity of the disease and the activity of a copper dependent enzyme may be related. Further work will be necessary to determine the cause of the abnormal copper homeostasis and whether correcting it has any bearing on the course of the disease.

Evaluating copper lysine and copper sulfate sources for heifers.

The effects of feeding different sources and quantities of Cu to heifers were evaluated in a 211-d experiment. Forty crossbred predominantly Brahman x Hereford heifers averaging 13.5 mo of age and 301 kg were initially depleted of Cu. The depletion diet was fed for 70 d and consisted of low Cu and high antagonist minerals, Fe, S, and Mo at 1000 mg/kg, 0.5%, and 5 mg/kg (dry basis), respectively. On d 71, heifers continued to receive the antagonistic minerals and were allotted equally to five Cu treatments: 1) control, no additional Cu source; 2) 8 mg of Cu/kg from CuSO4; 3) 16 mg of Cu/kg from CuSO4; 4) 8 mg of Cu/kg from Cu lysine; and 5) 16 mg of Cu/kg from Cu lysine. When no notable change in concentration of Cu in the liver was observed, d 169, a second diet was formulated. The heifers were fed the same Cu treatments, but S and Mo were removed and Fe was lowered to 50 mg/kg. This diet was then fed for the final 42 d of the experiment. In addition to performance, concentrations of Cu, Fe, and Zn in the plasma and liver, plasma ceruloplasmin, hemoglobin, superoxide dismutase (SOD) activity of neutrophils and lymphocytes, and a cell mediated immune response (phytohemagglutinin-P, PHA) were measured. Heifers in this study had increased growth over time, but there were no treatment differences for growth and average daily gain. Liver and plasma Cu concentrations were not greatly influenced by different supplemental Cu sources. However, compared with other treatments, Cu lysine (16 mg/kg) increased liver Cu in cattle that were deficient and tended to increase plasma Cu in animals that were marginally deficient in Cu. Iron concentrations decreased over time in liver and plasma, but there was no difference in Fe and Zn concentrations in liver and plasma among treatments. Differences in ceruloplasmin and hemoglobin concentrations were significant over time but not among treatments. The SOD activity in neutrophils did not change over time, but SOD activity of lymphocytes increased over time. For the PHA immune response test, there was no effect of time or a time by treatment interaction. These data suggest that all Cu sources were available, but Cu at 16 mg/kg from Cu lysine was more beneficial than were other sources and particularly for heifers with low Cu status.

Relationship between dietary copper concentration and acetylcholine-induced vasodilation in the microcirculation of rats.

Dietary copper deficiency has been shown to significantly reduce acetylcholine (Ach)-induced vascular smooth muscle relaxation. The current study was designed to examine the relative relationship between dietary copper and the vasodilator response to Ach in the microcirculation of the rat. Male weanling rats were fed a purified basal diet supplemented with 6.0, 3.0, 1.5 or 0.0 microg Cu/g diet for 4 weeks to provide an adequate, two marginal, and deficient intakes of dietary copper. Arteriole dilation in response to increasing concentrations of acetylcholine (10(-7) to 10(-4) M) was measured in the in vivo cremaster muscle microcirculation for each dietary group. Liver copper and both aortic and erythrocyte Cu,Zn-SOD activity were used as indices of systemic copper status. Dilation to the increasing concentrations of Ach was only different in the 0 microg Cu supplemented group compared to the copper-adequate control values. However, the combined results showed an exponential increase in 10(-5) M Ach-induced vasodilation as liver copper concentration increases from 0 microg Cu/g dry wt. This relationship suggests that dilation is attenuated at liver Cu concentrations below 5 microg/g dry wt. The results indicate that Ach-induced vasodilation is copper-dependent but that the pathway is not very sensitive to short-term marginal restriction of copper intake.

Arrested maturation of granulocytes in copper deficient mice.

The objective of this study was to examine the role of copper in neutrophil development and function. Mice were made copper deficient by feeding dams a diet containing 1.05 microg copper starting at parturition. Control mice were fed the same diet containing 6 microg copper. The pups were weaned to the diet and killed when they were 5-6 wk old. Peripheral blood cell counts, margination and cell maturity were measured. The response to an intraperitoneal injection of lipopolysaccharide (LPS) was also determined. Copper deficiency resulted in twice as many neutrophils and fewer than half the number of lymphocytes. Half as many cells in copper-deficient mice expressed Ly-6G, a granulocytic marker of cell maturity. In addition, copper-deficient cells expressed only half the amount of Ly-6G per cell than was expressed by copper-adequate cells. This suggested that the cells were younger, or arrested in their maturation as a result of copper deficiency. An arrest of maturation has been proposed as the cause of neutropenia in human copper deficiency. Injection of LPS in copper-adequate mice resulted in twice as many Ly-6G-expressing cells in the periphery. LPS injection into copper-deficient mice resulted in a severe leukopenia but did not influence Ly-6G expression any more than did copper deficiency alone. LPS treatment caused an increase in myeloperoxidase activity associated with the lungs of copper-deficient mice. The results suggest that although the neutrophils of copper-deficient mice are immature, they can be sequestered by the lung when stimulated to do so.

Manganese enhances phosphorylation of a 47 kD protein in retinoic acid-induced HL-60 cells.

We previously observed that HL-60 cells treated with manganese (Mn) during differentiation displayed an enhanced oxidative burst. Since a Mn-dependent kinase has been identified and phosphorylation is involved in burst activation, the objective of this research was to identify proteins in retinoic acid-induced HL-60 cells whose phosphorylation after phorbol myristate acetate (PMA) stimulation was affected by Mn treatment. Cells received Mn during differentiation and were then harvested, labeled with [32]P-orthophosphate, and stimulated with PMA. Cytosolic proteins were separated by isoelectric focusing, SDS-PAGE, and two-dimensional (2-D) gel electrophoresis. Time studies showed that Mn treatment did not alter the rate of PMA activated phosphorylation. Isoelectric focusing revealed that PMA stimulation resulted in the appearance of three phosphoproteins at pI's of 6.8, 7.3, and 7.8. Size separation gels showed a 200% increase in phosphorylation of a 47 kD protein in Mn-treated cells after stimulation. The 2-D gels showed that the pI of this protein was 6.8. Therefore, Mn treatment resulted in greater phosphorylation of a 47 kD protein, pI 6.8, in phorbol ester-stimulated cells.

Copper and immunity.

The immune system requires copper to perform several functions, of which little is known about the direct mechanism of action. Animal models and cells in culture have been used to assess copper's role in the immune response. Some of the recent research showed that interleukin 2 is reduced in copper deficiency and is likely the mechanism by which T cell proliferation is reduced. These results were extended to show that even in marginal deficiency, when common indexes of copper are not affected by the diet, the proliferative response and interleukin concentrations are reduced. The number of neutrophils in human peripheral blood is reduced in cases of severe copper deficiency. Not only are they reduced in number, but their ability to generate superoxide anion and kill ingested microorganisms is also reduced in both overt and marginal copper deficiency. This mechanism is not yet understood. Neutrophil-like HL-60 cells accumulate copper as they differentiate into a more mature cell population and this accumulation is not reflected by increases in Cu/Zn superoxide dismutase or cytochrome-c oxidase activities. The identity of copper-binding proteins in this cell type may be useful in learning new functions of copper or assessing copper status. Neutrophils, because they are short-lived and homogeneous cell populations, are predicted to be an effective and valuable tool for assessing nutrient status in human populations.

Reduced copper enzyme activities in blood cells of children with cystic fibrosis.

Cystic fibrosis patients are at risk for nutrient deficiencies from malabsorption related to exocrine pancreatic insufficiency. This research examined the copper homeostasis of children with cystic fibrosis. Our objective was to measure cytochrome oxidase and copper-zinc superoxide dismutase activities in mononuclear cells, neutrophils, and erythrocytes of adolescents with cystic fibrosis, as well as plasma copper and ceruloplasmin. Thirteen adolescents with pancreatic insufficiency caused by cystic fibrosis were compared with 10 age- and sex-matched control subjects. Serum copper concentrations and ceruloplasmin measurements were not significantly different between the two groups. Cytochrome oxidase activity was significantly lower in the mononuclear cells and copper-zinc superoxide dismutase activity was significantly lower in the neutrophils and erythrocytes of the cystic fibrosis group. Other measures of trace element status such as hemoglobin concentration, serum ferritin, serum zinc, glutathione peroxidase activity, and manganese superoxide dismutase activity were not different between the two groups. Reductions in the activity of two copper-dependent enzymes suggest abnormal copper homeostasis in this population.

Effects of long-term moderate exercise on iron status in young women.

The impact of long-term (6-month) moderate exercise on the iron status of previously sedentary women was determined by randomly assigning 62 college-age women into one of the following four groups: 1) 50 mg.d-1 iron supplement, low iron diet (N = 16); 2) Placebo, free choice diet (N = 13); 3) Meat supplement to achieve 15 mg.d-1 iron intake (N = 13); and 4) Control, free choice diet (N = 20). All groups except the Control group exercised 3 d.wk-1 at 60%-75% of their heart rate reserve. VO2max was measured at baseline and week 24. Blood was sampled at baseline and every 4 wk thereafter for 24 wk to measure iron status and to elucidate the causes for alterations in iron status. Subjects had depleted iron stores throughout the study as indicated by their serum ferritin levels (< 15 ng.ml-1). Serum iron, total iron binding capacity and transferrin saturation were not compromised with exercise. Mean hemoglobin level in the Placebo/Ex group was significantly (P < 0.05) lower than the 50 Fe/Ex and the Meat/Ex groups by week 24. However, changes in serum albumin, haptoglobin, and erythropoietin data from the study cannot explain these changes.

Neutropenia caused by copper deficiency: possible mechanisms of action.

Although copper is an essential nutrient in the human diet, overt or severe copper deficiency is not a major public health concern in the United States. A marginal or borderline deficiency has been suggested by researchers, although the widespread nature of copper deficiency has yet to be established. Reports indicate that copper deficiency occurs secondary to gastric resection, unsupplemented total parenteral nutrition, high levels of zinc intake, or general malnutrition. An early clinical sign of copper deficiency is a reduction in the number of circulating neutrophils. Although copper is known to play a wide variety of roles in the organism and in the immune system, the molecular mechanism for copper-deficient neutropenia is not known. Very little data exist with which to examine this question. This paper will summarize our existing knowledge of the mechanism by which copper deficiency results in neutropenia. Although the data are scarce, analysis of this question will allow us to better understand additional molecular roles of this trace element, and, in turn, to promote an improved knowledge of immune cell functions and cellular differentiation.

Manganese stimulates the oxidative burst of differentiated HL-60 cells.

Incubation with manganese results in a twofold increase in the oxidative burst of differentiated HL-60 cells. This stimulation was characterized by examining the dose response, length of incubation time, and specificity of manganese. Manganese only stimulated the burst in cells induced to differentiated with retinoic acid and not in undifferentiated HL-60 cells. Incubation with manganese did not result in a greater number of differentiated cells. The maximum stimulation occurred at 0.2 mumol/L manganese. Stimulation of the oxidative burst required 96 h of incubation with manganese, since cells incubated with the same levels of manganese for the last 24 h of culture did not result in any stimulation. Magnesium, present in the incubation medium at physiological serum levels (820 mumol/L) also stimulated the oxidative burst, whereas iron (0.3 mumol/L), zinc (18 mumol/L), and copper (12 mumol/L) had no effect. To determine whether manganese and magnesium stimulated the burst differently, the initial rates of superoxide anion production was determined. The initial rate of the reaction proceeded rapidly in cells incubated with manganese, whereas there appeared to be a lag before magnesium-treated cells produced superoxide anion. Thus, manganese seems to stimulate the oxidative burst differently than magnesium.

Cu/Zn superoxide dismutase activity does not parallel copper levels in copper supplemented HL-60 cells.

The objective of this research was to develop a method for measuring Cu/Zn-superoxide dismutase (Cu/Zn-SOD) (E.C. 1.15.1.1) in HL-60 cells and subsequently examine the relationship between cellular copper levels and the activity of this copper-requiring enzyme. In cells such as the neutrophil or HL-60 promyelocyte cell line, the activity of Cu/Zn-SOD cannot be measured because of an increase in the oxidation rate of the substrate by some unknown compound in the cells. Others have utilized heat treatment to inactivate the responsible compounds, however, we found that heat treatment of HL-60 cells resulted in a loss of over half of the activity of the enzyme. The method described here utilizes sodium azide to inhibit the substance(s) that are responsible for the enhanced rate of pyrogallol's oxidation. Gel filtration data confirmed that the compound responsible for the enhanced rate of pyrogallol oxidation was sensitive to azide and did not affect Cu/Zn-SOD activity. When HL-60 cells were incubated with various levels of copper, Cu/Zn-SOD activity did not reflect the cellular copper levels.

Retinoic acid-induced HL-60 cell differentiation is augmented by copper supplementation.

The objective of this study was to determine how copper influences the ability of HL-60 cells to differentiate into cells of the granulocytic lineage. We hypothesized that granulopoiesis requires copper because copper-deficient humans become neutropenic. Differentiation of HL-60 cells along the granulocytic lineage with retinoic acid was enhanced by copper. The results showed a greater number of cells were more differentiated when copper was added to the medium for 96 h. The respiratory burst activity of retinoic acid-induced cells was increased by copper supplementation, but intracellular superoxide anion generation was not affected. Supplementation with copper resulted in more cell-associated copper in both noninduced and induced cells; however, the induced cells accumulated three times more copper than the noninduced cells. Even though the amount of copper associated with retinoic acid-treated cells was greater than in untreated cells, the activity of a copper-requiring enzyme, copper/zinc superoxide dismutase, was significantly lower. Copper supplementation increased the activity of this enzyme in both retinoic acid-treated and untreated cells. Cytochrome c oxidase activity was not affected by retinoic acid treatment or by copper supplementation. Copper seems to play a specific role during the early stages of granulocyte differentiation.

Copper is required to maintain Cu/Zn-superoxide dismutase activity during HL-60 cell differentiation.

The objective of these studies was to characterize the relationship between copper levels and Cu/Zn superoxide dismutase (Cu/Zn-SOD) during cellular differentiation. It was hypothesized that the decrease in Cu/Zn-SOD activity that accompanied differentiation would be reversed by supplementing the culture medium with copper. HL-60 cells, a human promyelocytic cell line, were induced to differentiate with retinoic acid and were concurrently supplemented with copper or a copper chelator, tetraethylenepentamine. The results showed that retinoic acid-treated cells contained more copper after differentiation. When the medium was supplemented with copper during retinoic acid treatment, the differentiating cells accumulated more copper than the nondifferentiating cells. Differentiation was accompanied by a significant reduction in Cu/Zn-SOD activity and a slight reduction in Cu/Zn-SOD protein. Activity returned to control values when an extracellular source of copper was provided. Incubation of retinoic acid-treated cells with the chelator showed that they lost proportionally less copper than the noninduced controls. Levels of Cu/Zn-SOD protein were not affected by the copper or chelator treatments. It was concluded that the requirement of differentiating HL-60 cells for copper is not related to providing copper for Cu/Zn-SOD activity. If a supplemental source is not supplied in the medium, then the cells may acquire copper from an intracellular source, namely Cu/Zn-SOD.

HL-60 cells can be made copper deficient by incubating with tetraethylenepentamine.

A system for studying copper deficiency was developed in a cell culture model. HL-60 cells were incubated with three chelators known to bind copper. One chelator, tetraethylenepentamine (TEPA), reduced cellular copper levels and the activities of two copper-requiring enzymes, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and cytochrome c oxidase. The specificity of the chelator was assessed by incubating cells with both copper and TEPA and, in other experiments, with zinc and TEPA. Copper levels, Cu/Zn-SOD activity and cytochrome c oxidase activity were restored to control values when copper and TEPA were added to cultures simultaneously, indicating the TEPA was responsible for reducing these aspects of copper metabolism. Incubating with both zinc and TEPA reduced copper levels relative to the control, but did not reduce Cu/Zn-SOD activity to the same extent as TEPA alone. The chelation of copper was a time-dependent process that was stable for at least 4 d. Cell growth and viability were not affected by TEPA. Respiratory burst activity, an indicator of differentiation, was not affected by TEPA, demonstrating that the reduction of Cu/Zn-SOD activity was due to copper chelation and not due to changes in Cu/Zn-SOD protein levels that occur during differentiation. Loss of copper, as well as a reduction of the activity of two copper-requiring enzymes, provides evidence that TEPA is a useful compound for creating a functional copper deficiency in cell culture.

Iron metabolism is modified by the copper status of a human erythroleukemic (K562) cell line.

Copper deficiency is known to result in a microcytic, hypochromic anemia. Red cells of copper-deficient animals have less hemoglobin than their copper-adequate counterparts. The objective of this work was to determine what role copper plays in maintaining hemoglobin levels. It was hypothesized that the primary defect lies in intracellular iron metabolism. The influence of copper supplementation on iron uptake and storage was examined in a cell line capable of hemoglobin synthesis. The results demonstrated that copper supplementation of human K562 cells was associated with higher cytosolic iron levels and ferritin levels. Copper supplementation of the cell culture altered the initial rate of iron uptake from transferrin and enhanced iron uptake in noninduced cells; however, in hemin-induced K562 cells, which express fewer transferrin receptors on the cell surface, copper appeared to reduce iron uptake. Subsequent studies showed that the cells were able to take up the same amount of iron from transferrin when incubated over a longer period of time (24 hr). In the noninduced (non-hemoglobin synthesizing) cells, proportionally more iron was associated with the ferritin. We concluded from these studies that copper affects both uptake and storage of iron and that copper supplementation reduces cellular iron turnover.