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Background: Multiple micronutrient (MMN) supplementation may result in interaction effects due to competing absorptive pathways of trace elements., Objectives: The aim of this study was to investigate the effect of MMN supplementation with or without iron on serum zinc, selenium, and copper concentrations in Cambodian women., Methods: In a 2 × 2 factorial double-blind randomized 12-wk trial, predominantly anemic, nonpregnant women (aged 18-45 y) received daily 60 mg of iron (Fe; n = 201); 14 other micronutrients including zinc (15 mg), selenium (65 μg), and copper (2 mg), but no iron (MMN; n = 202); 60 mg iron plus MMN (Fe + MMN; n = 206); or a placebo (n = 200). Fasting morning blood was collected at baseline and 12 wk from women in 26 villages in Kampong Chhnang province. Serum zinc, selenium, and copper concentrations (secondary outcomes of the randomized controlled trial) were measured using inductively coupled plasma mass spectrometry. Generalized linear regression was used to estimate intervention effects [β coefficient (95% CI)] for Fe (with or without MMN) and MMN (with or without Fe) after testing for the presence of an Fe × MMN interaction., Results: A total of 760 women completed the trial. Zinc deficiency prevalence at baseline was 45% (inflammation-adjusted serum zinc <10.7 μmol/L). A significant Fe × MMN interaction (P = 0.02) was detected in the 2 × 2 analysis with serum zinc concentration as the outcome: the MMN group had a higher mean serum zinc concentration at 12 wk (12.3 μmol/L; 95% CI: 12.2, 12.4 μmol/L) compared with all other groups, and the Fe + MMN group had a higher mean serum zinc concentration (11.6 μmol/L; 95% CI: 11.5, 11.7 μmol/L) compared with the Fe group (11.0 μmol/L; 95% CI: 10.9, 11.0 μmol/L) and the placebo group (11.2 μmol/L; 95% CI: 11.1, 11.4 μmol/L)., Conclusions: The inclusion of 60 mg iron in the daily MMN formulation may be interfering with the absorption and/or metabolism of supplemental zinc in Cambodian women. This is of particular concern when MMN supplementation is implemented in populations with risk of zinc deficiency. This trial was registered at clinicaltrials.gov as NCT-02481375., (Copyright © American Society for Nutrition 2019.)

Subclinical micronutrient deficiencies remain a hidden aspect of malnutrition for which comprehensive data are lacking in school-aged children. We assessed the micronutrient status of Nepalese children, aged 6 to 8 y, born to mothers who participated in a community-based antenatal micronutrient supplementation trial from 1999 to 2001. Of 3305 participants, plasma indicators were assessed in a random sample of 1000 children. Results revealed deficiencies of vitamins A (retinol <0.70 μmol/L, 8.5%), D (25-hydroxyvitamin D <50 nmol/L, 17.2%), E (α-tocopherol <9.3 μmol/L, 17.9%), K (decarboxy prothombin >2 μg/L, 20%), B-12 (cobalamin <150 pmol/L, 18.1%), B-6 [pyridoxal-5'-phosphate (PLP) <20 nmol/L, 43.1%], and β-carotene (41.5% <0.09 μmol/L), with little folate deficiency (6.2% <13.6 nmol/L). Deficiencies of iron [ferritin <15 μg/L, 10.7%; transferrin receptor (TfR) >8.3 mg/L, 40.1%; TfR:ferritin >500 μg/μg, 14.3%], iodine (thyroglobulin >40 μg/L, 11.4%), and selenium (plasma selenium <0.89 μmol/L, 59.0%) were observed, whereas copper deficiency was nearly absent (plasma copper <11.8 μmol/L, 0.7%). Hemoglobin was not assessed. Among all children, 91.7% experienced at least 1 micronutrient deficiency, and 64.7% experienced multiple deficiencies. Inflammation (α-1 acid glycoprotein >1 g/L, C-reactive protein >5 mg/L, or both) was present in 31.6% of children, affecting the prevalence of deficiency as assessed by retinol, β-carotene, PLP, ferritin, TfR, selenium, copper, or having any or multiple deficiencies. For any nutrient, population deficiency prevalence estimates were altered by ≤5.4% by the presence of inflammation, suggesting that the majority of deficiencies exist regardless of inflammation. Multiple micronutrient deficiencies coexist in school-aged children in rural Nepal, meriting more comprehensive strategies for their assessment and prevention., (© 2014 American Society for Nutrition.)

Micronutrient deficiencies are common in undernourished societies yet remain inadequately assessed due to the complexity and costs of existing assays. A plasma proteomics-based approach holds promise in quantifying multiple nutrient:protein associations that reflect biological function and nutritional status. To validate this concept, in plasma samples of a cohort of 500 6- to 8-y-old Nepalese children, we estimated cross-sectional correlations between vitamins A (retinol), D (25-hydroxyvitamin D), and E (α-tocopherol), copper, and selenium, measured by conventional assays, and relative abundance of their major plasma-bound proteins, measured by quantitative proteomics using 8-plex iTRAQ mass tags. The prevalence of low-to-deficient status was 8.8% (<0.70 μmol/L) for retinol, 19.2% (<50 nmol/L) for 25-hydroxyvitamin D, 17.6% (<9.3 μmol/L) for α-tocopherol, 0% (<10 μmol/L) for copper, and 13.6% (<0.6 μmol/L) for selenium. We identified 4705 proteins, 982 in >50 children. Employing a linear mixed effects model, we observed the following correlations: retinol:retinol-binding protein 4 (r = 0.88), 25-hydroxyvitamin D:vitamin D-binding protein (r = 0.58), α-tocopherol:apolipoprotein C-III (r = 0.64), copper:ceruloplasmin (r = 0.65), and selenium:selenoprotein P isoform 1 (r = 0.79) (all P < 0.0001), passing a false discovery rate threshold of 1% (based on P value-derived q values). Individual proteins explained 34-77% (R(2)) of variation in their respective nutrient concentration. Adding second proteins to models raised R(2) to 48-79%, demonstrating a potential to explain additional variation in nutrient concentration by this strategy. Plasma proteomics can identify and quantify protein biomarkers of micronutrient status in undernourished children. The maternal micronutrient supplementation trial, from which data were derived as a follow-up activity, was registered at clinicaltrials.gov as NCT00115271.

The tolerable upper intake levels (UL) for zinc for children were based on limited data and there is concern that the UL may be set too low. The first effect of excessive zinc intake is a reduction in copper status. The primary objective of this study was to examine the effect of zinc supplementation on copper status in children. Healthy, 6- to 8-y-old boys from Ontario, Canada were assigned to take a placebo (n = 10) or 5 mg (n = 10), 10 mg (n = 9), or 15 mg (n = 8) of zinc supplement daily for 4 mo in a double-blinded, placebo-controlled, randomized trial. Biochemical measures were evaluated at baseline and after 2 and 4 mo of supplementation. Food records were completed near the baseline and 4-mo visits. Age and anthropometric measurements did not differ (P > 0.05) between treatment groups at baseline. Mean zinc intakes from food alone (10.9-14.8 mg zinc/d) approached or exceeded the UL of 12 mg/d. Compared with the placebo group, the zinc groups had a greater change in the urine zinc:creatinine ratio at 4 mo (P = 0.02). Traditional (plasma copper and ceruloplasmin activity) and more sensitive biomarkers of copper status, including erythrocyte SOD1 activity and the erythrocyte CCS:SOD1 protein ratio, were unchanged in zinc-supplemented boys, demonstrating that copper status was not depressed. Serum lipid measures and hemoglobin concentrations were also unaffected and gastrointestinal symptoms were not reported. These data provide evidence in support of the need for reexamining the current UL for zinc for children.

The protein affected in Menkes disease, ATP7A, is a copper (Cu)-transporting P-type ATPase that plays an important role in Cu homeostasis, but the full extent of this role has not been defined at a systemic level. Transgenic mice that overexpress the human ATP7A from the chicken beta-actin composite promoter (CAG) were used to further investigate the physiological function of ATP7A. Overexpression of ATP7A in the mice caused disturbances in Cu homeostasis, with depletion of Cu in some tissues, especially the heart. To investigate the effect of overexpression of ATP7A when dietary Cu intake was markedly increased, normal and transgenic mice were exposed to drinking water containing 300 mg/L of Cu as Cu acetate for 3 mo. Cu exposure resulted in partial restoration of heart Cu concentrations in male transgenic mice. Despite the extended period of Cu exposure, Cu concentrations in the liver remained relatively unaffected, with a significant increase in male nontransgenic mice. Liver pathology was unremarkable except for small areas of fibrosis that were detected only in livers of the Cu-exposed transgenic mice. Intracellular localization of ATP7A in various tissues was not affected by Cu exposure. Plasma Cu concentration and ceruloplasmin oxidase activity were reduced in both Cu-exposed transgenic and nontransgenic mice. The expression levels of other candidate Cu homeostatic proteins, endogenous Atp7b, ceruloplasmin, Ctr1, and transgenic ATP7A were not altered significantly by Cu exposure. Overall, mice are remarkably resistant to high Cu loads and the overexpression of ATP7A has only moderate effects on the response to Cu exposure.

There are conflicting data on the associations between copper and glycemia, plasma lipids, and atherosclerotic diseases. Copper has both pro-oxidant and antioxidant effects. We performed a cross-sectional analysis to investigate the associations between dietary copper intake and metabolic variables and serum high-sensitivity C-reactive protein (hs-CRP) in asymptomatic subjects from a population-based cohort (n = 1197) and between serum copper concentration and markers of oxidative stress, including plasma nitrotyrosine (NT) and total antioxidant status (TAS), hs-CRP, and metabolic variables in a subgroup of men from this cohort (n = 231). In all subjects, diastolic blood pressure and circulating glucose, uric acid, and total and LDL-cholesterol concentrations significantly decreased, whereas the hs-CRP concentration increased, from the lowest to the highest tertile of copper intake. In the male subgroup, glucose and total and LDL-cholesterol and TAS decreased, whereas hs-CRP and NT concentrations increased from the lowest to the highest tertile of serum copper concentration. In multiple regression models, dietary copper intake was inversely associated with diastolic blood pressure (P = 0.002), fasting glucose (P < 0.001), total cholesterol (P < 0.001), LDL-cholesterol (P < 0.001), and uric acid (P < 0.001) and was directly associated with the hs-CRP concentration (P < 0.001). Serum copper concentrations were inversely associated with glucose (P < 0.001), total cholesterol (P < 0.001), LDL-cholesterol (P < 0.001), and TAS (P < 0.001) and were directly associated with hs-CRP (P < 0.001) and NT concentrations (P < 0.001). Marginal copper deficiency is associated with an unfavorable metabolic pattern, but copper supplementation might not be recommended in view of its association with inflammation and markers of oxidative stress.

Copper deficiency has been reported to cause a decrease in urinary taurine excretion in rats. We determined whether Cu deficiency would decrease taurine status and the hepatic activities of cysteine dioxygenase (CDO) and/or cysteine sulfinic acid decarboxylase (CSAD) in rats. Ten weanling male rats were assigned to either a Cu-adequate (+Cu) or Cu-deficient (-Cu) group. All rats consumed a Cu-deficient purified diet and water ad-libitum for 16 wk. The water for the (+Cu) group contained 20 mg Cu/L as CuSO(4). At wk 16, the groups differed (P < 0.05) in the following variables (means +/- SEM, -Cu vs. +Cu): body weight (BW), 375 +/- 19 vs. 418 +/- 2.9 g; food intake, 16.2 +/- 0.7 vs. 18.5 +/- 0.4 g/d; hematocrit, 0.294 +/- 0.027 vs. 0.436 +/- 0.027; hemoglobin, 95.2 +/- 9 vs 134 +/- 10 g/L; liver Cu, 8.7 +/- 2.0 vs. 65.9 +/- 2.5 nmol/g; plasma Cu, 0.38 +/- 0.09 vs. 13.4 +/- 0.61 micromol/L; plasma ceruloplasmin activity, 1.75 +/- 1.0 vs. 67.9 +/- 8.4 IU; relative heart weight, 0.56 +/- 0.04 vs. 0.35 +/- 0.02% BW; relative liver weight, 4.06 +/- 0.23 vs. 3.37 +/- 0.06% BW; and liver CSAD activity, 18.8 +/- 1.37 vs. 13.5 +/- 1.11 nmol x min(-1) x mg protein(-1). The groups did not differ at wk 16 in: plasma taurine, 249 +/- 14 vs. 298 +/- 63 micromol/L; whole blood taurine, 386 +/- 32 vs. 390 +/- 25 micromol/L; urinary taurine excretion, 82.5 +/- 15 vs. 52.0 +/- 8.3 micromol/d; liver taurine, 2.6 +/- 0.7 vs. 2.8 +/- 0.4 micromol/g; liver total glutathione, 6.9 +/- 0.48 vs. 6.3 +/- 0.40 micromol/g; liver cyst(e)ine, 96 +/- 7.1 vs. 99 +/- 5.3 nmol/g and liver CDO activity, 2.19 +/- 0.33 vs. 2.74 +/- 0.21 nmol x min(-1) x mg protein(-1). These findings support the conclusion that Cu deficiency does not affect body taurine status.

Lutein, a dihydroxycarotenoid, has antioxidant and immunomodulatory potential. Two 2 x 2 x 2 factorial designs examined effects of carotenoids during in ovo embryogenesis and, in the diet posthatch, on the systemic inflammatory response to lipopolysaccharide (LPS). In both trials, breeder hens were fed a carotenoid-replete (40 mg lutein/kg) or a carotenoid-deplete diet, eggs were collected, and chicks were hatched from carotenoid-deplete or carotenoid-replete eggs. Meat-type chicks (n = 160 and n = 144, respectively) were then fed diets containing 0 or 40 mg lutein/kg diet and either injected or not injected with LPS. LPS injection increased plasma haptoglobin and Zn (P < 0.01) and reduced plasma Fe and Cu (P < 0.01). Chicks hatched from carotenoid-deplete eggs had greater changes in plasma Fe and S post-LPS than chicks hatched from carotenoid-replete eggs (P < 0.05 for each). Compared with chicks fed 40 mg lutein/kg diet, chicks fed 0 mg lutein had greater body weight losses and higher plasma haptoglobin and relative thymus, bursa, and spleen weights post-LPS (P < 0.05). Data suggest that a lack of carotenoid exposure, either in ovo or posthatch, increases parameters of systemic inflammation.

Nondigestible inulin-type fructan intake can stimulate intestinal mineral absorption in both humans and animals. However, this stimulatory effect may depend on experimental conditions such as the duration of the experience, mineral levels in the diet, and the animal's physiological status. The aim of this study was to determine the effect of inulin intake on Zn and Cu absorption in rats at different ages. Male Wistar rats (n = 80) of 4 different ages (2,5, 10, and 20 mo) were randomly assigned to a control group or a group administered 3.75% inulin in their diet for 4 d followed by 7.5% inulin for 26 d. Absorption of Zn67 and Cu65 was determined on d 21 of the experiment by fecal monitoring using Zn67 and Cu65 isotopes. Zn and Cu status was also assessed. Absorption of Zn67 and Cu65 was significantly lower in 11- and 21-mo-old rats than in 3- and 6 mo-old-rats. Moreover, inulin intake significantly increased Zn67 and Cu65 absorption. In conclusion, age and dietary inulin intake can significantly affect intestinal absorption of zinc and copper in rats. Further studies are required to explore this effect over longer periods of inulin intake and to test the effects of inulin in humans.

The health consequences of mild copper excess in humans are unknown. In a previous study, 2 mo of supplementation with up to 6 mg Cu/L in drinking water did not induce detectable changes. Here we assessed a copper supplement at the upper level of dietary recommendations for "healthy" adults. The study was a prospective controlled trial; participants (women and men, 18-50 y old), represented the upper and lower 5% of the ceruloplasmin distribution curve obtained from a community-based sample of 800 healthy adults (n = 41/group, each approximately 50% men). Individuals received a single daily dose of 10 mg Cu for 60 d. Before and after supplementation, blood [copper, ceruloplasmin protein, homocysteine, liver aminotranferases, Cu-Zn -superoxide dismutase activity in erythrocytes (eSOD), and glutathione in peripheral mononuclear cells] and urine [copper excretion after a 5-h administration of a chelator 2,3-dimercapto-1-propano-sodium sulfonate (DMPS)] analyses were performed. After 2 mo, liver enzyme activities remained below the clinical cutoff value used to diagnose liver dysfunction, but had increased significantly in both groups and genders. These increases were no longer present 12 mo after the copper loading period was completed. Glutathione in mononuclear cells (mmol/g of protein) also increased after the 2-mo copper loading in both groups and in both genders (P = 0.01). eSOD activity, serum homocysteine concentration, and urinary copper excretion 5 h after DMPS administration were not affected. We conclude that copper administered as described induced a transient, mild, but significant elevation of aminotransferases.

Because infants and young children in many developing countries are deficient in both iron and zinc, and zinc can affect iron metabolism, evaluation of optimum strategies to simultaneously supplement iron and zinc is an important public health priority. This study evaluated the efficacy of weekly supplementation of iron or zinc or both on iron, zinc, and copper status in Bangladeshi infants. In a double-blind, randomized, controlled community trial, 6-mo-old infants were assigned to receive weekly supplements of 1 mg riboflavin (control, n = 82) or 1 mg riboflavin + 20 mg iron (n = 83), 20 mg zinc (n = 83), or both (n = 85) for 6 mo. Hemoglobin, serum ferritin, transferrin receptor, zinc, and copper concentrations were measured at baseline and at the end of intervention. Serum Zn increased in both groups receiving zinc; the increase was greatest among children with low baseline serum zinc concentration. Iron status indicators did not differ among the groups before or after 6 mo of supplementation. Supplementation with either zinc or iron decreased serum copper after 6 mo. Joint supplementation did not alter the individual effects of iron or zinc supplementation in these Bangladeshi children. However, the dosing regimen may not have been adequate to achieve the desired biochemical effects.

The mechanism for reduced Fe absorption in Cu deficiency is unknown, but may involve the intestinal Cu-dependent ferroxidase, Hephaestin (Hp). A 2 x 2 factorial experiment was designed to include Cu-deficient (CuD) and Cu-adequate (CuA) male and female rats. Weanling rats of both sexes were randomly divided into 2 groups each and fed an AIN-93G diet with low (<0.3 mg/kg; CuD) or adequate Cu (5.0 mg/kg; CuA). After 19 d, rats were fed 1.0 g each of their respective diets labeled with (59)Fe. Retained (59)Fe was monitored by whole-body counting for 12 d. Then, rats were killed for (59)Fe and Fe measurements in blood and various organs. Duodenal enterocytes were isolated for Western blot analysis of Hp. Signs of Cu and Fe deficiency were evident in both sexes. CuD male rats absorbed 60% as much Fe as CuA male rats (P < 0.001), whereas CuD female rats absorbed 70% (P < 0.001) as much as CuA females, with no difference between the sexes. Hp protein in enterocytes of CuD rats of both sexes was only 35% of that in CuA rats. The biological half-life of (59)Fe in CuD rats was only 50% (P < 0.001) of that in CuA rats, suggesting that Fe turnover was faster in CuD rats than CuA rats. Serum, spleen, and kidney Fe were lower (P < 0.001) in CuD rats than in CuA rats. Duodenal mucosa and liver Fe were higher (P < 0.01) in CuD male rats than CuA rats. Duodenal Fe but not liver Fe was higher in CuD female rats than CuA rats. Liver Fe was much higher (<0.001) overall in females than males. The data suggest that Cu deficiency reduces Fe absorption in rats through reduced expression of duodenal Hp protein.

Dietary nutrient interactions are important factors to consider in the study of nutrient status and requirements. Here, the effects of dietary interactions among copper (Cu), iron (Fe), manganese (Mn) and sulfur amino acids (SAA) on blood cell characteristics and enzyme activities were observed. Male rats (n = 8) were used in a 2 x 2 x 2 x 2 factorial design and fed an AIN-93G-based diet containing dietary Cu (<1 and 5 mg/kg), Fe (10 and 35 mg/kg), Mn (10 and 50 mg/kg) and either L-cystine (LCys) or DL-methionine (DLMet). Blood was analyzed by automated hematology cell counting and by flow cytometry. Severe Cu deficiency was verified by reductions in the activities of serum ceruloplasmin (1% of control), RBC superoxide dismutase (SOD1) (14% of control), liver cytochrome c oxidase activity (25% of control) and serum extracellular SOD (SOD3) activity (20% of controls). Because Cu is required for Fe utilization, many physiologic responses that require Fe were affected by both deficiencies, including lowered blood hemoglobin (Hgb), lower RBC volume and Hgb concentration, and an increased number of reticulocytes. Cu and Fe deficiencies together worsened some conditions, i.e., lower Hgb, lower RBC Hgb, increased RBC distribution width, increased number of reticulocytes and nucleated RBC, and a higher platelet count. Increasing dietary Mn had little effect on most variables, except to reduce serum Cu when dietary Cu was adequate but not when it was low, and to reduce RBC SOD1 activity when dietary Fe was low but not when it was adequate. Hgb concentrations were higher (P < 0.002) in Cu-deficient rats fed LCys than in those fed DLMet. There was no effect in Cu-adequate rats. Hgb was higher (P < 0.004) in Fe-adequate rats fed LCys than in those fed DLMet, with no effect in Fe-deficient rats. Although the anemia of Cu deficiency in AIN-93G-fed rats was not as pronounced as that reported in rats fed the AIN-76A-based diet, other manifestations of the deficiency were prominent.

The effects of long-term supplementation with pharmacologic doses of zinc oxide on serum levels of zinc, lipids and hematocrit have not been studied systematically to date. Eleven Clinical Centers enrolled 4757 participants from 1992 to 1998 as part of the Age-Related Eye Disease Study (AREDS). Of these, 3640 participants, aged 55-80 y, who had early-to-late age-related macular degeneration (AMD) were randomly assigned to daily supplementation with or without 80 mg of zinc as zinc oxide plus 2 mg of copper as cupric oxide to study the effects of zinc supplementation on the progression to late AMD. This paper reports on the effect of a 5-y supplementation with zinc oxide and cupric oxide on serum zinc, copper, lipids, and hematocrit for 717 participants from three clinical centers. At the 5-y exam, the median increase in serum zinc levels for participants assigned to zinc formulations was 17% compared with a 2% increase for participants not assigned to zinc (P < 0.001). The differential effect on serum zinc was observed at 1 y and remained fairly constant over the 5-y period. After 5 y, no significant differences in changes in serum hematocrit, copper or lipids were found between participants assigned to formulations containing zinc and copper, and those assigned to formulations without zinc and copper. Estimates from a modified Block Food-Frequency Questionnaire suggest the AREDS population at baseline had a zinc intake from diet similar to that of the general population.

Evidence supports a role for ceruloplasmin (ferroxidase I) in the release of iron to the blood from mammalian cells. However, recent studies with cultured cells have suggested that it has the opposite effect, and that iron deficiency enhances expression of ceruloplasmin. We therefore examined in rats how nutritional iron status would affect expression of ceruloplasmin. Groups of male Sprague-Dawley rats were reared on a low iron, starch-based diet for 6-8 wk; half were supplemented by injection of iron dextran. At killing, hematocrits of deficient rats were half normal. Supplemented rats had normal liver concentrations of ferritin and ferritin iron. No ferritin was detected in the livers of the deficient rats. Northern analysis showed that ferritin L and H mRNAs were present in the deficient livers, but expression was half that of the normal rats. There was also twice as much copper. Levels of circulating ceruloplasmin (measured by rocket immunoelectrophoresis) were not altered by iron deficiency, although p-phenylenediamine oxidase activity and plasma copper were reduced approximately 30%. In repeated studies, no differences in the expression of hepatic ceruloplasmin mRNA were detected. Treatment of rats of both sexes with additional iron (25 mg as iron dextran) 5-14 d before killing increased liver ferritin but did not alter liver ceruloplasmin mRNA expression or levels of circulating ceruloplasmin. We conclude that iron status is not an important factor in the expression of plasma ceruloplasmin made by the liver. However, it does have modest effects on steady-state levels of liver ferritin mRNA.

Red wines contain many components such as polyphenols and ethanol that may influence mineral absorption. We report on studies in a rat model that were designed to investigate the extent to which short- and long-term intake of red wine or ethanol may influence (67)Zn and (65)Cu absorption in rats. Rats (n = 96) were divided into three groups, a control group that received demineralized water, a group that received red wine diluted with water (v/v) and an ethanol group that received 6% ethanol. Half of each group was used for the short-term study; the others were used for the long-term study. After 3 d (short-term study) or 28 d (long-term study) of beverage consumption, the rats were gavaged with 2 mL of solution containing 2027 nmol (67)Zn and 902 nmol (65)Cu. Subsequently, 3-d urinary and fecal collections were performed and analyzed for total and isotopic Zn and Cu. In the long-term study, blood, tibia and liver were also sampled for mineral status assessment. Neither short- nor long-term intake of red wine altered (67)Zn or (65)Cu absorption. In contrast, long-term (but not short-term) ethanol consumption significantly increased both (67)Zn and (65)Cu absorption compared with the control and red wine groups. The long-term consumption of ethanol or red wine did not affect blood or tissue Zn or Cu levels. In conclusion, short- or long-term consumption of red wine did not have a negative effect on intestinal absorption or tissue levels of zinc and Cu in rats.

The objective of this study was to determine the copper requirement of female cats (queens) for gestation. Cuproenzyme activities were evaluated to identify a noninvasive indicator of copper status. This study used a depletion-repletion model. Specific pathogen-free queens (n = 28) were adapted to a purified diet; after consuming a copper-depletion diet (0.8 mg Cu/kg diet) for 4 mo, they were randomly allocated to three dietary treatment groups receiving copper sulfate at 4.0, 5.8 or 10.8 mg Cu/kg diet. Four queens underwent liver biopsies at two time points during the study. Plasma samples were analyzed for copper concentrations, extracellular superoxide dismutase, ceruloplasmin and diamine oxidase activities. Only liver copper concentrations were responsive to dietary copper intake. The dietary concentration of copper had a significant effect on the time taken for queens to conceive (P = 0.04). There was a negative linear relationship between dietary copper (x = Cu mg/kg diet) and the mean time (y = days) for queens to conceive (y = 43.38-2.87x; R(2) = 0.97). The current NRC recommendation of 5 mg/kg diet copper for cats appears marginal for optimal reproduction.

This study was designed to examine the interactions among dietary iron (Fe), copper (Cu), and zinc (Zn) and their effects on Fe status and oxidative stress in female rats. In a three-factor central composite response surface design, rats were assigned to 15 groups and fed modified AIN-93G basal diets with varying amounts of Fe and Zn (7.0, 15.5, 45.8, 135.6, or 300 micrograms/g diet) and Cu (0.5, 1.1, 3.2, 9.2, or 20 micrograms/g diet) for 6 wk. Variations in hemoglobin, hematocrit, and serum ferritin were mainly related to dietary Fe. Liver nonheme Fe was directly affected by dietary Fe and was slightly attenuated by interactions between Cu and Zn, and Zn and Fe. Serum ceruloplasmin activity was primarily determined by an interaction between Cu and Zn with substantial moderation by the quadratic effect of dietary Cu. Liver and heart total superoxide dismutase (SOD) and Cu/Zn SOD activities were directly affected by dietary Cu. Dietary Fe was the only significant, yet weak, predictor of liver thiobarbituric acid reactive substances (TBARS) and vitamin E content and serum triacylglycerols. Variability in serum Cu was mostly determined by the interaction between Cu and Fe, with modification from the quadratic effect of dietary Cu. Serum Zn varied with dietary Zn with a small negative influence from the interaction between Cu and Fe. In summary, Fe status was minimally influenced by dietary Zn or Cu, and Fe intakes 10-fold greater than required did not induce overt oxidative stress in female rats. In addition, measures of antioxidant capacity were primarily influenced by dietary Cu and were optimal at moderate intakes of this micronutrient.

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.

The metallothionein gene is transcriptionally regulated by zinc. Consequently, metallothionein has potential for serving as an index of dietary zinc status in humans. To examine this possibility, an enzyme-linked immunoassay (ELISA) based on a sandwich approach that utilizes monoclonal and chicken egg yolk antibodies was used to compare the response of erythrocyte metallothionein protein levels with the response of monocyte metallothionein mRNA levels as measured by competitive reverse transcriptase-polymerase chain reaction (CRT-PCR) during zinc supplementation. Young male subjects participated in an 18-d supplementation study in which zinc was provided at 50 mg/d. Control subjects received a placebo. The zinc supplement resulted in significantly greater erythrocyte metallothionein levels by d 8 of supplementation compared with controls. Monocyte metallothionein mRNA levels were significantly greater than those of controls by d 2 of supplementation. Both remained elevated through d 18. They returned to base line by 8 and 4 d after supplementation, respectively. The plasma zinc concentration was significantly greater than in controls by d 6 and had returned to control levels by d 22 of supplementation. The results presented here show that both monocyte metallothionein mRNA and erythrocyte metallothionein protein concentrations change in human subjects in response to elevated dietary zinc intake and that monocyte metallothionein mRNA responds more rapidly to elevation of dietary zinc status than erythrocyte metallothionein protein. Consequently, both erythrocyte metallothionein and monocyte metallothionein mRNA may prove to be measures useful for assessment of either zinc depletion or the bioavailability of zinc supplements.

Dietary sulfur-containing amino acids influence zinc and copper status. This study was conducted to investigate the relationship between the status of these elements and tissue metallothionein in rats fed diets supplemented with sulfur-containing amino acids. In a series of experiments, a diet containing 100 g casein/kg diet was unsupplemented or supplemented with L-cystine (3-50 g/kg diet) or L-methionine (3 or 30 g/kg diet). Kidney concentrations of zinc and copper in rats fed the diet supplemented with high levels of cystine (25 or 50 g/kg) were significantly higher than those in rats fed the unsupplemented diet. Kidney concentrations of metallothionein and metallothionein mRNA were also significantly higher in rats fed cystine-supplemented diets. There was a correlation (r = 0.838, P < 0.01) between the levels of zinc and metallothionein in kidney of rats fed the diets with different levels of cystine and zinc. However, in the rats fed the diets with different levels of cystine and copper, the correlation was not as strong (r = 0.587, P < 0.01). The changes in kidney metallothionein concentration due to the addition of 3 or 30 g/kg cystine were associated with parallel changes in serum zinc concentration and in apparent absorption of zinc, but not in serum copper concentration or apparent absorption of copper. Addition of 3 or 30 g methionine/kg diet to the diet did not affect kidney concentrations of metallothionein or zinc. These results indicate that supplementation of cystine to the diet can induce kidney metallothionein through a mechanism involving altered zinc metabolism.

Male Wistar rats were pair-fed from weaning for 6 wk either a copper-deficient diet or a control diet containing sucrose. Hypercholesterolemia in copper-deficient animals was associated with a significantly greater plasma apolipoprotein B concentration and modifications in the lipid composition of triglyceride-rich lipoproteins containing apolipoprotein B. The proportion of triglycerides was elevated and the proportion of cholesterol was reduced in the VLDL + LDL fractions from copper-deficient rats compared with control animals. When the VLDL + LDL fractions were subjected to in vitro copper-induced oxidation, the lipoprotein fractions from copper-deficient rats were more susceptible to oxidative damage than lipoprotein fractions from control rats as indicated by the rate of diene conjugation. Hydroperoxide and thiobarbituric acid-reactive substance (TBARS) measurements performed 3 h after the induction of peroxidation were in agreement with the greater formation of conjugated dienes. Concentrations of TBARS were significantly greater in heart homogenates from copper-deficient rats compared with control rats. After exposure of tissue homogenates to iron-induced lipid peroxidation, TBARS were significantly higher in liver and heart from copper-deficient rats compared with control rats. Of particular importance is the observation that copper deficiency has a harmful effect on lipid peroxidation in the cardiovascular system.

The pharmacokinetics of D(-)-penicillamine (PA) and the effects of PA on the nutritional status of Cu and Zn in chicks were studied. When administered by subcutaneous or intravenous injection, PA was eliminated from the plasma with biphasic kinetics: i.e., a fast (halftime: 6-7 min) and a slow (halftime: 52-55 min) component. The elimination of PA from the liver or pancreas showed only the slow (halftime: 42-46 min) component. When PA was administered orally, plasma PA concentrations responded over time in bimodal pattern, but the maximal level was only one-fifth that achieved by injection. The subcutaneous injection of PA elicited differential, transient changes in the concentrations of Zn and Cu in the plasma: plasma Zn concentration was decreased, and plasma Cu concentration was increased. Analyses of urine collected from ureter-cannulated chicks showed that PA was rapidly excreted via that route, and that urinary PA excretion was associated with increased urinary excretion of both Zn and Cu. These results show that PA treatment can alter tissue distributions particularly of Zn and, to a lesser extent, of Cu. J. Nutr.

The effects of hyperoxia and the response of oxygen free radical defense enzymes in the lung and extracellular environment of the lung were measured in Zn-deficient rats. Although lung was the target organ as indicated by the increased lung:body weight ratio in all hyperoxia-exposed rats regardless of dietary regimen, 85% oxygen exposure seemed to impose a stress on the whole animal as indicated by decreased feed intake and body weight in ad libitum-fed rats. Hyperoxia exposure superimposed on Zn deficiency did not further reduce the feed intake or body weight of Zn-deficient rats. After 7 d of hyperoxia exposure, the Zn-repleted and ad libitum-fed groups consistently had increased activity of lung CuZn-superoxide dismutase (CuZnSOD), glutathione peroxidase and catalase; but changes in CuZnSOD activity were not related to lung Cu or Zn concentrations. Although Zn-deficient and pair-fed rats were unable to increase CuZnSOD activity, they had an increased lung Zn concentration compared with their air-exposed counterparts. Hyperoxia exposure also caused an increase in ceruloplasmin activity of pair-fed and ad libitum-fed control rats. We concluded that dietary Zn repletion started at the beginning of 85% oxygen exposure was effective for increasing the activity of the lung oxygen free radical defense enzymes, thus preventing hyperoxia-induced lung damage in Zn-deficient rats.

Accumulation of 64Cu in the 14-d mouse fetus was measured following intravenous injection of the dam with 64CuHis2. Concentration of 64Cu in the placenta increased rapidly over the first 4 h, thereafter remaining constant. Transfer to the fetus was linear over 48 h with little evidence of storage in the liver. Maternal serum levels decreased initially, concurrently with increased 64Cu levels in the maternal liver, but did not subsequently increase. Immediately following injection, as much as 40% of the radioactivity was in the nonalbumin fraction, and approximately 37% of that fraction (18% of total) was greater than 30,000 molecular weight. After 24 h, up to 60% of the 64Cu was still found in the albumin peak. The data suggest that the fetus can obtain its Cu from maternal ceruloplasmin but does not exclude the possibility that transfer occurs from the exchangeable (albumin/amino acid) Cu pool of the maternal plasma. In a second experiment, mice at different stages of gestation were injected with 64Cu and killed 4 h later. Total Cu levels and 64Cu uptake into the maternal tissues or into the placenta did not change with increasing gestation. Both total Cu and 64Cu uptake in the fetus and fetal liver increased to a maximum on d 16. Levels in the liver decreased thereafter to term, whereas levels in the rest of the fetus remained approximately constant. The pattern was similar whether the results were expressed per organ or per gram of fresh weight.(ABSTRACT TRUNCATED AT 250 WORDS)

The bioavailability of copper from isolated soybean protein was evaluated with growing rats. Copper depletion and repletion feeding techniques were used to evaluate the biologically available copper. Serum and liver copper contents were used as physiological indication to determine the bioavailability of copper in the diet. Sprague-Dawley male weanling rats were fed a diet low in copper for 21-42 days and then transferred to the test diets containing different levels of copper from copper carbonate or isolated soybean proteins for 7-14 days. Statistical analyses of serum and liver copper by using the slope-ratio bioassay were performed to determine the relative potency of the test products to the copper carbonate standard. The results from four feeding experiments indicate that 1) hypocupremia was produced by feeding a low copper basal diet to weanling rats after 28 days, 2) the time required to replete serum and liver copper was 7 days and 3) copper was available equally from isolated soybean protein and copper carbonate.

The effect of histidine-supplemented stock diets on plasma cholesterol and on the amelioration of the hypercholesterolemia by simultaneous copper supplementation was examined in rats. Weanling male Sprague-Dawley rats, five groups of 10 animals each, were fed ground stock diets unsupplemented or supplemented with 8% L-histidine, 8% L-histidine plus 222.8 micrograms Zn/g and 8% L-histidine plus 56.6 micrograms Cu/g. On day 46 plasma and liver cholesterol, copper and zinc concentrations were determined by colorimetry and atomic absorption spectrophotometry. Histidine supplementation produced hepatomegaly, depressed growth, a 131.8% (P less than 0.001) increase in plasma cholesterol and significant reductions in plasma copper and zinc and liver copper concentrations. The simultaneous feeding of histidine and zinc-supplemented stock diet failed to alter the hypercholesterolemia despite elevation in plasma zinc to values in excess of control animals. The simultaneous feeding of histidine and copper-supplemented stock diets abolished the hypercholesterolemia, reduced the hepatomegaly observed with histidine alone and elevated tissue copper concentrations to values in excess of control animals. Tissue copper and cholesterol concentrations showed significant negative correlations. No correlations between zinc and cholesterol were observed. The results suggest that histidine-induced hypercholesterolemia is due to changes in copper status, and is abolished by simultaneous copper supplementation.

The temporal response of zinc and copper metabolism to endotoxin administration was examined in Syrian hamsters over a 144-hour period. Serum copper was significantly elevated at 12, 24 and 72 hours after endotoxin, whereas serum zinc was reduced 4-48 hours after treatment. A brief elevation (8 hours) in liver copper concentration and a sustained (72 hours) increase in liver zinc concentration were also observed. The amount of zinc associated with liver metallothionein (MT) progressively increased with time, to a plateau by 24 hours and persisted at the elevated level until 72 hours after endotoxin treatment. In vitro translation of poly (A)+ RNA from liver polyribosomes showed that following endotoxin treatment MTmRNA activity was maximally elevated 6 hours after endotoxin administration and remained elevated 24 and 48 hours thereafter. Slab gel electrophoresis of serum proteins indicated changes in a stainable protein comigrating with purified ceruloplasmin after endotoxin administration. Pooled gingival tissue from endotoxin-treated hamsters demonstrated a consistently elevated copper content 12-144 hours after treatment. Endotoxin isolated from Bacteroides melaninogenicus was more effective in elevating gingival and serum copper and gingival zinc than Escherichia coli endotoxin. It was concluded that endotoxin administration elicits responses that result in enhanced metaollthionein mRNA activity. In addition, Cu and Zn concentrations in serum, liver and gingival tissue are influenced by different endotoxins to different degrees.

Several methodologies were evaluated to study iron, zinc and copper interactions. In three studies, rats were fed diets containing adequate levels of iron (33-35 micrograms Fe/g diet) and zinc (15-25 micrograms Zn/g diet) and diets with excessive levels of zinc (2441-2470 micrograms Zn/g diet) or iron (1408-3042 micrograms Fe/g diet). Consumption of excess iron for several weeks or in one test meal did not alter tissue levels of zinc or apparent absorption of 65Zn. In contrast rats fed excess zinc throughout the three studies had depressed hematocrits and serum copper levels, apparently absorbed 59Fe less efficiently and retained less 59Fe in livers and tibias than control animals. Nutritional status with regard to iron or zinc, not just the intakes of iron and zinc, also influenced the interactions between zinc and iron. Rats fed excess iron for several weeks retained less 65Zn from a test meal in their tibias than rats fed excessive iron in only the test meal. Although chronic ingestion of excess zinc depressed apparent absorption of 59Fe from a test meal, the ingestion of one test meal with excess zinc did not. Investigators studying nutrient interactions should not rely solely on studies in which unadjusted humans or animals are given a single dose of test substances because acute responses do not reflect all the changes induced by chronic feeding.

Red cell superoxide dismutase (SOD) activity was evaluated as a biochemical index of copper nutrition in a double-blind study of 17 infants recovering from malnutrition and receiving marginal copper intakes. Children were paired on admission by sex, birth weight, nutritional status and antecedents of diarrhea and breast feeding. Nine served as controls receiving a copper sulfate supplement (80 micrograms/kg daily for 120 d; eight received a placebo and were supplemented only if plasma copper levels dropped below 90 micrograms/dl or on d 90 for at least 30 d. After copper supplementation there was a significant rise (paired t-test; P less than 0.05) in plasma copper (96 vs. 165 micrograms/dl); ceruloplasmin (33 vs. 50 mg/dl) and SOD (1073 vs. 1371 U/g Hb). After supplementation these values were similar to those of the controls. SOD was correlated with plasma copper (r = 0.78; P less than 0.001) and not with weight-for-age or weight-for-length. Addition of copper in vitro did not modify the SOD activity. Red cell SOD is a good marker of copper nutrition in humans and correlates well with plasma copper.

The effects of starvation and refeeding of 2-wk-old turkey poults on serum and tissue levels of zinc, copper and iron were investigated. Serum concentrations of zinc and copper declined during 4 d of starvation. Refeeding for 24 h following a period of starvation restored serum copper to levels similar to those in the fed controls but failed to elevate zinc levels. Liver concentrations and total quantities of zinc, copper and iron increased throughout starvation. Refeeding the starved poults reduced hepatic metal concentrations but caused a further increase in total metal content. This was apparently related to the large increase in liver mass, and the effect was most pronounced in poults starved 1 d prior to refeeding. Starvation also caused an increased zinc concentration and content and a reduced copper content in the pancreas, duodenal mucosa and kidney. Iron content of the pancreas and kidney increased during starvation, but that of the duodenal mucosa declined. Starvation evoked a progressive increase in the cytosolic zinc concentration from liver, pancreas, duodenal mucosa and kidney. A major part of this increase was accounted for as zinc bound to metallothionein (MT). Refeeding rapidly reduced cytosolic and MT-bound zinc in each of these tissues. It was concluded that starvation and refeeding had major effects on tissue trace metal status. A function is proposed for MT during starvation as a mechanism for the conservation of body zinc stores. Zinc, released as a consequence of tissue catabolism, is repartitioned into a soluble storage site (MT), which can be rapidly mobilized to meet the demands of new tissue synthesis once anabolic metabolism resumes.

Serum selenium as well as serum zinc, copper, magnesium, calcium and manganese were investigated in a control group of adult males and in 11 groups of patients in various disease states. Not only the change of each trace element but also the possible association between elements was studied in the various groups. All patients were fasting when sampled and studied only after the acute phase of the disease was corrected. Trace metal determinations were performed by atomic absorption spectrophometry (Mg, Ca, Cu, Zn) and by neutron activation analysis (Se, Mn). All patients showed low serum zinc when compared to controls. Cirrhotic patients had a low serum selenium level as well as low calcium, magnesium and zinc. Emphysemia and cancer patients had an elevated serum copper concentration while copper and manganese levels were elevated in congestive heart failure, infection and pschoses. To our knowledge this is the first time low serum selenium values have been demonstrated to be associated with the low serum zinc, calcium and magnesium levels found in cirrhotic patients.

Female guinea pigs were fed diets varied in ascorbic acid and iron concentration for 21 days. Tissue concentrations of ascorbic acid, iron (total, ferritin and hemosiderin) and copper were determined in blood, liver and spleen. High dietary ascorbic acid (10 times control) increased tissue ascorbic acid levels and produced a decrease in liver ferritin and hemosiderin, without altering liver or plasma total iron. Conversely, splenic total iron increased with no changes in ferritin and hemosiderin iron. The increased ascorbic acid did lower copper levels in blood and liver, 39% and 52%, respectively. In guinea pigs maintained on an ascorbic acid-free diet for 21 days, a decrease in hepatic ferritin and total iron was observed, as well as an increase in splenic hemosiderin and total iron. No change in plasma iron was observed nor were any of the copper pools altered. Intake of high dietary iron (10 times control) increased hepatic iron stores and produced a reciprocal decrease in hepatic copper. Even though splenic iron increased, no significant change in copper resulted. The significance of these nutrient interactions is discussed.

The objective of this study was to evaluate the effect of phytic acid on copper (Cu) bioavailability. Male weanling rats were fed a Cu-deficient diet (less than 1.0 micrograms/g) for 4 wk and then were divided into 12 groups (n = 8) in a factorial design. Cu-deficient rats were then fed diets containing 1.4, 3.0, 5.2 or 10.5 micrograms Cu/g (CuCO3) and 0, 0.4 or 0.8% phytic acid as sodium phytate at each Cu level. All diets contained 30 micrograms Zn/g. After 3 d of Cu repletion, liver copper (LCu), liver Cu-Zn superoxide dismutase (LSOD) activity, serum Cu (SCu) and serum ceruloplasmin (CP) concentrations were determined. These parameters were used as indexes of Cu bioavailability. The addition of phytic acid to diets fed to Cu-deficient rats significantly enhanced Cu bioavailability compared to that of rats fed diets without phytic acid. Coefficients of determination (r2) were calculated for each response parameter versus dietary Cu concentration. The r2-values for pooled LCu and LSOD values were 0.31 and 0.30, respectively, between 1.4 and 5.2 micrograms Cu/g. At low dietary Cu concentrations, liver Cu parameters (i.e., LCu and LSOD) were more responsive indexes of Cu status than SCu and CP. Each index of Cu status was found to correlate with the other indexes of Cu nutriture. Phytic acid is postulated to enhance Cu utilization by its ability to bind other dietary components, such as Zn, that compete with Cu at the site of intestinal absorption.

Twenty-seven adult female mongrel dogs were studied to evaluate whether histidine is an essential amino acid. Dogs were tube-fed isocaloric, isonitrogenous amino acid diets which provided either no histidine or 67 mg histidine/kg body weight/day. The histidine-free diet was fed to 10 dogs for 5.6 +/- 3.6 (SD) days and to six dogs for 59.2 +/- 6.0 days. In the short-term studies, there were no differences between the responses of the dogs fed the histidine-free and histidine-replete diets. In the long-term studies, dogs fed the histidine-free diet developed a significant decrease in plasma and muscle histidine, muscle carnosine, body weight, hematocrit and serum albumin. The dogs fed the histidine-free diet tried to avoid the feedings, and after several weeks, they often manifested reduced activity and listlessness. One dog died on the 72nd day. None of these manifestations occurred in the dogs fed the histidine-replete diet in the long-term studies. Plasma zinc and copper were not different in the two groups of dogs. However, at the end of the long-term studies, the dogs fed the histidine-free diet had significantly lower final whole blood zinc and copper concentrations as compared to the histidine-replete dogs. These findings indicate that histidine is an essential amino acid in adult female dogs. The syndrome associated with histidine-deficiency tends to develop slowly over many days to several weeks.

Studies were conducted in suckling mice to investigate copper-dependent anemia. Brindled (Mobr/y) mice, which have a genetic defect that affects copper metabolism, were compared to their normal brothers (Mo+/y) as well as to anemic suckling mice that were copper-deficient (-Cu) because their dams were consuming a diet low in copper and to a fourth group of control suckling mice (+Cu) from copper-supplemented dams. Mice were given a subcutaneous injection of NaCl, FeCl2 or CuCl2 providing 50 micrograms of Na, Fe or Cu, respectively, when 7 days old and were killed 5 days later. Injection of FeCl2 into -Cu mice elevated liver iron 2.7-fold and raised hemoglobin levels to those observed in the +Cu and Mo+/y mice. Ceruloplasmin activity remained low at 5% of control levels. Injection of CuCl2 into -Cu mice resulted in significant increases in body and brain weight, elevations in serum copper and ceruloplasmin activities and hemoglobin levels. Liver copper rose and iron fell, both to levels observed in +Cu and Mo+/y mice. Brain copper and norepinephrine concentrations rose to control levels. Injection of CuCl2 into Mobr/y mice, although resulting in darker pigmentation and normal serum copper and ceruloplasmin levels, failed to stimulate growth or change brain weight. Furthermore, liver and brain copper levels did not rise to normal levels despite significant improvements. Similarly, brain norepinephrine levels rose, but were still below normal. Brindled mice do not respond to copper therapy to the same degree as do -Cu mice. Iron therapy was successful in reversing the anemia of -Cu mice but was without effect on growth or brain development.

Indicators of copper and iron metabolism were studied in pregnant rats and their 90-d-old offspring fed copper-sufficient or copper-deficient diets containing marginal or adequate levels of iron from the beginning of pregnancy until the offspring were 90 d of age. Offspring had more severe signs of copper deficiency (including anemia, hypertrophy of the heart, decreased activity of ferroxidase I and II, depression of growth and death) than the dams. In both dams and offspring, copper deficiency resulted in anemia when dietary iron was marginal but not when it was adequate. Liver iron was elevated in copper-deficient male offspring, but not in female offspring. Anemia and growth retardation were more pronounced in copper-deficient males than in females, despite similarly low levels of ferroxidase I and II. Iron absorption was reduced by copper deficiency only in female offspring. Activity of 59Fe in various tissues 6 or 48 h after gavage did not reveal any other effect of copper deficiency on iron metabolism. Thus age at the time copper-deficient diets were introduced, sex and dietary iron strongly influence the effect of copper deficiency.

The effect of depot parenteral injections of zinc (110 mg Zn/kg body weight) on copper metabolism in young, male rats was investigated. Individually caged rats, fed known amounts of stock diet and deionized-distilled water, were injected s.c. weekly for the first 4 weeks and biweekly for the next 17 weeks with zinc in sesame oil or the oil vehicle only. No significant differences in body weight, hemoglobin, hematocrit and fecal copper excretions were observed between treatments. However, 2 weeks after the initial injections, urinary copper excretion was elevated in the zinc-injected animals and remained elevated throughout the rest of the study. Plasma copper concentrations were significantly higher in the zinc-injected animals from week 2 to 8 of the study, and plasma zinc concentrations of these injected animals were elevated (P less than 0.05) from week 2 and throughout the remainder of the study. Zinc concentrations were significantly higher in the liver, heart, kidney and spleen (P less than 0.05) and copper concentrations were lower in the liver (P less than 0.07), kidney and spleen (P less than 0.05) of zinc-injected animals compared to the vehicle-treated control animals. The data indicate that when zinc is administered by a non-gastrointestinal route, the fecal excretion of copper, the major route of copper excretion, is not altered. Thus, a negative copper balance is not initiated by high levels of zinc administered by the depot technique, in contrast to the negative copper balance stimulated by the gastrointestinal administration of zinc.

Angiotensin-converting enzyme (ACE) is a zinc-dependent peptidyldipeptide hydrolase found in blood and in the endothelium of many tissues. This study was designed to determine the effects of dietary zinc deprivation in rats and guinea pigs on ACE activity in serum. Rats were fed zinc-deficient (less than 1 mg/kg) or zinc-adequate (25 mg/kg) diets for 3 d. Guinea pigs were fed zinc-deficient (less than 1 mg/kg) or zinc-adequate (100 mg/kg) diets for 3 wk. The plasma zinc concentration in the zinc-deprived rats was 26% and that of the deprived guinea pigs 35% of the control values. When serum was diluted by one-half in the assay medium, the respective ACE activities were 42 and 59% of controls. In spite of decreased ACE activity there were no differences in plasma angiotensin II levels in either species or in bradykinin levels in the rat. To determine the effect of zinc on the kinetics of serum ACE, rat serum was treated to remove low-molecular-weight components and diluted 30-fold in an assay medium that contained a final zinc concentration of 0.68 microM. Supplemental zinc (total, 24 microM) increased both Vmax and Km. EDTA inhibited the enzyme activity, and the inhibition was totally reversible by zinc. Copper at 30 microM had no effect on ACE activity. The results of this study show that ACE activity in serum of rats and guinea pigs is highly sensitive to the dietary intake of zinc and suggest that metabolism of the vasoactive hormones, angiotensin II and bradykinin, might be affected in vivo.

The effects of an immune response on growth and feed efficiency in chicks and the role of interleukin-1 (IL-1) and corticosterone (Cort) as mediators of the response were investigated. Daily injections of either sheep red blood cells or the inflammatory agent Sephadex resulted in significantly (P less than 0.05) lower rates of weight gain, feed intake and efficiency of feed utilization than controls fed ad libitum, indicating an immunologically mediated stress. Feeding control chicks the same amount of diet as that consumed by immunologically challenged chicks did not completely equalize rates of weight gain. Injections of a crude preparation of IL-1, but not Cort, resulted in weight gain, feed intake and efficiency of feed utilization that were similar to those of immunologically challenged chicks. The concentrations of IL-1 and Cort, measured by bioassay and radioimmunoassay, respectively, in serum from immunologically challenged chicks were significantly higher than in nonchallenged chicks. To determine the influence of IL-1 and Cort on protein accretion in skeletal muscles, the extensor digiti communus and ulnaris lateralis were incubated in the presence of these two hormones at concentrations similar to that seen in serum after an immunologic challenge. Cort did not affect the rate of protein degradation but resulted in rates of protein synthesis that were significantly lower than controls. IL-1 did not affect the rate of protein synthesis but resulted in rates of protein degradation that were about 24% greater than controls.

Regulation of metallothionein gene expression by dietary zinc and copper was examined in rat liver, kidney, intestine and brain using a 3 X 3 factorial design. Purified diets containing 5, 30 and 180 mg Zn/kg and 1, 6 and 36 mg Cu/kg were fed for 2 wk. Serum concentrations of copper and zinc were lower at the lowest intakes of either metal than at normal or supplemental levels. Kidney metallothionein levels were proportional to dietary zinc, being 50% less in the 5 mg Zn/kg group than in those fed the highest zinc intake. Metallothionein mRNA was measured by dot blot hybridization to a 32P-labeled oligonucleotide DNA probe representing the terminal 5' sequence of the metallothionein gene. In kidney the number of metallothionein mRNA molecules per cell increased four- to five-fold (from 4 to 29 molecules per cell) with increasing dietary zinc. A less pronounced effect on metallothionein mRNA was observed in response to dietary copper. At the lowest copper intake level and highest intake of zinc intestinal metallothionein mRNA was sevenfold greater than in any other group. Liver and brain did not respond appreciably to the dietary levels of copper and zinc that were fed. Chromatography showed that copper and zinc content of renal metallothionein was directly related to the dietary levels fed. In kidney, both metallothionein-1 and -2 genes were expressed.

Three experiments involving 52 baby pigs were conducted to determine the minimum copper requirement of baby pigs fed purified diets. Diets were supplemented with anhydrous cupric sulfate to yield the following copper concentrations (ppm, by analysis) when the three experiments were combined: 0.6, 0.9, 1.3, 1.9, 2.0, 2.8, 3.2, 4.0, 4.9, 5.6 and 9.3. Parameters examined include weight gain, hematocrit, hemoglobin concentration, mean corpuscular hemoglobin concentration, plasma ceruloplasmin activity, plasma copper concentration, copper balance, brain and erythrocyte superoxide dismutase activity, copper concentration of liver, kidney, spleen, heart, brain, femur and hair, liver ferritin-iron and total iron concentration, strength characteristics of the femur, and gross and histological appearance at necropsy. Weight gains were subnormal at dietary copper concentrations below 1.9 ppm; plasma ceruloplasmin activities, and plasma and tissue copper concentrations were depressed at dietary copper levels below 2.8 ppm. Bone histopathology was evident at dietary copper levels below 3.2 ppm, and copper balance was low at dietary copper levels below 4.9 ppm. Some evidence of anemia was present at dietary copper levels below 5.6 ppm. Under the conditions of this study, the copper requirement of the baby pig fed a purified diet was judged to be approximately 5.6 ppm (6 ppm copper, dry basis).

The influence of dietary iron and the sex of rats on suppressed lymphocyte functions caused by copper deficiency was examined. Male and female weanling Lewis rats were fed two concentrations of copper (0.6 or 5.6 micrograms Cu/g diet) and iron (50 or 300 micrograms Fe/g diet) for 42 d. Regardless of dietary iron concentrations, male and female rats consuming low copper diets had lower serum ceruloplasmin activity and serum and liver copper concentrations than those fed the high copper diet. However, hemoglobin and hematocrit levels were higher in copper-deficient females than in copper-deficient males and were unaffected by copper deficiency in females fed the high iron diet. Copper-deficient females also had higher serum and liver iron concentrations than copper-deficient males. Proliferation of concanavalin A (Con A)-stimulated spleen lymphoid cells (SLC) was suppressed in copper-deficient males and females, but the suppression was less in the females. Thus, the primary cause of suppressed SLC proliferation in copper-deficient rats is poor copper status; poor iron status induced by copper deficiency had little influence on proliferation.