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Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, MN 55812
2To whom correspondence should be addressed. E-mail: jprohask{at}d.umn.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • copper deficiency • mice • development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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As with many micronutrients, the trace metal copper is essential for development of mammals. Not long after its seminal role in sustaining hemoglobin levels was discovered, copper was also found to be needed to prevent neonatal ataxia in lambs born to ewes grazing on pastures low in copper (1
). Copper is also important in reproduction. More than 70 y ago Keil and Nelson (2
) reported that copper, in addition to FeCl3, needed to be added to a milk diet so female rats could reproduce. Several other groups have carefully shown that copper deficiency can lead to reproductive failure and fetal resorption if the deficiency is imposed before mating (3
–5
). The limiting factor is likely copper itself because copper added to serum from copper-deficient rats can reverse the malformations observed in rat embryo cultures (6
). This role for copper in supporting fetal development has recently been highlighted when two independent groups reported death during midgestation in mice that were missing CTR1, the gene coding for the high affinity copper transport protein (7
,8
).
In humans a role for copper and reproduction is less well established. The primary potential developmental problem is the risk of copper deficiency in preterm infants 3–6 mo after birth (9
,10
). The recent dietary reference intakes established for copper were largely based on studies with adult men and postmenopausal women and suggest an adult recommended dietary allowance (RDA) of 900 µg for copper (11
). The RDA for pregnancy is 11% higher, 1000 µg and for lactation 44% higher, 1300 µg.
Is this extra copper sufficient? The purpose of the rodent experiments described herein was to evaluate the needs of copper for adult women compared with those during late gestation/lactation. Results indicate that the needs for copper to support perinatal development are far greater than for adult copper homeostasis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Adult male and female ND4 Swiss Webster mice were purchased commercially (Harlan Sprague Dawley, Indianapolis, IN). Mice received one of two dietary treatments, copper-deficient (-Cu)3
or copper-adequate (+Cu), consisting of a copper-deficient purified diet (Teklad Laboratories, Madison, WI) and either low copper drinking water or copper-supplemented drinking water, respectively. The purified diet was similar to the AIN-76A diet (12
,13
). It contained the following major components (g/kg diet): sucrose, 500; casein, 200; cornstarch, 150; corn oil, 50; cellulose, 50; modified AIN-76 mineral mix, 35; AIN-76A vitamin mix, 10; DL-methionine, 3; choline bitartrate, 2; and ethoxyquin 0.01. Cupric carbonate was omitted from the AIN-76 mineral mix. The purified diet contained 0.30 mg Cu/kg and 43 mg Fe/kg by chemical analysis. Mice on the -Cu treatment drank deionized water, whereas +Cu treatment groups drank water that contained 20 mg Cu/L by adding CuSO4. Mice were given free access to food and drinking water. All mice were housed at 24°C with 55% relative humidity on a 12-h light cycle (0700–1900 h). All protocols were approved formally by the University of Minnesota Animal Care Committee.
In expt. 1, four breeding units were set up two to three females/male. Males were rotated to each unit and then removed. Females were randomly divided into two groups 2 wk after the units were first set up. The day of parturition was considered embryonic d 21 (E21) or postnatal d 0 (P0). Day of birth, litter size, and survival were recorded. In expt. 1, dams were killed at P13 because -Cu pups were all dead. Experiment 1 was repeated partially with four females placed on the -Cu treatment after the first trial ended with poor pup survival.
In expt. 2, four breeding units were set up and dietary treatments were delayed until E19. Offspring were weaned when 3 wk old and maintained on the same treatment as their respective dams for an additional wk after transfer to stainless steel cages. A total of nine litters (four +Cu and five -Cu) were sampled. Male offspring were killed at P28 to establish copper status. This paradigm is similar to that described previously (14
).
An additional study, expt. 3, was conducted in which 10 age-matched, adult female mice were placed on either +Cu or -Cu treatments for 3 wk to compare with the dams in expt. 1. This time-frame corresponds with E13 to P13 of expt. 1.
Mice were anesthetized with diethyl ether and decapitated. A sample of blood was collected to measure hematocrit and hemoglobin. Livers were removed and processed for biochemical analysis.
Biochemical analyses.
Plasma from the hematocrit tubes was used to measure ceruloplasmin activity by following the oxidation of o-dianisidine (15
). Approximately 1-g portions of liver and diet were weighed to the nearest 0.1 mg and were wet-digested with 4 mL of concentrated HNO3 (TraceMetal grade; Fisher Scientific, Pittsburgh, PA), and the residue was brought to 4.0 mL with 0.1 mol/L HNO3. Samples were then analyzed for total copper and iron by flame atomic absorption spectroscopy (Model 2380; Perkin-Elmer, Norwalk, CT). The method was checked with a certified standard, U.S. National Bureau of Standards 1577 bovine liver (Gaithersburg, MD).
Statistics.
Dietary treatment effects were evaluated by Student’s t test after variance equality was tested or by factorial ANOVA and Scheffe’s test. Data were analyzed using a personal computer and statistical software (Statview 4.5; Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, all the pups from the three -Cu litters were dead by P13, whereas all three litters of +Cu pups were healthy and survived to weaning. In expt. 2, when the start date for treatments was delayed until E19, there were no differences in pregnancy rate, litter size, or survival to weaning between +Cu and -Cu dams (Table 1)
. We repeated expt. 1 (treatment onset E13) with four additional -Cu females. Two of four delivered pups, but none survived beyond P17.
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). Compared with P28 +Cu males, -Cu males were anemic (lower hematocrit), had near total loss of ceruloplasmin diamine oxidase activity, had a 50% lower liver copper concentration, and had a robust increase in liver iron concentration. These features are prominent characteristics of rodents after dietary copper deficiency (14
).
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).
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. There were significant interactions between diet and physiological state because the -Cu dams were clearly different from the other three treatment groups. Body weights differed between dams and nonpregnant mice, as expected. We estimate that the -Cu dams and nonpregnant female mice were consuming 
0.05 mg Cu/kg body. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In that study, poor pup survival was mentioned but not documented when -Cu treatment began at E14. What is the critical copper-dependent process?
It is generally believed that the biological functions of copper are expressed via specific cuproenzymes. It is difficult to pinpoint which cuproenzyme might be limited by dietary copper restriction and explain the outcomes in the current studies. Experiments with mutant mice can clarify this process somewhat. Mice not expressing the cuproenzymes ceruloplasmin, Cu, Zn-superoxide dismutase, or tyrosinase survive and show no major reproductive problems. Mice not expressing certain homeostatic genes such as the membrane transporter CTR1, the chaperone Atox1, or the efflux transporter ATP7A die in utero or shortly after birth (7
,8
,17
,18
). Likewise, mice devoid of dopamine-ß-monooxygenase die in utero (19
). The consequences of total loss of cytochrome c oxidase, peptidylglycine 
-amidating monooxygenase or lysyl oxidase via null mice have not yet been published; however, their deletion would likely be lethal. One or more key cuproenzyme reductions could explain the outcome of the present studies.
It could be questioned whether mice are a good mammalian model for perinatal copper nutrition. Previous studies have indicated that longitudinal changes in milk copper in mice parallel those of humans more closely than rats (20
–22
). In both mice and humans, milk copper changes little throughout lactation, whereas in rats, milk copper drops rapidly from a higher initial level. Mouse milk copper concentration drops significantly after dietary copper deficiency (23
). The copper status of infants who are drinking formula rather than breast milk can be affected by factors such as thermal processing and iron supplementation (24
). Thus, it is possible to influence copper status of human infants by alteration in diet or infant formula.
It could be argued that the changes we observed in mice were due to severe copper deficiency. Recall, however, that the same protocol that failed to provide enough copper for perinatal survival did not alter any biochemical (clinical) features of adult females. Thus, the copper requirement in mice for pregnancy and lactation must far exceed that for the nonpregnant adult. We estimated that our -Cu female mice were consuming 0.05 mg Cu/kg body. This exceeds the copper intake of a 60-kg woman consuming the copper RDA (0.9 mg) or a 65-kg pregnant woman consuming 1.3 mg of copper. Both result in an estimate of 0.02 mg Cu/kg. Comparisons between species based on body weight may not be apropos. A 2500 kcal/d (10.46 MJ/d) human diet containing 500 g of dry matter and the RDA of copper, 0.9 mg, corresponds with 1.8 mg Cu/kg. This would be considered a marginally copper-deficient diet for reproducing rodents. Perhaps the target group most susceptible to altered copper status is young females who become pregnant. Hunt and Meacham (25
) reported an average daily copper intake for this group of 720 µg, which is below the RDA of 890 µg. Because it is currently difficult, if not impossible, to detect biochemically marginal Cu deficiency, efforts should be made to develop suitable methods for this purpose and to encourage adequate copper intake to support development of the fetus and infant.
It should also be pointed out that alterations to the immune system and cardiovascular system of rats were documented in offspring of dams maintained on marginal copper treatments of 2.8 mg Cu/kg (26
,27
). In fact, no biochemical differences were detected between control rats and those on chronic marginal Cu intake. The brain development of rats can also be impaired by moderate copper deficiency of 1.8 mg Cu/kg body (28
). If copper deprivation is continued throughout lactation, the offspring demonstrate persistent irreversible behavioral changes even when repleted with copper for 5 mo (29
). It is important to determine the copper requirement for human fetal development and for human infants. It is equally important that we recognize that copper supplementation may be necessary during gestation and lactation to provide the copper RDA or to provide extra copper in case the current RDA is set too low.
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FOOTNOTES |
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3 Abbreviations used: +Cu, copper-adequate; -Cu, copper-deficient; E, embryonic day; P, postnatal day; RDA, recommended dietary allowance.
Manuscript received 22 May 2002. Initial review completed 3 July 2002. Revision accepted 15 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Bennetts, H. W. & Chapman, F. E. (1937) Copper deficiency in sheep in Western Australia: a preliminary account of the etiology of enzootic ataxia of lambs and an anemia of ewes. Aust. Vet. J. 13:138-149.
2. Keil, H. L. & Nelson, V. E. (1931) The role of copper in hemoglobin regeneration and in reproduction. J. Biol. Chem. 93:49-57.
3. Dutt, B. & Mills, C. F. (1960) Reproductive failure in rats due to copper deficiency. J. Comp. Pathol. 70:120-125.[Medline]
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5. Beguin, D. P., Kincaid, R. L. & Hargis, A. M. (1985) Fetal death in copper-deficient rats. Nutr. Rep. Intl. 31:991-999.
6. Mieden, G. D., Keen, C. L., Hurley, L. S. & Klein, N. W. (1986) Effects of whole rat embryos cultured on serum from zinc- and copper-deficient rats. J. Nutr. 116:2424-2431.
7. Kuo, Y. M., Zhou, B., Cosco, D. & Gitschier, J. (2001) The copper transporter CTR1 provides an essential function in mammalian embryonic development. Proc. Natl. Acad. Sci. USA 98:6836-6841.
8. Lee, J., Prohaska, J. R. & Thiele, D. J. (2001) Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development. Proc. Natl. Acad. Sci. USA 98:6842-6847.
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11. Trumbo, P., Yates, A. A., Schlicker, S. & Poos, M. (2001) Dietary reference intakes: vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. J. Am. Diet. Assoc. 101:294-301.[Medline]
12. Anonymous (1977) Report of the AIN Ad Hoc Committee on standards for nutritional studies. J. Nutr. 107:1340-1348.
13. Anonymous (1980) Second report of the AIN Ad Hoc Committee on standards for nutritional studies. J. Nutr. 110:1726.
14. Prohaska, J. R. & Bailey, W. R. (1993) Persistent regional changes in brain copper, cuproenzymes and catecholamines following perinatal copper deficiency in mice. J. Nutr. 123:1226-1234.
15. Prohaska, J. R. (1991) Changes in Cu,Zn-superoxide dismutase, cytochrome c oxidase, glutathione peroxidase and glutathione transferase activities in copper-deficient mice and rats. J. Nutr. 121:355-363.
16. Prohaska, J. R. & Bailey, W. A. (1993) Copper deficiency during neonatal development alters mouse brain catecholamine levels. Nutr. Res. 13:331-338.
17. Hamza, I., Faisst, A., Prohaska, J., Chen, J., Gruss, P. & Gitlin, J. D. (2001) The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis. Proc. Natl. Acad. Sci. USA 98:6848-6852.
18. Levinson, B., Vulpe, C., Elder, B., Martin, C., Verley, F., Packman, S. & Gitschier, J. (1994) The mottled gene is the mouse homologue of the Menkes disease gene. Nat. Genet. 6:369-373.[Medline]
19. Thomas, S. A., Matsumoto, A. M. & Palmiter, R. D. (1995) Noradrenaline is essential for mouse fetal development. Nature 374:643-646.[Medline]
20. Reis, B. L., Keen, C. L., Lonnerdal, B. & Hurley, L. S. (1991) Longitudinal changes in the mineral composition of mouse milk and the relationship to zinc metabolism of the suckling neonate. J. Nutr. 121:687-699.
21. Casey, C. E., Hambidge, K. M. & Neville, M. C. (1985) Studies in human lactation: zinc, copper, manganese and chromium in human milk in the first month of lactation. Am. J. Clin. Nutr. 41:1193-1200.
22. Keen, C. L., Lonnerdal, B., Clegg, M. & Hurley, L. S. (1981) Developmental changes in composition of rat milk: trace elements, minerals, protein, carbohydrate and fat. J. Nutr. 111:226-236.
23. Prohaska, J. R. (1989) Effect of diet on milk copper and iron content of normal and heterozygous brindled mice. Nutr. Res. 9:353-356.
24. Lonnerdal, B., Kelleher, S. L. & Lien, E. L. (2001) Extent of thermal processing of infant formula affects copper status in infant rhesus monkeys. Am. J. Clin. Nutr. 73:914-919.
25. Hunt, C. D. & Meacham, S. L. (2001) Aluminum, boron, calcium, copper, iron, magnesium, manganese, molybdenum, phosphorus, potassium, sodium, and zinc: concentrations in common western foods and estimated daily intakes by infants; toddlers; and male and female adolescents, adults, and seniors in the United States. J. Am. Diet. Assoc. 101:1058-1060.[Medline]
26. Hopkins, R. G. & Failla, M. L. (1995) Chronic intake of a marginally low copper diet impairs in vitro activities of lymphocytes and neutrophils from male rats despite minimal impact on conventional indicators of copper status. J. Nutr. 125:2658-2668.
27. Wildman, R. E., Hopkins, R., Failla, M. L. & Medeiros, D. M. (1995) Marginal copper-restricted diets produce altered cardiac ultrastructure in the rat. Proc. Soc. Exp. Biol. Med. 210:43-49.[Abstract]
28. Hunt, C. D. & Idso, J. P. (1995) Moderate copper deprivation during gestation and lactation affects dentate gyrus and hippocampal maturation in immature male rats. J. Nutr. 125:2700-2710.
29. Prohaska, J. R. & Hoffman, R. G. (1996) Auditory startle response is diminished in rats after recovery from perinatal copper deficiency. J. Nutr. 126:618-627.
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