Nutritional Anemias, edited by S. J. Fomon and S. Zlotkin, Nestle Nutrition Workshop Series, Vol. 30, Nestec Ltd., Vevey/Raven Press. Ltd., New York © 1992. Copper Deficiency in Term and Preterm Infants Jonathan C. L. Shaw Department of Paediatrics, Faculty of Clinical Sciences, University College London, The Rayne Institute, London, England, United Kingdom In 1928 Hart et al. (1) demonstrated that copper was essential for erythropoiesis in the rat. They showed that rats fed a milk diet developed an anemia that could not be corrected by iron alone. It was cured, however, by the addition of ash of liver, lettuce, or corn. By precipitating copper from acid extracts of the ash with hydrogen sulfide, they were able to show that it was the copper in the ash that was essential for the full reversal of the anemia. As a result of this work, copper deficiency has been recognized as a cause of anemia in man, although the full syndrome has a number of other features. It is now known that copper is a component of several enzymes, some of which are listed in Table 1. These enzymes generally bind between 1 and 8 gram-atoms of copper per mole, and the presence of copper is essential for their activity. Many of them engage in oxidation-reduction reactions. Some, but not all, of the features of copper deficiency can be explained by the functions of these enzymes. THE ANEMIA OF COPPER DEFICIENCY The anemia of copper deficiency has been dealt with in a number of reviews which should be referred to as a source of references (2-6). Most of the investigations on the etiology of the anemia in copper deficiency have been carried out in experimental animals, and the difference between species means that their results must be inter- preted with caution. Nevertheless, it seems probable that the anemia results from defects in at least two points where copper enzymes interact with iron metabolism. Release of Iron from Iron Stores In copper-deficient swine, ceruloplasmin levels fall to less than 1% of normal and there is an associated fall in the serum iron. Iron absorption is diminished and iron accumulation is observed in the intestinal mucosal cells, in the adjacent macro- 105 106 COPPER DEFICIENCY IN INFANTS TABLE 1. Some copper enzymes in humans Cytochrome c oxidase Ceruloplasmin Superoxide dismutase Tyrosinase Dopamine p hydroxylase Lysyl oxidase phages, and in the parenchymal cells of the liver, suggesting a difficulty in the release of iron from iron stores. Injection of iron in the form of colloidal iron or storage- damaged erythrocytes results in trapping of the iron in the iron stores and reticu- loendothelial cells of the copper-deficient animals. Injection of ceruloplasmin causes a prompt rise in the plasma iron concentration, indicating a release of iron from iron stores. These data are consistent with the hypothesis that ceruloplasmin acts as a ferroxidase oxidizing ferrous (Fe2+) iron to ferric (Fe3+) iron prior to its incorpo- ration into plasma transferrin (2,5). The data do not, however, account entirely for the anemia of copper deficiency for a number of reasons. In the first place, parenteral iron does not fully correct the anemia of copper deficiency (7) and perfusion with ceruloplasmin does not augment the transfer of iron from the serosal surface of isolated perfused intestine of copper- deficient rats (8). Also in Menkes' disease and Wilson's disease, where the ceru- loplasmin is low, anemia is not a feature (6). Finally there is considerable variability of ceruloplasmin oxidase activity amongst different species, and it is said to be vir- tually absent in the plasma of the turkey, the peacock, and certain crocodiles (4). Clearly much remains to be explained. Synthesis of Heme The final stage of heme synthesis involves the insertion of iron into protoporphyrin IX, and this takes place within the inner mitochrondrial membrane. It requires the participation of ferrochelatase and a supply of electrons to reduce ferric (Fe3+) to ferrous (Fe2+). Though there has been some evidence that ferrochelatase activity may be reduced in copper deficiency (9), there has been no recent confirmation of this. The anemia of copper deficiency is a sideroblastic, microcytic, hypochromic ane- mia. The bone marrow shows vacuolated erythroid and myeloid precursors with ringed sideroblasts which accumulate iron in the cytosol. Such anemias are char- acteristic of ineffective erythropoiesis, implying a defect in iron metabolism, heme synthesis, or globin chain synthesis. In copper deficiency there is no defect in pro- phyrin synthesis, and because both iron and protoporphyrin accumulate in the er- ythroid cells of copper-deficient animals the precursors of heme synthesis are not lacking. Iron uptake by copper-deficient reticulocytes from transferrin has been COPPER DEFICIENC Y IN INFANTS 107 shown to be 52% of normal, whereas heme synthesis was reduced by 33% (10). Synthesis of heme from protoporphyrin IX and Fe2+ is not impaired in tissues from copper-deficient animals, but synthesis from protoporphyrin IX and Fe3+ is. Syn- thesis of heme from protoporphyrin IX and ferric iron is dependent on a supply of substrate for electron transport. Inhibitors of the citric acid cycle and electron trans- port chain such as malonate, rotenone, antimycin A, and cyanide profoundly inhibit heme synthesis (10), whereas inhibitors of ATP synthesis such as 2,4-dinitrophenol do not. This has been interpreted as indicating that intact electron transport rather than ATP synthesis is the essential function. Reduction in the activity of cytochrome c oxidase is a regular finding in copper deficiency, and it is probable that the defect of heme synthesis contributes both to the reduced activity of cytochrome c oxidase and to the sideroblastic anemia. COPPER DEFICIENCY IN TERM AND PRETERM INFANTS The features of copper deficiency in infants given below are based on 51 cases reported in the pediatric literature since 1956 (11-32). Of the 51 cases, 49 were 18 months or less at the time of diagnosis (11,12,13-15,17-32). Those regarded as full term include seven infants who are presumed to be full term but whose birth weight or gestation are not given. The low-birth-weight infants are infants whose birth weight is given or who are stated to be of low birth weight (<2.5 kg). Their median birth weight was 1.040 kg (range 0.68-2.3 kg). The features of the syndrome of copper deficiency in infants are as follows: • Psychomotor retardation • Hypotonia • Hypopigmentation • Prominent scalp veins in palpable periosteal depressions • Pallor • Sideroblastic anemia, resistant to iron therapy. Bone marrow shows vacuolated erythroid and myeloid cells with iron deposition in the vacuoles • Hepatosplenomegaly (a feature of sideroblastic anemia) • Neutropenia, usually <1.0 x 109/l • X-ray changes of osteoporosis, blurring and cupping of the metaphyses, sickle- shaped metaphyseal spur formation, subperiosteal new bone formation and fractures • Plasma copper level usually <6.3 (imol/1 (<40 jJLg/dl), and ceruloplasmin <130 mg/1 The etiology of the sideroblastic anemia has been discussed above. The prominent scalp veins and bone changes are thought to result from impaired collagen and elastin cross-linking due to depressed lysyl oxidase activity. The consequent reduction in strength of the bone collagen causes fragile bones that fracture easily. The hypo- pigmentation is probably due to depressed tyrosinase activity and impaired melanin synthesis. The cause of the neutropenia is not understood. 108 COPPER DEFICIENC Y IN INFANTS PREDISPOSING FACTORS In the published reports, one or more of a number of predisposing factors have been present in every case. These are discussed below. Low Birth Weight Forty percent of reported cases were less than 2.5 kg at birth (11,12,14,15,18,26, 27,29,30,32). Low-birth-weight infants have lower body stores of copper at birth than do full-term infants [55 (imol (3.5 mg) per kilogram fat-free body weight at 20 weeks' gestation rising to 76 |imol (4.8 mg) at term (33)]. During the last 3 months of gestation they accumulate copper at a daily rate of about 0.8 nmol (51 |xg) per kilogram (4). If, however, they are born prematurely they may be in negative copper balance for up to 6 weeks after birth (34). Full-term infants also have a period of negative balance following birth (35). Though this will cause a transient decline in the total body copper, it is not thought to cause copper deficiency if the subsequent dietary copper intake and absorption are sufficient. The full-term infant at least has a high concentration of copper in the liver (36), and the current evidence suggests that full-term infants have stores sufficient for at least 5 months and that low-birth- weight infants have them for at least 2 months. Dietary Deficiency of Copper Total Parenteral Nutrition Twenty-three percent of the infants received total parenteral nutrition (12,13,20- 22,24,29,31). In eight of the cases the solutions were deficient in copper. In the three other cases (29) copper had been added to the solutions, but other predisposing causes, namely prematurity and copper-deficient milk, were present. Copper-Deficient Milk Fifty-four percent of infants were fed exclusively or predominantly on cow's milk with very short periods of breast-feeding (12,15,16,19,23,26,28). A further 12% were fed formulas (11,14,17,18,32) of which at least one was copper-deficient (11). The changes in the concentration of copper in breast milk are given in Tables 2 and 3. Cow's milk contains a much lower concentration of copper than does breast milk. The copper content of cow milk is stated to be 3.1 jtmol /I, range 1.6-4.7 (20 jxg/dl, range 10-30) (39), and in one report values as low as 0.9 jimol/l (5.7 jjug/dl) were found (26).