GEORGE E.CARTWRIGHT, CLARK J.GUBLER AND MAXWELL M.WINTROBE ^†

Introduction. For the past eight years studies on the role of copper in erythropoiesis have been conducted in our laboratory in collaboration with Drs. Gubler and Wintrobe. We have been assisted in this work by M.E.Lahey, M.S.Chase, J.A.Bush, W.N.Jensen, J.W.Athens, Helen Ashenbrucker and H.Markowitz and the results of our work have been published in detail in a series of articles.^1–8 The purpose of the present paper is to summarize these studies. Our research confirms in great part and extends the much earlier observations of the Wisconsin group.^9,10 Pertinent literature on this subject has been reviewed in previous publications.^1–8

A deficiency of copper has been produced in swine by feeding a diet of homogenized evaporated milk to which a liberal amount of “copper-free” iron was added (30 mg./kg. body weight daily). Control animals were given 0.5 mg. of copper/kg. of body weight daily in addition to iron. The animals were two to ten days of age at the start of the experiment.

Description of the Anemia. During the first month of the copper-deficient dietary regime, there is generally little or no decline in the hemoglobin or volume of packed red cells (V.P.R.C.) (fig. 1). Thereafter, a precipitous fall in these values occurs. If the animals are not treated, the hemoglobin decreases from 15 to 2 gm./100 ml. and the V.P.R.C. from 40 to 8 ml/100 ml in about 40 to 50 days. The animals become extremely pale and weak, the respiratory rate increases, and death supervenes, apparently as a result of tissue anoxia.

FIG. 1.

Showing the development of microcytic, hypochromic anemia, leukopenia and neutropenia in a pig deficient in copper and the response of the blood to an oral administration of 0.5 mg. of copper/kg. of body weight/day. M.C.V., mean corpuscular volume; M.C.H.C., (more...)

The type of anemia which develops is microcytic and hypochromic, as indicated by a substantial decrease in the mean corpuscular volume (M.C.V.) and in the mean corpuscular hemoglobin concentration (M.C.H.C.) as well as by marked microcytosis and hypochromia of the erythrocytes in the blood smear. The anemia is not accompanied by a significant reticulocytosis (table I).

TABLE I

SUMMARY OF THE MORPHOLOGIC CHARACTERISTICS OF THE ERYTHROCYTES OF CONTROL, COPPER-DEFICIENT AND IRON-DEFICIENT SWINE.

Examination of the marrow of copper-deficient pigs reveals hyperplasia of erythroid elements. The normoblasts are predominantly polychromatophilic. Thus, the anemia associated with a deficiency of copper in the pig is morphologically similar in all respects to that due to a deficiency of iron (table I).

Blood and Tissue Copper and Response to Copper. Within 14 days of the start of the experiment the plasma copper level of the copper-deficient swine decreases from the normal value of about 186 µg./100 ml. to 0 to 25 µg./100 ml. and remains in this range until the animals are treated with copper (fig. 2). The copper in the red cells is depleted somewhat more slowly and to a lesser degree than is plasma copper (table II). Normal corpuscles of swine contain about 61 µµµg of copper per red cell. Erythrocytes from anemic copper-deficient swine contain about 26 µµµg of copper per cell. As would be expected, the amount of copper in the tissues is greatly reduced (table III).

FIG. 2.

Showing the development of hypoferremia, hypocupremia, microcytic, hypochromic anemia, leukopenia and neutropenia in a copper-deficient pig and the response to the oral administration of 0.5 mg. of copper/kg. of body weight/day. PFe, plasma iron; PCu, (more...)

TABLE II

A COMPARISON OF THE BIOCHEMICAL CHARACTERISTICS OF COPPER AND OF IRON DEFICIENCY.

TABLE III

TISSUE COPPER IN CONTROL, COPPER-DEFICIENT AND IRON-DEFICIENT SWINE.

The anemia responds rapidly and completely following the addition to the diet of 0.5 mg. of copper/kg, body weight/day. Three to eight days following the initiation of such therapy, the reticulocyte increase ranges between 18 and 45 per cent. Simultaneously with the increase in reticulocytes, there is an increase in the mean corpuscular volume to values within or above the normal range. A rapid increase in the erythrocyte count, hemoglobin and

V.P.R.C. ensues, and by three weeks after the initiation of therapy the blood has returned to normal. In general, the hemoglobin level increases more slowly than does the V.P.R.C., with the result that the microcytosis disappears before the mean corpuscular hemoglobin concentration returns to normal. The plasma copper level increases significantly within 24 hours and reaches the normal level in about five days.

Iron Metabolism. Because of the morphologic similarities between the anemias of copper and of iron deficiency, various aspects of iron metabolism have been investigated in copper-deficient pigs. In spite of the fact that the copper-deficient pigs received 30 mg. of iron/kg, body weight/day from the beginning of the experiment, the level of iron in the plasma was reduced to levels comparable to those observed in iron-deficient swine (table II). Maxi-mal reduction in the plasma iron level occurs early in the course of the experiment and persists throughout the duration of the copper deficiency (fig. 2). The hypoferremia is accompanied by an increase in the total iron-binding capacity of the plasma, with the result that there is a marked reduction in the per cent saturation of transferrin with iron (table II).

Analyses of the livers and kidneys of copper-deficient pigs for iron reveal that there is a distinct decrease in the amount of iron in these organs (table IV). The amount of iron in the spleen is increased; the amount in the heart is not significantly altered. Since most of the iron in the animal is normally contained in the hemoglobin in the circulating erythrocytes, and since there is a great reduction in the amount of circulating hemoglobin in the copper-de-ficient animals, it is apparent that the total amount of iron in the body is greatly reduced. Since the copper-deficient animals were fed the same amount of iron over the course of the experiment as were the litter-mate control pigs, this observation suggests that the absorption of iron by copper-deficient pigs is impaired.

TABLE IV

TISSUE IRON MG/ORGAN.

In order to demonstrate more conclusively that the absorption of iron is impaired by a deficiency of copper, two copper-deficient and one control pig were given oral radioiron daily for 12 days. The animals were sacrificed 14 days later and the amount of radioactivity in the liver, blood, spleen, kidney and heart was determined. Six per cent of the radioactivity administered was recovered in these tissues of the control pig and only two per cent in the tissues of each of the copper-deficient animals. A similar type of experiment has been performed in rats, and again it was possible to demonstrate that in the absence of an adequate amount of copper, iron is not absorbed at the normal rate.³

That the anemia associated with a deficiency of copper is not due to failure to absorb iron can be readily demonstrated by the fact that the development of the anemia is neither prevented nor alleviated by the intravenous administration of large amounts of iron (tables V and VI).

Although it has been suggested in the past by ourselves² and by others^9,10,11 that the mobilization of iron from tissue stores is impaired in copper deficiency, recent studies in our laboratory do not substantiate this suggestion (table VII). The plasma iron turnover rate in copper-deficient pigs is even greater than in normal pigs and the amount of iron turned over through red cells per day was about twice as great in the deficient pigs as in the normal control animals. Furthermore, if the mobilization of iron were impaired by a deficiency of copper, then it might be expected that the activity of all heme-containing enzymes would be reduced. Such is not the case (table VIII). Although the cytochrome oxidase activity of heart is greatly reduced, the cytochrome C activity of heart muscle, the catalase activity of renal tissue, and the myoglobin content of muscle are not reduced, even in severely deficient animals. Thus, it would seem that although copper is involved in the absorption of iron, it is not concerned directly with the movement of iron between the body compartments.

TABLE VII

FERROKINETIC STUDIES IN CONTROL AND COPPER-DEFICIENT SWINEAND IN SWINE WITH HEMOLYTIC ANEMIA.

TABLE VIII

HEMIN CHROMOPROTEINS % OF NORMAL VALUE.

Erythrocyte Survival Studies. Calculation of erythrocyte survival time from ferrokinetic data indicates that the survival time of erythrocytes from copper-deficient pigs is shorter than normal time (table VII). Measurement of the erythrocyte survival time by the use of radioactive chromium confirms this observation (table IX). The fact that erythrocytes from a normal pig, when transfused into a copper-deficient pig, do survive a normal period of time suggests that the shortened life-span of the copper-deficient cells is not due to an extracorpuscular abnormality.

TABLE IX

SURVIVAL OF RED CELLS TAGGED WITH RADIOACTIVE CHROMIUM.

On the other hand, if the decreased life span were due to an intracorpuscular cause, one would expect that the copper-deficient cells would not survive for a normal period when transfused into a normal recipient. Such is not the case. When cells from a copper-deficient pig are transfused into a normal pig, the survival time approaches normal. An explanation for this observation is that copper may enter the “copper-deficient” red cells from normal plasma and correct the intracorpuscular defect. In support of this explanation, it has been demonstrated that radiocopper, when added to plasma either in vitro or in vivo, is taken up by erythrocytes within several hours.⁷

Role of Copper in Erythropoiesis. The vital role of copper in erythropoiesis is confirmed by these studies. However, the manner whereby copper so profoundly influences erythropoiesis is obscure.

Since the daily hemoglobin (or red cell) production of normal pigs may be increased fourfold under the stimulus of anemia, and since the rate of hemoglobin (or red cell) production in copper-deficient pigs is only 1.1 to 1.3 times greater than in normal animals, it is apparent that the ability to produce hemoglobin is greatly impaired in copper-deficient swine. Furthermore, both ferrokinetic studies and chromium erythrocyte survival studies indicate that the life-span of the erythrocyte in copper deficiency is shortened. It seems, therefore, that anemia develops in the absence of copper because of a limitation of the capacity of the marrow to produce cells and because of a shortened erythrocyte survival time.

A possible explanation for the decreased survival time of the erythrocytes is that the copper is an essential component of adult red cells and when the copper concentration of the erythrocyte is below a certain minimal, critical level, the survival time of the cells is shortened. There are several observations which are compatible with this hypothesis. Copper is a normal constituent of the adult red cell (table II). In copper-deficient pigs, the concentration of copper in the erythrocytes decreases from the normal value of 100 µg/100 ml. of packed cells to 67. Furthermore, when the anemia is severe there is a tendency for the concentration of copper within the red cells to increase slightly (fig. 3). One possible explanation of the latter observation is that the cells with the least amount of copper have been selectively destroyed.

FIG. 3.

Showing the development of anemia (V.P.R.C., volume of packed red cells), and decrease in plasma copper (PCu) and red cell copper (RBC Cu) in copper-deficient swine (mean of 7 pigs).

The role of copper within the erythrocytes is unknown. Dr. Harold Markowitz in our laboratory has recently isolated a copper protein from human erythrocytes.¹² A few of the physical characteristics of this protein are given in table X. We have chosen to call this compound erythrocuprein, rather than hemocuprein, the name originally used by Mann and Keilin.¹³ Its physical characteristics seem to be slightly different than those of the protein isolated by them and the name, erythrocuprein, clearly distinguishes this red cell protein from another “hemocuprein” ceruloplasmin, the copper protein of plasma.¹⁴ At least 80 per cent of the copper in erythrocytes is accounted for in the erythrocuprein fraction. It is possible that the shortened erythrocyte survival time of copper-deficient red cells is due to deficiency of this copper protein. Studies on the precise function of erythrocuprein in the metabolism of red cells are now under way in our laboratory.

TABLE X

PHYSICAL AND CHEMICAL PROPERTIES OF ERYTHROCUPREINAND CERULOPLASMIN.

Because of the striking morphologic and biochemical similarities between the anemias of iron and of copper deficiency, we have suggested that copper may in some manner influence the metabolism of iron. It is apparent from our studies that copper profoundly influences the absorption of iron. This suggests that a copper protein may be involved in some manner in the mechanism by which iron is absorbed from the gastro-intestinal tract. However, copper does not exert its influence on erythropoiesis by virtue of this effect on iron absorption since the anemia is neither prevented nor alleviated by the intravenous administration of iron. That the activity of all heme chromoproteins is not uniformly depressed suggests that not all of the metabolic pathways of iron in the body are impaired. Furthermore, our recent observation that the daily turnover rate of iron through the plasma and into the red cells is increased rather than decreased in copper-deficient swine suggests that movement of iron within the body is not curtailed. The relation of copper to iron metabolism must be in some way concerned with the role of copper in red cell synthesis.

Summary. Swine deficient in copper develop a severe anemia which is morphologically indistinguishable from the anemia of iron deficiency. It appears that anemia develops in the absence of copper because of the limited capacity of the marrow to produce cells. It is suggested that deficiency of the erythrocyte copper compound erythrocuprein, is in some way related to these alterations in erythrocyte production and survival.