Cover Image

Not for Sale



View/Hide Left Panel
Click for next page ( 101


The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement



Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.

OCR for page 100
THE ROLE OF COPPER IN ERYTHROPOIESIS~ GEORGE E. CARTWRIGHT, CLARK J. GUBLER AND MAXWELL M. WINTROBEt Introduction. For the past eight years studies or the role of copper In erythropoiesis have been conducted in our laboratory in collaboration with Drs. Gubler and Wir~trobe. We have been assisted in this work by M. E. Lahey, \1. S. Chase, .r. A. Bush, W. N. Jensen, l. W. Athens, Helen Ashenbrucker and H. Markowitz and the results of our work have been published in detail in a series of articles.~~S The purpose of the present paper is to summarize these studies. Our research confirms in great part and e~cter~ds the much earlier observations of the Wisconsin group.9 A Pertinent literature on this subject has been reviewed ire previous publications.~~S . A deficiency of copper has heen nrodllced 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 ma. of copper/kg. of body weight daily in addition to iron. The animals were two to tern days of age at the start of the experiment. Description of the Anemia. During the first month of the copper-defic- ient dietary regime, there is generally little or no decline in the hemoglobin of- 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 de- creases from 15 to 2 gm./100 ml. and the N7.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 anoxla. The type of anemia which develops is microc~rtic and hypochromic, as in- dicated by a substantial decrease in the mean corpuscular volume (M.C.V.) and in the mean corpuscular hemoglobin concentration (M.C.fI.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). 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 morpho- logically 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 These investigations have been supported in part by a research grant (C-2231) from the National Institutes of Health, Public Health Service, and in part by a con- tract (AT (11-1)-82, Project 6) between the United States Atomic Energy Com- mission and the University of Utah. This paper was presented by Dr. Cartwright. 100

OCR for page 100
COPPER IN ERYTHROPOIESIS CARTWRIGHT ET AL. 1Ol ~ _ _ 70 64D >35 ~ ~ .C.V 30 ~~ tM.~,_~ ~ id_ 20 40 60 ways Q{^ge BE 100 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 ma. of copper/kg. of body weight/day. M.C.~., mean corpuscular volume; M.C.H.C., mean corpuscular hemoglobin con- centration; V.P.R.C., volume of packed red cells; R.B.C., red blood cells; Hb, hemo- globin; W.B.C., total leukocytes; P.M.~., polymorphonuclear leukocytes. (Lahey et al., Blood 7: 1053, 1952. By permission.) swine decreases from the normal value of about 186 ~g.~100 ml. to 0 to 25 ~g.~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 Agog of copper per red cell. Erythrocytes from anemic copper-deficient swine contain about 26 Agog of copper per cell. As

OCR for page 100
loo PART II. BIOSYNTHESIS OF HEMOGLOBIN- 300 t00 ~9 0~ ~ 60t ~,60 `5 40 H] ~3 S 20 CUT '13 ß13 8 2D ~ ID Pi9 22-3 ~ Pi . PFe ~ \_._- ~CU ~ ~ ._ ∑ -'---_ , relics ' , I `t 1 ~ ~1 a 40 60 80 100 110 Days olAge FIG. 2. Showing the development of hyp~oferremia, hypocupremia, microcytic, hy- pochromic anemia, leukopenia and neutropenia in a copper-deficient pig and the re- sponse to the oral administration of 0.5 ma. of copper/kg. of body weight/day. PFe, plasma iron; PCu, plasma copper; M.C.V., mean corpuscular volume; V.P.R.C., volume of packed red cells; W.B.C., total leukocyte count; P.M.N., polymorphonuclear leukocytes. (Lahey et al., Blood 7: 1053, 1952. By permission.) would be expected, the amount of copper in the tissues is greatly reduced (table III). The anemia responds rapidly and completely following the addition to the diet of 0.5 ma. 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

OCR for page 100
COPPER IN ERYTHROPOIESIS CARTWRIGHT ET AL. 103 TABLE I SUMMARY OF THE MORPHOLOGIC CHARACTERISTICS OF THE ERYTHROCYTES OF CONTROL, COPPER-DEFICIENT AND IRON-DEFICIENT SWINE V.P.R. C. ml 1100 ml. M.C.V. ,u3 M.C.H.C. per cent Reticulocytes per cent Bone Marrow L:E Ratio Copper Iron Control Deficient Deficient 42 55 33 5 1.8 21 29 4 0.6 21 36 28 9 0.8 V.P.R.C. refers to volume of packed red cells M.C.~. refers to mean corpuscular volume M.C.X.C. refers to mean corpuscular hemoglobin concentration L :E ratio lenl~ocyte-erythroid ratio TABLE II A COMPARISON OF THE BIOCHEMICAL CHARACTERISTICS OF COPPER \NTD OF IRONS D EFICIENC, Plasma Cu g/100 ml. R.B.C. Cu g/100 ml. Plasma Iron g/100 ml. T.I.B.C. pcg/100 ml. F.E.P. g/100 ml. Copper Iron Control Deficiency Deficiency 15 207 186 110 175 511 118 67 38 628 110 30 864 127 T.I.B.C. refers to total iron-binding capacity of the plasma. F.E.P. refers to free erythrocyte protoporphyrin. TABLE III TISSUE COPPER IN CONTROL, CCPP ER- D EFICIENT AND IRON -D EFICIENT SWIM E Copper Iron Control Deficient Deficient Liver 8.1 0.6 31.6 Spleen 0.04 0.02 0.04 Kidney 0.7 0.3 1.2 Heart Q.4 0.2 0.5 The values are expressed in ma. per organ.

OCR for page 100
104 PART II. BIOSYNTHESIS OF HEMOGLOBIN 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 disap- pears 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 ma. 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 ex- periment 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 TABLE IV TISSUE IRON MG/ ORGAN Copper Control D efficient Iron Deficient Blood 961 189 IS5 Liver 87 27 5 Kidney 7 3 1 Spleen 6 12 3 Heart 4 9 3 ~ _ Total (mg/kg.) 40 15 9 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 corner-deficient animals were fed the same amount < ~ , . ~ r 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 1mPaire( .. 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

OCR for page 100
COPPER IN ERYTHROPOIESISóCARTWRIGHT ET AL. 105 and heart was determined. Six per cent of the radioactivity administered was recovered in these tissues of the control pig and only taco 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 adminis- tration of large amounts of iron (tables NT and VI). Although it has been suggested in the past by ourselves' and by others I" ii TABLE V FAILURE OF INTRAVENCUS IRON TO PREVENT THE DEVELOPMENT OF ANEMIA IN COPPER-DEFICIENT SWINE Plasma Plasma Liver V.P.R.C. Copper Iron Iron Group ml./100 ml ~g/100 ml ~g/1OO ml ma. - Contro l 175 Copper- 19 8 15 27 D efficient Copper- 13 14 144 631 Deficient +I.V. Iron* * These animals were given one gram of iron intravenously at the beginning of the experiment, prior to the development of anemia. TABLE VI 186 FAILURE OF IRON ADMINISTERED INTRAVENOUSLY TO INDUCE A HEMOPOIETIC RESPONSE IN ANEMIC, COPPER-DEFICIENTT SWINE After Deficient V.P.R.C. ml/100 ml Reticulocytes per cent Iron* 21 10 M.C.V. 46 48 ,u3 M.C.H.C. 30 28 per cent Plasma Cu 14 18 g/100 ml Plasma Iron 48 140 ~g/100 ml T.I.B.C. 627 578 * 200 ma. of venously. ~g/lOO ml iron in the form of colloidal saccharated oxide of iron were administered intra- 1 efers to volume of slacked red cells. refers to mean corpuscular volume. refers to mean corpuscular hemoglobin concentration. refers to total iron-bin ding capacity of the plasma. ~ .~.~.C. M.C.V. M.C.H.C. T.I.B.C.

OCR for page 100
106 PART II. BIOSYNTHESIS OF HEMOGLOBIN 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). TABLE NIII FERROKINETIC STUDIES IN CONTROL AND COPPER-DEFICIENT SWINE AND IN SWINE WITH HEMCLYTIC ANEMIA Copper Hemolytic Control Deficient Anemia* Number of Pigs 20 4 3 Plasma Iron, ~g/100 ml 166 39 159 T-~/2 hours ~ 1.2 0.5 0.3 Plasma Iron Turnover Rate 1.1 I.7 4.9 mg/kg/24 hours To Injected Dose Incorporated 91 67 86 Into RBC RBC Iron Turnover Rate 0.6 1.1 4.1 mg/kg/24 hours Erythrocyte Survival 61 13 5 days * Induced with Phenylhydrazine. ~ Time required for one-half of the isotope to disappear from the plasma. 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 er~zymes would be reduced. Such is not the case (table VIII). Although the TABLE VIII HEMIN CHROMOPROTEINS % OF NORMAL VALUE Copper Iron Deficiency Deficiency Cytochrome C ( Heart) 124 46 Cytochrome Oxidase (Heart) 10 Catalase (Kidney) Myoglobin (Muscle) 65 121 104 99 25 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 com- partments. Erythrocyte Survival Shoddies. Calculation of erythrocyte survival time from ferrokinetic data indicates that the survival time of erythrocytes from cGpper-deficient pigs is shorter than normal time (table VII). Measurement of the erythrocyte survival time by the use of radioactive chromium con-

OCR for page 100
COPPER IN ERYTHROPOIESISóCARTWRIGHT ET AL. 107 firms 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 Life Span Donor Recipient No. Pigs Mean T 1/2 (Calc.) - Normal Normal 4 17 + 3.6 64 Cu-Deficient Cu-Deficient 10 9 + 1.9 33 Normal Cu-Deficient 4 16 + 3.6 58 Cu-Deficient Normal 5 13 + 2.2 49 Fe-Deficient Fe-Deficient 3 18 + 4.2 69 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 observa- tion is that copper may enter the "copper-deficient" red cells from normal plasma and correct the intracorpuscular defect. In support of this explana- tion, 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 Erythropolesis. The vital role of copper in erythro- poiesis 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 hemo- globin ~ 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 pro- duce 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 observa- tions which are compatible with this hypothesis. Copper is a normal con- stituent of the adult red cell (table II). In copper-deficient pigs, the con- centration of copper in the erythrocytes decreases from the normal value of 100 ,ug/ 100 ml. of packed cells to 67. Furthermore, when the anemia is

OCR for page 100
PART II. BIOSYNTHESIS OF HEMOGLOBIN 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 ob- servation is that the cells with the least amount of copper have been selectively destroyed. 120 Cod To 50 ~ 80 40 :~O :30 Y4D ~ 20 10 \ VP.~.C. ~ .. . . _ x\: ,x - ax - An<- _ _` 20 40 60 SD DID Days 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-de- ficient swine (mean of 7 piers). The role of copper within the erythrocytes is unknown. Dr. Harold Marko- . . ~ . . . . . WltZ In our I erythrocytes.1~ 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.43 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 aboratory has recently isolated a copper protein from human TABLE X Property Color Absorption Max. Mol. Wt. % Cu Atoms Cu / Mol. Isoelectric Point PHYSICAL AND CHEMICAL PROPERTIES OF ERYTHROCUPREIN AND CERULOPLASMIN Erythrocuprein Colorless None 3 5,000 .32 - .34 2 5.3 Ceruloplasmin Blue 605 my 151,000 .32 - .34 8 4.4

OCR for page 100
COPPER IN ERYTHROPOIESISóCARTWRIGHT ET AL. 109 . ~ . Ot plasn~.li At least 80 per cent of the copper in erythrocytes is accounted for in the erythrocuprein fraction. It is possible that the shortened erythro- c~te survival time of copper-deficient red cells is due to deficiency of this copper protein. Studies on tile precise function of erythrocuprein in the me- tabolism of red cells are now- under way in our laboratory. 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 sug- gests 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 intra- venous 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 in- creased rather than decreased in copper-deficient swine suggests that move- ment 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. Seminary. 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 arid survival. REFERENCES 1. Lahey, M. E., Gubler, C. J., Chase, M. S., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. II. Hematologic manifestations of copper deficiency in swine, Blood, 7: 1053, 1952. 2. Gubler, C. J., Lahey, M. E., Chase, M. C., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. III. The metabolism of iron in copper-de- ficient swine, Blood, 7: 1075, 1952. 3. Chase, M.S., Gubler, C. J., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. IV. The influence of copper on the absorption of iron, J. Biol. Chem., 199: 757, 1952. 4. Chase, M. S., Gubler, C. J., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. V. Storage of iron in liver of copper-deficient rats, Proc. Soc. Exper. Biol. and Med., &0: 749, 1952. Cartwright, G. E., Gubler, C. J., Bush, J. A., and Wintrobe, M. M.: Studies on copper metabolism. XVII. Further observations on the anemia of copper de- ficiency in swine, Blood, 11: 143, 1956. 6. Follis, R. H. Jr., Bush, J. A., Cartwright, G. E., and Wintrobe, M. M.: Studies