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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 101
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 102
loo
PART II. BIOSYNTHESIS OF HEMOGLOBIN-
300
t00
~9
0~
~ 60t
~,60 `5
40 H]
~3
S
20 CUT
'13 §13
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 103
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 104
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 105
COPPER IN ERYTHROPOIESIS—CARTWRIGHT 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 106
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 107
COPPER IN ERYTHROPOIESIS—CARTWRIGHT 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 108
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 109
COPPER IN ERYTHROPOIESIS—CARTWRIGHT 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
Representative terms from entire chapter:
mean corpuscular
