Studies

on

Copper of Iron

Metabolism in Copper
%(J), iSI. M.
\\INTROBE,

III.
By C. J 0.

The
GUBLER,

Metabolism
PH.D.,
CAItTw-RIGIIT,

Deficient
S.

Swine
CInAsE,t

NI.

E. M.1).

LAIIEY,* AND

M.D.,

E.

M.1).

I
mia

THE PREVIOUS copper, like swine with normoblastic iron-binding protoporphyrin states are
were

PAIER it was pointed out. that swine deficient. in deficient in iron, develop a microcytic hypochiomic anehyperplasia of the 1)01w marrow, hypoferremia and iii-

creased of free ficiency

res)ectively,

capacity of the plasma, without alteration in the quantity in the erythrocytes. In all of these respects these two (ICsimilar in spite of the fact that ample amounts of iron or copper,
furnished

These
deficient
iron.

morphologic
animals the

and
anemia

the copper i)iochemical

may be the

and the similarities

result
this

iroim

deficient suggest.
could

that
l)e

animals. in the
the failure by result to (a)

copperof
of utilize abnorto to (1)

of a defect
defect

in the
or with of there (2)

metabolism

From
to for

a hypothetical
absorb

point
from

of view

inability

iron

iron
mality

hemoglobin

synthesis.

the gastro-intestinal The latter defect.

in the liver

tract;

could
tissues inability is that

l)e caused!
resulting

in the iron
the
iron.

storage from
storage

of iron the
arid

or other or
of iron
(b)

failure

mobilize
utilize absorption,

storage
hypothetical mobilization

tissues;

the

bOne on

maiow the

A third

possibility as

is a defect

well

as inability
pigs in the
is

in the part of

the
irons

nucleated
ring.

red
or

cells even

in the
the plasma increased

hone
of

marrow
degree,

to incorporate
a defect.

porphyrin mechanism The earlier

Since

copper-deficient

irons into capable

iron

the protoof binding

transport-

to a normal

plasma

ing

would
literature storage,

seem to be an unlikely contains mansy reports

mobilization

mechanism. concerning

the

role

of copper
review-

iii

the
ing gested stated,
ali(I

absorption,

this

literature with the

in 1935, hemoglobin.
evidence

concluded
of that hint, that

and that,
iron

utilizationi In the
but with is not

of iron. Elvehjem,2 animal body it (copper)

the transformation
d)f

is not
the in-

concerned

assimilation indicates

iron
Much storage

into

Schultze,3

reviewing
copper it. facilitates

the
or

literature
for

five
t.he

years the

later, utiliza-

necessary

absorption

of irons

iii tissues

is essential

for

From cine, This Institutes No. Submitted The Salt

the

Departments City, Utah. was Public University 22, milk the July

of

Medicine in

and part and for

Pediatrics, by and the publication a research in part U.

University grant

of (RU Energy 28, 1952.

Utah 25) (AT

College from (1l-l)-82) the

of Medi-

Lake investigation

supported Health accepted in these of Utah

National
Project

of Health,

Service

by a contract
August

6, between evaporated

S. Atomic was

Commission. supplied by Mr.

1952;
usedl

experiments

generously

W. C.
Miss for

Cross,
We helen technical
*

Carnation Milk Company, are indebted to Miss Betty

Ashenbrucker, assistance. Research Post-doctoral Council Fellow Mrs. Jean

Los Angeles, Calif. Tatting, Miss Doris

Van Fellow in the Dilla, in Mr. George

Kurtli,
Trappett.

Miss and

Jean Mr.

Robinson, Ocie Hadlev

National A.E.C.

the
1075

Medical
Sciences,

Sciences,

1949-1951.

Medical

1949-1952.

1076
tioni tissues. In of iron by the

STUDIES

ON

COPPER

METABOLISM.

III

blood-forming

organs

and

for

mobilization

of iron

from

the

the were

regard to the older literature, either

effect of copper on which the or were not

on the absorption above conclusions in agreement..

of iron, the were based levels the

experiments unfortunately,

in

inconclusive

Elvehjem and Sherman4 fec! to anemic rats deficient the blood proportional remained to the unchanged amount

found that when graded in both iron and copper, and of ironi the fed. amount In the

of inorganic hemoglobin

iron were contenit of

of iron presenice

stored in the liver was of copper, the rate of

hemoglobin formation no iron storage until that

tions

was dependent 0.3 mg. or more deficient in both to find that in the content question
was made

upon the iron intake amid the liver showed of iron was fed. It should be pointed out iron and copper and that. This under these condithe oral administration of iron in the liver. or not iron storage was made copper of iron was accomtype of experiment iron absorp-

the

rats

were

it is not

panied does not

surprising by an increase answer the

as to whether with no allowance

influences

tion since no comparison of these elements alone synthesis.

and

in animals deficient in each for iron used iii hemoglobin

Josephs5 determined the total body iron of milk fed rats given iron alone or copper in addition to iron. He found no significant difference in total body iron between the two groups and concluded that copper has no effect on iron retention. This conclusion has since been widely quoted. Unfortunately, the rats fed milk supplemented with iromi but receiving no copper were quently, as he pointed out, could not have been depleted studied of rats. copper the His effect of copper results, although diet had the not anemic and conseof copper. Cunningham6

feeding on the iron content of the livers and the bodies far from conclusive, suggested that the inclusion of effect of lowering the iron content in the liver and incarried out iron balance studies in found that, When iron was given led to a diminished retention but an studies of rats fed diets by Honk, given oral high in copper, Thomas and Sheriron and fec! diets have indicated of iron. and Nelto increase in such However,
ill

in the

creasing that of the body. Barer and Fowler7 10 patients with hypochromic anemia and in moderate amounts, the addition of copper increased utilization man8 on the retention low that in copper, of iron. More recently, of iron in the bodies with animals

as compared

the addition of copper resulted in a marked In reference to the utilization of parenterally and Eveleth,
in both

increase in the retention administered iron, Keil iron a rather suggest intraperitoneally striking that tract.

son9
deficient

Bing iron

and and

Myers#{176}administered copper, iron from and the observed these workers of this,

rats the

hemoglobin there

level.

As a result to absorb

animals Elveh-

is a failure

gastro-intestinal

jem2 observed only a slight increase in hemoglobin following the intraperitoneal administration of pure iron, and in a later paper by Keil and Nelson only a temporary and incomplete alleviation of the anemia was observed. Since both Elvehj em2 and Bing, Saurwein and Myers2 found an increase in the body copper content of animals given iron parenterally, questionable. examination of copper, iron the significance rather utilized of the large partial hemoof earlier formapoietic response to iron becomes There can be no doubt from literature that., in the absence

of the is not

amount

for hemoglobin

GUBLER,

LAHEY,

CHASE,

CARTSVRIGHT

AND

WINTROBE

1077 movement
incorporation other effect

tioni
inon

amid that
into in the

the

administration
That this molecule

of copper
effect rather than

is followed
is related attril)utable

by

a rapid
to the to some

of
of on

hemoglobin. hemoglohini

of copper

iron

hemoglobin

synthesis,

is suggested

also

by

our

experiments1 on the
swine.

in copper ai)sorption,
Two

deficient utilizationi.

swine to which reference has been made already. It is the purpose of this paper to present studies
storage ports and of this mobilization work have of iron appeared.#{176}

mi copper

deficient

preliminary

re-

14

METHODS Details concerning the animals, their care and digesting acid appeared. of nitric acid and diet as well as the methods used, in a 30 ml. nitric and were 0.5 with digestion acid to added. 1.0 until ml. The the

exception
Tissue flask charring
of

of those
iron occurred cent was
solution

described
were concentrated or until of

below,
performed

have
by sulfuric

been

given

in the
5 ml. of

previous
of tissue concentrated were 2 ml.

paper.

analyses

1 to 2 Gm. The and flasks 1 to

with 70 per

2 ml.

white acid,

fumes 1 ml.

cooled of water

perchloric then

mixture

made
per

up to volume.
cent

heated until To a 1 ml.

of 0-phenanthroline,

clear, aliquot

and transferred to a 10 or 25 ml. was added 0.1 ml. of thioglycolic

and, after mixing, sufficient

volumetric flask and acid, 0.4 ml. of a 0.1

sodium ace-

saturation

water redistilled in an all glass distilling apparatus) to make a final volume of 3 ml. or more, depending upon the concentration of iron. The color of the solution was determined by the use of a Beckman spectrophotometer at wave lenigth 510 mt and cuvettes with a 1 cm. light path. A reagent blank was prepared in the same mannier as the sample. Radioiron measurements of tissue were made by the method of Greenberg et al.5 except that the plating solution and the amperage were t.hose recommended by Vosburgh et al.6 The Fe59 activity was counted by the use of a thin mica end-window Geiger tube with a counting efficiency of 17 per cent. Four thousand and ninety-six counts were made on each
tate solution to make the sample was part tion It and prepared corrections from the applied iron for chloride background solution and coincidence to t.he loss. A standard During iron the sample latter administered animals.

1H basic

to congo

red paper. was added

Iron

free

water

(distilled

of the studies radioiron measurements of blood were made counter.7 Two ml. of i)loOd were counted without any prior has been stated that tissue fernitin is soluble in saline8

with
and

a vial
that

sample

preparation

scintillaof the sample. is not.

hemosidenini

Hemosidenin iron can be extracted from the saline-insoluble residue into dilute hvdrochionic acid.9 On the basis of this difference in solubility, an attempt was made to measure ferritin and hemosiderin iron in the liver and spleen of control and copper-deficient pigs, copper-deficient animals given parenteral iron injections, and i ron-deficient swi tie. Since these two fractions undoubtedly did not contain only fernit.in or hemosidenin, and since no attempt to identify unequivocally the two iron storage compounds was made, the two fractions will be referred to as the soluble-iron fraction and the insoluble-iron fraction. The method used was essentially that of Finch et al.B Ten Gm. of liver or spleen were homogenized in a Waring blender for 2 minutes with 40 to 50 ml. of 0.9 per cent saline. The homogenate was transferred to four 15 ml. centrifuge tubes and centrifuged at 2200 X g. for 30 minutes. The supernatant fluid was decanted off into 100
ml.
Waring

volumetric

flasks.

Approximately

40 ml.

of 0.9

per

cent

saline

was

used

to

wash

out the

and transferred to the centrifuge tubes containing the saline-insoluble residue. After thorough mixing, the tubes were again centrifuged as above arid the supernatant fluid added to the volumetric flasks containing the first supernate. The supernatant fluid was then ma(1e up to volume with 0.9 per cent saline. After the addition of 2 ml. of acetate buffer (pH 4.5) and approximately 40 mg. of sodium hydrosulfite to a 1 ml. aliquot in a centrifuge tube, the mixture was heated to 90 to 95 C. while stirring, cooled, centrifuged and the supernatant solutions decanted off. To a 1 to 2 ml. aliquot was added 0.1 ml. thioglycolic acid (Eastman, practical grade), 0.4 ml. of an 0.1 per cent aqueous solution of 0-phenanthroline, 0.5 ml. of a saturated solution of sodium acetate, and lastly water to

blender

1078 make
tissue
a final volume of

STUDIES

ON

COPPER

METABOLISM.

III

3 ml.

The

iron

content

was

then

measured

as described

above

for

iron.

Ten ml. of 10 per cent iron-free hydrochloric acid were added while stirring to each centrifuge tube containing the saline-insoluble residue. The mixture was heated to 90 to 95 C., cooled and centrifuged as noted above. The supernatant fluid was transferred to a 100 ml. volumetric flask and the residue was re-extracted with 10 per cent hydrochloric acid without
further heating until spot-plate tests with potassium ferrocyanide for ferric iron became negative. The washings and the original supernate were combined and diluted to 100 ml. with saturated sodium acetate solution. One or 2 ml. aliquots were taken, 0.1 ml. thioglycolic acid, 0.5 ml. of 0.1 per cent 0-phenanthroline, anid sufficient saturated sodium acetate to make the solution basic to congo red were added. After making up the solution, to a final volume of 3 ml. with iron-free water, the irons content was determined as noted above for
the determination, of tissue iron.
RESULTS

Studies

on the Absorption

of Iron

from

the Gastro-intestinal

Tract

To determine whether the absorption of iron from the gastro-intestinal tract of animals deficient in copper is impaired, four rather different types of experimerits were performed. In the first experiment the iron content of several organs as well as that of the blood the findings in litter-mate was determined and the results were controls and in irons-deficient animals. compared In the with second and from pigs sevthe

experiment radioactive iron was administered and a litter-mate control and the radioactive eral organs
was

orally to two copper-deficient irons content of the blood experimenit., irons was omitted

determined.

In

the

third

diet of anemic, copper-deficient animals and, five days later, the animals were given adequate quantities of copper ins order to determine whether the tissues contained sufficient iron to allow a satisfactory hemopoietic response to occur. In the fourth experiment, the increase ins plasma iron after the oral administrations of irons was studied were treated first with first with iron amid then ins animals deficient in both copper and then with iron with copper. irons and copper. while tsvo ot.hers Two were aniimals treated

Experiment

I A pigs (12-27, 12-30 and 12-31), all litter-mates, on the ninth day of age. Thirty mg. of orally
amount one

Three
beginning

were
irons/Kg.

given body

the

milk

diet were

weight experiment.

administered
the served same

in capsules
of iron on a

six

days

each
weight 88

week.
basis

Thus,
during of age,

each
the

animal weight/day the

32

received
In

body

addition, trationi The

and by

(12-27) was given 0.5 mg. as a control. When the animals were amid total blood volume were and total cent)

pig

of copper/Kg.
days

body latter by

and
concen-

hemoglobin

determined, the irons contenst amount of iron X total the in total studied.
blood

the

method.

ansimals were viviperfused heart was measured. The

the formula: Hg

of the liver, in hemoglobin

volume

kidneys, spleen was calculated X 0.0034. The difweight

(Gm.

per

results fered,
found

are the
in the

presensted results blood are plus

ins table also the four

1. Because expressed organs

body mg.

weights of

of the iron/Kg.

animals body

GUBLER,

LAIIEY,

CHASE,

CARTWRIGHT

AND

WINTROBE

1079

Experiment Five

I B additional pigs (12-62, 12-63, 12-65, 12-66 andl 12-68), all litter-mates,

were fed the milk diet, beginninig at 8 days of age. Both irons (30 mg./Kg. body weight/day) and copper (0.5 mg./Kg. body weight/day) were added to the diet of the two control ansimals (12-62 and 12-63). Iron only was added to the diet of the
deficient

copper-deficient in iron

by

pig (12-68). including only

Two ansimals copper ins the

(12-65 and diet. Because

12-66) were matte the iron-deficient pigs, the two for three days the development on viviperfused
and Copper-

animals became anemic more rapidly than dud the copper-deficient iron-deficienst animals were given 30 mg. of iron/Kg. body weight (days of the hundred
TABLE

62,

63, and

64),

an amount. Animals 12-62, day

of Iron

which 12-65 of age;

in the

would and animals

Blood Deficient and

suffice 12-68 12-63

Several

only were

to delay viviperfused were

of

ansemia.

the

one on

sevensth
Content

amid 12-66
Tissues

h-The

Control

Swine
Control Copper-Deficient
12-30 12-31

Group Pig. No.

G
isa.

n2-27

#{149}

mg/
Tissue ,g.

G
m.

*
,

mg/ Tissue
g.

G
m.

mg/
Tissue

Blood

Liver
Kidney

497t 56
18

293t
10

191t 30
15

140t 9
2

163t 38
15

lost
11

Spleen Heart Total, mg Bodywt.,Kg Total

Body
*

53 20

1 1 306
6.7

115 28

4 3

136 31

158
10.3

3 4 124 9.8

mg. wt.,

Fe Kg.
#{149}

45.7

15.3

12.6

Wet

tissue. data miumber hundred for content.

of iron

t The
in

in

blood in

are

expressed

as

/4g./mi.

of

whole

l)lood

and

total one

of fig, amid

of iron thirteenth

circulating day

the

blood. Blood ansdl tissue irons were measured

the as

of age.

described

In globin

both plus

above. Experiments the four

Th

results are presensted I A and I B the total was appreciably

ins table amount less ins the

2. of iron

founid

in the

Isemothami ins

organs

copper-deficient

the control pigs. This difference was due mainily to a reduction ins the hemoglobin iron. As can be noted ins Experiment I B there was as great a depletion of iron ins the copper-deficient. animals as ins the iron-deficient ones. Combining the results of the two experiments, the mean total iron comstent of the blood plus liver, spleens, kidiseys amid! heart, expressed ins mg/Kg. body weight, for the 3 control pigs was 50.5; 2 iroms-deficienit animals, Experiment The fourteens II 3 animals days (12-27, of the 12-30 amid were 12-31) given in Experiment 12 doses
of

for the 13.1.

three

copper-deficient

animals,

13.6;

ansd

for

the

I A, durimsg 10 mg. each

the

last

experiment

of radioac-

1080 tive irons/Kg. body unsit*

STUDIES

ON

COP1ER

METABOLISM.

III

weight.

The

radioactive of Fe55

irons

was

prepared

l)y
dhstilled

dilutimsg
water

ans to a

irradliations concentration The amount

constainimsg

a mixture

amid Fe59 with

of 100 mg. of iroms/ml. Each mg. of irons solutions to be a(iminiistered and givens The animals
of Iron in the

of irons contained to each pig was

0.1 c of Fe59. measured! out

into a gelatins capsule throat of the animal.

TABLE

immediately were then

Blood and

by forcinsg the capsule down the allowed to coissume the diet.. At the
Tissues Swine
Copper Deflcient
12-68 12-63

2.-The

Content

Several

of Control,

Copper-Deficient

and Iron-Deficient
Group Pig No. 12-62 mg./ Tissue g./ Gm. Control 12-63 mg./ Tissue

Iron-Deficient
m2-66

Gm.

sg./ Gm.

mg./ Tissue

g./ Gm.

mgi Tissue

/Lg./ Gm.

mg./
Tissue

Blood
Liver

490t 162
90

67Sf
70
12

450f

58Sf 59 4 12 2 662 11.3

105f 68 20 289 47

145t 33 3 16 3 200 15.5

194t
19

269t

121t

117f

206
43 468 25

8
1 3 2 283 17.4

13
7

6
1 3 2

Kidney
Spleen Heart Total, Body mg wt., Kg

8 90 16

558
31

23 4 784 16.6

104
15

129
12.9

Bodymg.Fe

Bodywt.,Kg
*

47.2

58.6

12.9

16.3

10.0

Wet

tissue.
data for content of mg. of iron
Iron

t The total
TABLE

of iron

in 1)100(1 are in the

of the Blood ist ration (For details

expressed blood.
and of see Several

as /4g./ml.

of

whole

blood

amid in

number

circulating
Content

3.-Radioactive

Tissues

after

the

Oral

Admin-

Fe55
text)
Copper-Deficient 12-30 mg. Fe 12-31 mg. Fe

Organ

Control 12-27 mg. Fe9

Liver Spleen
Kidney

4.50 0.16 0.29

0.21

4.46 1.52 0.66

0.71
19.85

4.70 1.13

Heart T.V.P.R.C.* Total

Amt. given

0.56 0.46
15.86
22.71

47.46 52.62 878 6.1

27.20

1416 1.9

1245 1.8

Percent
*

Total of the

circulating fourteen for are

volume day presensted was

of packed

red

cells.

end blood

period

the iron ins table for

the

animals were
in

were and liver,

viviperfused onse-teisth per kidneys,

anti cent heart

tissue of the and

anid total blood

alsalyses results administered

Obtained on

radioactive accounted
from

performed. the
Atomic

The dose
*

3. Six

spleen,

allocation

U. S.

Energy

Commissioms.

GUBLER,

LAHEY,

CHASE,

CARTWRIGHT

AND

WINTROBE

1081 fri these organs

of the in the

control
two

pig.

Oiily

1.9 and
pigs.

1.8 per

cent

were

accounted

for

copper-deficient

Experiment Iron was

III
withd!rawnl
from

the

diet

of two

ansemic,

copper-deficieist

pigs.

Both of mg. was

ansimals had the experiment of copper/Kg.

received body

30 mg. weight.)

(sixty-nine

of irons/Kg. days). Five

was

body weight/day days later, oral

A reticulocyte

from the beginning copper therapy (0.5

peak of

beguns.

14 per

cent

20
FIG. per after 1.-Showing irons had the been failure withdrawn

40
DAYS
of a copper-deficient from the

60
OF
diet

80
AGE
pig for five to respond days by

100
prior the to

120
to the of copiron, giving copper.

hemopoietically administration

After discontinuing even though the copper therapy.

the aninsals

copper, the anemia was alleviated had received 30 mg. of iron/Kg.

body

weight

for 69 days

prior

to

observed there therapy, weight/day) crease rapid! the

ins 1)0th a significant the

animals
increase

following
in

copper

administration

but

ins neither

pig

was body in-

the diet.

V.P.R.C. five In both

After days animals

twenty-one later iron a prompt and limits. (30

days

of

copper

copper was

was added
cent,

discontimsued; to the
respectively)

mg./Kg. reticulocyte

(20 ansd 25 per increase ins the two amsimals

V.P.R..C.

to within

was observed the normal 1.

this was followed by a The course of omse of

is showni

in detail

ins figure

1082 Experiment The tiorn

(12-96)
administrations

STUDIES

ON

COPPER

METABOLISM.

III

I V changes ins plasma and

of The This

irons during
follovinsg

the
oral

finst 24
a(lmusistnat

lsoinns

aften

the

oral
to

adminsistraan

of ironi

theni
inn both irons

the

ions of copper 2. In tisis

l)lasma

animal

(leficient

elensents, iso
significant for

are

sisown
increase

ins figure
ins the days arid

pig,
irons

following
level was

the obby

was
given

served. mouth. per cenit

irons was
was

(onsi inssned by

14 mone

thens

coppen iron

followed hours.

a modest

rise

ins the l)lasma

level

to

105

g.

ins four

0
Fzu. animals copper

16

2 immediately
i ron.
One

16

Hours
2.-Showing the plasm:s iron values initially deficient in hot Ii copper anti and the other (12-96) first with iron.
animal

.J(ocirs
following
(12-98)
oral

therapy
t rented first

two

was

wit ii

A second with
copper

aninsal

(12-98)
significant was

deficient

ins 1)0th

iron

arid
iron

copper

syas

treated

first

copper.

No Two

increase tonstiniuecl the l)lasma ironn

was

ins the for

l)lasma fourteens was

level arid absence

pigs

was tisens

observed. iron
.tg.

The
givers

administrations

(lays ins the

was

by mouth. Thus in the Similar ficienst

results

hours seem
d)f

later that little

irons level al)sorhed quarntities

two

founnd

to be 700

pen cennt.
whereas

it would presence
studies

of copper, ins the l)lasma.

copper,
were

appreciable
performed on

appeared ins the same riot

a(iditionial

(12-97

amid 12-99)

(leThe

in both were

iron entirely

arid

copper arid

ant! for

treated this

fashion, presented

as

above.

similar

neasois

are

ins detail.

GUISLER,

LAHEY,

CHASE,

CART\VRIG1IT

AND

WINTROBE

1083 for Ilemoqiohin

Studies

on

the

Utilization

of

Parenlerally S!Intlnesis

Administered

Iron

To mensts

determimie were

tise

(legree

to which
irons for

(opper-(leficienst
hemoglobins synthesis,

annimals
three

are

a1)le
types

to utilize
of experi-

parensterally adminsistened

adiminsistered

performed!.

Ins the

first,
prior

natural
to t.he could!

irons ins the

occinrrence

form
of

of irons ascorbate
copper deficiency

was
to

instraperitonseally

determimie if the development meat large d!OseS of (Olloidal ministered the amiemia intravenously could to be alleviatec!

of amsemia

be prevented.

irons ins the anemic by such

form therapy.

of the

In the seconsd experisaccharated oxide were adswinse to learn whether, a small as third experimenit

copper-deficient Ins the

amounst of radioactive irons was givens intravenously to amsemic copper-deficient pigs to determine the extent of utilizations of irons for hemoglobins synthesis, compare(! with that of a normal pig amid that of ani irons deficient, pig.
TABLE

4.-Failure

of

lion

Given in

In1raperitoneallj Copper-Deficient

to

Prevent

the

Development

o,f .1 neunia

Pigs givers 12-33, initraperitoneally

12-34

Fifty mg. of iron in the form of the ascorhate were for 6 weeks, beginning at 3 weeks of age, to animals 12-36, 12-37 arid 12-38 served as nregative controls.
Age g
0.

twice 12-35.

weekly

arid
Copper

Litter-nsates
Plasma Iron

V.P.R.C.
ml./n0oml.

Prasma
pg.

Days

12-33

97

22

240

12-34
12-35

97
97 97
92

12
25 15
14

14
12 16
9

140
50 143

Mean 12-36
12-37 12-38 Mean

38
33 35

92 92

15 21

6 17

92

17

11

35

Experiment Six pigs,

V all litter-mates, were

fed the

milk

diet

with

added

irons

begimsnsinsg

at

9 days of age. Beginmsing at the age form of the ascorbate was injected

of 3 weeks, 50 mg. twice weekly into

of elemenstal the peritonseal

irons ins the cavity of

three pigs (12-33, 12-34 ansd 12-35). The injectionss were continued for a total of six weeks so that each animal, in additions to the iron givers orally, received 600 mg. of iron parensterally. The remainimig 3 animals (12-36, 12-37 and 12-38) served toneal miormal
vented,

as controls. The results are presented in table 4. Although the intraperiadministrations of irons effectively increased the plasma iron to withiss the range ins 2 of the 3 animals, the developmenst. of amsemia was neither predelayed VI mg.
kindly
to

or modified

ins severity.

Experiment

Onse humsdred was

*

of irons in the on
furnished

form
Dr. G.

of the
M. Bayne,

colloidal days
Sharp

saccharated to each
arid Dohme, material.

oxide
Glenolden,

of irons* copperPa.

givers
Proferrin, are

intravenously
I)r.

two
by
for

consecutive

of 5 anemic,

We

indebted

Bavne

spectroscopic

analysis

of this

1084 deficient comitain

venotnsly

STUDIES

ON

COPPER

METABOLISM.

III

swimse. 33 g.
in two

Sinsce of copper,
injections

this

amoumst as a colstrol
to each

of iron

was

found

by spectroscopic was

analysis given intra-

to

a total

of 33 gig. of copper

All of the weight17day values

respectively,

pigs had received, by mouth from pigs 12-76

after

of 3 (13-00, 13-15, 13-16) copper deficienst pigs. arid! comstiisued to receive, 30 mg. of iron/kg. body the beginsnsing of the experiment (see table 5). The for days, the

for animals

the

and 12-79 are given for twenty-eight and forty-three second imsjection of irons. The values presensted fourteen neither the administration days after the second imsjections imstravenous of 200 admimsistration mg. of iron

other

were

detemminiec!

of irons.

As cans be seems ins this table, copper msor the imstravemsous quantity
TABLE

of 33 j.g. of constainsing this response.

a Hernopoielic

of copper
5.-Failure of

was

effective

ins elicitinsg

a significant
Intravenously

hemopoietic
to Induce Swine

Colloidal Iron Administered

Response in
ins

4 nem

ic,

Copper-Deficient
saccharated

Two

hundred

nsg. and 13-16 mg.

of

iron

the
by

forns
12-76,

of colloidal
12-79, 13-02,

oxide

of

irons

were

adpigs

ministered 13-00, contmsined

-

intravenously

to

pigs

13-03

and

13-04.

As

controls,

13-15
in

received,
of

200

the
V.P.R.C.

iron

intravenous preparation.
Retics.

insject ion,

that

amount

of copper

(33 ,g.)

mn./iOOmn.
PigNo.

_____
D e.

%
_____

M.C.V. cup
_____

M.C.H.C.

P1. Co

per

pg.
Def. After Iron

P1. Iron pg.%

____________

T.I.B.C.

_____

After Iron

C.

After Iron

C.

After Iron

C.

After Iron

After Iron,

D C. f

After Iron

12-76 12-79 13-02 13-03 13-04 Mean 13-00 13-15 13-16

20 16 28

22 16 22

10 3 5

18 15 2

60 44 40

54 52 40

28 31 30

26 29 29 31

3 9 27 17

11 14 17 27

57 77 43 32

236 120
140 57
,

667 500
693 635

33 23 24 29
24 29

32 15 21 22
11 19

3 3 5 2
1 4

6 7 10 5
3 3

45 41
46

46 46
48

30 29
30

27
28

14
14

20
18 30 17 15

32
48 48 37

145
140 58 67

640
627 598 675

666 520 540 632 530

578

38
44 42

40
47 39

28
30 29

29
29 29

29
20 15

658
567

Mean
V.P.R.C.,
mean

27

17

41

42

29

29

21

21

42

62

636 M.C.H.C., capacity

612

volume of packed corpuscular hemoglobin

red cells;
concentration;

M.C.V.,

mean

corpuscular
total

T.I.B.C.,

volume; iron-binding

of

the plasnsa. There

was

no signsificant

change

in the

V.P.11.C.

or the

c!egree

of microcytosis

or

hypochromia. A sigmsificant inscrease in reticulocytes but the reticulocytosis was isot. accompansied by worthy, however, that. the hypoferremia was

occurred in 2 of the 5 animals blood regeneration. It is noteins 4 of 5 animals level, given iso sig-

corrected

the irons parenterally. In spite of the increase in the plasma irons nificant decrease in the total iron-bindinsg capacity was observed. Experiment Radioiroms* iron-deficient deficient
*

VII as ferric chloride was injected imitravemiously imito one constrol, amid three copper-deficient. swine. The control pig and the were
ons

one iron-

pig

given

550
from

sg. of irons
the

corstaimiimsg
Energy

6.7

tic. (2.5

X 106 countable

Obtained

allocation

U. S. Atomic

Cornnsission.

GUn5LER,

LAIIEY,

CHASE,

(AItTWRIGHT

AND

WINTROBE

1085 j.g.
by

(.p.m.)
the use

of Fe59
of

activity.

The

of inoniconstainiinsg 6.7

tic. Thse
to 5 ml.

three col)pel-deficielst total circulating of

1)100(1

red

)igs cell

wese each givemi ill mass was detenminsed

iron aIR! at the enid

at thse tinse pemiod. days the

Since

of injection

of radioactive

of the

sampling activity. total total red

Two aften

wene withdhawns isotopic iron duminig ins weight

two, and the from

foum, six, analyzed sampling the

two

eight, period, deternsinmed

tens the

arid fotnrteens

the

injections

of tise
was

for radio-

pigs

weme inicneasing
on a givers day
The

red cell voltnme cell volumes.

ins terpolated

results

ane l)nesenit.ed

ins figure 3.

FIG.

3.-Showing hemoglobin data for are ins total

the of one presente(1

per

cent in terms

uptake

of intravenously

administered

Fe5 dose

into

the

circuacthat

lat inig The (ounited

control,

one
hemoglobin.

irons-deficient
per (erA The amount

amid three
of the

copper-deficient.
was

swine.
of radioiron such Since

of the

administered of iroms administered

circulating

it was
lwanse phisnia figs thesis

hound
(lisperse(l iron

by
level

the
was

free

iron-binding
comisisting lower

protein
of the ins the

of the
plasnsa

plasma.
iron plus

Therefore,
the and tagged in

the
irons.

tagged iron-deficient

irons
the

in, a 1)001

considerably aniinsals, pigs

copper-deficient

the

than in the control in the (opl)er-deficienit

the actual over-all utilization was probably evens less than

of irons for hemoglobin the above data indicate.

synFor

the

experinsental

details

see text.

counsts this

Ins the copper appeared 93 per l)eIiod

deficienst amiimals ins the hemoglobin cent of the Inn the admimsisteredl irons

onsly
fraction

20
counts

to 25 per cemst of the admimsistered its ten days whemeas, in t.he control
were

animal,

found 83 per

ins tise hemoglobins cent was takers up

ins ins

of time.

deficient

animals, Iron

hsemoglobims.

Studies Irois dleficieist

*

on Storaqe of 5 control,
U. S. Atomic

ins the animals

on

livers

arid!

spleemis
200
from

5 copper-deficient ins the

Comnsission.

and form

4 copper-

givems
allocation,

mg.
the

of irons instnavensously
Energy

of tise sacchar-

Obtaimsed

1086 ated solul)le portiorss. ins table

In the

STUDIES

ON

COPPER

METABOLISM.

III

oxide

of irons, (presumably

and

of 4 iron-deficient ferritin) amid sacrificed

pigs, saline-insoluble

was

fractionated (presumably The results

into

the

saline-

hemosiderims) are livers presented of the irons

All animals 6.
livers of the

were

by viviperfusioms.
amsimals as

coppen-dleficienst

well

as ins the

deficient fractions

pigs, than

there was a proportionately greater reduction ins the saline-inssoluble fractioss. Whets large pigs, there thams ins the of these there ansimals was
than
in

ins saline-soluble doses of iroms were

administered intravenously to copper-deficient greater increase ins the irssoluble-irors fractions though comparable
storage In

was a proportiomsately soluble fraction. Evens increased to a level greater soluble. amid ironsfrac-

the

total to

iron that the

contenst ins the reverse

of the corstrol situation

livers livers, oc(urred.

a comparatively the saline copper-deficient ins the

and Spleen

of irons ins the saline-insoluble

compartmenst

the

spleems pigs

Ins the

deficiemit
TABLE

there

was

a pmoportionsately
and Saline-insoluble

greater
Fractions

reductioms
of Liver Iron
details
,

imisoluble
Iron

6.-Saline-soluble and Copper-deficient

in Control and

Pigs,

Copper-deficient Iron-deficient

Pigs Given Pigs

tissue. For

Intravenously,
see text. Spleen
Sot. Insol.

The

results

are
Group

expressed

in /4g./Gm.
No. of
Pigs

of wet

______-__.._-__Sol.

Liver
S/I

Insol.

S/I

Control
Copper-def.

5 5

Ill (94-l39) 42
(32-64)

28 (17-42) 21
(11-36)

4.0 (3.0-5.4) 2.0

(l.1-2.9)(76-142)

155
(60-293)

126
(16-257/

1.2
(1.1-2.5)

99

57
(19-91)

1.7
(1.1-4.2)

Copper-def.

i.v.

iron

79 79 (33-129)/25-136)(0.8-1.3) 9

1.0

175
(128-253)

104
(62-144)(1.5-2.0)

1.7

Iron-def.

10

(8-11)

(7-13)

0.9 (0.8_1.2)

49 (46-52)

20 (19-21)

2.4 (2.1-2.6)

tions

thais

ins the

soluble

fractions.

After

the

imstravenious

administration
greater

of irons

to the copper-deficient saline-soluble irons Studies Ins order

tissues,

pigs, there was a proportionately fractions than ins the inssoluble fractions. on the Mobilization the influensce were of copper measured of Iron ors the at two from

imserease ins the

the Tissues of irons from following the the

to study irons

mobilization hour intervals

plasma

levels

administrations of copper to animals deficient irs this element. The copper in the liver of irons-deficient amsimals before amitl at imitervals therapy was also studied to determine whether copperS is mobilized!
liver along with irons.

contenit of after irois from the

Experiment

VIII a single oral dose of (opper (0.5 mg. daily oral supplement of irons (30 mg.

Eight copper-deficiemst swimse were given of copper/Kg. body weight.) along with the

GUBLER,

LAHEY,

CHASE,

CARTWRIGHT

AND

WINTROBE

1087

of isois/Kg. the experiment. alsd 48 hours the per the

body after

weight) The
the

which of iron
administrations

they
in

had the

beemi receivisig plasma per was

As showis

siisce
iii

the

begimimsimug of

level

determined figure

of copper.

0, 2, 4, 6, 8, 24 4 (Group B), of 150 occur iron pig. with (30

plasma irons level incmeased from 37 tg. cent in 8 hours. To determimse whether a similar rise in the admimuistrationi pigs at the of iron same without instervals copper, after time

cent

to a maximum iron level iron would level was of only

plasma the the plasma

measured!

ins two

administration

zoo

Grocip Copper .Zeficier Givez: A. Oral Zroi2 (30 t29/Jc9) B Oral Iron -#- Copper (ct5.Pxz/Jca.) C Ho 1D022 !orlieceedinq 5J3 Then Oral Copper Oi2151
-

210
FIG. iron

4.-Plasma the

iron

levels

immediately

following

tise and

administrations curve B represents

of iron

(Group

A),

and copper
C represent

(Group

B),
meami

and
value

copper
for

only
2 animals

(Group
each

C) to copper-deficient

swine.
the

Curves
mean of

A
8

and animals.

mg./Kg.).

Ins these

amiimals

(figure

4, Group

A) there

was
in

an increase

only

from

45 Mg. per cent to 60 g. In order to determinse from the gastro-intest.inal diet

were

per cent. whether the irons appearing t.ract or from the tissues,

iron

the plasma was comiisg was omitted from t.he

iii

iron ceiviisg

of two copper-deficiemst swirse for given a single oral dose of copper level was of the same magnitude irons together IX data are on the copper in figure content 5. The with copper.

a period of five days and then (0.5 mg./Kg.). The increase as that observed in the eight

the animals the plasma animals re-

Experiment The pigs

of the mean

livers value

of 8 control
in

and

9 iron-deficient animals was 24.5

presented

the

control

1088

STUDIES

ON

COPPER

METABOLISM.

Ill

g./Gm. of mg.
deficient, ficient As
was

of wet of copper
21.7.

tissue per
The latter

ansd! in the liver,

figunes,

inons-dleficient valune
howeven, are

pigs,
somewhat

43.1/j.g. nol pigs

Expressed was
since

ins terms ins the ironsthe de-

the means on the

5, the

in the coat

misleadinsg

animals
be seen

weighed,
in figure

can not

average, inscnease values

20.9 Kg. and ins liver copper

were present.

the control pigs, 13.2 in the iron-deficieist

ins only 6 of 9 pigs.

Kg. pigs

consistent

since

elevated

75

Z5

Con frol #{149} Iron Del

0
FIG.

5.-Showing pigs. The liver. values

the are

comstenst presented

of copper both

in ins g,

the

livers

of eight

control of wet tissue

and

nine and

ironi-defimg. of cop-

cient per/total

of copper/Gns.

The tired at

concentration five day

of copper intervals after

in the the

liver

of 4 iron-deficient of irons.

pigs

was

meas-

ad!mimsistration

Laparotomy

was

performed under Gm. in size, was

istered intravenously. inon
We

ethen ansesthesia ansd a small portions of liven, approximately 3 removed.* Colloidal saccharated oxide of irons was then adminTwo pigs (13-06 and 13-07) received 100 mg. of irons while 13-10) were ins the diet givens 200 beginsnsing mg. the The
day

2 ansimals
given

(13-09 aisd (30 mg./Kg.)

last after

2 animals

weme

also
rfhe

the laparotomy.
of Surgery, University

Utah

are indebted to Dr. William Moretz, Associate Professor College of Medicine, for performing the serial liver biopsies.

of

GUBLER,

LAIIEY,

CHASE,

CARTWRIGHT

AND

WINTROBE

1089

livers resunlts The

wene

agaimi

biopsied

five,

tens amid! fifteen d!ays ins tise livers

after

the

irsitial animals

biopsy.

The

are presented ins figunre 6. insit.ial conscenstrations of copper

of 2 of the

ssas marklimits. corscens-

edly increased. In all 4 ansimals trations greatly. 53.6 tions per

were

Ins the other 2 pigs the initial there was ans imicrease irs liver irons therapy insitially high Ins the 2 with

valunes were within irons audi a decrease

nsormal ins the

of copper following In the 2 with the cent., was respectively.

onsly 7.2

although the degree of chansge varied values there was a decrease of 37.1 anal insitial values within Plasma normal copper after limits, determinsathe instrathe

decrease

arid! 14.4 per cenit, respectively.

performed!

ons all 4 pigs

at 0, 2, 4, 6, 8, 24 and

48 hours

300

13-06

13-07

13-0?

13-10

zoo
t

#{149}

TPOZ2

t#{176}#{176}
50

copper

-s

s.
I I I

5
FIG.

10 15 .D2qAfer
the changes

10

15

5
ins liver

10
iron

15

10 15
administra-

Znraveriocis
in liver pigs. For copper details and of the experiment

Iron
following see the

6.-Showing

tions of irons to 4 iron-deficient venous adminsistrations

text. of copper ins the

of ironi.

No

increase

in

the

conscenstrations

plasma in the

was urine

observed amid fedes

in any
was

of the animals. determimsed daily

In ad!dlitiomi, the excretion for the fifteens day period!.

of copper
However,

due to variation specimemss with olism penis, the sensted.

in the dnetary intake, comst.amimsat.ioms diet, as well as contamination from the results obtained were msot reliable amid,

of the urinse ansd stool galvansized irons metabtherefore, are not pre-

DISCUSSION

The several

studies

reported

here

insdicat.e metabolism

that

ins copper-deficiemst namely:

swimse

t.here absorption

are

abnormalities

ins the

of iron,

(1) impaired

1090 of irons from the tissunes; hemoglobin ins normal The

absorption

STUDIES

ON

COPPER

METABOLISM.

III

the

gastro-instestimsal (3) evens suggests

the

tract whets this that fed

; (2) incomplete pamensterally is presented elememst there

mobilizations admimsistered to the

of iron irons bonse

from for

ansd finally syrsthesis quantities. which

from of irons

inability

to utilize

marrow impaired!

evidlensce

is, ins (oppem-defi(ient

tract. may quanstity

swimse,

gast.ro-imstestinal

( 1) ins copper-deficient
of time
(as

animals, constrol

the

same there

be summamized of iron fom the ins total

as follows: same period body irons to levels of radiooral adamsimals;

as litter-mate the to those

inn the (!eficient

ansimals,

was

a reductions

insdicated comparable
iron founsd! ministrations

by

sunm of 1)100(1, liven, seems ins irors-deficienst

tissues irons, responided of sas

kidmsey, swinse;

spleen and heart iron) (2) the total amoumut

sivinse, less followiisg thams ins control the

above swinse

copper-deficient significamstly to

of radioactive

( 3) copper
irons iron ins the

was
to

insciuded
allosv for

ins the
more

(liet,
thars

insdicatinsg
minimal

the that

administration thetissuesdid
formations

of copper only whemi isot constaims sufficient

; whens
arid!

hemoglobin

plasma

iron giveis

level with

was inn both

observed copper

immedliately
amid! irons,

following
whemeas

the the there

(4) mio inscrease adlmirsistrations same amount amid was a rapid

of irons to pigs of irons sas marked evidence ficienst given determine reported
foumsd that,

deficiemst

or following

copper

administrations,

increase in the plasma irons level. Firsally, will be presented that the total body rats copper total from
as

ins the next paper radioinons constent as compared neasons, are it was with

ins this series2m of copper-dIethat of rats to those msot possible

fed

radioiroms ins the body Aunstralia

the

is (onssiderably For obvious these deficient irons ins pigs. out. that in sheep msormal

reduced teehnsical stindies

diet.

It is of interest

to poirst
deficiency

ins strikinig

(onitrast

to

progressed,

us copper.22 Ins these amsimals it was the conscenstrations of irons inn the liver to 0.4 mg. of iron/Gm. of dry
tissue

rose

steadily

from

the

rarsge

of 0.2

to levels which exceeded fined to a copper-deficiemut differensde

experimenits

35 mg./Gm. fodder for It may

ins the livers of animals two years or more. The however, to species, deficiemscy to respond that

that had explansations conditions

beess consfor this of the two

is not
are

obvious.

be nsote(!, ins regard of copper animals

possible

that

quite dissimilar the maisifestatious components. of copper-deficient

diet and durations. It is also ane markedly influensced by hemopoietically to large

other dietary The failisre amounsts

of irons admimsistered

either decreased to utilize radioiron was large

imstravemsously

or intraperitoneally,

indicates

that, ins additions to the impairmemit. in the ability The

cent

rate of absorption irons onsce it has was adminsistered determined, amounsts

of iron, there is marked gainsed access to the body. imst.ravensously arid the this per consor substanstiates either instraperitonseally

experiment

ins which ins the

uptake clusioms. In 8 of the intravenounsly, maintained

globin

erythrocytes givens

further of irons,

10 aisimals the there

plasma irons level for a considerable occurred. This

increased to within normal limits amid! was period of time. Ins spite of this, no hemowould seem to imsdicate that there must arsimals be a after
to store

regenerationi

defect
The

ins the studies

utilizations of irons withins the bonse marrow itself. oms the storage of iron irs the livers of copper-deficient adminsistratiomi
of irons suggest.

the

instravenoins

that

there

was

a tendency

GUBLER,

LAHEY,

CHASE,

CARTWRIGIIT

AND

WINTROBE

1091 with consthe the that

mone trol
livens. stonage

of the amsimals
However, forms

irons as hemosidemins witis

of

amid! less the

specificity studied

as fernitiii,

as compared!

approximately
because inons was the not

same
of ann!

comscemstnatioms of total inons in the method unsed ins sepanatinsg

because of the technucal difficulties

encountered, a redluctioni
as well The as

insterpretatiois ins the

ins the

of these

data

is nsot possible.

It should us the
the

be nsoted

ratio
that

sohnble-iroms,/imssoluble-irons
ansimals. deficierst animals ins the

occunmed
most of

inon-deficienst
of I)ody

dopper-deficienst

observation

depletions

in.ons took place ins the hemoglobins irons ins the liver, spleen, kid!nsey all, reduced amid yet. the animals evi(Ience that Furthermore,
tions of copper

compartmemst, amid heart was wene invariably

amid that the concentratioms onsly slightly, or at times riot hypoferremic, the plasma pigs after plasma as rose
irons

of at as

cams be takems was decreased. the adminsistrafrom 8 hours. as high

was

movemenst of iron fnom the tissues insto the first change msoted ins copper-deficient
was a rapid!

mobilizations cotnld plasma

regardless

of irons be detected

insto the as soon

the after

tissues. givinsg Mg.

An

increase

ins plasma so observed,

This rise

irons the
at

2 hounrs

copper
pen cent.

by mouth

ansd generally
occunrred tract

reached

a maximum irons conscenstratiors

of whethem

ins about
or nsot

Ins 2 of the as 300

ins the

10 animals
gastro-instestimsal ans increase

present

Since similar
absorption,

hemoglobins ansimals depleted

in

the time of the administrations of copper and preceded by a comssiderable period of time. of coppen develop ans anemia which is morphologically anemia
amid

to iron-deficienscy ins the

mobilizations

and capacity

sinsce

it is accompansied plasma,

by

hypoferremia, of impaired

an inscrease

irons-bimsding

of the

amid evidence

utilizations of irons, it seems logical to conclude that the amsemia is the comisequensce of ans imsability to utilize iron. The fact that copper insfiuensces irons metabolism ins such diverse sites as the mueosal cell, the liver and the borne wherever completely standing marrow, suggests that copper may, ins some basic mannser, amid whensever inoms moves. Kmsowledge concermsing this lackimig of the role at this of copper time arid is l)adly Iseeded nutritioms. for a more ins mammaliams
SUMMARY

be coiscernsed mechanism is complete unsder-

Studies anemic animals

are

described

which

deal

with

the

metabolism

of irons

ins swimse

made

by feeding a milk diet there is (1) impaired

low ins copper. Evidensce ability to absorb irons

is presented that ins such from the gastro-instestimsal tissunes; amid (3) insability synsthesis evens whens to it is

tract; (2) incomplete mobilizations of irons from the utilize parenterally admirsistered irons for hemoglohins presented to the l)omse marrow ins msormal quaistities.
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