STUDIES IN NUTRITIONAL. ANEMIA

QUANTITATIVE VARIATIONS IN IRON, COPPER, AND MANGANESE SUPPLEMENTS *

BY HELEN S. MITCHELL AND LILA MILLER

(From the Nutrition Research Laboratory, Battle Creek Sanitarium, Battle Creek)

(Received for publication, April 16, 1931)

The question of the optimum mineral supplement for hemoglo- bin regeneration in rats suffering from milk anemia has been at- tacked from several angles by different investigators. The first essential, an adequate supply of available iron, has most frequently been furnished by 0.5 mg. daily in the form of some soluble iron salt. Differences in response of anemic rats to this or similar quantities of iron have been attributed to possible contamination of the commercial salts with other metals (l), or to differences in availability of the various forms of iron used (2). With care- fully purified iron salts Waddell and coworkers (3) failed to cure milk anemia in rats even when as high as 10 mg. of iron were fed daily. Beard and Myers (4), however, report a slow but steady increase in hemoglobin when pure iron salts furnished the only supplement to the milk ration.

The specificity of copper as a supplement to iron in hemoglobin regeneration has been demonstrated by Hart, Steenbock, et al. (5), Krauss (6), and Lewis et al. (7) while others maintain that although more effective than other minerals copper is not specific in this capacity. Our previous paper from this laboratory (2) also ex- pressed this opinion. Drabkin and Waggoner (8) report copper aa non-essential for hemoglobin regeneration, but are working with a synthetic ration which may introduce other factors not encoun- tered in a simple milk anemia. Titus and Hughes (9), Goerner (lo), and Beard and Myers (4) have attributed to manganese an

* Presented at the meeting of the Medical and Biochemical Divisions of the American Chemical Society at Indianapolis, April 1, 1931.

421

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422 Nutritional Anemia

effect similar to that of copper but somewhat less marked. The latter investigators have also found a variety of other minerals effective as supplements to iron in hemoglobin regeneration. Variations in experimental technique, strains of rats, milk supply, and other factors make it diihcult to reconcile or explain the dis- crepancies in the results reported and the conclusions reached by these several investigators.

The present paper reports an extensive series of experiments undertaken in an attempt to throw further light on some of these questions. What is the nature of the response to iron alone at different levels of intake? Is manganese really effective as a supplement to iron in milk anemia? If so what is an optimum level of iron, copper, and manganese for the promptest and highest hemoglobin response? Are other metals present in the ash of plants significant in this capacity? Throughout this work em- phasis has been placed on carefully standardized technique and a sufficiently large number of animals to overcome if possible the errors due to individual variations.

Procedure

Experimental Animals

Between 3660 and 4600 hemoglobin determinations on over 306 rats are reported in this paper. Many more used in preliminary tests are not included here. Severe secondary anemia was pro- duced in young rats by the feeding of fresh milk from the time of weaning. To insure that none of the stock ration was consumed by the young while they were still with the mother, milk was the only food given to the family after the litter was 2 weeks old. A depletion period on milk alone averaging 5 to 6 weeks after weaning was usually sufficient to produce the desired degree of anemia. Fresh milk from a certified herd has been used through- out the experiment. Aluminum and block tin were the only metals which came in contact with the milk before it was fed. Rats have been kept in galvanized metal cages of which the raised screen bottoms and food cups were washed daily.

Hemoglobin Determinations

Hemoglobin determinations were made weekly on each animal until a normal level was reached after which biweekly determina-

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H. S. Mitchell and L. Miller 423

tions were considered sufficient. The Newcomer method with a Bausch and Lomb hemoglobinometer with blue filter was em- ployed throughout the experiment. A detailed discussion of the precautions observed is given in a subsequent article being pub- lished elsewhere (11).

Normal hemoglobin in our colony ranges between 15.3 and 16.6 gm. per 100 cc. of blood after the rats are 7 weeks of age. Com- plete regeneration is assumed when the hemoglobin level has reached I5 gm. or above.

Mineral Supplements

The iron, copper, and manganese salts used in these experiments were of the highest grade obtainable (Baker’s analyzed) and were further subjected to most careful purification in this laboratory. Iron obtained as ferrous sulfate (FeS04.7Hz0) was dissolved in double distilled water and subjected to H2S precipitation, after the acidity of the solution had been adjusted to 0.3 X. The salt was crystallized from the filtrate, redissolved, and again subjected to HPS precipitation. Three subsequent recrystallizations from double distilled water yielded the salt which WRS finally used for the feeding tests. This procedure proved more satisfactory and yielded a product of greater purity than that prepared from iron wire. Manganous sulfate (JInS04.4H20) was treated in the same manner as the iron salt. Copper sulfate (CuS04.5H20) was recrystallized five times from double distilled water. All water used in these manipulations and in solutions for feeding was dis- tilled the second time from a complete glass apparatus to avoid all mineral contamination. Table I shows the various amounts and combinations of iron, copper, and manganese fed as daily doses 6 days a week.

The solutions of salts were prepared in such dilutions that 0.2, 0.5, 1, or 2 cc. daily would supply the desired amounts of the minerals. Such volumes were easily measured and did not greatly reduce the concentration of the milk fed. The supplements were added to the morning feeding which was small in amount and was consequently completely consumed within a few hours. Later in the day additional milk was given ad libitum.

In one short series of tests salts of aluminum, tin, antimony, and zinc were fed as supplements to 0.5 mg. of iron. The unqucstion-

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424 Nutritional Anemia

ably negative results obtained in every case prompted us to con- centrate attention on the iron, copper, and manganese only. These findings also reassured us that the metals with which the milk or the rats came in contact were not serious sources of error in the present investigation.

TABLE I

@antities and Combinations of Iron, Copper, and Manganese Fed as Supple- ments to a Milk Ration

W.psr~Y

0.5 0.25

0.1 0.5

0.25 0.1

0.5 0.5 0.5 0.5

0.25 0.25 0.25 0.25

0.1 0.1 0.1 0.1

0.5 0.5

copper (copper sulfate)

cnq. pa h 0.1 0.05 0.0235 0.01 0.1 0.05 0.025 0.01 0.1 0.05 0.025 0.01

0.1 0.05

0.025 0.01

0.1 0.05

0.025 0.01

0.1 0.05

0.025 0.01

12 4 6 8

- 1

-

0.1 0.05 0.1 0.1 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05 0.1 0.05

12

0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01 0.025 0.01

4

Initial Hemoglobin

A uniformly low hemoglobin at the time when mineral addi- tions were to be made was found to be of prime importance. Only under such conditions could it be assumed that the physiological stores of the essential elements had been equally depleted. It frequently happened that one or two rats in a litter tailed to de- velop as severe anemia in a given time as had the litter mates. As it was believed that a uniform age for supplementing the milk ration of litter mates was desirable in some of our earlier work, these less anemic rats had received the mineral additions at the

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H. S. Mitchell and L. Nller 425

same time as the majority which were more anemic. The prompter recovery of those which started at a higher level made us realize that a low initial hemoglobin was of far more importance for consistent results than the exact age of the animal. Without this precaution the hemoglobin responses to the same or to graded quantities of mineral supplements were neither constant nor con- sistent. At best there is a natural variation in animals which must be taken into account. Table II shows the hemoglobin response over a period of 10 weeks of two groups of rats, receiving exactly the same mineral supplement (0.5 mg. of Fe), the only difference between the groups being that the initial hemoglobin values in the first ranged between 4.5 and 6 gm. per 100 cc. of

TABLE II

Weekly HemogloEin Averages Shou-ing Diflerence in Response Due to Initial Hemoglobin Leael

The Hb values are given in gm. per 100 cc. of blood for rats fed milk plus 0.5 mg. of Fe daily.

Initial Hb between

Difference due to initial Hb.. . . . . . 2.3

Week of experimental period

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th ----------

7.4 8.910.311.311.713.113.113.014.113.8 4.3 5.8 6.1 7.1 8.2 8.3 8.8 9.410.911.2

-- ---- ----

2.9 3.1 4.2 4.2 3.5 4.8 4.3 4.2 3.2 2.6

blood while in the second they ranged from 3 to 4.5 gm. This initial difference is reflected in the later response in hemoglobin level. All animals which had shown an initial hemoglobin of more than 4.5 gm. were subsequently discarded from our data as soon as the importance of this precaution was observed. The difference in speed of hemoglobin regeneration due to such initial variation may easily have been responsible for the inconsistent results and different conclusions reported by other workers. In an early paper from the Wisconsin (12) laboratories 4.23 f 0.15 gm. of hemoglobin per 100 cc. of blood were recommended as a suitable degree of anemia for a starting point, but in the next series of papers initial hemoglobin values ranging from 3.0 to 10.0 were apparently considered satisfactory. In more recent work,

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Nutritional Anemia

however, a lower and narrower range has again been employed. Exact figures are not always given in the published reports but the approximate range (calculated from graphs or figures) of the initial hemoglobin values used by workers of several other labora- tories may be significant.

Beard and Myers (4). . 4.0-6.0 gm. per 100 cc. Titus and Hughes (9). _. _. . . . 2.3-9.0 “ “ 100 “ Drabkin and Waggoner (8). . . . 5.5-8.0 “ “ 100 “ Krauss (6). . . . . . . . . . . 2.0-5.0 “ I‘ 100 “

Lewis cl al. (7) . . . . . . . . . . . . . . . . 3.0-6.55 “ “ lofj I‘

McGhee (13). . . . . . . . . . . . . . . . . . . 75 per cent (standard not given)

We wish to emphasize the necessity of uniformly low initial hemoglobin values in experimental animals when any factor pur- ported to have a stimulating effect on hemoglobin synthesis is being investigated.

DISCCTSSION

Space does not permit the publication in detail of the large volume of data accumulated during these experiments but com- posite tables and charts afford a means of summarizing the results and of arriving at fair and unprejudiced interpretation thereof.

Response Due to Iron Alone

The feeding of a pure iron salt as the only supplement to milk has in the present series as well as in our previous experience re- sulted in a very slow but unmistakable increase in hemoglobin concent,ration. In the present instance when the iron salt was of such purity as to preclude possibility of significant copper con- tamination it seems most logical to attribute the slow response observed to the traces of copper available from the milk supply or stored in the animal body. As the former item may vary in differ- ent localities it is at least a plausible explanation of thevariations observed in several laboratories concerning the ability of the rat to synthesize hemoglobin on a ration of milk and a pure iron salt. The average difference in the hemoglobin response to three levels of iron (0.5, 0.25, and 0.1 mg. daily) is shown in Chart I and Table III.

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H. S. Mitchell and L. Miller 427

Response Due to Iron and Copper

The rapid synthesis of hemoglobin when even the lowest level of copper (0.01 mg. daily) was fed in addition to 0.5 mg. of iron confirmed our previous results as well as those of several other

TABLE III

Weekly Hemoglobin Averages on Diflrent Levels of Iron and copper &QQk?7?WltS

The Hb values are given in gm. per 100 cc. of blood.

6 5 6

Supplement

Fe Cu --

nw. mg.

0.5 0.25 0.1

0.5 0.1 0.5 0.05 0.5 0.02; 0.5 0.01

0.25 0.1 0.25 0.05 0.25 0.02! 0.25 0.01

0.1 0.1 0.1 0.05 0.1 0.02; 0.1 0.01

0.5 1.0 0.5 2.0 0.5 4.0 0.5 6.0 0.5 8.0

7 4 5 4

4 4 4 5

7 4 7 5 5

-

nitia Hb

3.6 4.2 3.8

3.8 3.8 3.8 3.8

3.9 3.8 3.7 3.3

Average weekly Hb for

1st 2nd 3rd 4th 6th 6th 7th 8th 9th 10th wk. wk. wk. wk. wk. wk. wk. wk. wk. wk. ----- -- ---

5.3 8.0 8.1 9.1 8.9 9.2 9.910.412.011.6 4.0 4.4 5.1 5.1 5.8 6.4 6.7 7.3 8.6 8.7 3.7 4.2 4.7 4.6 4.9 5.5 6.0 6.0 6.2 7.0

8.9 11.8 14.1 14.5 15.915.615.715.715.715.5 9.1 12.8 13.9 14.1 14.914.714.714.514.914.9 8.3 13.8 13.2 13.5 15.716.415.916.315.915.5 7.3 10.6 12.2 13.3 14.814.915.015.316.015.6

7.5 10.8 13.2 14.2 15.216.016.015.915.916.1 6.3 9.0 10.9 13.2 14.614.315.715.615.115.4 8.7 11.6 13.4 14.2 15.916.015.915.915.916.2 5.3 7.2 8.7 10.1 11.413.313.414.314.312.6

6.5 8.5 10.2 11.8 13.314.415.215.815.915.9 6.4 8.4 9.9 11.0 11.313.013.514.314.415.1 5.3 7.1 8.5 9.2 9.910.912.012.612.713.3 4.8 5.7 6.8 7.3 8.3 9.210.311.211.311.7

9.6 13.6 14.4 15.3 17.016.616.416.517.016.5 10.1 14.9 14.5 15.1 14.715.015.215.114.914.7 8.6 12.8 12.6 15.2 14.814.914.915.114.614.4 9.1 12.4 14.8 14.7 16.216.114.914.914.213.7 8.5 14.9 15.2 16.3 16.316.115.214.815.416.0

3.6 3.8 3.5 3.8

3.8 3.7 3.6 3.4 3.8 -

investigators that copper is exceedingly potent as a supplement to iron. Table III gives the averages for the first series of iron and copper tests. The full significance of Chart I which summarizes these results along with other data subsequently available will be

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428 Nutritional Anemia

discussed later. Numerous tests were made with 0.1, 0.05, 0.025, and 0.01 mg. of copper daily. Larger quantities of copper (1,2,4, 6, and 8 mg. daily) were also fed to determine whether any addi- tional increment of response or possible toxic effects might be noted. Rats refuse to drink milk containing high concentrations of copper. Attempts at dilution with larger volumes of milk often resulted in incomplete consumption of the total mineral dosage. Thus results from this latter group must be considered approximate but at least no toxic effect was noted. From these data it may be concluded that the optimum level of copper for the rat is between 0.1 and 1.0 mg. daily. No increase in rate or height of hemoglobin regeneration was observed with amounts greater than 1.0 mg. but rather was there a suggestion of less rapid re- sponse with the higher levels of copper. With daily doses of less than 0.1 mg. there was slight but distinct retardation in hemo- globin synthesis which was more evident when the daily iron allow- ance was low (0.25 or 0.1 mg. daily).

Response Due to Iron, Copper, and Manganese

The various doses of manganese noted in Table I were fed as supplements to each of the quantities of iron and to each of the combinations of iron and copper. It was conceivable that some combination of these three elements might result in optimum hemoglobin synthesis. 211 rats in all were fed on the four doses of manganese (0.1, 0.05, 0.025, and 0.01 mg. daily). The apparently inconsistent and variable results obtained led us to analyze these data from every possible angle. Individual varia- tion in animals is so great as to necessitate very appreciable differ- ences before conclusions may be drawn. The apparent slight beneficial effect of manganese noted in some of our earlier work and in those rats receiving higher levels of manganese (1, 2, and 4 mg. daily) was observed in a limited number of cases in all of which a uniformly low initial hemoglobin did not obtain. Further experiments are being conducted on this phase of the problem but difficulty has repeatedly been encountered in inducing rats to take the higher doses of manganese. As in the case of higher copper levels results are unreliable when incomplete mineral supplement and less than an adequate quantity of milk are consumed.

The responses of groups of rats receiving different mineral sup-

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H. S. Mitchell and L. Miller

plements may conveniently be compared by subtracting the average figures week by week for gm. of hemoglobin per 100 cc. of blood. These differences may be interpreted as due to the difference in mineral supplement supplied. If such a comparison is made between differences due to copper and those due to man- ganese at exactly the same level of intake the differences due to manganese are much less than those due to copper. But it might

TABLE IV

Weekly Hemoglobin Averages Shozving Response to Diflerent Levels of Manganese with Constant Iron and Copper Supplements

The Hb values are given in gm. per 100 cc. of blood.

Average weekly Hb for

_-------------- w. w. w. 0.5 0.01 0.1 4 3.7 8.311.713.715.615.615.6 15.215.4 15.616.7 0.5 0.01 6 3.8 7.310.612.213.314.814.9 15.015.3 16.015.6

----------- Difference. . . . . . . -0.1 1.0 1.1 1.5 2.3 0.8 0.7 0.2 0.1 -0.4 1.1

----------- 0.5 I 0.01 I 0.05 4 3.2 8.511.814.014.614.815.3 15.715.6 16.016.5 0.5 0.01

I 6 3.8 7.310.612.213.314.814.9 15.015.3 16.015.6

--------- -- Difference. . . . . . . -0.6 1.2 1.2 1.8 1.3 0 0.4 0.7 0.3 0 0.9

~----~----- 3.4 7.711.413.114.515.015.4 14.315.3 15.615.8 3.8 7.310.612.213.314.814.9 15.015.3 16.015.6

----------- -0.4 0.4 0.8 0.9 1.2 0.2 0.5-0.7 0 -0.4 0.2 --------- --

3.3 7.912.514.514.915.816.5 16.115.6 15.916.1 3.8 7.310.612.213.314.814.9 15.015.3 16.015.6

-------- --- Difference. . . . . . . -0.5 0.6 1.9 2.3 1.6 1.0 1.6 1.1 0.3-0.1 0.5

be argued that these smaller differences were still significant if it were not for the ,fact that a volume of additional data does not substantiate even this slight beneficial effect. A similar calcula- tion showing the effect of four levels of manganese fed as supple- ments to 0.5 mg. of iron and 0.01 mg. of copper is given in Table IV. This low level of copper would presumably give manganese a chance to show its effect if there were any. The differences due

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Nutritional Anemia

to manganese in this instance seem to be negligible as they fall largely within the range of experimental error. More convincing, however, than either of these is the evidence presented in Table V. The differences listed here are those due to 0.1 mg. of manganese daily fed in addition to the iron and copper doses listed in the left- hand column. Since there is no consistent or significant variation correlated with the graded manganese dosages nor any summation effect due to manganese plus copper it seemed logical to take the algebraic total of these differences to detect if possible any general trend. The average difference due to 0.1 mg. of manganese ob- tained by this calculation resolves itself into distinct insignificance. Three similar tables, not published here, demonstrate that the lesser doses of manganese (0.05, 0.025, and 0.01 mg. daily) are equally ineffective in stimulating hemoglobin regeneration in milk anemia in rats. It may therefore be concluded from these data that manganese, in doses of 0.1,0.05,0.025, and 0.01 mg. daily, has a negligible or at least insignificant effect in the synthesis of hrmo- globin in the rat, either when used alone with iron or in addition to iron and copper.

It must be remarked, however, that manganese appears to have some stimulating effect on growth and food intake; and it yet remains to be demonstrated what physiological effects the higher levels of manganese may have if the rats can be forced to take these amounts.

Assuming then that the effect of these levels of manganese on hemoglobin regeneration is negative, we are justified in using all of the data accumulated in the groups fed on iron, copper, and manganese for making a more extensive study of the response of rats to graded quantities of iron and copper disregarding the man- ganese additions. Chart I represents the averages of the larger number of rats thus available on each of the levels of iron and copper indicated. Over 900 hemoglobin determinations are rep- resented in each of the three groups of curves. A careful study of this chart reveals several interesting facts. With 0.5 mg. of iron even the lowest of the’four levels of Cu is almost as effective as the highest level in its supplementary action. The cont,rast is striking, however, between the response to 0.01 mg. of copper and that to 0.5 mg. of iron. With half the quantity of iron (0.25 mg. daily) the higher doses of copper succeed in stimulating a synthesis of

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TABL

E V

Diffe

renc

es

in

Hem

oglob

in Le

vels

Due

to 0

.1

Mg.

oj

Mau

gane

se

Fed

in

Addi

tion

lo

Vario

us

Qua

ntitie

s of

Iro

n an

d Co

pper

Su

pplem

ents

The

Hb

value

s ar

e giv

en

in

gm.

per

100

cc.

of

blood

.

Supp

lemen

t8

Fe

fw.

0.5

0.5

0.5 0.5

0.5

0.25

0.

25

0.25

0.

25

0.1

0.1

0.1

0.1

0.1

-7 CU

w.

0.1

0.05

0.

025

0.01

0.

1 0.

05

0.02

5 0.

01

0.1

0.05

0.

025

0.01

- I No.

of ra

ts In

itial

ifhen

ce

11

0.4

12

-0.2

12

-0

.1

8 -0

.1

10

-0.1

11

-0

.1

8 -0

.3

9 0.

2 8

0.3

9 -0

.2

9 0.

3 9

-0.2

10

0.

3 10

-0

.3

Alge

brai

c to

tal..

. .

. -0

.1

Aver

age

diffe

renc

e.

. .

0

! - _ - -

let

2nd

3rd

4th

5th

6th

7th

8th

9th

10th

1.2

0.1

1.5

1.1

1.1

1.0

1.1

1.4

1.0

0.9

-0.2

-1

.0

-3.1

1.

2 1.

6 1.

6 1.

2 1.

0 0.

9 1.

2 0

-0.1

0.

1 1.

7 0.

2 1.

0 1.

6 1.

4 1.

0 1.

5 -0

.5

-1.9

0.

8 1.

3 -0

.1

0.6

1.1

0.7

0.9

1.7

1.0

1.1

1.5

2.3

0.8

0.7

0.2

0.1

-0.4

1.

1 -0

.2

-0.1

-1

.0

-0.8

-0

.7

-0.5

-0

.4

-0.1

0.

7 -0

.1

0.8

1.6

2.1

0.6

0.4

1.3

0.1

-0.3

0.

9 0.

1 -3

.9

-3.9

-1

.6

-2.3

-2

.9

-2.4

-1

.6

-0.9

-0

.6

-0.2

-0

.7

-0.1

0.

5 0.

9 1.

6 0.

3 0.

8 0.

3 1.

0 2.

7 0.

6 0.

3 0.

4 0.

8 0.

3 -0

.3

-0.6

-0

.6

0.4

-0.7

-0

.9

-1.3

-2

.1

-2.1

-3

.0

-2.4

-2

.6

-0.9

-0

.8

-2.0

-1

.0

-1.8

-2

.3

-1.8

-0

.5

-1.3

-1

.5

-1.4

-1

.0

-0.3

0.

6 1.

4 2.

6 3.

0 3.

8 2.

9 2.

2 1.

4 2.

2 2.

2 -0

.8

-0.7

-0

.6

-0.1

-0

.7

-1.2

-1

.6

-3.0

-3

.0

-3.7

_

-3.7

-0.3

-6.4

-1

.2

-0.1

5.8

1.9

0.1

1.3

0.1

0 0

-0.9

-0.1

3.2

-0.5

0.

4 0.

2

_ -

_ _

-

4.4

0.3

- W

eek

of ex

perim

enta

l pe

riod -

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Q 0

,0as

lw+

0 0.

01 m

g.C

u .

no C

u I

I I

I I

6 t

* 6

r

CEAR

T I.

Aver

age

hem

oglo

bin

resp

onse

to

di

ffere

nt

leve

ls

of i

ntak

e of

iro

n an

d co

pper

su

pple

men

ts

to

milk

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H. S. Mitchell and L. Miller 433

hemoglobin to the normal level in 8 weeks while on the smaller doses the normal level is not reached in 10 weeks. The difference between 0.01 mg. of copper and none, however, is more pro- nounced than with the 0.5 mg. of iron.

With 0.1 mg. of iron the differences in supplementary action of the various doses of copper are even more pronounced and the slope of all the curves has changed approaching a straight line. The natural tendency for rapid recovery followed by a plateau at the normal hemoglobin level has been partially inhibited. This would indicate that the iron as well as the copper has become a limiting factor at this level. Furthermore, none of these averages reaches the normal during the whole experimental period of 10 weeks. It may therefore be concluded that 0.1 mg. of iron daily is decidedly inadequate for the growing rat although it may serve as a convenient device for demonstrating the slight differences in response due to a so called catalytic supplement such as copper.

Comparing these three groups of curves it would appear that 0.25 mg. of Fe daily is adequate for the rat if sufficient copper is present in the ration. At this level also the differences are easily noted between the various doses of copper and none. It is there- fore suggested that 0.25 mg. of iron daily is a convenient level to use when testing the supplementary action of other minerals, food, etc.

SUMMARY

1. There is slow but definite hemoglobin response to pure iron salts. The response is directly proportional to the amount of iron fed within the limits tested in this experiment.

2. The supplementary action of copper is confirmed. The optimum daily dose for a rat appears to be between 0.1 and 1.0 mg. daily.

3. Manganese fed in 0.1, 0.05, 0.025, and 0.01 mg. doses daily has a negligible supplementary action to iron in hemoglobin syn- thesis, but may have a slight stimulating effect on growth and food intake. Higher doses of manganese are being investigated further.

4. The optimum daily iron requirement for a rat is around 0.25 mg. if sufficient copper supplement (0.1 mg. daily) is provided. The 0.5 mg. of Fe daily used by many investigators affords a

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434 Nutritional Anemia

margin of excess which, however, may not be desirable when differ- ences in the supplement,ary action of graded doses of other factors are being demonstrated.

5. When recovery from a nutritional anemia is the experimental procedure a uniformly low initial hemoglobin in experimental animals is essential for consistent results.

Acknowledgment is made of the technical assistance of Mrs. Ethel Cox and Mrs. Eleanor Turner.

BIBLIOGRAPHY

1. Waddell, J., Elvehjem, C. A., Steenbock, H., and Hart, E. B., J. Biol. Chem., 77, 777 (1928).

2. Mitchell, H. S., and Vaughn, M., J. Biol. Chem., 76, 123 (1927). Mit- chell, H. S., and Miller, L., J. Biol. Chem., 86, 366 (192930).

3. Waddell, J., Steenbock, H., and Hart, E. B., J. Biol. Chem., 83, 243 (1929).

4. Beard, H. H., and Myers, V. C., J. Am. Med. Assn., 93, 1210 (1929); J. Biol. Chem., 87, p. xxxix (1930).

5. Hart, E. B., Steenbock, H., Waddell, J., and Elvehjem, C. A., J. Biol. Chem., 77, 797 (1928); Waddell, J., Steenbock, H., Elvehjem, C. A., and Hart, E. B., J. Biol. Chem., 63, 251 (1929); Waddell, J., Steen- bock, H., and Hart, E. B., J. Biol. Chem., 84,115 (1929).

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10. Goerner, A. J., J. Lab. and Clin. Med., 16, 119 (1929). 11. Mitchell, H. S., and Miller, L., Bull. Ballle Creek Sanitarium and

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Helen S. Mitchell and Lila MillerSUPPLEMENTS

COPPER, AND MANGANESE IRON,QUANTITATIVE VARIATIONS IN

STUDIES IN NUTRITIONAL ANEMIA:

1931, 92:421-434.J. Biol. Chem. 

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