IRON

I. NUTRITIONAL UTILIZATION BY E. B. HART, (From the Department

IN

NUTRITION.

ANEMIA ON WHOLE MILK DIETS AND THE OF INORGANIC IRON IN HEMOGLOBIN BUILDING.* H. STEENBOCK, WADDELL. C. A. ELVEHJEM,
University
AND

J.

of Agricultural Chemistry, Madison.)

of Wisconsin,

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(Received

for publication,

June 2, 1925.)

Von Bunge was the first to show (1) that milk is very low in iron. He also showed that mammalian young (the guinea pig excepted) are born with extra stores of iron in the spleen and liver and that these reserves become sources of iron for hemoglobin building during the early periods of life when milk is the sole article of the diet. Abderhalden demonstrated (2) that if the animal was kept for a prolonged period of time on a diet consisting only of milk, anemia would result with a marked decrease in the hemoglobin content of the blood. He further demonstrated that the addition of inorganic iron to the milk diet did not result in an increasein the hemoglobin, although he seemedto find some favorable effect upon the growth of the animal (3). Since the time of these experiments by Abderhalden, it has been assumed that inorganic iron cannot take part in hemoglobin building although Abderhalden himself held no such view. In his well known text on physiological chemistry he states:
Enough has been said to show that the formation of hemoglobin does not solve the question as to the part that iron plays in its formation. The We cannot hope kernel of the whole question has not yet been attacked. for a solution of the problem until we understand clearly the formation of hematin. The mere fact that the addition of iron to nutriment poor in iron * Published with the permission tural Experiment Station. of the Director 67 of the Wisconsin Agricul-

68

Iron in Nutrition.

does not have any distinct influence upon the formation of hemoglobin in no way speaks against the participation of inorganic iron in the synthesis of hemoglobin in the case of normal nutrition, but it indicates that other building material is wanting as well as the iron.

That same view has again been expressed by Robertson his text on The principles of biochemistry when he says:
There is every reason for supposing that the pyrrole group

in

-c-cII - \/
N

II c-

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cannot be synthesized by animals but must be obtained by them preformed; that is to say from the tissues of plants or from the tissues of animals which acquired it from plants. This pyrrole grouping is contained in small amounts in the majority of proteins and it forms a very important component of chlorophyll, the green coloring matter of plants, which as we shall see is very closely related chemically to hemoglobin. It is not improbable, therefore, that inorganic iron salts added to an exclusive milk diet are not utilized for building up hemoglobin simply for the reason that other component parts of the hemoglobin molecule as essential as iron itself are either lacking altogether in the milk diet or present therein in insufficient amount to subserve the needs of the blood forming tissues and those of the other tissues of the body as well.

It is unfortunate that in several recent investigations with the rat the original fact observed by Abderhalden that inorganic iron added to a milk diet of some animals will not improve the ration in respect to its capacity to build hemoglobin has not been given the consideration it deserves (4, 5). In these investigations by Mattill and associates, Anderegg and Nelson, and Sure, no data have been presented in reference to the normality or abnormality of the blood, especially in respect to its content of erythrocytes and hemoglobin. In the work of the aforementioned investigators divers natural materials have been added to the diet with great improvement in the conditions of the rat, especially in respect to its capacity to reproduce. The conclusion that they reached was that vitamin E was deficient in the milk and was introduced by the inclusion of various materials such as wheat embryo a&l green lettuce leaves. It is, however, true that in most of the recent

Hart, Steenbock, Elvehjem,

and Waddell

69

investigations on the nutritive properties of milk an exclusive, milk diet has not been used, but generally one in which a whole milk powder constituted 50 per cent or more of the ration and of which the remaining portion consisted of lard, corn-starch, and a salt mixture. This was the ration used by Mattill and Stone. The ration used by Anderegg and Nelson contained whole milk powder varying from 5 to 99.8 per cent. When 99.8 per cent was used the remainder of the ration was a citrate of iron. Where the lower levels of milk powder were used, casein, agaragar, starch, or dextrin constituted the rest. It is not to be expected, on the basis of Abderhaldens work, that an animal receiving a ration of 99.8 per cent of whole milk powder fortified with 0.2 per cent of citrate of iron will be able to build or maintain a blood stream normal in respect to its hemoglobin content. Admitting this to be a fact, we may well ask the question: Should we expect normal rates of reproduction from animals suffering from anemia? This immediately brings up the matter of the availability of inorganic iron as compared with iron in organic combination. Of course, citrate of iron is no more organic so far as its relation to hemoglobin building is concerned than is the oxide of iron. It has seemed to us, for some time, that the whole problem of what was involved in normal hemoglobin building needed intensive study, involving the use of inorganic iron as well as the use of complex organic iron compounds; and that in addition there should be studied the r81e in this problem of simple and complex structures such as chlorophyll, which is entirely free from iron. The relation that these factors would have to the recent studies on the nutritive properties of milk mentioned above; to vitamin E itself; and to the large practical problem of the relation of diet to an anemia of suckling pigs observed in England by McGowan and Crichton (6) and very probably related to a similar trouble observed in this country in spring born litters and known as thumps is worthy of intensive consideration.
EXPERIMENTAL.

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In this paper data are presented which support the original hypothesis of Abderhalden; namely, that hemoglobin can be built from inorganic iron in the diet only when t.hat diet is accom-

70

Iron in Nutrition.

panied by certain organic structures. And, further, that milk is not only very low in iron, as is well known, but that it is also low in the organic complex or complexes that make possible the building up of hemoglobin by the organism in the presence of inorganic iron. For our first experiments rabbits were used. They are easy to bleed from the ear and are very sensitive to a nutritional anemia induced by whole milk feeding. On such a diet an initial hemoglobin content of 85 to 95 per cent and an erythrocyte content of 7 to 8 million per c.mm. will be reduced to 50 to 60 per cent and to 3 to 4.5 million in the course of 5 to 6 weeks. Yet at the same time growth of the animals continues at an approximately normal rate. Placed on the liquid whole milk diet at an initial weight of 400 to 500 gm. they will reach weights of 1000 to 1200 gm. in 6 weeks although the blood stream is progressively deteriorating. These increments in live weight, however, will not be continued indefinitely as anemia progresses. When the hemoglobin content of the blood reaches the lower levels of 50 to 55 per cent, growth will cease and death supervene. The remarkable fact is the apparent well being and growth of the animal for such a considerable period of time although the blood stream is abnormal. In our work we took the rabbits at weaning time (4 to 5 weeks old and 300 to 500 gm. in weight) and placed them in suitable In practically all expericages on 3 inch mesh screen bottoms. mental work with small animals this procedure is now followed It prevents consumption of feces and is in this laboratory (7). Liquid cows milk (whole) plus sodium citrate highly sanitary. was the only food allowed as the basal ration. The sodium citrate was added at the rate of 3 gm. per liter to prevent the formation of large curds in the stomach and reduce the possibility of gastritis. Very few animals suffered from gastrointestinal disturbances on the diet of whole milk alone, but it nevertheless was a regular procedure in our work to incorporate the sodium citrate, at least for the first 4 weeks of the experimental period. The milk used was from cows that were housed all the time Whether the diet of and received no fresh green plant tissue. the cow would influence the milk in respect to its capacity to build hemoglobin we do not know at present.

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Hart, Steenbock, Elvehjem,

and Waddell

71

Hemoglobin was estimated by the Fleischl hemometer, modified by Miescher. In later work we have also used the Dare hemoglobinometer. The erythrocytes were determined in a standard Thoma-Zeiss hemocytometer. The recovery type of experiment was the one followed in our later work although in some of the earlier experiments the method of preventing anemia by incorporating various materials with the diet at the time of the initiation of the experiment was followed. Table I shows a few of our earlier results where the method of prevention rather than that of cure was followed. In these earlier experiments iron only (Fez03), or fresh cabbage, or fresh cabbage plus iron (Fez03) was used as a supplement to the milk diet. 50 gm. of cabbage were allowed daily. This amount of cabbage introduced 0.0002 gm. of Fe, as determined by a method which will be published later. On milk plus sodium citrate the animals invariably came down with anemia. When cabbage was fed as a supplement at a level of 50 gm. daily per individual, the animals often escaped anemia or were When iron (0.015 gm. per individual very slow in coming down. daily) was fed in addition to the milk-cabbage ration, the blood stream remained normal and there was never any tendency toward an anemia. When iron only (0.015 gm. of Fez03 per individual daily) was added to the milk diet at the initiation of the experiment, anemia developed in most cases; although the animals were slower in coming down than on milk only. Occasionally an animal would not become anemic on the basal ration supplemented with iron, indicating a possible reserve of hemoglobinbuilding material in the body or a very efficient use of the small amounts of such material that may be contained in the milk. We soon learned that we could dry the cabbage at 65C. for 24 hours and still have it effective as a preventative of anemia when fed with inorganic iron. Data showing results with this material are also incorporated in Table I. It was fed at a level of 5 gm., equivalent to 50 gm. of fresh cabbage per individual per day. In the administration of the fresh unextracted cabbage a small amount of iron was also given and it became clear that this type of an experiment would not conclusively answer the

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72

Iron in Nutrition.

question: Are we introducing some organic complex containing iron in traces which can be directly used in hemoglobin building or can the organic complex be iron-free and in the presence of inorganic iron be utilized in hematin formation? Consequently we resorted to the use of certain plant extracts.
TABLE I.

Record

of Hemoglobin and Administration

Erythrocyle Conlents of Blood of Cabbage and Iron Oxide.

as Modified

by

Ei
1

Date.

Diet.

1HemoIglobin. t ET cent

Erythrocytes. million per c. mm.

Weight. pm.

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Jan. Feb. Jan. Feb. Jan. Feb. Jan. Feb. Jan. Feb. Jan. Feb. Jan. Feb. Jan. Feb. Jan. Feb. Jan. Feb.

6 17 6 17 6 17 6 17 6 17 6 17 6 17 6 17 6 17 6 17

Whole milk citrate. I I

only

+ I I

sodium

83 59 93 70 89 98 94 92 104 72 117 96 + 85 96 83 89 93 96 98 94

7,175,OOO 4,200,OOO 8,300,OOO 5,400,OOO 7,270,OOO 7,650,OOO 6,650,OOO 6,450,OOO 7,550,OOO 5,450,OOO 9,850,OOO 8,800,OoO 7,4&~,000 7,300,OOO 6,850,OOO 6,525,OOO 7,150,OOO 6,500,OOO 7,cJOO,OOO 6,025,OOO

440 loo0 615 740 475 1320 490 1440 510 1240 620 1240 610 1320 555 1360 475 1330 630 1260

Whole milk + 50 gm. cabbage daily. I I Whole milk + 15 mg.

fresh

+ sodium citrate Fe,O, daily.

Whole milk + 15 mg. Fe203 50 gm. cabbage daily. I I

10

Whole milk + 15 mg. Fe203 + 5 gm. dried cabbage daily. I I

-

In Table II is given the detailed record of a rabbit started on milk plus sodium citrate only. The record is a typical one and depicts the development of anemia; the failure to restore nor-

Hart, Steenbock, Elvehjem,

and Waddell

73

mality of the blood stream by ferric oxide administration (this administration was continued for 2 weeks); and the final success in the correction of the anemia when in addition to the ferric oxide there was administered the alcoholic extract of dried cabbage equivalent to 50 gm. of fresh cabbage daily. This table illustrates very clearly the general situation that occurs. With an anemia induced by a milk diet only, the administration of inorganic iron will not restore the blood stream to a normal condition; but if in addition to the inorganic iron certain organic structures are added to the diet, recovery is rapid and complete.
TABLE II.

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Record

of Hemoglobin Normal
Date.

and Erythrocyte with Alcoholic

Diet.

Contents of Blood. Extract of Cabbage.

Restoration

to

Iemolobin. er cenl

_
million per c. mm.

Weight. Qm.

75

Jan. Feb. ( Mar.

6 13 21 29 4 11 17 24 2

Whole

milk + sodium citrate. + ( + + ( + + 15 mg. Fe203 added daily. 15 I

9 16 23 27

25 mg. alcoholic extract dried cabbage equivalent to 50 gm. fresh cabbage + Fe,Ol. I I I

83 75 73 76 66 68 59 59 64

7,175,OOO 5,775,OOO 4,375,OOO 4,000,000 4,200,OOO 4,425,OOO 4,200,OOO 3,925,ooo 3,450,OOO

440 615 790 850 840 1000 940 870

68 76 79 81

4,775,OOO 5,450,OoO 5,450,OOO 5,950,ooo

-

970 1050

In our later experiments the recovery type of experiment has been employed. When the animals showed a hemoglobin content of 50 to 60 per cent and at the same time the erythrocytes were down to 3 to 4.5 million, the particular material to be tested was added to the diet. This was done by triturating the material with a small quantity of milk in a mortar. Material prepared in this manner was readily consumed in most cases. Further experiments were made to determine if extracts of desiccated cabbage would be as potent as the unextracted plant

'74

Iron in Nutrition.

tissue in restoring an anemic blood stream to a normal condition, particularly in the presence of inorganic iron. In this respect the experiments were completely successful. The extract was prepared from desiccated cabbage by extracting it for 24 hours at room temperature with 95 per cent alcohol. The alcoholic solution was filtered off and evaporated before a fan at a temperature of about 40C. A syrupy mass was obtained which was found practically free from iron as estimated by our method which will accurately determine as small an amount as 0.0001 gm. The determination of iron by the method in use is finally dependent upon the development of a color with potassium thiocyanate. It took 1 gm. of the extract or 40 times the daily administration to show a faint color with thiocyanate. This extract was fed at a daily level equivalent to 50 gm. of fresh cabbage and appeared as potent a carrier of the organic complex concerned in hematin building as the unextracted cabbage. Similar experiments have now been carried out with an alcoholic extract of yellow corn-meal, giving results similar to those obtained with desiccated cabbage. Evidently these two different types of plant material carry certain substances soluble in alcohol, and free from iron, which are intimately concerned in hemoglobin formation. Whatever their nature, they appear to be low in amount or absent from whole milk. See Tables III and IV for the records of these data. Since it has been suggested that the pyrrole nucleus may be concerned in hematin building, we naturally turned to chlorophyll itself. It is, of course, true that the pyrrole nucleus is contained in the proteins of milk, but apparently from free proline alone hematin formation in the animal cannot take place. It is Direct experiments to determine this point are in progress. entirely possible, however, that the organic substance concerned here is much more complex than a single pyrrole molecule, if it is a pyrrole structure at all; for example, it may consist of a multiple of these molecules in definite union such as has been projected for the structure of both chlorophyll and hematin. The possibilities are many and very inviting and will be studied as time permits. Chlorophyll was prepared by the well known method of Will-

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TABLE

III

Record
Rabbit1 No.

of Hemoglo?)in Normal
/ Date

and Erythrocyle with Alcoholic

Diet.

Contents of Blood. Extract of Cabbage.

I g P er cent

Restoration

to

Srgthrocytes.
million per c. mm.

veigllt. 0m.

.i

/ .!:.r. Apr.

16

8
4 8

15 22 7 18 3 15 22 3 15 18 27 4 16

+ sodium citrate. + + alcoholic extract 25 mg. cabbage + 15 mg. Fe203. I milk + sodium citrate. + + I + -I + + 25 mg. alcoholic extract cabbage + 15 mg. Fe203. I
TABLE IV.

Whole

milk

79 66

6,275,OOO 3,775,OOO

260 330 410

83 81 81 69 69 77 70 68 53

6,550,OOO 5,950,ooo 5,500,OOO 5,400,OOO 4,800,OOO 6,050,OOO 4,975,ooo 4,725,OOO 3,500,OOO

456 530 430 550 660 765 850 920 1070

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12

Nov. Dec. Jan. Feb.

Whole I

75 89 89

5,175,OOO 5,250,OOO 5,375,OOO

1100 1200 1320

Record

of Hemoglobin
Normal
Date.

and with

Erythrocyte Alcoholic
Diet.

Contents of Blood. Extract of Corn-Meal.

-

Restoration

to

Weight. 0m.

RFc!Ti
33 Apr. May 29 Mar. Apr. ( May

- _

Henlo. Iglobin.

Erythrocytes. milzion per c. mm.

1Pm cm

15 7 I 28 4 8 30 10 25 25 30 11 13

Whole

milk

+ sodium

citrate. 59 3,425,OOO 61Q 746 820 920 320 380 470

+ ( 25 mg. alcoholic extract corn-meal + 15 mg. Fez03. I milk + sodium citrate. + + 25 mg. alcoholic extract corn-meal + 15 mg. Fe208.
-

68

77 75 81 47

3,700,OOO 6,950,OOO 6,700,OOO 5,875,OOO 3,750,OOO

Whole

77 64 73

5,950,ooo 5,700,OOO 5,600,000

710 800 920

75

Iron in Nutrition.

statter from tomato leaves desiccated in the dark and before a fan. The ground dried material was extracted on a Buchner funnel with 80 per cent commercial acetone, allowing the solvent to pass through the powder once and sucking dry after each addition. The acetone solution was added in two separate portions to an equal quantity of petroleum ether, and with each portion, water was added to constitute one-eighth the volume of the petroleum ether. The mixture was gently rotated and mixed to transfer the chlorophyll, xanthophyll, and carotins to the petroleum ether layer. The watery acetone was run off each time and the petroleum ether further washed with two additions of 80 per cent of acetone which was also run off. The trace of acetone remaining in the petroleum ether was removed by washing four times with water. The petroleum ether solution was next washed with three successive additions of 80 per cent of methyl alcohol which removed the xanthophyll. The remaining petroleum ether solution was washed again with four additions of water to remove the last traces of acetone and methyl alcohol. At this stage the chlorophyll was precipitated as a fine suspension in the petroleum ether. This suspension was shaken with dry sodium sulfate and talcum powder and filtered on a Buchner funnel through a layer of talc. The chlorophyll on the Buchner funnel was dissolved in anhydrous, alcohol-free ether, concentrated in the drying room, and finally dried in a desiccator under diminished pressure. The preparation was free from iron as determined by our method. No further determinations of chemical or physical constants were made to establish its degree of purity. This chlorophyll was fed at the level of 25 mg. daily per individual plus the inorganic iron and the whole milk. The results show that the chlorophyll functioned in the same way as fresh cabbage, as the alcoholic extract of dried fresh cabbage, or as the alcoholic extract of corn-meal, in correcting the anemic condition induced by whole milk only. We presume that every phytochemist will admit that there are chlorophyll precursors in such plant tissues as etiolated cabbage leaves or corn-meal, but at present there is no evidence as to their nature. Table V gives a part of the records secured with chlorophyll. In the work SO far done, about 75 rabbits have been used, but only a small percentage of the records are here presented.

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Hart, Steenbock, Elvehjem,

and Waddell

77

Are These Complexes of Plant Origin and Concerned in Hematin Building in the Animal Related to Any of the Known Vitamins? From the fact that nutritional anemia is produced on a whole milk diet which is well supplied with vitamin A, it is evident
TABLE V.

Record

it &k?_ -

of Hemoglobin and Erythrocyte Normal with Addition

Date. Diet.

Contents of Blood. of Chlorophyll.

Restoration

to

lemo,lobin. er

Erythrocytes.
million c. mm. per

Veight.

__ Cm t

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l7m.

15

Mar. Apr. May

-.

16 26 4 6 15 22 28 6 16 26 1 10 17 18 22 29 6 15 27 1 2 8 13

Whole milk citrate. I I

only

+ I

sodium 73 57 + 15 mg. 7,000,000 4,975,ooo 210 325 370 370 390 360 420

25 mg. chlorophyll FelOt. I Whole milk citrate. I I I only

I I + I I + I 15 mg. sodium

81 68 77 85

3,625,OOO

2,f3@3,f3c@ __
3,850,OOO 6,725,OOO

14

Mar. Apr. May

--

79 81 79 66

6,625,OOO 6,425,OOO 5,575,OOO 4,325,OOO

25 mg. chlorophyll FelOt. I Whole I

170 250 310 340 400 460 500 560 680 830 850 830

53 81 83

3,500,000 7,050,OOO 6,425,OOO

34

Apr. May

milk + sodium citrate. + + 25 mg. chlorophyll + 15 mg. Fe203. I I

62 62

5,375,OOO 3,225,OOO

77 81

5,050,OOO 5,050,OOO

830

that this compound can be dismissed as being concerned in the problem. The same statement can be made with respect to

78

Iron in Nutrition.

vitamin B. Whole milk is not classed as a material rich in vitamin B, but there were no symptoms indicating such a deficiency. With respect to vitamin C, there is also no reason to believe that it is concerned in this problem. The rabbit is not sensitive to scurvy1 and if it were, the amount of milk consumed would have fully protected it. Consumption of 120 to 150 cc. of milk daily was the record for these rabbits and half of that amount of winterproduced milk will fully protect a guinea pig of 300 to 400 gm. from scurvy. Further confirmation of the non-relation of vit,amin C to this problem is found in the fact that nut,rit.ional anemia is readily induced in chicks on whole milk diets. This animal is certainly not dependent on preformed vitamin C in the diet.
TABLE VI.

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Record of Hemoglobin 5 to Y Months Old

Rat NO.

and Erythrocyte and of Rats Kept

Stock ration. I 3rythrwytes. - million per c. mm.

Contents of Blood oj Normal Rats 6 to Y Months on a Synthetic Diet.

Synthetic Hemoglobin. per cent ration.

Hemoglobin. per cent

Erythrocytes.
million c. mm. per

145 153 132 128 128 128 128

9,950,ooo 10,700,000 9,850,ooo 9,500,ooo 9,700,ooo 9,6@J,@Jo 10,550,000

96 117 117 119 134 130 142

10,050,000 7,600,000 8,450,OOO 6,300,OOO 7,650,OOO 9,450,ooo 11,800,OOO

As to vitamin D, that also can be dismissed. Anemias on a milk diet were as readily induced when the animal was subjected to ultra-violet irradiation as without. With respect to the relation of vitamin E to our problem we are not so clear. In the publications of Evans and Bishop (8, 9) or of Sure (5) nothing has been said as to the normality or the abnormality of the blood stream of their animals (rats). Reproduction was reestablished by the addition of just such natural materials as have cured the nutritional anemia described in this paper. It is, of course, readily admitted that vitamin E and the complexes concerned in nutritional anemia induced by a milk
1 Unpublished data.

Hart, Steenbock, Elvehjem,

and Waddell

79

diet may be different, but definite proof of this fact will be necessary. In some preliminary work with rats kept for 6 to 7 months on a synthetic diet consisting of casein 18, salt mixture 4, yeast 6, agar-agar 2, dextrin 70, and cod liver oil 2, the hemoglobin and erythrocyte contents of the blood were determined. These data are given in Table VI. In addition there are given the hemoglobin and erythrocyte contents of the blood of rats kept on stock rations and presumably normal. The data are too limited to decide definitely that these rats are or are not anemic on synthetic diets. From the data there is a suspicion that some of the animals on the synthetic rations were not normal in respect to the hemoglobin content of the blood. On the other hand, some of them on the synthetic diet appeared to have a blood stream just as rich in hemoglobin as those receiving the natural food mixture. Possibly all of the figures are within the range of variation. In one of their publications (9) Evans and Bishop raise the question as to whether they are dealing with chlorophyll or other plant pigments in restoring lost fertility in rats on synthetic diets by the use of green leaves, etc. With the use of a commercial preparation of chlorophyll-Mercks Phyllosan-they obtained results which are interpreted by them as excluding chlorophyll as having a r81e in their problem. Should further investigations absolutely exclude vitamin E as a factor concerned in hematin building, then it appears probable that in addition to the protein mixture of milk and the known vitamins there must occur, preformed in the diet, certain complexes needed for hematin formation.
SUMMARY.

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1. Rabbits limited to a whole milk (cows)-sodium citrate diet develop a nutritional anemia characterized by low hemoglobin and erythrocyte contents of the blood. 2. Inorganic iron (Fez03) added to the basal ration will not per se correct this anemia. 3. Inorganic iron in the presence of fresh cabbage or an alcoholic extract of desiccated cabbage or an alcoholic extract of yellow corn-meal will prevent or cure such an anemia. These extracts are free from iron.

Iron in Nutrition.
4. Chlorophyll, free from added inorganic iron, correct whole milk-sodium citrate stances to vitamin E and to nutritive properties of milk

iron, will also, in the presence of such an anemia as is induced by a diet. The relation of these subcertain recent investigations on the are discussed.

BIBLIOGRAPHY.

1. 2. 3. 4.

5.

6. 7. 8. 9.

von Bunge, G., Z. Biol., 1901, xlv, 532. Abderhalden, E., Z. Biol., 1900, xxxix, 193. Abderhalden, E., Z. Biol., 1900, xxxix, 483. Mattill, H. A., and Stone, N. C., J. Biol. Chem., 1923, Iv, 443. Mattill, H. A., Carman, J. S., and Clayton, M. M., J. BioZ. Chem., 1924, lxi, 729. Sure, B., J. BioZ. Chem., 1924-25, lxii, 371. Anderegg, L. F., and Nelson, V. E., Id. and Eng. Chem., 1925, xvii, 451. Sure, B., J. BioZ. Chem., 1923-24, lviii, 693. McGowan, J. P., and Crichton, A., Biochem. J., 1924, xviii, 265. Steenbock, H., Sell, M. T., and Nelson, E. M., J. BioZ. Chem., 1923, Iv, 399. Evans, H. M., and Bishop, K. S., J. Metabol. Research, 1922, i, 319, 335; 1923, iii, 201. Evans, H. M., and Bishop, K. S., J. Metabol. Research, 1923, iii, 233.

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