COBALT IN ANIMAL NUTRITION*

BY F. J. STARE AND C. A. ELVEHJEM

(From the Department of Agricultural Chemistry, University of Wisconsin, Madison)

(Received for publication, November 10, 1932)

The biological significance of cobalt has attracted the attention of relatively few investigators. McHargue (1) studied the oc- currence of certain inorganic elements including cobalt in soils, plants, and animals, but the only definite values reported for cobalt were a trace in blue-grass, soy bean leaves, soy bean seeds, and 1.5 parts per million in Kentucky virgin soil. Fox and Romage (2) from spectrographic observations of the tissues of Archidoris tuberculata report the presence of 0.0003 per cent cobalt in the liver, and of smaller amounts in certain other tissues of polychaetes. Dutoit and Zbinden (3) in a spectrographic analysis of the ashes of human organs observed cobalt in the pancreas and traces of cobalt in some of the other organs, but none in the liver.

Bertrand and coworkers are the only investigators, so far as we know, who have studied cobalt in an extensive manner. Bertrand and Mokragnatz (4) in studies on the zinc content of certain French soils encountered small amounts of cobalt in several of the soil samples. This led to a more detailed study of the cobalt content of soils and also of the cobalt content of plants (Bertrand and Mokragnatz (5)) and animals (Bertrand and Macheboeuf (6, 7)). Bertrand (8) was impressed with the amount of cobalt observed in the pancreas in comparison to other organs and concluded that cobalt may somehow be connected with the elaboration of insulin. In experiments on the influence

* This work was aided in part by a grant from the University of Wiscon- sin Research Fund.

Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station.

473

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474 Cobalt in Animal Nutrition

of cobalt on the action exercised by insulin in rabbits (9), and later in dogs (lo), Bertrand and Macheboeuf concluded that cobalt increases the hypoglycemic power of insulin. Bertrand and Nakamura (11) have conducted a few feeding experi- ments with mice in an at.tempt to study the physiological im- portance of cobalt. It is impossible to reach any conclusion from this work because the animals were placed on diets deficient in factors other than cobalt and survived only about 3 weeks.

Mokragnatz (12) studied the effect of cobalt in the development of cultures of Aspergillus niger but observed no beneficial effect. He found that in concentrations of cobalt greater than 2 parts per million growth was retarded, and in concentrations of 400 parts per million growth was completely inhibited.

The recent reports from several laboratories concerning poly- cythemia produced by cobalt have revived interest in the bio- chemical importance of this element. Waltner and Waltner (13) observed in a study of the toxicity of various metals that cobalt when added to the diet of rats produced a marked increase in the number of red blood cells. An increase in erythrocytes due to the feeding of cobalt to dogs has been reported by Mascherpa (14).

Myers, Beard, and Barnes (15), and more recently Orten, Underhill, Mugrage, and Lewis (16), in studies on hemoglobin regeneration, reported a polycythemia produced by the addition of cobalt to a milk-iron-copper diet. Similar effects have also been observed in this laboratory (17).

These results led us to a study of the cobalt content of normal rats and those in which a polycythemia had been produced by cobalt feeding. Later we studied the distribution of cobalt in the organs of pigs which had received cobalt in their diet. These studies necessitated the development of a method for the estima- tion of small amounts of cobalt.

We present in this paper, first, a rapid, convenient, and accurate calorimetric method for the estimation of cobalt in biological materials which contain amounts of cobalt ranging from 0.01 to 0.5 mg. per sample; secondly, data showing that if cobalt does occur in the normal animal organism it does so in extremely minute amounts; and, thirdly, data revealing the distribution of cobalt in the bodies of rats and pigs fed a diet containing cobalt.

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F. J. Stare and C. A. Elvehjem 475

Method

Previous workers have utilized gravimetric methods designed for the general macro inorganic determination of cobalt. In the usual chemical reactions cobalt behaves quite like nickel, and in the ordinary gravimetric or volumetric analysis the problem is to separate the nickel and cobalt. Nickel can be effectively removed by precipitation with dimethylglyoxime and the cobalt remaining in the filtrate may be determined by precipitation as cobalti- nitrite in acetic acid solution. Bertrand, in his early work on the cobalt content of soils and plants, used the potassium nitrite method. In his later work with Macheboeuf he used a colori- metric method as follows (7): The mother liquor after the pre- cipitation and separation of nickel (Ni precipitated by dimethyl- glyoxime) was evaporated to dryness and heated to dull redness in a muffle furnace. The residue was taken up in hot concentrated hydrochloric acid and the solution evaporated to dryness and taken up in water. To this was added 0.2 cc. of a 1 per cent alco- holic solution of dimethylglyoxime and 2 drops of 10 per cent ammonia. The cobalt was estimated calorimetrically in the resulting brownish yellow solution; sensitivity was reported as 0.01 mg.

When nitroso-/3-naphthol is added to a hydrochloric acid solution of cobalt, a precipitate of cobaltinitroso-@-napthol is formed; nickel, if present, remains in solution. McHargue used this method in all of his work.

The methods mentioned above, or slight modifications of them, are neither convenient nor readily adaptable for the estimation of small amounts of cobalt in biological materials, since they all require the removal of iron, copper, and certain other elements. The precipitations required may involve loss of cobalt through co-precipitation, and for small amounts of cobalt the solubility factor of cobaltinitrite becomes an important factor. In the case of the nitroso+-naphthol method, often a small amount of nitroso- naphthol is formed which is not dissolved by boiling in nitric acid, and which may obscure any color due to cobalt,

We have developed a convenient and simple calorimetric method for the estimation of small amounts of cobalt based upon the observation recorded by Van Klooster (18) that the nitroso deriva- tive of R salt, sodium-2,3,6-fl-naphtholdisulfonate, when treated

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476 Coba*lt in Animal Nutrition

with cobalt salts forms a red dye of the composition of (CloHSNOsSzNa~)&o. The color formed is exceedingly stable and is not destroyed by heating with acids. Other cations such as Fe, Ni, Cu, and Zn, form colored dyes with the R salt but these colors are destroyed by boiling with an excess of nitric acid. The color is best developed in a solution made slightly alkaline with sodium acetate, and then by adding nitric acid and boiling, the red color formed by cobalt is produced.

Reagents Standard Cobalt Solution-l cc. = 0.1 mg. of cobalt. A solution

of pure CoClz.6Hz0 (0.2017 gm. per 500 cc.) in redist.illed water. Indicator Solution-l gm. of the nitroso R salt’ made up t,o 100

cc. with redistilled water. c. P. sodium acetate. Saturated solution of potassium hydroxide. Hydrochloric acid. Nitric acid. Procedure-Samples containing preferably between 0.05 and

0.5 mg. of cobalt are thoroughly ashed at dull red heat and the ash taken up in a small amount of 1: 1 hydrochloric acid. To this solution, whose volume for convenience should not be more than 20 to 25 cc., are added 2 drops of phenolphthalein, 2 cc. of indicator reagent, and about 2 gm. of sodium acetate. The mixture is warmed to about 70” and thoroughly stirred. Then while the stirring is continued, saturated potassium hydroxide is added slowly, drop by drop, until the reaction medium is just alkaline, as indicated by the phenolphthalein. The contents of the beaker are then heated to boiling and concentrated nitric acid added drop by drop until there is a distinct excess of the acid; the boiling is continued for 1 to 2 minutes. A permanent change in color toward red indicates the presence of cobalt. The colors produced by iron, nickel, and other metal salts are destroyed by oxidation with nitric acid. The solution is allowed to cool and made up in a 50 cc. volumetric flask with distilled water. This is compared in a calorimeter against a standard cobalt solution containing a somewhat similar concentration of cobalt and prepared in an analogous manner. By this procedure we have been able to determine as little as 0.01 mg. of cobalt,

’ Obtained from Eastman Kodak Company.

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F. J. Stare and C. A. Elvehjem 477

Results are presented in Table I to show the recovery of cobalt through this method. All the samples used in this study had been analyzed previously and found to contain less than 0.01 mg.

TABLE I

Recovery of Cobalt Added to Biological Materials before Ashing

Material

Beef liver.. . . . . . . . . . . . . . . . . “ “ . . . . . . . . . . . . . . . . . . . . . . . . .

Lobster.. . . . . . . . . . . . . . . . . . . Dog liver.. . . . . . . . . . . . . . . . . Beef “ . . . . . . . . . . . . . . . . . . . . . . . . .

“ ‘I . . . . . . . . . . . . . . . . . . . . . . . . .

Solution of distilled

water containing

m7.

0.2 co

0.2 co 1.0 Ni

0.2 co 5.0 Ni

0.2 co 10.0 Ni

0.2 co 10.0 Ni

0.2 co 5.0 cu

-

--

- Weight of

sample

!Tm.

5 5 5 5 5 5

Co added Co found Recovery

%l.

0.2 0.3 0.2 0.2 0.5 0.05

m&l.

0.195 0.3 0.194 0.194 0.495 0.05

per cent

97.5 100

97 97 99

100

TABLE II

Effect of Other Cations on Recovery of Cobalt

Amoo;mt indioata

added

cc.

2

-7

Co recovered

-

w?.

0.2

0.2

0.2

0.15

0.2

0.2

per cent

100

100

100

75

100

100

Solution of di;;;led

containing

mg.

0.3 co 5.0 Mn

0.2 co 5.0 Zn

0.2 co 0.5 Fe 0.5 cu

0.2 co 0.5 Mn 0.5 Zn 0.5 Fe 0.5 cu

AmoOfuunt ndioator added .

Co recovered

ml.

0.2

0.2

0.2

0.195

per cent

100

100

100

97.5

of Co per sample used (see Table III). It is readily seen that added cobalt can be quantitatively recovered from biological materials by this method.

As was previously mentioned, the nitroso R salt forms colored

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478 Cobalt in Animal Nutrition

compounds with other cations besides cobalt, but on boiling with an excess of nitric acid these colors are destroyed and only the red color of cobalt remains. To insure complete recovery of cobalt one must have an excess of indicator so that if other cations are present there will be sufficient indicator to unite with the cobalt. Table II demonstrates the recovery of cobalt in solutions con- taining known amounts of other cations. It is observed from Table II that cobalt was quantitatively recovered in the presence of nickel, manganese, zinc, iron, and copper. When other ele- ments are present in relatively large amounts, it is necessary to increase the amount of R salt added. For example, 2 cc. of indicator added to a solution of 0.2 mg. of cobalt and 10 mg. of

TABLE III

Cobalt Analysis of a Few Biological Products Each sample contained less than 0.01 mg. of cobalt.

Material

Rat liver.................... “ “ . . . . . . . . . . . . . . . . . . .

Abbott’s liver extract. Lobster..................... Dog liver.. .

W%ht sample

Qm.

1.984 2.073 5.0 5.0 5.0

Material I

Beef liver .................. Canned peas ............... Milk. ...................... Yeast, ..................... Lettuce ....................

W%ht sample

gm.

5.0 5.0

100.0 10.0 20.0

nickel gave only a 75 per cent recovery; however, when 4 cc. of indicator were used, recovery of cobalt was complete.

A few precautions should be taken to insure the best results. Ashing should be done in platinum dishes and at a temperature not exceeding 500”. The addition of calcium carbonate to materials which ash with difficulty decreases the time of ashing. The reaction of the nitroso R salt with cobalt takes place most readily and most thoroughly in the presence of sodium acetate and at a slightly alkaline pH; thus one should slowly make the solution alkaline, with the indicator present and with constant stirring to insure thorough mixing of the indicator with the solution. Blank determinations should be run frequently to check any possible source of con- tamination.

The results obtained when this method was applied to a few

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F. J. Stare and C. A. Elvehjem 479

biological substances are given in Table III. It is observed that cobalt, if it occurs in biological substances, does so in extremely small amounts, amounts less than 0.01 mg. for the weight of sample used. At present, efforts are being made to increase the sensitivity of the method so that we may determine smaller amounts of cobalt.

Since we have been unable to detect cobalt in the animal organs studied, and since it is known that cobalt when administered to rats produces a polycythemia, we were interested in determining whether or not we could detect cobalt in the body of an animal which had been fed cobalt, and if so in what tissues the cobalt was distributed.

Animal Xtudies

For the analysis of the total body, rats were used that had been employed in polycythemia studies. All these rats were fed a basal diet consisting of milk fortified with iron, copper, and manganese in amounts sufficient to supply each rat 0.5 mg. of Fe, 0.05 mg. of Cu, and 0.04 mg. of Mn per day. Some of the rats remained on the basal ration alone, others were given cobalt varying in amount from 0.1 to 2.0 mg. of Co per rat, daily. When the polycythemia studies were concluded, the rats were killed without loss of blood and the digestive system removed.

Table IV includes the data obtained by the analysis of the whole bodies and digestive systems of these rats, and the amount of cobalt which each rat received. In no case was cobalt definitely detected in concentrations above 0.01 mg. in the body or digestive Oract of a rat not receiving added cobalt, and in each case it was detected in those rats receiving added cobalt. The amount of cobalt retained in the body is proportional to the amount of cobalt administered in the diet. For example, Rat 594 received 0.6 mg. of cobalt per day and the amount of cobalt found in its body was 0.150 mg. Rat 601 received 1.0 mg. of cobalt per day and the amount detected in its body was 0.208 mg.

With young rats receiving more than 0.6 mg. of cobalt per day definite toxic effects were observed. At levels of 1.0 and 1.5 mg. per day the rats rapidly lost weight, and at 2.0 mg. per day the rats survived only for a period of 2 weeks. Rat 606, receiving 2.0 mg. of cobalt per day, was very feeble at the time it was taken for

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480 Cobalt in Animal Nutrition

analysis, and the amount of cobalt found in the entire body was 0.290 mg. With older rats no toxic effects were observed at levels less than 1.0 mg. per day.

For the study of the distribution of cobalt in the tissue, pigs were utilized because they could be fed larger amounts of cobalt and the organs would offer larger samples for analysis. Four pigs were used and given only whole cow’s milk ad &turn, to

Analysis ( ?f Rats Administered Cobalt and Amount of Cobalt Fed

Rat No

351 352 341 340 434 437 450 451 452 453 454 594 595 601 604 606

1

0

-

Lge wheI placed

8n experi. merit

wks.

3 3 3 3

8-10 S-10

3 3 3 3 3 3 3 3 3 3

TABLE IV

- t

- I

_-

-

Time on

experi- ment

Co fed per day

,aks. ml.

10 0.0

10 0.5 13 0.0 13 0.5 6 0.0 6 1.0

18 0.0 18 0.1 18 0.0 18 0.1 18 0.1

3 0.6 3 0.6 3 1.0 2 1.5 2 2.0

- - Weight R.b.c. when when killed killed

gm.

140 93

280 173 258 278 218 210 149

34 42 37 40 28

millions Pm

c.mm.

7.8 12.6

7.4 11.5

8.7 10.9

8.9 11.1

9.0 13.3 14.9

-

_

.

.

-

Co in Weight of

body d$%&l&

WJ. gm.

:0.01 0.0726

:0.01 0.0645

:0.01 0.146

:0.01 30.0 0.0484 31.0

co.01 28.0 0.0517 35.0 0.039 20.0 0.150 5.0 0.105 5.0 0.208 6.0 0.222 6.0 0.290 3.0

Co in digestive system

<O.Ol 0.125

<O.Ol 0.122 0.101 0.129 0.0715 0.102 0.29 0.067

which were added daily 25 mg. of Fe, 5 mg. of Mn, and 2 mg. of Cu. Two of the pigs received 25 mg. of cobalt in addition. The pigs were 2 weeks old when started on the experiment; one from each diet was killed at the age of 7 weeks, and the remaining two were killed at 12 weeks.

Results are presented in Table V which show the distribution of cobalt in the tissues of pigs that have received cobalt in their diet, and the relative absence of cobalt in similar tissues of pigs that have not received cobalt. It is noted that cobalt is distrib- uted to many tissues of the pig with the greatest amount found

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F. J. Stare and C. A. Elvehjem 481

in the liver, pancreas, and spleen. The vertebrae and ribs con- tained 1.98 mg. of cobalt per kilo of dry material, whereas the long bsnes contained an amount undetectable. It may be that the vertebrae and ribs are more active in erythrocyte formation and this may be where cobalt exerts its effect in producing a poly- cythemia.

We have stated that samplesof tissues from animals not subjected to cobalt treatment were found to contain less than 0.01 mg. of Co

TABLE V

Anal&s o.f Certain Tissues of Pigs on a Basal and Basal Plus Cobalt Diet I _

Pig No ............................ Age when placed on experiment,

wk.3 ............................. Time on experiment, wks .......... Co fed per day, mg ................ R.b.c. at beginning of experiment,

millions per c.mm ................ R.b.c. at end of experiment, mil-

lions per c. mm .................. Co per 10 gm. (dry), mg.

Liver ........................... Pancreas ........................ Spleen .......................... Heart ........................... Blood ........................... Vertebra and ribs ............... Skin. .......................... Muscle .......................... Long bones .................... Lung ..........................

.l 22 125

2 2 5 5

25.0 0.0

4.3 5.7

12.9 8.2

0.084 <O.Ol 0.080 <O.Ol 0.050 <O.Ol 0.0397 <O.Ol 0.0382 <O.Ol 0.0198 <O.Ol 0.011 <O.Ol 0.009 <O.Ol

:0.01 <O.Ol (

Co-fed Pig

Co-fed Pig

23

2 10 25.0

5.1

10.1

0.0318

0.025

0.0297

NOrId control

121

2 10 0.0

5.5

9.4

<O.Ol

<O.Ol

<O.Ol

per sample. However, a color sufficient to indicate a trace of cobalt was never observed in the analysis of any of these tissues. It is impossible for us to state that normal animals do not contain cobalt and that this element is unnecessary for normal develop- ment, but when a normal rat weighing 250 gm. contains much less than 0.01 mg. of cobalt it must be concluded that if this metal is essential it is active in extremely small amounts.

That cobalt in traces so small as to be undetected by the method described in this paper may be active physiologically is indicated

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Cobalt in Animal Nutrition

by the small amount necessary to produce polycythemia. The presence of 0.04 to 0.05 mg. of Co in the entire body of a rat is sufficient to produce a decided polycythemia (see Rat 454, Table IV). The results obtained for the cobalt content of the different tissues in cobalt-fed pigs indicate that this element does not con- centrate in any particular organ. If the polycythemia is pro- duced by the action of cobalt in a specific organ or tissue the amount active in producing the condition would be still less.

SUMMARY

1. A method for the estimation of cobalt in biological materials has been outlined. It is applicable to samples containing 0.01 to 0.5 mg. of cobalt.

2. The results obtained when this method was applied to a few biological substances are given.

3. The entire body of rats fed a milk, iron, copper, and man- ganese diet was found to contain less than 0.01 mg. of cobalt. Definite amounts of cobalt were found in all rats fed a similar diet plus cobalt, and the quantity present was proportional to the amount fed. The presence of 0.04 to 0.05 mg. of cobalt in the entire body of a rat was sufficient to produce a decided poly- cythemia.

4. No cobalt was detected in tissues taken from pigs fed a milk, iron, copper, and manganese diet, but definite quantities were detected in practically all the tissues taken from pigs fed a similar diet plus cobalt.

BIBLIOGRAPHY

1. McHargue, J. S., J. Agric. Research, 30, 193 (1925). 2. Fox, H. M., and Romage, H., Nature, 126, 682 (1930). 3. Dutoit, P., and Zbinden, C., Compt. rend. Acad., 190, 172 (1930). 4. Bertrand, G., and Mokragnatz, M., Compt. rend. Acad., 176,112 (1922). 5. Bertrand, G., and Mokragnatz, M., Compt. rend. Acad., 176, 458 (1922);

190,21 (1930); Bull. Sot. chim., 37,554 (1925). 6. Bertrand, G., and Mdcheboeuf, M., Compt. rend. Acad., 180,138O (1925). 7. Bertrand, G., and Mdcheboeuf, M., Compt. rend. Acad., 180, 1993

(1925). 8. Bertrand, G., Science, 64, 629 (1926). 9. Bertrand, G., and MBcheboeuf, M., Compt. rend. Acad., 182,1504 (1926).

10. Bertrand, G., and Mdcheboeuf, M., Compt. rend. Acad., 183, 5 (1926). 11. Bertrand, G., and Nakamura, H., Compt. rend. Acad., 186, 321 (1927).

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F. J. Stare and C. A. Elvehjem

12. Mokragnatz, G., Bull. Sot. chim. biol., 13, 61 (1931); Bull. Sot. chim. roy. YougosZav., 2, 9 (1931).

13. Waltner, K., and Waltner, K., Klin. Woch., 8, 313 (1929). 14. Mascherpa, P., Arch. ital. biol., 82, 112 (1930); Chem. Abet., 24, 3280

(1930). 15. Myers, V. C., Beard, H. H., and Barnes, B. O., J. Biol. Chem., 94,

117 (193132). 16. Orten, J. M., Underhill, F. A., Mugrage, E. R., and Lewis, R. C., J.

Biol. Chem., 96, 11 (1932). 17. Elvehjem, C. A., unpublished data. 18. Van Klooster, H. S., J. Am. Chem. SOL, 43, 746 (1921).

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F. J. Stare and C. A. ElvehjemCOBALT IN ANIMAL NUTRITION

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