ADDITIONAL OBSERVATIONS ON THE CHICK ANTIANEMIA VITAMIN*

BY BOYD L. O’DELL AND ALBERT G. HOGAN

(From the DepartwLent of Agricultural Chemistry, University oj Missouri, Columbia)

(Received for publication, May 20, 1943)

While attempting to devise a simplified ration that is adequate for the chick, Hogan and Parrott (1,2) observed that on certain rations the number of red blood cells was distinctly subnormal. The anemia was of the hyper- chromic, macrocytic type, and it was due to the lack of an unrecognized vitamin which was designated vitamin B,. An aqueous extract of liver is one of the best sources of the vitamin. It is adsorbed from acid solution on fullers’ earth and is easily destroyed by heat in acid solution. The essential features of the above report were confirmed by Mills, Briggs, Elvehjem, and Hart (3) who also have shown that chicks maintained on a simplified diet require an unrecognized factor to promote growth, hemo- globin formation, and normal feathering.

The purpose of the present investigation was (I) to improve the diet used for producing anemic chicks, (2) to develop a technique for the assay of vitamin B,, and (3) to obtain additional information on its chemical properties.

EXPERIMENTAL

Day-old white Leghorn chicks, obtained from the University poultry de- partment, were divided into groups of eight and placed in electrically heated batteries. Water and the experimental ration were supplied ad libitum. The chicks were weighed at regular intervals. When they were 3 weeks of age, and each week thereafter, blood samples were taken to determine the degree of anemia. Very few chicks were anemic at 2 weeks of age. The chicks which had not become anemic by the end of the 5th week were discarded and the red cell volume was recorded as normal.

Riological Studies

Criteria of Anemia-Hogan and Parrott (2) used erythrocyte counts and hemoglobin content to determine the degree of anemia, but counting eryth- rocytes is quite laborious and there was considerable variability in read- ing the visual calorimeter used at the time in making hemoglobin determina-

* Contribution from the Department of Agricultural Chemistry, Missouri Agri- cultural Experiment, Station, Journal Series, No. 894. Aided by a grant from Parke, Davis and Company.

323

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324 CHICK ANTIANEMIA VITAMIN

tions , In an effort to eliminate these objectionable features, determination of the red cell volume by use of the Van Allen hematocrit tube (4) was found to be quite rapid and efficient,. In this determination a measured volume of blood is mixed in a Van Allen tube with a 1.3 per cent solution of sodium oxalate and centrifuged until the erpthrocytes are solidly packed. This is accomplished by centrifuging for 20 minutes in an International centrifuge, size 1, at a speed of 2500 R.P.M.

In order to determine the relation between red cell count, hemoglobin content, and the hematocrit value, these three determinations were made on over 60 samples of blood taken at random from chicks on anemia- producing rations. The results by all of the methods showed quite close correlation. An att.empt to derive a mathematical relation between the red cell volume in per cent and the red cell count, according to the method of Mills (5), gave the equation, C = 8.42 + 10.42E, in which C is the red cell volume in per cent and E is the erythrocyte count in millions per c.mm. The coefficient of correlation is 0.85. A similar relationship between the red cell volume and the hemoglobin content is expressed by the equation, H = 0.66 + O.lW, in which H is the hemoglobin content in gm. per 100 cc. of blood and C is the red cell volume in per cent. The coefficient of corre- lation is 0.94, showing that approximately the same degree of anemia is indicated by these two methods.

The relation between the cell volume and the erythrocyte count, and between the cell volume and hemoglobin content, is shown in Figs. 1 and 2.

Fig. 1 shows that as the anemia develops the rate of decrease in cell volume is accelerated. This indicates that the cells become larger when the anemia begins to develop, though there is no reason to suppose that the cells continue to increase in size as the anemia progresses. Presumably the same t,ype of curve would be obtained if the large cells disappeared more rapidly than those that are smaller. Fig. 2 indicates that as the hemo- globin content decreases t.he cell volume decreases at a constant rate.

During the first part of the investigation a chick was considered anemic when the hematocrit value was 25 per cent or less. It was found that assays for the curative agent, were more reliable if the cell volume was allowed to fall still lower; therefore in the latter part of the study a chick was not classed as anemic until the red ccl1 volume reached the arbitrary value of 20 per cent by volume. This corresponds to a hemoglobin value of 4.26 gm. per 100 cc. of blood and to an erythrocyt,e count of 1.11 millions per cmm. The normal henlatocrit value for thirty-five chicks 3 to 5 weeks of age, maint,ained on a practical diet of natural foodstuffs, was 32 per cent. Calculated by the formulae given above this would correspond to an eryth- rocyte count of 2.26 millions per c.mm. and a hemoglobin content of 6.42 gm. per 100 cc.

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H. I,. O’DELL ASD A. G. HOGAS 325

Assay Technicyue--When t,he red cell volume fell to 20 per cent, or less, t.he chicks were used to assay experimental materials. They were con- tinued on the basal diet, and the preparation to be assayed was adminis- tered orally every other day for a 12 day period. The final hematocrit reading was taken on the 14th day. Dry preparations mere administered in gelatin capsules and liquids by pipette. Roth positive and negative controls were maintained on the basal diet during the assays. The chick unit of vitamin 73, is defined as the amount, that must be administered every

35

30

25

2 s 20 ::

3

2 ’ 15 2 =: 3

P 1 10

5

1

2.55 2.10 1.65 1.35 0.90 0.60 0.15 Erythrocyte Count (Millions per cu. mm.)

FIG. 1. Red blood cell volume is not R linear function of the red cell count

other day for six doses to bring the hematocrit reading from 20 per cent or below to 30 per cent or above in at least 60 per cent of the chicks. Chicks which did not survive the first 5 days of the assay period mere disregarded, but those which died after the 5th day were classified as failures.

Rations Used for Production of Anemia-The diets mere of the simplified type, consisting of casein, corn-st,arch, salts, lard, vitamins, and a liver ext,ract fraction. The diet of Hogan and Parrott contained tikitiki and a 95 per cent. alcohol extract (Liver Extract 3703) of dried pork liver as sources

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326 CHICK ASTIANEMIA VITAMIN

of the B vitamins other than thiamine and riboflavin. As other synthetic vitamins became available, it was found that more uniform results were obtained when tikitiki was omitted and pyridoxine was included. How- ever, the alcoholic liver extract could not be dispensed with until quite recently, when it was found that it could be replaced by biotin and other known members of the vitamin B complex. As previously mentioned, during the first part of this investigation the chicks were considered anemic when a hematocrit value of 25 per cent by volume was reached instead of

$0 6.80 5.90 5.30 4.40 3.80 2.90 2.30 Hemoglobin Content (grams per 100 cc)

FIG. 2. Red blood cell volume is a linear function of the hemoglobin cont,ent

the present standard of 20 per cent. The data will therefore be presented in two sections, corresponding to the standard by which the chicks were classified as anemic.

1. A red cell volume of 25 per cent was the standard. Table I gives the composition of diets employed during this part of the study. Tikitiki was first replaced by pyridoxine in Ration 6636. This diet contained only 100 y of pyridoxine in addition to that supplied by Liver Extract 3703. Optimum growth is not supported by this amount, but large amounts

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13. L. O’DELL AND A. G. HOGAN 327

of the vitamin apparently decrease the incidence of anemia. This is indicated by a comparison of the response of chicks from a flock restricted to dry feed which received Rations 6636 and 6637, shown in Table II.

TABLE I

Composition of Rations

Constituents --

Casein........... .._...... _...,....,...._ Corn-starch. __...., ., ,, .._..__.... .._..._..__... Salts*................................................. Celluflour............................................ Lard.................................................. Cod liver oil. . Wheat germ oil........................................ Liver extract 3703..................................... Vitamins added per 100 gm. ration

Thiamine hydrochloride, Riboflavin........................................... Pyridoxine hydrochloride. Calcium pantothenate. Choline chloride.....................................

-

_ -

-

Ration 6636 Ration 5056

per cent

35 43

4 3 7 2 4 2

w. 0.2 0.4 0.1

.- fer cent

35 45

4 3 7 2 4

m. 0.2 0.4 0.3 1.0

100.0

* A modification of Salt Mixture 351, described by Hubbell, Mendel, and Wake- man (6). 2.5 gm. of manganous sulfate, MnS04*4HzO, were added to 100 gm. of Salt Mixture 351.

TABLE II

Incidence of Anemia As Affected by Diet of Laying Flock and of Experimental Chicks

“~2 Source of chicks No of $;;; chicks*

MOP All+

3rd wk: tality mitt -__ ______--

w. per cent gcr cent

6636 Winter flock allowed open range 348 74 27 62 6636 summer “ I‘ “ ‘I 70 87 34 40 6636 L‘ ‘I restricted to dry feed 74 73 20 70 6637 “ 1‘ I‘ ‘I ‘I I( 16 90 6 31 5056 Winter “ allowed open range 67 70 45 45

* Number surviving at the end of the 1st week. t A red cell volume of 25 per cent or less at the end of the 5th week.

Ration 6637 was the same as Ration 6636 except that it contained 300 y of pyridoxine. Approximately 70 per cent of the chicks from the summer flock maintained on Ration 6636 became anemic, while only 31 per cent became anemic on Ration 6637. The higher level of pyridoxine markedly decreased the mortality rate.

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Ration 6636 was first employed in the winter mont,hs, and during this time about 62 per cent of the chicks became anemic. However, as the spring and summer months came on, the incidence of anemia decreased, dropping to 40 per cent of the total, as is shown in Table II. Although the hens from which the eggs were obtained were allowed free range throughout the year, they would not be able to obtain as much green feed during the winter as during the summer. It was suspected, therefore, that the anti- anemia factor was being stored in the egg from the green feed which the hens consumed during the summer months. To test this hypothesis part of the summer flock was kept indoors and fed a practical laying ration of natural feeds. Ration 6636 was fed to chicks from this flock and the inci- dence of anemia increased to 70 per cent, which is slightly better than was obtained during the winter months with the flock on open range. Thus it appeared that the factor is abundant in green feeds and is stored in the egg. Thereafter the chicks were obtained from hens restricted to dry feed.

As more synthetic vitamins became available, an effort, was made to dispense with Liver Extract 3703. Ration 5056, containing thiamine, riboflavin, pyridoxine, pantothenic acid, and choline, but no liver extract, is an example of such a,n attempt. The rate of growth was about the same as on the other type of diet, but the mortality rate was excessive. As will be shown later, this was probably due at least partially to a biotin defi- ciency.

2. A red cell volume of 20 per cent was the standard. It is unnecessary to point out that the use of this standard decreased to some extent the percentage of chicks which were classed as anemic. The composition of the rations used during this phase of the investigation is shown in Table III.

In an effort to produce a larger number of chicks that were useful for assay, Ration 6636 was modified in various ways. The level of pyridoxine was increased to 0.3 mg. per 100 gm. of diet, though the change decreases slightly the incidence of anemia. However, the mortality rate is reduced, and since the survivors are superior test animals the over-all effect is ad- vantageous. Furthermore, the wheat germ oil and cod liver oil mere re- placed by 2-methyl-1,4-naphthoquinone, a-tocopherol, and a vitamin concentrate which contained vitamins A and D. The effect on the inci- dence of anemia due to the addition of sulfaguanidine to the diet was also studied. Our observations are summarized in Table IV.

It will be noted that in a,ddition to the column showing the percentage of anemic chicks, a column is included which indicates the percentage of chicks which mere usable for assay purposes. A useful chick is defined as one which has a red cell volume of 20 per cent or less and survives the first 5 days of the period during which supplements are administered. This mformation is helpful in evaluating a basal ration, since a diet may produce

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TABLE III Composition of Rations

Constituents

Casein ................................... Gelatin ................................... Corn-starch .............................. Dextrin .................................. Cellu flour. .............................. Lard ..................................... Soy bean oil .............................. Wheat, germ oil. .......................... Salts* .................................... Liver Extract3703 ........................ Mixture 6092 (vitamins A, D, E, K)t. ..... Vitamins added per 100 gm. ration

Thiamine hydrochloride. ............... Riboflavin .............................. Pyridoxine hydrochloride. .............. Calcium pantothenate. ................. Choline chloride ........................ Nicotinic acid. ......................... Inositol................................

Biotin (S. M. A. No. 200). .............

* See foot-note to Table I.

- -

-

6091 6385

ger cent fier cent 35 35

43 43

3 3 12.5 12.5

4 2 0.5

w. 0.4 0.8 0.3 2

200

4 2 0.5

w. 0.4 0.8 0.3 2

200

Ration No.

6554 6582

per cent per cent 25 25 10 10

56 56

4.5

4

0.5

m. 0.4 0.8 0.3 2

200 10

100 Y

10

4.5 4

0.5

mg. 0.4 0.8 0.3 2

200 10

100 Y

10

t 0.5 per cent of Mixture 6092 furnished 7250 I.U. of vitamin A, 725 I.U. of vitamin D, 0.1 mg. of 2 methyl-1,4-naphthoquinone, and 8 mg. ofor-tocopherol. Natola, sup- plied by Parke, Davis and Company, was dissolved in corn oil and served as the source of vitamins A and D, while vitamins E and K were added as synthetic compounds.

TABLE IV Incidence of Anemia As Affected by Diet of Experimental Chicks

Ration No.

6091 6091 6385 6385 6385 6554

T .

per cent

0 1 0 0.5 0.25

-

No. of chicks*

206 92 88

137 85 57

gm. per cent per cent per cent

95 21 23 21 68 23 60 39 81 13 24 14 67 28 37 21 73 10 53 46 92 15 51 51

Mortality Anemict Usable for assay

* Piumber surviving at the end of the 1st week. t A red cell volume of 20 per cent or less at the end of the 5th week.

329

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330 CHICK ANTIANEMIA VITAMIN

a high proportion of anemic chicks but be impractical because of a high mortality rate during the assay period.

It should be stated that there is considerable variation in the incidence of anemia. This is probably due to at least two factors: (1) the chicks themselves, and (2) the alcoholic extract of liver, No. 3703. That the source of the chicks affects the incidence of anemia has already been shown. Even when the diet of the hen is controlled, there is variation in the chicks from one hatch to another. Although the liver extract is prepared in as nearly the same manner as possible each time, the chicks do not respond to different preparations in exactly the same way. Ration 6091, which contains 0.3 mg. of pyridoxine per 100 gm. of diet and the vitamin A, D, E, and K mixture instead of the oil carriers, produced a rather low incidence of anemia, 25 per cent. However, it should be mentioned that although relatively few chicks were anemic according to the arbitrary standard many of the others had low hematocrit values and might be classed as anemic under less severe standards. Ration 6385 is similar to Ration 6091 except that it does not contain choline. The incidence of anemia on this ration was about the same as on Ration 6091, but the percentage of usable chicks was somewhat lower.

The inclusion of sulfaguanidine in some of the rations was based on the theory that bacterial synthesis in the alimentary tract might make the antianemia factor available to the chick and thus reduce the incidence of anemia. Since Black, McKibbin, and Elvehjem (7) have used sulfa- guanidine successfully to prevent bacterial synthesis in the intestine of the rat, this drug was added to the chick rations at the levels indicated in Table IV. When it was added to Ration 6091 at a level of 1 per cent, the incidence of anemia was increased from 23 to 60 per cent without an increase in mortality. There was a decrease in weight, presumably because of a reduction in the number or amount of vitamins that were available to the chick. The chicks would still respond to treatment with the vitamin B, concentrates, which indicates that it was not entirely a toxic effect. The addition of 0.25 per cent sulfaguanidine to Ration G385 increased the anemia from 24 to 53 per cent. The addition of 0.5 per cent did not prove as useful as 0.25 per cent because of a higher mortality rate.

Almquist, Stokstad, Mecchi, and Manning (8) and Hegsted et al. (9) have shown that chicks require more glycine and arginine than is supplied by casein. Since our basal diet appeared to be deficient in these amino acids, 10 per cent gelatin was substituted for that amount of casein in Ration 6554. This change improved the growth rate and feathering, and thus produced a better test animal. Another improvement was made by discarding Liver Extract 3703, and adding a biotin c0ncentrate.l This modification eliminated one of the variables, the liver extract, and made it

1 Concentrate No. 200, obtained from the S. M. A. Corporation.

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13. L. O’DELL .\SD .l. G. El0ci.W 331

possible to obtain a high incidence of anemia without the USC of sulfa- guanidine. Furthermore the chicks produced rere superior in appearance and the mortality rate was reduced.

When the new type of diet, Ration ci554, containing the biotin concen- trate was first tried, soy bean oil was used as a source of fat. Although this diet produced a fairly high proportion of anemic chicks, our past esperience indicated that the substitution of wheat germ oil for soy besn oil increased the incidence of anemia. It was decided thcreforc to study the effect of various sources of fat on the incidence of anemia among chicks under ob- servation at the same time. These data, not, included in Table IV, arc summarized in Table V.

Evidently chicks may at times become severely a.ncrnic on rat,ions that, contain soy bean oil, but up to t,he present t.he results indicate that the anemia develops much more consistently jrhen the soy bean oil is replaced by wheat germ oil. Ration 6632 is similar to Ration ci.582 cxccpt that the

TABLE V Incidence of tine~wiu As Injuenced by Source of Pat in Diet

Ration No. Source of fat / No. of

chicks 1

6554 6582 6632

--

Soy bean oil, 4.5% Soy bean oil, 4.5% 47 47 Wheat germ oil, 4.5y0 Wheat germ oil, 4.5y0 j j 46 46 Lard, 4.5% Lard, 4.5% / 15 / 15

AWage weiiht, 3rd wk.

w.

9i j 87 67 i

hlortality

per cent

21 15 33

-.-..- -

Anemic Usable for assay

-__ per cm1 per 0x1

38 1 30 65 ~ 59 66 i 20

wheat germ oil is replaced by lard. When lard It-as used as a source of fat, all of the surviving chicks became anemic, but the growth rate was low and the mortality rate was quite high. Since many of the chicks which became anemic died within a week, the percentage usable for assay was very low. It appears that the oils contain a factor essential for chicks maintained on this type of diet and that it is more abundant in soy bean oil than in wheat germ oil. It may not be identical with vitamin Is,.

Although t,he chicks develop anemia more readily n-hen the liver extract is omitted, it is also true that they respond t,o treatment less readily. This difference may be due to the vitamin B, content of the liver extract, or to some other limiting factor which is supplied by this extract.

FractGrnation P~ocedures2----An aqueous extract of beef liver seemed to be the most potent source of the vitamin. In most of t,he fractionation studies

? A preliminary unnounccment of the isolation of crystalline vitamin 13, was re- cently reported (10).

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332 CHICK ANTIANEMIA VITAMIN

an extract prepared according to the following procedure was used. 50 pounds of fresh, ground beef liver were stirred with 15 gallons of boiling water adjusted to pH 4.0. The temperature was maintained at 90” for 5 to 10 minutes; then the solution was allowed to settle. The supernatant liquid was decanted off and the residue filtered. This process was repeated with 10 gallons of water. The combined extracts (No. 4978) were con- centrated in vacua to approximately 4 liters, and sufficient 95 per cent al- cohol was added to make a 50 per cent solution. The precipitate from this treatment was washed three to four times with 50 per cent alcohol and the washings were added to the original filtrate. The 50 per cent alcohol- soluble portion (Extract 4979) was concentrated to a syrup in vacua, and constituted the source material for further fractionation. A curative unit

Ground beef liver

Aqueous extraction at pH 4

Residue Extract (No. 4978) Alcohol added to

50% by volume

50’% alcohol-soluble 50% alcohbl-insoluble portion portion (Extract 4979) (Extract 4980)

of Extract 4979 was 400 mg. In more recent work an aqueous extract of pork liver corresponding to Extract 497S3 has been used.

The procedure for the preparation of a concentrate of vitamin B, is as follows: 1 kilo of Extract 4979 is dissolved in 10 liters of water and the solution is adjusted to pH 1 with dilute sulfuric acid. Any insoluble residue is removed by decantation and centrifuging. 200 gm. of English fullers’ earth are added to the filtrate and stirred for 1 hour. The earth is removed, washed three times with water, and the process is repeated with another 200 gm. of fullers’ earth. The combined adsorbates are then eluted with 0.2 N ammonium hydroxide, three portions of 3 liters each being used. The combined eluates are concentrated to a syrup under reduced pressure. The material contains 1 unit in 40 mg. and represents a yield of about 45 per cent of the activity in the original extract. It is then dissolved in water to make a 5 per cent solution, adjusted to pH 1, and the flocculent material that does not dissolve is removed by centrifuging. The clear

3 Supplied by Parke, Davis and Company.

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IS. L. O’DELL AND A. G. HOGAl\ 333

filtrate is then adsorbed three times with a total amount of Super Filtro14 equivalent to twice the weight of solids. After washing, the filter cake is eluted with 1 per cent ammonia in 50 per cent alcohol, with three portions of 800 cc. each. The eluates are combined and concentrated to a con- venient volume. A unit of this material is about 15 mg., corresponding to a recovery of about 80 per cent of the activity of t,he previous fraction. In some cases precipitation with phosphotungstic acid was introduced at this point. The material is dissolved in water to make a 5 per cent solution and is adjusted to PI-I 1. A 10 per cent sodium phosphotungstate solution is added to the point of maximum precipitation. The mixture is allowed to stand in the refrigerator overnight, and the precipitate is centrifuged off and washed several times with water. The precipitate is decomposed by grinding in a mortar with a paste of barium hydroxide, and then extracted

TABLE VI

Concentrates Prepared from Liver Extract 4979

Description

Eluate from readsorption with Super Filtrol. 10% ammonia eluate of norit adsorption at pH 3.. . . 10% “ “ “ Amberlite IR4* adsorption at pH

4.5................................................... Barium hydroxide ppt.. Zinc sulfate ppt......................................., Phosphotungstic acid ppt.. Residue from cold methanol extraction.. Methanol ppt. of phenol solution.

-

-

Concentration effected

20

7

4 2 4 5 2 5

-

Recovery

per cenl

36 22

40 25 36 50 60 95

* The use of Amberlite IR4 was suggested by Dr. J. J. Pfiffner.

with a hot solution of barium hydroxide. This process is repeated until no more color is removed. The barium is removed from both the filtrate and the decomposed precipitate as barium sulfate. A unit of the phospho- tungstic acid precipitate is 7 mg. and approximately 70 per cent of the activity of the previous fraction is recovered. This is about the same degree of concentration as is obtained by a second readsorption on Super Filtrol. The over-all yield is about 25 per cent, and the factor has been concentrated 55 to 60 times.

Other procedures which have effected some concentration of the factor when applied to Extract 4979 are presented in Table VI, but in most cases the recovery was too low to make the procedure useful for the purpose of

4 The Super Filtrol was obtained from the Filtrol Corporation, 315 West Fifth Street, Los Angeles.

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preparing a concentrate. Phosphotungstic acid precipitation offers some promiw, l)ut, t,hn use of t.his reagent would not be practical in the precipita- tion of crude estrncts. ‘l’hc prwipitation of a phcnolic solution of the anti-pernicious anemia factor wit,h a large volume of methanol was found useful by I’nglcy (11) in preparing concentrates of that factor. This procedure also proved helpful in concentrating vitamin 13 C.

Propertics of T’itamin H,. StabiZity~-The factor is much more stable in alkaline than in acid solution. Approximately 50 per cent of the activity is lost by allowing a solution at pH 1. to stand at room temperature for 72 hours. At, least 80 per cent of the activity is lost by acid autoclaving for 2 hours, whereas less than 50 per cent is dest,royed by alkaline autoclaving. Less than 30 per cent of the factor is destroyed by standing in a 5 per cent solut#ion of hydrogen peroxide at room t,emperature for 12 hours.

Prccipitu.n,ts----The factor is partially precipitated from a 10 per cent solu- tion of Extract, 4979 with salts of lead, mercury, barium, zinc, and silver. Considerable loss is associat,ed with lead and mercury precipitation. How- ever, no loss is sust,ained in precipitation with zinc, but less than 40 per cent of the factor is precipitated.

Phosphot.ungstic acid gives almost complete precipitation. Volubility---The factor as it occurs in crude concentrates is insoluble in

the common organic solvents such as ether, chloroform, pyridine, ethanol, butanol, and acetone. It is very slightly soluble in dioxane and quite soluble in glacial acetic acid, phenol, and hot methanol.

Adsorbon,ts-----l’he factor is much more completely adsorbed on fullers’ earth from acid than from alkaline solution, but there is considerable loss associated \vit,h acid adsorption. Since the factor is quite labile to acid, the loss during adsorpt,ion can be at least partially attributed to destruction by hydrogen ions. However, acidification following the preliminary ad- sorption does not lead to such marked losses. In view of the fact that the factor is destroyed at pH 1, attempts were made to adsorb it on fullers’ earth from more alkaline solutions. Less than one-third as much of the activity was recovered by adsorption at pH 3.4 as at pH 1, owing to in- complet,e adsorpt,ion. Practically none is adsorbed at pH 7. Readsorp- tion was carried out effectively with Super Filtrol which gave a 2- to 3-fold concentration and recovered a.pproximately 80 per cent of the activity. The factor is completely eluted from fullers’ earth with a dilute ammonia solution.

Korit, Kuchar, and Marco will remove the factor almost completely from solution at pH 5, but the recovery was unsatisfactory. It was not deter- mined whether the loss was due to destruction or to incomplete elution. The factor is elated with difficulty, 5 to 10 per cent ammonia being requirecl for the most complete elution.

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13. L. O’DELL ASD A. G. HOG.\S 335

hmberlite IR4, an acid adsorbent described by Myers, Eastes, and Myers (12), adsorbs the vitamin, and it may be cluted nith 5 per cent ammonia to give a 40 to 50 per cent recovery. Apparently aluminum oxide adsorbs the factor, but it has not yet been possible to recover it. Permutit and kaolin have not shown any promise ‘as adsorbents.

Comparison, of Vitamin R G with, Other Antianemia Factors-Simmons and Norris (13) have reported that xanthopterin will cure a nutritional anemia which develops in Chinook salmon maintained on a high protein diet. Inasmuch as the properties of xanthopterin resembled those of vitamin B,, it was prepared synthetically according to the method of Purrmann (14). 1 t showed no curatiye action on the chick anemia.

h sample of a pot,ent fraction5 containing the anti-pernicious anemia factor showed no act’ivity for the chick.

DISCT’SSIOS

This investigation confirms the conclusions reported by Hogan and Parrott (2), but there is one discrepancy that may require some comment. The incidence of anemia they observed was higher than that given in this report. In explanation it may be stated that (1) the percentage of anemic chicks reported by Hogan and Parrott was based on the number of chicks that survived, while in this paper it is based on the total number started; (2) in order to have chicks that are more suitable for assay purposes the standard for anemia was made more severe; (3) all of the observations of Hogan and Parrott were made in the winter and early spring, when pre- sumably the laying flock did not have access to green feed; the data reported in this paper were collected throughout the year.

Some of the known vitamins seem to influence the production of the anemia. For example t,here is some reason to believe the incidence is higher when pyridoxine is supplied at a low level. Presumably this vitamin does not exert a specific effect, but it is possible that it aids in the bacterial synthesis of the antianemia vibamin in the intestine.

Evidence is presented to show that the factor is transmitted through the egg to the chick and that it. is more abundant in green than in dry feeds, Ii’or this reason it is advantageous to restrict the hatching flock to dry feeds only, in order to obtain more suitable experimental chicks.

The recent work of Black, Overman, Elvehjem, and Link (15), in which they fed sulfaguanidine to rats, shows that there is another factor required by the rat when bacterial synthesis in the intestine is inhibited. Our data show that the chick develops anemia more readily when sulfaguanidine is supplied in the diet. However, since sulfaguanidine may be slightly toxic, it, is preferable t,o omit it if a suit)able diet can be formulated that does not

5 This frnction was kindly supplied hy Dr. Y. Subbmow.

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33G CHICK ANTIANkiMIh VITAMIN

contain the drug. Such a diet can be prepared by including wheat germ oil as a source of fat, by adding biotin to the vitamin combination, and by omitting the alcoholic extract of liver.

The properties of the substance described by Black et al. (7) as being required by the rat are very similar to those exhibited by vitamin B,. The properties of the antianemia vitamin as indicated by its adsorption behavior and by salt formation with various bases indicate an acid. The precipitation with phosphotungstic acid indicates basic properties, although it is possible that the factor is merely adsorbed on the precipitate formed by the phosphotungstic acid. The factor is very labile to acid, but some- what more stable toward alkali. It is not easily destroyed by oxidation. One of its salient characteristics is insolubility in the common organic solvents.

Mills, Briggs, Elvehjem, and Hart (3) have called attention to the similarity in the properties of the eluate fraction required by Lactobacillus casei E, the factor U of Stockstad and Manning (IB), folic acid described by Mitchell, Snell, and Williams (17), and vitamin B,. The properties of the factor required for lactic acid bacteria, which was described by Hutchings, Bohonos, and Peterson (18), are very similar to those of vitamin B, in all points investigated. Both factors are quite labile to acid, are precipi- tated by heavy metals, are adsorbed on fullers’ earth and norit, and are insoluble in the common organic solvents.

SUMMARY

1. A diet has been developed which will produce a severe anemia in at least 50 per cent of the chicks.

2. A technique has been devised for the assay of the factor, essential in chick nutrition, which prevents the anemia.

3. Sulfaguanidine increased the incidence of anemia under the experi- mental conditions imposed at that time.

4. Vitamin B, is acidic in nature and forms salts with heavy metals. It is destroyed by mineral acids (pH 1) but is more stable to alkali. It is adsorbed from acidic solution by a variety of adsorbents and can be eluted by ammonia. It is insoluble in the common organic solvents.

BIBLIOGRAPHY

1. Hogan, A. G., and Parrott, E. M., Proc. Am. Sot. Biol. Chem., J. Biol. Chem., 128, p. xlvi (1939).

2. Hogan, A. G., and Parrott, E. M., J. Biol. Chem., 132, 507 (1940). 3. Mills, R. C., Briggs, G. M., Jr., Elvehjem, C. A., and Hart, E. B., Proc. Sot.

Exp. Biol. and Med., 49, 186 (1942). 4. Van Allen, C. M., J. Lab. and Clin. Med., 10, 1027 (1925). 5. Mills, F. C., Statistical methods, New York, 355 (1938).

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6. Hubbell, R. B., Mendel, L. B., and Wakeman, A. J., J. Nutrition, 14, 273 (1937). 7. Black, S., McKibbin, J. M., and Elvehjem, C. A., Proc. Sot. Exp. Biol. and Med.,

47, 308 (1941). 8. Almquist, H. J., Stokstad, E. L. R., Mecchi, E., and Manning, P. D. V., J. Biol.

Chem., 134, 213 (1940). 9. Hegsted, D. M., Hier, S. W., Elvehjem, C. A., and Hart, E. B., J. Biol. Chem.,

139, 863 (1941). 10. Pfiffner, J. J., Binkley, S. B., Bloom, E. S., Brown, R. A., Bird, 0. D., Emmett,

A. D., Hogan, A. G., and O’Dell, B. L., Science, 97,404 (1943). 11. Ungley, C. C., Lancet, 2, 1513 (1936). 12. Myers, R. J., Eastes, J. W., and Myers, F. J., Znd. and Eng. Chew, 33, 697 (1941). 13. Simmons, R. W., and Norris, E. R., J. Biol. Chem., 140,679 (1941). 14. Purrmann, R., Ann. Chem., 646, 98 (1941). 15. Black, S., Overman, R. S., Elvehjem, C. A., and Link, K. P., J. Biol. Chem., 146,

137 (1942). 16. Stokstad, E. L. R., and Manning, P. D. V., J. Biol. Chem., 126, 687 (1938). 17. Mitchell, H. K., Snell, E. E., and Williams, It. J., J. Am. Chem. Sot., 63, 2284

(1941). 18. Hutchings, B. L., Bohonos, N., and Peterson, W. I-I., .T. Biol. Chem., 141, 521

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Boyd L. O'Dell and Albert G. HoganCHICK ANTIANEMIA VITAMIN

ADDITIONAL OBSERVATIONS ON THE

1943, 149:323-337.J. Biol. Chem. 

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