ON THE EXISTENCE OF TWO ACTIVE FACTORS IN THE VITAMIN B COMPLEX. II.*

BY W. D. SALMON, N. B. GUERRANT, AND I. M. HAYS.

(From the Laboratory of Animal Nutrition, Alabama Polytechnic Institute, Auburn.)

(Received for publication, December 12, 1927.)

A recent paper (1) from this laboratory described the prepara- tion of a beriberi-preventing solid, obtained by treating an extract of velvet bean seed with fullers’ earth. Despite the efficacy of this solid as a preventive of beriberi, it apparently had no growth-pro- moting action. Two preparations from the leaves of the velvet bean were also described: one preparation possessed marked beriberi-preventing and slight growth-promoting properties; the other was ineffective as a prophylactic for beriberi in pigeons and, when fed alone, it was capable of producing only a very irregular growth in rats that were receiving an otherwise vitamin B-free diet. A combination, however, of the latter fraction with the beriberi-preventing fraction from either the leaves or the seed enabled rats to make normal growth, when the combination was used as the sole source of vitamin B. More recently Chick and Roscoe (2) found that an extract prepared by the Peters method showed marked beriberi-preventing but no growth-promoting ac- tion in the dosages fed. Nevertheless, the addition of this product to autoclaved yeast, which in itself was incapable of supporting growth, provided an adequate source of vitamin B for normal growth of rats. Hassan and Drummond (3) also reported a sig- nificant acceleration in the rate of growth of rats, caused by the addition of an alkali-treated extract of yeast to a diet fortified by 5 mg. daily of Seidell’s concentrate as the only other source of vitamin B.

It seems that the results of these independent investigations

*Published with the permission of the Director of the Alabama EX- periment Station.

487

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488 Vitamin B Complex. II

furnish sufficient proof to remove any doubts that may have existed regarding the complex nature of vitamin B. Apparently there are at least two active substances in the complex: one of these prevents the onset of experimental beriberi but is not cap- able of inducing growth, when it is the sole addition to a vitamin B-free diet; the other is impotent as a beriberi prophylactic and is also in itself inactive as a growth accelerator but is an essential accessory of the beriberi-preventing factor in supporting growth. The accessory growth-promoting substance is identical or asso- ciated with a substance which protects rats against a syndrome believed by Goldberger and associates (4) to be the analogue of pellagra. Since there is at present no conclusive proof that the accessory growth-promoting substance consists of more than one active factor, this substance is designated tentatively as the P-P factor which is the term suggested by Goldberger for the pellagra-preventing substance. In harmony with this terminology factor B-P is used for the beriberi-preventing substance, while the term vitamin B is retained for the complex.

In the following pages are presented data bearing upon three points which have received further consideration in theinvestiga- tion of the vitamin B complex now in progress in this laboratory. These are (3 ) the relative adsorption of the B-P and the P-P factors by fullers’ earth; (2) the further purification of the P-P fraction; (3) the retardation of growth by an insufficiencyof the B-P factor.

EXPERIMENTAL.

Relative Adsorption of the Two Factors by Fullers’ Earth.

It was shown in the first paper (1) that, when an acidulated aqueous extract of velvet bean leaves was treated with fullers’ earth at the rate of 50 gm. per kilo of air-dried leaves, the resulting “activated solid” was more effective in preventing beriberi than in stimulating growth. However, 0.40 gm. of this solid, Extract L-1-50, per rat daily produced slow but continuous gains. This was a dosage of the solid slightly in excess of the amount necessary to protect pigeons completely against beriberi and at least 8 times the daily dosage required to maintain the weight of 50 gm. rats for a period of 10 weeks following a preliminary deple- tion period of 2 weeks on the basal diet a.lone. No symptoms of

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Salmon, Guerrant, and Hays 489

beriberi or pellagra were manifest on dosages varying from 0.05 to 0.40 gm. per rat daily.

In order to determine whether the use of a smaller amount of fullers’ earth would result in a more effective separation of the two factors, Extract L-1-10 was prepared. The method was similar to that used for Extract L-1-50, except that the aqueous extract was passed through a supercentrifuge before the fullers’ earth was added and only 10 gm. of the earth were used instead of 50 gm.

Varying amounts of Extract L-1-10 were supplied to rats re- ceiving Diet 2B (the technique was identical with that given in the first paper). The test period was 9 weeks following the usual depletion period of 2 weeks. Typical results are illustrated by the curves of Rats 812, 814, and 828 in Fig. 1. No symptoms of beriberi developed on a daily dosage of 0.01 gm. That this extract contained less of the P-P factor than the previous preparation, Extract L-l-50, was shown by the fact that practically no growth was obtained on as much as 0.10 gm. daily or approximately IO times the minimum protective dosage for rats (or the equivalent of the daily dosage required to protect pigeons completely).

Tests on pigeons showed that the protective dose of the velvet bean leaves was about 3.8 gm. On this basis 1 kilo of leaves furnished 260 protective doses. About 100 protective doses were removed from the extract by the 10 gm. portion of fullers’ earth. Thus, it is certain that more than one-third of the B-F factor was taken up by the 10 gm. of adsorbent. On the other hand the proportion of the P-P factor adsorbed must have been negligible since the amount carried by the solid was too small to be demon- strated by the tests on rats.

Further Purijication of the P-P Fraction.

The residue, Extract L-1-500, which was used as a source of the P-P factor in the previous experiments reported from this labora- tory (1) apparently contained some of the B-P factor. This was indicated by the tests on rats although the tests on pigeons gave negative results with this residue. The requirement of the pigeon apparently is so high that this animal cannot be used to determine an extremely low concentration of the B-P factor. The rat, which requires a smaller protective dose of this substance, will apparently

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490 Vitamin B Complex. II

FIG. 1. Representative weight curves of rats. The depletion period of 2 weeks is not included in the curves. The daily dosage of the extracts is shown below.

Rat No. Extract L-1-10. Fraction II

828 814 812 832 822 821 864 863 866 831 818 816

902 0.05 904 0.05 906 0.10

yn. 0.10 0.05 0.01

0.10 0.05 0.01

B-P solid, NO. 50.

gm.

1.00 0.50 0.25

0.50 0.50 0.50

0.25

F _-

-

rsction II auto&wed.

gm.

1.00 0.50

0.25

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Salmon, Guerrant, and Hays 491

respond to a correspondingly lower concentration. Nevertheless, it seemed probable that, if all of the B-P factor could be removed from the residue, the purified product would not delay the onset of beriberi in rats and would not produce any growth unless it was further supplemented by a sufficient amount of the missing B-P factor.

Osborne and Wakeman (5) precipitated a fraction which was very efficient for the growth of rats, by pouring an aqueous extract of yeast into sufficient alcohol to make the total concentration about 78 per cent aIcoho1 (by weight). On the other hand, Gold-

_ _ _. ..----- -beraera.ndmh’ .~ JR nnlleta~ues (filfoundthe BPstistance tobe readilq. . _._ ~__ removed from maize meal with 79.4 per cent alcohol (by weight). Thus, it seemed that a combination of the selective action of fullers’ earth with a fractionation of the filtrate by alcohol might offer possibilities for the preparation of a fraction that would be rich in the P-P factor and free of the B-P factor.

Accordingly, after the solid was removed in the preparation of Extract L-l-10, the filtrate was concentrated by vacuum dis- tillation to a volume of 2 liters for each kilo of leaves used. The concentrated solution was treated with two successive 30 gm. portions of fullers’ earth. Each portion of fullers’ earth was removed by a supercentrifuge and washed with water and alcohol. The centrifugate and the washings from the solid were combined and again concentrated to 2 liters per kilo of leaves extracted. 2 liters of the concentrated solution were then poured into 3 liters of 93 per cent alcohol, which gave a total alcoholic concentration of about 51 per cent (by weight). The precipitate, Fraction I, was washed with 51 per cent alcohol and rejected.

The filtrate and washings were concentrated to 300 cc. and poured into 3 liters of 93 per cent alcohol, which made an alcoholic concentration of about 82.7 per cent (by weight). The precipi- tated material was allowed to settle and the supernatant liquid decanted. 300 gm. of Argo corn-starch were then worked into the precipitate and the resulting product was macerated in absolute alcohol. It was then filtered out on a Buchner funnel, washed with two small portions of absolute alcohol, and dried in an oven at 50-60’. The dried product, Fraction II, was light brown in color and very slightly hygroscopic.

The alcoholic washings and the decanted extract were combined,

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492 Vitamin B Complex. II

reduced to a small volume by vacuum distillation, taken up on 100 gm. of Argo corn-starch, and dried. The dried product, Fraction III, was darker and markedly more hygroscopic than Fraction II.

The average weights of the various fractions per kilo of leaves were: Fraction I, 18.0 gm.; Fraction II, 419.0 gm., including the 300 gm. of corn-starch added; Fraction III, 177.0 gm., including the 100 gm. of corn-starch. In other words, of the 214 gm. of solids in the crude extract, 8.4 per cent was precipitated in Frac- tion I, 55.6 per cent in Fraction II, and 36.0 per cent in Fraction III.

Rats 821 and 822 in Fig. 1 show that no growth was produced by daily doses of 0.25 or 0.50 gm. of Fraction II as the only addi- tion to the basal diet. Severe beriberi invariably developed and thelength of life was practically the same as in the negative controls on the basal diet alone. A daily dosage of 1.0 gm. did not result in appreciable growth but did prolong life to some extent, although severe beriberi developed eventually. That Fraction II retained significant amounts of the P-P factor is shown by the excellent growth illustrated by Rats 818 and 831 that received 0.05 and 0.10 gm. respectively of Extract L-1-10 in addition to a daily dosage of 0.50 gm. of Fraction II. Rats that received only 0.01 gm. of Extract L-1-10 in addition to 0.50 gm. of Fraction II per day made very little growth (Rat 816, Fig. 1) as compared with those receiving more of the B-P factor in the larger dosages of Extract L-1-10.

In the feeding of Fraction II it was noted that it was consumed much more readily than the crude residue, Extract L-1-500 described in the first paper. It was also evident that the very pronounced laxative properties of the crude product were absent from Fraction II.

Other investigators (2, 3, 6, 7) have reported the P-P factor to be more thermostable than the B-P factor. In order to determine whether Fraction II still contained some of the latter substance that could be inactivated by heat, a portion was moistened, auto- claved for 4.5 hours at 15 pounds pressure, and dried. Rats that received 0.50 or 1.0 gm. of the autoclaved material per day did not survive longer than the negative controls. A dosage of 2.0 gm. was tried but dependable results could not be obtained due to,

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Salmon, Guerrant, and Hays 493

fluctuations in the amounts consumed. Apparently there was no significant impairment of the growth-promoting activities of the extract due to the heat treatment. (Compare Rats 902 and 904, Fig. 1.)

Although Fraction III was not subjected to as detailed a study as Fraction II, the results that were obtained indicate that a large part of the P-P factor, originally contained in the crude extract, was concentrated in the 55 to 56 per cent of solids in Fraction II. If the growth-promoting activities of the two fractions were com- pared on the basis of their total net solids (i.e. excluding the added starch), Fraction II seemed to be 3 to 4 times as efficient as Fraction III, when an adequate supply of B-P factor was added to each fraction. The tests further showed that Fraction III contained somewhat more of the B-P factor per unit of net solids than did Fraction II. It was also found that Fraction III had the marked laxative action that had been noted previously from the crude residue. Since these findings showed that Fraction III was a far less desirable source of the P-P factor than was Fraction II, the studies on the former fraction were not continued further.

Retardation of Growth by an Insuficien,cy of B-P Factor.

It was noted above that when rats received 0.01 gm. per day of Extract L-l-10 they made very little growth even when an ade- quate amount of Fraction II was supplied. However, when the dosage of Extract L-1-10 was increased to 0.05 gm. per day, the dosage of Fraction II remaining constant, a rapid rate of growth occurred.

Since some dried leafy materials had been found to b.e poor sources of the B-P factor, the effect of adding an “activated” fullers’ earth, which was rich in the B-P factor and very poor in the P-P factor, to dried kudzu (Pueraria thunbergiuna) leaves was determined. This B-P solid, No. 50, was prepared by extracting white maize meal with 80 per cent alcohol (by weight), concentrating the extract by vacuum distillation, removing the precipitated protein, and adsorbing the B-P factor on a small amount of fullers’ earth. As may be seen by the weight curves for Rats 906, Fig. 1, and 1006, Fig. 2, which were typical controls on this solid as the sole addition to the basal diet, the amount of the P-P factor carried by 0.05 to 0.10 gm. of

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494 Vitamin B Complex. II

the B-P solid was negligible. Rat and pigeon tests, however, showed that it possessed marked B-P properties.

FICA 2. Representative weight curves of rats. The depletion period 2 weeks is not included in the curves. The daily dosage is shown below.

Rat ‘No.

919 916 914 912 911

Kudsu.

Qrn.

1.00 0.50 0.25 0.20 0.15

Rat No.

1000 990 987 985

1001 1003 1006

Kudsu.

elm.

1.00 0.50 0.25 0.20 0.15 0.10

of

P.

0.05 0.05 0.05 0.05 0.05 0.05 0.05

Two series of rats were used in the tests. One series received 0.15, 0.20, 0.25, 0.50, and 1.0 gm. respectively of dried kudzu

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Salmon, Guerrant, and Hays 495

leaves per rat daily. The other series varied from 0.05 gm. of B-P solid,No. 50, per rat daily without any kudzu to 0.05 gm. of the solid added respectively to doses of 0.10,0.15,0.20,0.25,0.50, and 1.0 gm. of dried kudzu leaves. The same sample of kudzu leaves was used in the tests on both series.

Typical results of these tests are shown in Fig. 2. Although the rats had steadily declined and died of beriberi on 0.15 gm. of the dried leaves alone, those on 0.10 gm. of the leaves and 0.05 gm. of the B-P solid made slight but consistent gains. In all cases the rats that received the 0.05 gm. of B-P solid, No. 50, in addition to the leaves, excelled those receiving corresponding doses of leaves without the solid. As the experiment progressed the weight curves of the rats receiving the leaves as the sole source of vitamin B, rapidly flattened out and a decline soon set in for all dosages of 0.25 gm. or less of the dried leaves per day. Ontheotherhand, the rate of growth was rather uniformly sustained throughout the test, when the leaves were further supplemented by the B-P solid. It was evident that the leaves carried an excess of the P-P factor in proportion to their content of the B-P factor. Consequently, the latter substance became the first limiting factor either for maintenance or for growth on the small dosages and was not sup- plied in quantities sufficient for optimum growth even by as much .as 1.0 gm. of the kudzu leaves per day.

DISCUSSION.

It is evident that fullers’ earth has a tendency under some experi- mental conditions to adsorb selectively the B-P factor from ex- tracts containing both this substance and the P-P factor. This may occur despite a preponderance of the P-P factor in the solu- tion being treated. The data presented in the preceding pages show that for this selective action to occur, there must be such a relation between the concentration of the B-P factor in the solu- tion and the amount of fullers’ earth used that the solid may be- come saturated with the B-P factor. Under the experimenOa1 conditions reported, this apparently necessitated the leaving of a considerable portion of the B-P factor in solution. Many factors may influence the adsorption of the two substances. (A detailed study of the effect of hydrogen ion concentration will be reported in a later paper.)

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496 Vitamin B Complex. II

Three successive treatments of the extract of velvet bean leaves with small amounts of fullers’ earth were not sufficient to effect a quantitative removal of the B-P factor. Further treatment with fullers’ earth did not seem desirable due to the probability of losses of the P-P factor. A fractionation of the concentrated extract (after the previous treatments with fullers’ earth) by alcohol furnished an improved product but one which still contained some of the B-P factor. It was necessary to resort to inactivation of the remainder of this factor by heat in order that a potent source of the P-P factor without any B-P activity might be obtained. Al- though the work of other investigators which has been cited has indicated that all of the B-P factor originally present in an extract may be inactivated by heat or treatment with alkali, it seems more desirable to remove as much of this factor as possible to avoid the inactivated or decomposition products that would otherwise remain in the treated extract.

The demonstration of a relation between the amount of the B-P factor available and the rate of growth of rats indicates that pres- ent methods in which rats are used for measuring the amount of vitamin B in food materials are entirely inadequate. It is evident that a low concentration of either the B-P factor or the P-P factor may limit the rate of growth. It is also conceivable that a food product\might be a highly efficient source of either factor and yet produce very little growth due to an insufficiency of the other factor. This points to the conclusion that any future attempts to determine the vitamin B content of a product, which do not take into consideration the complex nature of the vitamin, may well be regarded as useless.

SUMMARY.

1. .Fullers’ earth adsorbed both the B-P factor and the P-P fac- tor of the vitamin B complex, but under certain conditions the former factor was more completely adsorbed than the latter.

2. A combination of treatments with small amounts of fullers’ earth, of fractionation with alcohol, and of heating, furnished a preparation from an extract of velvet bean leaves, that retained marked properties of preventing pellagra-like symptoms and also of accelerating growth when it was fortified with sufficient B-P factor. The treated preparation alone did not produce any growth

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Salmon, Guerrant, and Hays

and did not prolong the life of rats beyond the average for rats on the basal diet alone.

3. A relation between the amount of E-P factor available and the rate of growth was found to exist. The concentration of the B-P factor in a sample of dried kudzu leaves was too low to permit of a complete utilization of the P-P factor.

BIBLIOGRAPHY.

1. Salmon, W. D., J. Biol. Chem., 1927, lxxiii, 483. 2. Chick, H., and Roscoe, M. H., Biochem. J., 1927, xxi, 698. 3. Hassan, A., and Drummond, J. C., Biochem. J., 1927, xxi, 653. 4. Goldberger, J., and Lillie, R. D., Pub. He&h Rep., U. S. P. H. S., 1926,

xii, 1025. 5. Osborne, T. B., and Wakeman, A. J., J. Biol. Chem., 1919, xl, 383. 6. Goldberger, J.,Wheeler, G. A., Lillie, R. D., and Rogers, L. M., Pub.

Health Rep., U. S. P. H. S., 1926, xli, 297. 7. Smith, M. I., and Hendrick, E. G., Pub. Health Rep., U. S. P. II. S.,

126, xii, 201.

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W. D. Salmon, N. B. Guerrant and I. M. HaysCOMPLEX. II

FACTORS IN THE VITAMIN B ON THE EXISTENCE OF TWO ACTIVE

1928, 76:487-497.J. Biol. Chem. 

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