STUDIES ON VITAMIN G (Bz)

I. YEAST AND LIVER PREPARATIONS AS A SOURCE OF

VITAMIN G (I&)*

BY RICHARD J. BLOCKt AND LUCILLE REED FARQUHAR

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(From the Department of Physiological Chemistry, Yale University, and
the Biochemical Laboratory of the Connecticut Agricultural Experiment
Station, New Haven)
(Received for publication, October 20, 1933)
A number of recent observers have obtained unusually rapid
ratea of growth of albino rata by including in the mixed dietary
certain natural foodstuffs such as liver, lettuce, or yeast. Osborne
and Mendel (1) and Mendel and Cannon (2) employed a diet that
contained a mixture of casein 35 per cent, salts 4 per cent, starch
37 per cent, butter fat 9 per cent, and lard 15 per cent, supple-
mented by 0.2 gm. of yeast and 40 gm. of lettuce daily, and ob-
served gains in weight at the rate of 5 gm. per day between the
limits of 60 and 200 gm. of body weight. Even more rapid gains
in weight (6.1 gm. per day) were demonstrated by Anderson and
Smith (3) in the case of rats fed a highly complex diet of natural
foodstuffs which included large amounts of yeast and lettuce.
These records far surpass those reported by earlier workers
(Donaldson (4), King (5), and Smith and Bing (6)). The stimu-
lus for such rapid rates of growth may have been supplied by the
ample amounts of proteins, carbohydrates, fats, minerals, and
vitamins presented to the rats, or by some hitherto undiscovered
growth-promoting substance in the natural foods (cf. Mapson
(7)). To test these alternatives a highly purified diet, adequate
in all the known essentials, has been prepared. Difficulties in
securing an even relatively pure source of vitamin G (BS were
* The expenses of this investigation were shared by the Connecticut
Agricultural Experiment Station, the Carnegie Institution of Washington,
and were furbher aided by a grant from Eli Lilly and Company.
t Standard Brands Incorporated Fellow, 1932-34.
643
644 Studies on Vitamin G (B,). I

encountered, and this communication deals wit,h the growth-

promot,ing propert,ies of various preparations derived from yeast
and from liver.
The basal diet, as shown below, contained purified, vitamin-
free caseinl supplemented with cystine, a partially hydrogenated
vegetable oil (Crisco) fortified with linoleic acid, sucrose, and
Osborne-Mendel salts t,o which small amounts of cupric chloride
and manganous sulfate had been added. The vitamins were sup-
plied separately with the exception of vitamin E, which was con-

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tained in the vegetable oil. This source of vitamin E was sup-
Basal Diet

Casein.. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.0
Crisco.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.9
Linoleic acid. . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Osborne-Mendel salts*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.0
Sucrose. . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48.9
Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1

100.0
Vitamins were fed separately in the following amounts.
Vitamin A and D concentrates 20 mg. per rat per week
E concentrate 35
I B (BJ concentrate 10 day
I G (B2) as indicated in Table I
* Supplemented with 2.5 mg. per cent of copper and 1.6 mg. per cent of
manganese.
plemented during the preliminary experiments with 35 mg.
weekly of the non-saponifiable fract,ion of wheat germ oil dissolved
in peanut oil. The requirements for vitamins A and D were met
by 20 mg. weekly of t,he non-saponifiable fraction of cod liver oil.
The antineuritic vitamin was derived from an extract of rice
polishings and consisted of an active fraction prepared by ad-
sorption on fullers earth and elution therefrom. The amount of
1 The casein was prepared from skim milk by precipitation with dilute
hydrochloric acid, the precipitate was dissolved in sodium hydroxide,
reprecipitated by acetic acid, redissolved in sodium hydroxide, filtered
through paper pulp, and again precipitated by acetic acid. The casein
was then extracted with hot alcohol, with high grade gasoline, again with
boiling alcohol, and was finally dried in a current of warm air (So-90) for
2 days.
 R. J. Block and L. R. Farquhar 645

vitamin G (Bz) in this concentrate was found by suimble tests to

be very small. Concentrates of liver and yeast were used t,o
supply the vitamin G (B,). The pot.ency of t,hese preparst,ions,
as judged by growth-promot.ing properties, was compared t,o the
potency of whole dried liver and of bakers yeast. Various liver
concentrates, sold commercially for use in the treatment of per-
nicious anemia, were also used as sources of vitamin G (Bz).
The potencies of digested extracts of liver and yeast were com-
pared with those of the original extracts.

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EXPERIMENTAL

Albino rats that weighed more than 40 gm. at 21 days of age

were fed the basal diet, and all vitamin supplements with the
exception of a source of vitamin G (B2). If, after 10 to 20 days,
the animals had declined in body weight, or had maint,ained an
unchanged body weight, a source of vitamin G (Bz) was added to
t,he diet for 30 days and the total gain in weight was recorded.
Two to seven rats, no two of which were litter mates, were used in
each group. The growth of twenty-one negative controls was also
recorded. Five rats were given 25 times the usual amount of the
vitamin B (Bl) supplement to determine whether any vit.amin G
(Bz) was present. The results of all tests are presented in Table I.

Sources of Vitamin G (I?,)

The preparations of dried, and of autolyzed liver, were made as
follows: Half of a fresh beef liver, previously freed from the larger
blood vessels, was ground, suspended in 50 volumes of 0.1 N hy-
drochloric acid, and heated to 100 for 30 minutes to destroy the
autolytic enzymes. The suspension was then concentrated, and
the product was dried in a current of warm air and subsequently
made into tablets each of which was equivalent to 1 gm. of raw
liver. The other half of the liver was suspended in 50 volumes of
0.1 N hydrochloric acid, allowed to autolyze at 37 for 10 days in
the presence of chloroform, and was then treated in the same
manner.
The liver concentrate, No. 343, manufactured by Eli Lilly and
Company for use in the treatment of pernicious anemia, was tested
at various levels of intake. The pernicious anemia factor has
been report,ed to be increased by 3 to 4 times (Walden and Clowes
646 Studies on Vitamin G (BJ. I
TABLE I
Average Rate of Growth of Rats Fed for 30 Days on Highly Purified Diet
Supplemented with Various Inver and Ieaut Preparations As
Source o/ Vitamk G (I
--
Daily doesee :nitis l+ml
Source of vitamin (: (IS>) of vitamin G body
zk:i u~r~e~~Ple- vcighl
MY
weight

mg. gm.
Beef liver
Dried. . . 5 290 147

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Autolysed and dried 5 290 177
Liver concentrate
Lilly, No. 343. 4 40 54 138
Same 5 80 61 180
. . ... . . . 3 160 47 185
. ... . . . 5 160 53 166
digested with human gas-
tric juice . .. . 5 40 54 126*
Same, digestedwith pig stomach
(extralin) . . .. . .. 5 40 133
Same, digeeted with pig stomach
(extralin) - 160 45 191
Same, treated with cold alkali. ; 40 46 143
(noadded vitamin B (B,)) t, 5 40 44 95
I I I 5 160 51 151
i.
Lederle, No. 24OH235B. .. . . . 5 46.4 51 101
I 187H35T (oral). . 5 14.9-59.51 45 72
255H88A (paren-
teral). . . . . . . . . . . . . 5 35.5142.1! 50 85
Chsppel (oral). . . . . . . . . . . . 5 79 52 106
(for injection). . . . 4 8 45 96
Yeast
Fleischmann, No. lL-9902. . . 5 400 50 209
Same, heated 2 wks.. 5 440 45 186
I ( 4 ( . . 5 440 47 202
Yeast concentrate
Fleischmann, No. lL10617.. 5 36 53 108
Same . . . . . 3 360 55 216
. . . .. . . . . 2 360 48 156
digested with human gas-
tric juice.. . .. . . . . 2 36 41 98
Same, digested with human gas-
tric juice.. . . .. . . 7 360 52 236
Negative controls. . . . . .. 21 51 7911
with 25 X sup-
plement vitamin B (B,) . . . . . 5 44 16QTI
-~--__ -- - -
 R. J. Hock and L. R. Farquhar 647
TABLE I-Concluded
* Owing to lack of mat.erial the experimental period was reduced from
30 to 21 to 28 days in this group.
t Source of vitamin B (B,) as well as vitamin G (Bz).
$ The dosage was increased after 15 days from 14.9 to 59.5 mg. daily.
0 The dosage was increased after 15 days from 35.5 to 142.1 mg. daily.
11Seven rats died in from 12 to 28 days; the averages are for fourteen rats.
7 Experiment was run for 42 days.

(8)) by digestion of the concentrate with scrapings of pig stomach.

The vitamin G (B,) potency of this preparation (extralin) is

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herein reported, together with that of a preparation of concentrate
No. 343 which had been digested with 15 times its weight of fresh
human gastric juice at 37 for 22 hours.
The amount of vitamin B (B1) present in the liver concentrate
No. 343 was tested by omitting the source of vitamin B (B,)
from the dietary and feeding the liver concentrate alone.
Treatment of the concentrat,e No. 343 with alkali in the cold
was undertaken to ascertain whether the vitamin G (Bz) content
was thereby altered. Unpublished experiments of Cowgill and
Block have shown that the antineuritic potency is destroyed under
these conditions.
The liver concentrates prepared by Lederle and by Chappel
were fed in doses equivalent to 0.8 gm. of raw liver as calculated
from data supplied by the manufacturers. These dosages were
approximately equal to 160 mg. of concentrate No. 343.
Bakers yeast and an aqueous extract thereof, concentrated to a
paste, were fed as sources of vitamin G (&). The concentrate
was digest.ed with twice its weight of diluted (1: 1) normal human
gastric juice for 24 hours at 37 and then tested.
Recently Elvehjem, Kline, Keenan, and Hart (9) have reported
a 50 per cent destruction of vitamin G (B2) in dried yeast that had
been heated at 95-100 for 144 hours. In order to test this point
we have also fed, as a sole source of vitamin G (Rz), two samples
of bakers yeast which had been dried in a desiccator and then
heat.ed in the above manner for 2 and 4 weeks respectively.

Results
The results as summarized in Table I indicate that good and
even rapid growth was obtained with certain of the liver and yeast
648 Studies on Vitamin G (13,). I
concentrates. Daily administration of 160 mg. of the liver con-
centrate No. 343, or of 160 mg. of No. 343 digested with scrapings
of pig stomach, permitted good growth. Supplements of 360 mg.
daily of the yeast concentrate, or of 360 mg. daily of the concen-
trate digested with human gastric juice, permitted rapid growth.
Daily doses of 400 mg. of unheated yeast or 440 mg. of yeast
heated for 4 weeks were similarly effective; evidently, therefore, no
destruction of vitamin G (Bz) by dry heat had taken place.
Liver concentrate No. 343 is a good source of vitamin G (Bz).
Treatment with cold alkali was without effect upon the vitamin G

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(B,) content of the concentrate, although according to unpublished
experiments of Block and Cowgill the antineuritic vitamin is
completely destroyed.
The liver concentrates prepared by Lederle and by Chappel con-
t,ained little if any vitamin G notwithstanding their high content
of the pernicious anemia factor. These results are in accord with
those of Brand, West, and Stucky (10). Furthermore, digestion
of the liver concentrate No. 343 with the scrapings of pig stomach,
or with normal human gastric juice had no influence upon the
vitamin G (B,) content of the preparation, whereas the anti-
anemic potency is known to be increased in this way (11). How-
ever, the digestion of the yeast concentrate with human gastric
juice, and the autolysis of fresh beef liver, seem to have actually
increased the growth-promoting power of the respective products.

SUMMARY

Gains in weight of from 4.5 to 6 gm. per day were made by male
albino rats fed on an adequate but highly purified diet in which
vitamin G (B.J was supplied by yeast or liver concentrates.
These studies do not support the view that there is a simple
relationship between vitamin G (B,) and the substance effective
in the treatment of pernicious anemia.
Yeast and liver preparations show no decrease in vitamin G
(Bg) potency after autolysis, digestion, treatment with alkali in the
cold, or prolonged heating after being previously dried.

We are exceedingly grateful to Mr. W. H. Berg of the Health

Products Corporation, Newark, for the vitamin A and D con-
centrates; to Dr. Guy W. Clark of the Lederle Laboratories.
 R. J. Block and L. R. Farquhar 649
Dr. A. E. Meyer of Chappel Brothers, and Dr. H. W. Rhodehamel
of Eli Lilly and Company for the generous supplies of liver con-
centrates; and to Dr. C. N. Frey of Standard Brands Incorporated,
for the yeast.
BIBLIOGRAPHY

1.Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 69, 661 (1926).
2. Mendel, L. B., and Cannon, H. C., J. Biol. Chem., 76, 779 (1927).
3. Anderson, W. E., and Smith, A. H., Am. J. Physiol., 100, 511 (1932).
4. Donaldson, H. H., Boas anniversary volume, New York, 5 (1906).

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5. King, H. D., Anat. Rec., 9, 751 (1915).
6. Smith, A. H., and Bing, F. C., J. Nutrition, 1, 179 (1928).
7. Mapson, L. W., Biochem. J., 26, 970 (1932).
8. Walden, G. B., and Clowes, G. H. A., J. Biol. Chem., 97, xi (1932).
9. Elvehjem, C. A., Kline, 0. L., Keenan, J. A., and Hart, E. B., J. Biol.
Chem., 99, 309 (1932-33).
10. Brand, E., West, R., and Stucky, C. J., Proc. Sot. Exp. Biol. and Med.,
30, 1382 (1933).
11. Strauss, M. B., and Castle, W. I~.,-Lancel, 333, 111 (1932).
 STUDIES ON VITAMIN G (B2) : I.
YEAST AND LIVER PREPARATIONS AS
A SOURCE OF VITAMIN G (B2)
Richard J. Block and Lucille Reed Farquhar
J. Biol. Chem. 1933, 103:643-649.

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