tors exist in the vitamin B complex. Other investigators (3, 4)

14 Pages·2003·0.58 MB·English

FURTHER EVIDENCE OF THE COMPLEX NATURE OF
VITAMIN B
II. EVIDENCE THAT A THIRD FACTOR EXISTS*
BY CHARLES H. HUNT AND WILLARD WILDER
(From the Department of Animal Industry, Ohio Agricultural Experiment
Station, Wooster)
(Received for publication, September 16, 1930)
In previous papers (1, 2) it was shown that at least three fac-
tors exist in the vitamin B complex. Other investigators (3, 4)
have also presented evidence that a third and possibly a fourth
factor exists. Much of the evidence presented in favor of the
existence of the third factor has been based upon the effect of
heat, in the presence of different pH values, upon yeast. In
this paper additional evidence is presented to show that a third
factor exists in this exceedingly complex vitamin B. A large
quantity of the two fractions has been prepared and studied.
The data in this paper were obtained by feeding the prepared
fractions from yeast and rice polishings alone and with natural
products. The methods of preparing the fractions are given
below.
Method
From Yeast-Dry powdered yeast was suspended in 0.1 per
cent acetic acid in the proportion of 1 kilo of yeast to 4 liters
of acidulated water and allowed to stand 24 hours. The super-
natant liquid was then siphoned off and 2000 cc. of 0.1 per cent
acetic acid were added and well shaken. It was allowed to stand
another 24 hours when the supernatant liquid was siphoned off
again. This was repeated for a total of fourteen times. The
resulting residue was dried before an electric fan, at room tem-
* Published with the permission of the Director of the Ohio Agricultural
Experiment Station.
279
This is an Open Access article under the CC BY license.
Vitamin B Complex. II
perature. The extract was concentrated to about 5 liters
before an electric fan at room temperature, and its pH value
adjusted to about 4.5. It was then supercentrifuged to remove
the suspended solids. The solids were discarded while the clari-
fied extract was treated twice with fullers’ earth, in the propor-
tion of 50 gm. per 1000 cc. of the extract. After shaking occa-
sionally for several hours it was filtered on a Buchner funnel.
The earth was washed several times with 0.1 per cent acetic
acid. The filtrate was concentrated before an electric fan until
1 cc. was equivalent to 1 gm. of the original yeast. In this
paper it will be designated as the B or non-adsorbable fraction.
Another fraction of the vitamin B complex was removed from
the moist fullers’ earth by means of a 2.5 per cent solution of
barium hydroxide. The liquid was separated from the earth
by filtering and washing with suction on a Buchner funnel.
This extraction was repeated five or six times, and washed thor-
oughly with water. The filtrate was removed frequently from
the suction flask and acidified with H&304. The filtrates were
combined and the BaS04 was removed by centrifuging. The
liquid was then concentrated before an electric fan until 1 cc.
was equivalent to 1 gm. of the original yeast. This fraction is
designated as the A fraction, and contains the antineuritic
vitamin (vitamin B or F). The fullers’ earth residue was dried
at room temperature.
From Rice Polishings-2 kilos of rice polishings were sus-
pended in 6 liters of 0.1 per cent acetic acid, and allowed to
stand 24 hours, when the supernatant liquid was siphoned off.
About 4 liters of 0.1 per cent acetic acid were added to the solids,
stirred, and allowed to stand another 24 hours, when the super-
natant liquid was siphoned off. This procedure was repeated
four times. The extracts were combined and evaporated at
room temperature before an electric fan to an approximate
volume of twice the original weight of the polishings. The ex-
tract was then treated with a solution of lead acetate, as sug-
gested by Rosedale (5). The antineuritic fraction (non-pre-
cipitated fraction) prepared by this method had a potency
equivalent to that prepared from yeast. It is also designated
as vitamin B or vitamin F.
C. H. Hunt and W. Wilder
EXPERIMENTAL
Rats 24 days of age and weighing from 50 to 60 gm. were used
as the experimental animals. They were kept individually
in screen bottom cages. The basal vitamin B-free diet con-
sisted of casein 18, starch 64, Salt Mixture 185l 4, Crisco 10,
agar-agar 2, and cod liver oil 2. The vitamin preparations were
fed daily and separately. In a previous communication (6) it
was shown that a similar synthetic diet containing 25 per cent
of wheat, plus 0.4 gm. of autoclaved yeast, daily, supplied the
necessary vitamins in sufficient quantity for normal growth
and prevention of pellagra. This raised the question, “Does
autoclaved yeast contain any other vitamin or vitamins, aside
from vitamin G (antipellagra or P-P factor)?” All attempts to
produce growth that anywhere approaches normal with vitamin
B (adsorbable fraction) and the non-adsorbable fraction (treated
with fullers’ earth) from yeast extract have failed. As a rule,
rats on a diet of 25 per cent of wheat and the non-adsorbable
fraction (fraction B) from yeast extract failed to grow, but when
the non-adsorbable fraction was fed in addition t,o the yeast
residue, growth was excellent. It is evident from this that the
non-adsorbable fraction is not equivalent to autoclaved yeast,
but that the non-adsorbable fraction with the yeast residue, or
the yeast residue alone, is equivalent to autoclaved yeast in pre-
venting pellagra (Chart 1). This suggests that in the prepara-
tion of an acidulated water extract of yeast something essential
remains adsorbed on the protein of the residue, and which is
very difficult to remove, although, as shown later, some of it
dissolves when yeast is extracted with 0.1 per cent acetic acid.
This also suggsts that there are at least two fractions, a growth
fraction and the antipellagra fraction, other than the anti-
neuritic fraction, present in yeast, and two fractions in auto-
claved yeast. Chart 1 also shows the growth curves of rats
receiving in addition to the basal diet 0.4 gm. of autoclaved
yeast plus 1 cc. of the antineuritic fraction, and 2.0 gm. of auto-
claved wheat plus 1 cc. of the antineuritic fraction, respectively.
1 McCollum’s Salt Mixture 185 (McCollum, E. V., and Simmonds, N.,
J. Biol. Chem., 33,63 (1918)).
282 Vitamin B Complex. II
This may indicate that autoclaved yeast contains some factor
or factors that are absent, or practically so, in wheat.
f f
12
200
180
160
i
120
IO0
SO
60
f8
IlO
WO
120 ‘
B
IO0
80
Go
40
CHART 1
An extract of yeast of proper concentration produces excellent
growth in rats, placed on a diet adequate in every respect except
the vitamin B complex, while the adsorbable and non-adsorbable
C. H. Hunt and W. Wilder 283
fractions (treated with fullers’ earth) from the yeast extract
failed t,o produce growth, as was shown in a previous paper (2)
I I I I I I I I I I I I
CHART 2
as well as in Chart 2. It was concluded that the fullers’earth
had removed something from the extract that was essential for
the growth of t,he animal (Chart 2). Excessive washing of the
284 Vitamin B Complex. II
fullers’ earth with water and 80 per cent alcohol, respectively,
has failed materially to remove the factor. This is shown by
the growth curves in Chart 2, and is in agreement with the data
presented by Narayanan and Drummond (7). Other reagents
'*' I lob-’A
/ , /
1OE-A
i/j-//
ci I eadh/ A&I B ftrI ackiodI s I
CHART 3
for removing this fraction from the fullers’ earth residue are
being tried. The fact that t,he animal has no trouble in remov-
ing this fraction indicates the complexity of the process taking
place in the animal’s digestive tract.
In a previous publication (2) it was shown that the factor
C. H. Hunt, and W. Wilder 285
which was present in the yeast residue was thermostable.
Further investigation has confirmed this and also shown that
there are two factors present in yeast and fullers’ earth residues,
respectively, one of which is thermostable in either an acid or
alkaline medium, while the other is thermolabile in an alkaline
medium only. It is believed that the thermolabile factor is the
B or non-adsorbable fraction which remains adsorbed as an
impurity. For that matter both factors are evidently im-
purities but one is more easily removed than the other. This
GWI.
200
180
160
JYO
120
IO0
80
60
CHART 4
conclusion is supported by the growth curves presented in
Charts 3 and 4. Further confirmation will be shown later.
In order further to confirm or reject the hypothesis that an
essential factor exists in the yeast residue, and to attempt to eluci-
date the fact that the yeast extract when administered to animals
in proper concentration, or amounts, causes them to grow nor-
mally, it was thought that the problem might be attacked by cen-
trifuging the extract of yeast. The thought was that an extract of
yeast contained suspended matter which had growth-promoting
qualities. For this purpose an extract of yeast was divided into
two portions, one of which was run through a supercentrifuge,
operating at 36,000 to 40,000 revolutions per minute. The other
286 Vitamin B Complex. II
portion was allowed to stand 12 hours, or longer, and the upper
layer was decanted off. The two portions were fed at two con-
-
-
/
I I I I I I rl
1 ICC. bxtdact I of heah der I 1 ICC.h en
-
-
,-
/
I-
-
-
I-
, -
P-
,-
CHART 5
centrations to rats as the sole source of the vitamin B complex,
with results as shown in Chart 5. The effect of adding the yeast
residue to the centrifuged portion on the growth of rats is also
C, H. Hunt and W. Wilder 287
shown in Chart 5. The results seem to indicate that the yeast
extract contains some substance in suspension that stimulates the
+ lo .ertra:t cf ysast. per lcc.au o%l ve extract of ytdst
per ra da ly
1 I I I I I I,, I I I I I
CHART 6
growth of rats and which can be partially removed by super-
centrifuging.
As is well known, yeast extract has been found to be an ade-
288 Vitamin B Complex. II
quate source of the vitamin B complex, for rats grow normally
when the extract and a diet adequate in every respect, except the
complex vitamin B, are fed. This gives assurance that the three
factors, and possibly others of this complex, are present in the
extract. It is true that one factor is heat-labile under one condi-
tion, and another one is heat-stable under all conditions, etc.;
then one should be able to differentiate them by autoclaving
different portions of the extract under different pH conditions.
An extract of yeast was prepared from dry whole yeast, by the
method previously described. This extract was divided into
three portions. One was fed as prepared, the second portion was
CHART 7
autoclaved as prepared (pH 5.1), and the third portion was made
alkaline (pH 9.4), autoclaved for 5 hours at 15 pounds pressure,
and then made slightly acid. The autoclaved acid extract was
fed alone andwith the addition of vitamin B (antineuritic vitamin).
The autoclaved alkalized extract was fed with the addition of the
vitamin B alone, and with the vitamin B and the non-adsorbable
fullers’ earth fraction, respectively. It is evident from the re-
sults shown in Charts 6 and 7 that one fraction, vitamin B, of the
extract is heat-labile in an acid medium, another one (non-ad-
sorbable fraction) is heat-labile in an alkaline medium, and the
third fraction (antipellagric or vitamin G) is thermostable under
all of the conditions tried in this laboratory. It will be noticed