STUDIES ON THE RELATIVE EFFICIENCY OF VITAMIN D

FROM SEVERAL SOURCES

I. INFLUENCE OF VITAMIN D OF DIFFERENT ORIGINS ON BONE
ASH AND BODY WEIGHT OF THE CHICKEN

BY JOHN T. CORRELL AND E. C. WISE

(From the Research Laboratories, The Upjohn Company, Kalamazoo)
(Received for publication, August 26, 1938)

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Within the last few years the dual assay for vitamin D, with
both rats and chickens, has become a popular method for dis-
tinguishing between the several forms of the antirachitic vitamin
as they are found in numerous fish liver oils and other sources.
The ash content of either the femur or the tibia of the chicken has
been used as the basis of interpreting the responses obtained.
By the use of this technique Massengale and Nussmeier (1) and
other workers were able to demonstrate that the vitamin D of
irradiated ergosterol and that of cod liver oil were two separate
entities.
Further information on the vitamin D problem was contributed
by Waddell (2), who reported that upon the rat unit basis the
vitamin D of irradiated cholesterol was more effective for the
chick, as measured by the bone ash procedure, than the vitamin
D of irradiated ergosterol. Furthermore, the irradiated cho-
lesterol compared favorably in that respect with the vitamin D
of cod liver oil. This demonstration was an incentive for the
study of cholesterol as a precursor of vitamin D. The interest
in this relationship culminated in 1936 in the isolation by Windaus,
Schenck, and von Werder (3) of an antirachitic substance, vitamin
Da, as the crystalline dinitrobenzoate and allophanate, from the
irradiation product of 7-dehydrocholesterol. In the same year
Brockmann (4) reported the isolation, as the cryst,alline dinitro-
benzoate, of the same substance from tuna liver oil, and Grab (5)
reported a similarity in the antirachitic efficacy, for the chick, of
vitamin D1 from the two sources.
573
574 Vitamin D Efficiency. I

These results were followed in 1937 by the isolation by Brock-

mann (6) of vitamin DS from halibut liver oil. This was the only
antirachitic substance the presence of which was detected in this
oil. Also, Brockmann and Busse (7), working with liver oil of the
blue-fin tuna from Japanese waters, concluded that the vitamin
D of that species is identical with the antirachitic vitamin of the
tuna and halibut liver oils previously reported.
In the meantime additional information had accumulated
relative to the comparat,ive physiological response of the rat and
the chick to vitamin D from various sources. Rygh (8) reported

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in 1935 that the vitamin D of seventeen species of fish was equally
effective for the chicken, and Dols (9) found that the vitamin D of
tuna liver oil was as effective as that of cod liver oil upon the rat
unit basis.
Thus, although it was recognized that the vitamin D of fish
liver oils varied considerably in concentration, in general it was
thought to consist only of one substance until Bills, Massengale,
and Imboden (10) reported that the vitamin D of blue-fin tuna
liver oil is only about one-sixth as active as the antirachitic factor
of cod liver oil when measured on chickens. Also, Haman and
Steenbock (11) and Black and Sassaman (12) indicated the
inefficiency of tuna liver oil as compared with cod liver oil under
similar conditions.
This confusion has been admirably explained by Bills, Massen-
gale, Imboden, and Hall (13). They point out in an extensive
study of fish liver oils that tuna oils vary, according to species,
from those which are about equal to those which are only about
one-sixth as effective as cod liver oil, rat unit for rat unit, as
demonstrated by the bone ash content of chickens. Evidence of
this nature indicates that the vitamin D of fish liver oils may con-
sist of more than one antirachitic substance.
The present studies were undertaken to determine the relative
efficiency of several different fish liver oils as compared with cod
liver oil by means of the rat-chicken technique. We were anxious
also to ascertain whether other criteria besides chicken bone ash
might further establish the differences or similarities in the physio-
logical response of the various forms of vitamin D.
It is the purpose of this paper to report briefly our findings on
approximately 150 chicken assays, the results of which confirm
 J. T. Correll and E. C. Wise 575
the conclusions of Bills and others (13) concerning tuna liver oil.
AIso, we submit data on groups of chicks which have been raised
on vitamin D supplements of varying origin for a considerably
longer period of time than is necessary for the ordinary chicken
assay. These latter investigations demonstrate the growth-
promoting effect of vitamin D and the differences in growth
response of the chicken to vitamin D from several sources.

EXPERIMENTAL

All oils were first assayed in our own laboratories for vitamin D

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with the rat by the standard U.S.P. XI method. For the most
part our samples of fish liver oils were taken from relatively large
lots and are representative of the oils of commerce and, therefore,
do not represent oils from strictly selected species.
For the chicken tests, single comb white Leghorn chicks with a
uniform nutritional background were used in the prophylactic
type of experiment. The birds were started on assay when 1
day old and the bone ash experiments terminated on the 21st day.
The growth investigations were prolonged for 8 weeks. In both
studies the chicks were weighed and freshly supplemented diets
mixed each week. In all cases the basal diet employed was that
of Hart, Kline, and Keenan (14), modified to contain 57 parts
of maize and 2 parts of dried yeast.
The oils to be assayed were incorporated into the basal ration
in quantities calculated to furnish definite dosages of vitamin D
in each 100 gm. of diet. When necessary, maize oil was added
to the vitamin-bearing adjuvant to bring the total oil supplement
of the feed to 1 per cent. The negative control diets were sup-
plemented with 1 per cent of maize oil.
Data which we have accumulated from a large number of
chicken assays indicate that the bone ash response to a fixed dose
of vitamin D from cod liver oil varies from one time to the next,
even under the most uniform conditions. Thus the establish-
ment of a master response curve for vitamin D from cod liver oil,
to which the resuIts obtained on feeding vitamin D from other
sources could be referred, seemed impractical for this prophylactic
chick technique. The most satisfactory method appeared to be
the feeding of two or three parallel groups of chicks graded doses
of a reference cod liver oil, and the use of these data as a standard
576 Vitamin D Efficiency. I
of comparison. One group received no vitamin D and served as
a negative control. Griem (15) has arrived at similar conclusions
as to the use of the prophylactic chick test for vitamin D.
Twelve to fifteen chicks were started in each group; the sex was
fortuitous. Ash determinations were made on pooled left tibiae,
alcohol- and ether-extracted and moisture-free, from ten indi-
viduals.
TABLE I
Average Bone Ash from Groups of Ten Chickens, Supplemented with Diferent

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Sources of Vitamin D

Tibia ash from B~OUPS fed

- -
rUgilLp Irradiated
ergosterol
_
i?&lYka-
tional units
per cent per cent per cent per cent per cent
0 34.1
6 39.7 37.2 38.5
12 42.7 38.0 38.8
24 45.5 41.8 38.7 42.3
50 46.1 46.8
0 34.2*
9 37.8* 38.8t 3a.9t 36.0
18 44.4* 39.7t 39.87 39.7
27 46.7* 43.4t 43.3t 43.4
0 34.7
18 46.5 41.1 35.0
50 47.2 36.0
80 46.8
100 39.5
-
* Average of three consecutive assays.
t Average of two consecutive assays.

Results
Table I indicates the relative ineffectiveness of several different
tuna liver oils as compared with cod liver oil in the calcification
of bone. In general, they all appear to be from 40 to 60 per cent
as efficient, at the levels fed, as was cod liver oil. Irradiated
ergosterol fed at a 100 unit level gave an ash response equivalent
to that which would be produced by about 10 international units
of vitamin D from cod liver oil. These data substantiate the
 J. T. Come11 and E. C. Wise 577

reports in the literature which indicate the comparative ineffi-

ciency of some tuna liver oils and of irradiated ergosterol.
GMS I I I I
1 , , I

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I I I I I I I I I
12 6 7 8

FIG. 1. The figures represent average growth curves for groups of chick-
ens receiving the same basal ration plus the following supplements per 100
gm. of diet. A, Curve 1, 37 I.U. of vitamin D from cod liver oil, 120 I.U. of
vitamin A; Curve 2, 37 I.U. of vitamin D from cod liver oil, 652 I.U. of vita-
min A; Curve 3,lO I.U. of vitamin D from cod liver oil, 120 I.U. of vitamin A;
Curve 4, 0 unit of vitamin D and 652 I.U. of vitamin A; Curve 5, 0 unit of
vitamin D and 120 I.U. of vitamin A. B, Curve 1, 371.~. of vitaminDfrom
cod liver oil; Curve 2, 37 I.U. of vitamin D from Tuna Liver Oil 1; Curve 3,
100 I.U. of vitamin D from irradiated ergosterol; Curve 4, 10 1.1~. of vitamin
D from cod liver oil; Curve 5, 10 r.o.of vitamin D from Tuna Liver Oil 2;
Curve 6, 10 I.U. of vitamin D from Tuna Liver Oil 1; Curve 7, 0 unit of
vitamin D. The figures in parentheses denote the final average weights of
the groups.

In order to compare, in a different manner, these oils with cod

liver oil as sources of vitamin D, it was decided to feed similar
groups of chickens over a longer period of time and see whether
the oils would have any pronounced effect on body weight.
Hart and coworkers (16) early indicated that sunlight had a
578 Vitamin D Efficiency. I

growth-stimulating effect on chickens; since that time numerous

publications have mentioned the growth-promoting influence of
vitamin D. However, few data are available on the quantitative
relationships of vitamin D to growth of chicks. For this reason
a preliminary experiment was conducted as follows.
Chicks were fed the same basal diet as in the previous experi-
ments. This ration seemed to be adequate in all known vitamins
with the possible exception of vitamin A. Three groups of
chickens were started on this feed supplemented with 0, 10, and
37 international units respectively of vitamin D per 100 gm. of

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diet from cod liver oil. These diets were adjusted so that all of
the groups received 120 I. u. of vitamin A per 100 gm. of ration
in excess of that inherent in the basal mixture. Two additional
groups were started on supplement levels of 0 and 37 I. u. of
vitamin D, but these diets were adjusted to contain 652 I. u. of
vitamin A per 100 gm. above that in the basal ration.
The results of this experiment are shown by the growth curves,
A, Fig. 1. It can be seen from these curves that vitamin D,
in the presence of all other known vitamins, exerts a definite
influence on the body weight of the chick. Since those groups
which received the larger quantity of vitamin A showed the same
growth responses as the birds on the lower level, it seemed ap-
parent that the basal ration required little or no extra vitamin A
for satisfactory growth.
A similar series of investigations next was conducted in which
vitamin D from several sources was incorporated in the basal
ration. The sources and levels fed are recorded in the legend to
Fig. 1; the growth curves in B show the results obtained.
The data from this experiment indicate that rat unit for rat unit
the several sources of vitamin D tested are considerably less
effective in the promotion of growth with the chicken than is
cod liver oil.
SUMMARY

1. Several commercial samples of tuna liver oil have been shown

to be only 40 to 60 per cent as effective as cod liver oil, rat unit
for rat unit, as measured by the bone ash of chickens.
2. These same sources of vitamin D also have been demon-
strated to be much less efficient than the vitamin D from cod liver
 J. T. Come11 and E. C. Wise 579
oil in the promotion of growth in the chick during the first few
months of life.
3. It is indicated that vitamin D has a definite influence on
the body weight of the chick when supplemented in a diet ade-
quate in all other known vitamins.
BIBLIOGRAPHY

1. Massengale, 0. N., and Nussmeier, M., J. Biol. Chem., 87, 423 (1930).
2. Waddell, J., J. Biol. Chem., 106, 711 (1934).
3. Windaus, A., Schenck, F., and von Werder, F., Z. physiol. Chem., 241,

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100 (1936).
4. Brockmann, H., 2. physiol. Chem., 241, 104 (1936).
5. Grab, W., 2. physiol. Chem., 243,63 (1936).
6. Brookmann, H., Z. physiol. Chem., 246, 96 (1937).
7. Brockmann, H., and Busse, A., 2. physiol. Chem., 249, 176 (1937).
8. Rygh, O., Nature, 136, 552 (1935).
9. Dols, M. J. L., 2. Vitaminforsch., 6, 161 (1936).
10. Bills, C. E., Massengale, 0. N., and Imboden, M., Science, 80, 596
(1934).
11. Haman, R. W., and Steenbock, H., J. BioZ. Chem., 114, 505 (1936).
12. Black, A., and Sassaman, H. L., Am. J. Pharm., 108, 237 (1936).
13. Bills, C. E., Massengale, 0. N., Imboden, M., and Hall, H., J. Nutrition,
13,435 (1937).
14. Hart, E. B., Kline, 0. L., and Keenan, J. A., Science, 73, 710 (1931).
15. Griem, W. B., J. Assn. 08. Agric. Chem., 20,438 (1937).
16. Hart, E. B., Steenbock, H., Lepkovsky, S., and Halpin, J. G., J. Biol.
Chem., 68, 33 (1923-24).
 STUDIES ON THE RELATIVE
EFFICIENCY OF VITAMIN D FROM
SEVERAL SOURCES: I. INFLUENCE OF
VITAMIN D OF DIFFERENT ORIGINS
ON BONE ASH AND BODY WEIGHT OF
THE CHICKEN
John T. Correll and E. C. Wise
J. Biol. Chem. 1938, 126:573-579.

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