THE. LSE OF THE. PROPHYLACTIC
METHOD FOR THE DETERMENATZON
OF THE ‘v’ITAMN A
CONTENT OF FEEES

 

Thesis for the Degree. of M. :5

MICHIGAN STATE COLLEGE
Bernard R: Homrich
1938

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THE USE OF THE PROPHYLACTIC METHOD FOR
THE DETERMINATION OF THE VITAMIN A
CONTENT OF FEEDS

THE USE OF THE PROPHYLACTIC METHOD FOR
THE DETERMINATION OF THE VITAMIN A
CONTENT OF FEEDS

THESIS

Submitted to the faculty of Michigan State
College in partial fulfillment of the require-
ments for the degree of Master of Science.

BY
Bernard Romuald Homrich

1938

Acknowledgment
The author of this thesis is greatly indebted
to Dr.C.A.Hcppert, Professor of Chemistry, for his
helpful criticism and guidance in the course of this

study and in the preparation of the manuscript.

INTRODUCTION and HISTORY

In recent years some manufacturers of patented
foods,such as dog foods and other cereal products,
have experienced a great deal of difficulty in plac-
ing on the market a food which they could guarantee
to contain a definite vitamin A potency.This diff-
iculty was not so much in finding a source of vit-
amin A as it was in preventing the loss of the vit-
amin during the storage of the food.

Cod liver oil has been known to be a very good
source of this vitamin,and as such has been added to
foods to fortify the vitamin A potency.When kept in
the cold at O°C,cod liver oil is quite stable so far
as some of its constituents are ccncerned,but the
vitamin A.potency of cod liver oil kept at 0°C has
been found to decrease even at this low temperature.
The length of time the vitamin A potency of cod liver
oil will be retained has been found by Fraps and
Kemmerer (l) to vary with the temperature and the
food with which it is mixed.At higher temperatures
the stability of the vitamin is greatly decreased.

The vitamin A activity of yellow and green color-
ed plant materials has been traced to the presence of
carotenoid pigments alpha,beta and gamma carotene by
von Euler,Karrer,Hellstrom and Rydbom (2),and more

recently also to xanthophyll pigment,kryptoxanthin,

by Kuhn and Grundmann (8).These ”precursors" of
vitamin A have also been used as supplements in foods
and they too have been found to be unstable.Fraps and
Kemmerer (1) dissolved them in oil which was then I
mixed with various feeds.The mixtures were stored
under various conditions and examined periodically.
It was found that practically all of the vitamin A
value was lost after four weeks of storage.thn hydro-
quinone was used as a stabilizer the vitamin A.pct-
ency did not decrease as quickly,but even then most
of it was lost after three weeks of storage.Fraps
'and Kemmerer (I) believe that the addition of fish
oils or their concentrates to feed mixtures,for the
purpose of increasing the vitamin A potency,is of
little value unless the feed is used immediately.
They found that from 79% to 100% of the vitamin A
disappeared after four weeks of storage at either 7°C
or 38°C.Feeds stored in large amounts lost their
vitamin A from fish oils as rapidly as feeds stored
in small amounts.Fraps and Kemmerer (1) found also
that carotene dissolved in vegetable oil after being
added to feeds was more stable than the vitamin A in
cod liver oil,espeoially when the mixture was stored
at low temperatures.

Carotene in alfalfa and kryptoxanthin in yellow

corn were also found to be unstable,althcugh they

were not destroyed as rapidly as carotene dissolved
in oil.Carotene in alfalfa meal was found to be more
stable at 6°C than at room temperature.Alfalfa stored
at a temperature of 35°C lost carotene rapidly at
first and then quite slowly,indicating that some of
the carotene was easily destroyed,while a portion was
apparently protected so that it was less readily des-
troyed.Heywang and Titus (4) found that alfalfa meals
varied considerably in their vitamin A potency and
that it was unwise to rely on a supplement of less
than 5% of any type of meal unless its vitamin A
potency was known.The variability of commercial

meals in this factor is due in part to the length

of time the alfalfa is exposed to the sunlight.
Krizenecky (5) has expressed the opinion that the
vitamin A value is preserved best by artifical drying
and that the greatest losses occur when hay is dried
as usual in full sunlight.Russell (6) has done consid-'
erable work on-the effect of drying of alfalfa on the-
vitamin A value and his results substantiate those of
.the previous authors.Hs also found that yellow dent
corn was about 50%tmore potent with reference to its
vitamin A value than a white capped dent variety.
Esselen,Fellers and Isgur (7) found that the kernels
of yellow maize lost much of their.vitamin A potency

as they matured and dried out.

Fraps and Treichler (8) in studying the effect
of storage on the vitamin A value of dried foods ob-
served a gradual loss in the case of alfalfa leaf
meal,yellow dent corn and powdered whole milk.Grind-
ing corn before storage did not seem to increase to
any noticeable extent the loss of the vitamin A pot-
ency in the yellow corn meal as compared with the
whole grain.

Kon (9) tested certain patented dog foods on
rate and found that they differed considerably in
nutritive value.Even the best of them did not produce
the rate of growth obtained with the stock ration
used in his laboratory.Most of these dog foods appear-
ed tc be deficient particularly in vitamin A.The
effects of a continued diet deficient in vitamin A
are to be noted in experimental xerophthalmia.
Steenbock,Nelson and Hart (10) produced this con-
dition in dOgs after ninety four days.A knowledge
of the vitamin A value of dog foods and the effect
of storage are therefore important.Because of the
physiological importance of vitamin A and its in-
stability,the assay of foods and food mixtures is
obviously necessary.

The curative method for the biological assay of
foods for their vitamin A value is at present the

official method.Rats weighing from 40 to 50 grams

5
and less than twenty eight days old are fed a vitamin
A-free diet for a period of 24 to 45 days in order to
deplete them of their vitamin A reserves.The point of
depletion is indicated by the failure of the rats to
gain in weight.At this stage the rats are fed the test
material and the reference oil daily.The response of
the rats of the assay and the reference groups is cal-
culated on the basis of the difference between the
average weight of the surviving rats and the average
weight of the same rate on the day beginning the assay
period.Rats that gain between 13 and 60 gm. and which
consume the prescribed dose of oil or test material for
at least 23 days of the assay period are considered
valid.The units of vitamin A per gram of test material
are determined by dividing the daily dose in milligrams
of the reference oil necessary to produce in a refer-
ence group an average gain in weight,"G',of not less
than 12 gm. and not more than 60 gm.,by the daily dose
in milligrams of the test material that will produce
in an assay group an average gain in weight equal to
or greater than 'G',and multiplying the result of this
division by the units per gram of vitamin A contained
in the reference oil. '

Sherman and Todhunter (11) gave single feedings
of carotene,of cod liver oil,of kale or of calf liver

to standardized rats depleted of their surplus body

6
stores of vitamin A.The weight curves throughout the
period of survival after such feedings were then
charted on a fixed scale and the areas bounded by the
resulting curves were measured with a planimeter.With-
in limits amply sufficient for experimental work,in-
creasing the amount of the vitamin A or of the "precur-
sor“ given in a single feeding gave an approximately
preportional increase in the area under the curve.
Carotene curves were used as a standard of reference.
Direct comparison showed that the method of single.
feedings results in data which can be evaluated in
terms of the same units and with findings in close
agreement with those of the methods of feeding through
periods of four or more weeks as hitherto regarded most
accurate and conclusive.The single feeding method elim-
inates any possibility of loss of vitamin A in the
material when it is stored.Storage is necessary when
methods requiring subsequent feedings are employed.
Besides eliminating the storage of material the method
has another advantage in that it saves considerable
time.

In this work an attempt was made to determine
whether or not a prOphylactic method could be used
advantageously,that is,shorten the time period with-
out sacrificing accuracy.Richards and Simpson (13) are

of the opinion that the persistence of pathological

conditions that have been induced by vitamin A de-
ficiency,taken in conjunction with their diverse nature,
forms the strongest argument against the curative
method of assay.In the curative test,vitamin A can
influence growth only indirectly by effecting improve-
ment in the pathological conditions,and it is quite
obvious that no consistency in results can be expected
when these conditions vary so widely not only in their
nature but also in their intensity and curability.It
. has been shown that with a comparatively short deplet-
ion period there is more likelihood of a good response
to vitamin A dosage,but even a considerable reduction
in the length of this period cannot insure similarity
in the condition of the experimental animals.It was
suggested that any biological assay of vitamin A to
be satisfactory must be of the prOphylactic type.Its
object should be to determine not the amount of vit-
amin A which will produce fairly satisfactory weight
curves in animals of inferior type,but the amount
which will keep normal animals in a normal condition
as evidenced by the weight curves and also by exam-
ination of the tissues after death.

Accordingly,the object of this study was to deter-
mine whether a prophylactic method could be used for

the vitamin A assay of a variety of feeds.

EXPERIMENTAL

In carrying out these assays with albino rats,
care was taken in establishing the groups of test
animals so that all the groups were comparable.That
is,whenever an animal was assigned to one group,a
comparable animal of the same sex,age,litter,weight
and dietary history was assigned to each other group.
Each group was composed of two males and two females.
Since the stock diet was relatively rich in vitamin A
it is certain that the animals used in this study had
a reserve of vitamin A at the beginning of the exper-
imental period.This reserve was undoubtedly acquired
in the following three ways: a) by passage through
the placenta to the foetal circulation; b) by ingestion
post-natally by suckling; c) by direct consumption of
the stock ration.Due to this storage of the vitamin A
it seemed advantageous to remove the young rats from
their maternal environment just as soon as they became
old enough to be used for experimental purposes.It
was determined that animals weighing from forty to
fifty grams and from twenty to twenty two days old
were most suitable for the assay,and animals of that
weight and age were used throughout this work.

The rats were kept in individual cages during the

entire experiment.The cages,made of wire mesh,were

9

about ten inches in diameter and ten inches in height.
The bottoms of the cages were raised and the mesh was
large enough to allow the feces and the urine to pass
through and be collected in a pan.A metal feeding cup
was used for the vitamin A-free diet.

Instead of depleting the rats of their vitamin
A store and then supplementing the basal diet with
the test material,as is the procedure in the curative
method of assay,the rats were fed the vitamin A—free
diet "ad libitum",and had access to all the water
they cared to drink during the experimental period of

six weeks.0nce each week the rats received in addition

Vitamin A-Free Diet

Dextrin -------------------------- 67.0%
Casein (Vitamin-Free) - ----------- 18.0%
Crisco --------------------------- 5.0%
Dried Brewer's Yeast-— ------------ 5.0%

I ' ' (Irradiated) - 1.0%

Salt mixture (Steenbock's 40B) --~ 4.0%

to the above diet a definite amount of the test
material.When it was found that the supplement was
not relished by the rate it was mixed with the basal
ration and then fed.The approximate vitamin A potency
of the foods was known before the assays were begun,

thus eliminating much preliminary experimentation.

10
Six feeds were assayed,three being mixed dog foods
and the others,alfalfa leaf mea1,yellow corn meal
and dried whole milk powder.

The Cro-Pup dog food was indicated to contain
1560 International (U.S.P.) units of vitamin A per
pound of food.A representative sample was taken and
fed at the following two levels as the supplement,
4.45 grams to one group of rats and 8.90 grams to
another.These amounts of food were expected to con-
tain a vitamin A potency of 15 and 30 units (U.S.P.)
respectively.Each rat in one group received 4.45 grams
of the food once a week,while each rat of the second
group received 8.90 grams of the food weekly.The Fox
Cube and the Vita Ration # 1 dog foods were expected
to contain 5400 units (U.S.P.) of vitamin A.per
pound.0n this basis two grams of either of these
dog foods should contain 15 units (U.S.P.) of vitamin
A and four grams 30 units (U.S.P.) of vitamin A.
These quantities of the two dog foods were fed to
four other groups of rats.

The vitamin A potency of alfalfa varies consid-
erably.Fraps,Copeland and Treichler (13) found a sam-
ple to contain about 32.5 units (U.S.P.) per gram.0n
the basis of their results it was decided that the
alfalfa be fed at three levels,that is 0.25 gram,0.50

gram and 1.0 gram weekly.In the case of the yellow

11’
corn meal and the whole milk powder there was also
considerable uncertainity as to the probable vitamin
A potency.Fraps and Treichler (14) found the vitamin
A content of corn meal to vary from 4 to 7 units (U.S.P.)
per gram.00nsequently it was decided to feed the corn
meal at three levels,1.0 gram,2.0 grams and 5.0 grams.
Rice and Munsell (15) found that dried whole milk
powder contained from 8 to 28 units (U.S.P.) of vit-
amin A per gram,while Fraps and Treichler (14) found
a sample to contain from 9 to 14 units (U.S.P.) per
gram.With this food it was also decided to feed
supplements of 1.0 gram,3.0 grams and 5.0 grams weekly.

For comparison there was used a standard refer-
ence oil distributed by the Board of Trustees of the
United States Pharmac0poeial Convention.This oil
contained 3000 units (U.S.P.) of vitamin A per gram
of oil and was diluted with Wesson oil in such a
proportion that 1 cc. of the diluted standard oil
contained 30 units (U.S.P.) of vitamin A.A few crystals
of hydroquinone were added as a preservative and the
oil was stored in a refrigerator at about 0°C.this 011
was fed at levels of 10,15,20 and 50 units weekly
to four groups of rats.A group receiving no supple-
ment was included in this series.

All of the supplements were fed in separate,

flanged dishes so as to minimize wasting of the feed.

12
The dishes were left in the cages until all of the
supplements were eaten.The rats were weighed once
each week.The gains in weight of each group were
averaged and appear as the average total gain in
Table # l.The total gains of the controls over the
six week period were plotted against the number of
units of vitamin A in the supplement.Then by referr-
ing the average total gains of the groups of rats
receiving the feed supplements to this curve,the units
of vitamin A of each supplement could be determined
quite accurately.By dividing the number of units by
the number of grass fed,it was possible to determine
the number of units of vitamin A per gram of the
feed.The results obtained from each level were then

averaged and they appear in Table # 2.

Table # 1
Controls
Supplement Units of Vitamin A Total Gain
0.00 cc. of St'd oil 0 55 grams
0.53 " ” " " 10 102 '
0.50 ' ” ' ” 15 116 '
0.66 ' ' " " 20 125 '

1.00 " ” " " 50 141 '

7.-
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Feed

Gro-Pup

Fox Cube

Vita Ration # l

Alfalfa

Corn meal

Milk powder

Table i 3
Feeds

Total
Gain

77g. 4.45g.
93g. 8.90g.

108g.
181g.

2.00g.
4.00g.

71g. 2.00g.
107g. 4.00g.

100g. 0.35g.

134g. 0.50g.

141g. 1.00g.

102g. 1.00g.
2.00g.

5.00g.

136g.
141g.

140g.
176g.
i74g.

1.00g.
2.00g.
5.00g.

Supp't

Units/g.
(U.S.P.)

0.67
0.73

6.0
4.4

1.0
2.9

36.0
38.0
30.0

9.8
13.1

13

Avera e
Units5g.

5.3

1.95

34.6

9.6

29.25

14
DISCUSSION of RESULTS

The curve obtained by plotting the gains in
weight of the rats receiving the different levels
of the official reference oil was very smooth and
therefore appeared to be suitable as a standard for
comparison in the evaluation of the vitamin A potency
of various food stuffs.

The milk powder used in this experiment had a
much higher vitamin A content than expected.Conse--
quently the amounts of the milk powder used as supp-
lements were too large for a comparison of the growth
data with those of the reference oil groups.The growth
obtained with the two and five gram levels was approx-
imately the same,indicating that 2 grams of milk powder
contained sufficient vitamin A to give maximum growth
when fed in conjunction with the basal ration.The
levels for the corn meal and the alfalfa on the other
hand were very satisfactory because the growth re-
sponses fell within the range of those given by the
reference oil.

Fraps,Copeland and Treichler (13) found alfalfa
to contain 32.5 units (U.S.P.) per gram,while by the
prophylactic method used in this study the content
was found to be 34.6 units (U.S.P.) per gram.Fraps
and Treichler (14) found the vitamin A potency of

corn meals to vary from 4 to 7 units per gram.By the

15
prophylactic method an average value of 9.6 was ob-
tained.In the case of the milk powder,of which only
one level of the supplement could be used for com-
parison,a potency of 29 units per gram was obtained
by the prOphylactic method,whereas Rice and Munsell
(15) found that samples contained from 8 to 28 units
per gram,and Fraps and Treichler (14),9 to 14 units
per gram.As a result of the fairly close agreement
of the results,it is reasonable to believe that the
prOphylactic method of assay is dependable for the
determination of vitamin A in these types of feeds.

From the results it is evident that the vitamin
A content of the dog foods was not as high as antic- .
ipated.This was shown also by a comparative feeding
itest in which alfalfa was used to increase the vitamin
A supply.Although it is not definitely known how long
the dog foods had been stored before they were used
in this assay,it is certain that storage decreased
the vitamin A potency because known amounts of vit-
amin A (Cod liver oil) had been added when the food
was prepared.The Vita Ration # l was prepared in the
form of a meal while the Fox Cube was prepared in the
form of compressed cubes about % inch in diameter and
slightly more than A inch in length.Both foods origin-
ally had the same vitamin A potency,yet upon biological

assay after a few months of storage it was found that

16
the exclusion of air by preparing the food in the
cube form apparently greatly decreased the loss of
the vitamin A potency.Similar conclusions on differ-
ent foods were made by Taylor and Russell (16) and
also by Fraps and Kemmerer (l),who found that large
compact samples of feeds lost their carotene at a
less rapid rate than small samples loosely packed.

The units of vitamin A per gram of GroePup,Vita
Ration # l and Fox Cube were found to be .7,l.95 and
5.2 respectively.In comparative feeding tests carried
out previously,eaoh of the dog foods was supplemented
with alfalfa,an excellent source of "pro-vitamin A“,
and the growth responses were tabulated.After ten
weeks it was noted that the supplement affected the
greatest increase in growth response with the Gro-Pup,
less with the Vita Ration # l and least with the Fox
Cube.This substantiates the results obtained by the
prOphylactic method and indicates that the method in
question may be used for the determination of the vit-
amin A potency of a wide variety of foods and food

mixtures.

17
SUMMARY

1. The prophylactic method of assay,when used in
conjunction with an official reference oil as a stand-
ard for comparison,gave results with alfalfa,corn meal
and milk powder which are in close agreement with those

‘obtained by the official curative method.

2. The prophylactic method was also applied with
satisfactory results to the determination of the vit-

amin A potency of several complete dog food mixtures.

1.

3.

5.

18
BIBLIOGRAPHY

Fraps,G.S.,and Kemmerer,A.R. "Losses of Vitamin
A and Carotene From Feeds During Storage."

Texas Agric. Exper. Sta. Bull. 557,1937.

. Euler,H.von,Karrer,P.,Hellstrom,H.,and Rydbom,M.

Helvetica Chim. Acta 14: 839-842. illus.,l931
Cited by Daniel and Munsell. U.S.D.A.Miscellaneous
Publication 275,1937.

Kuhn,R.,and Grundmann,C.

Ber. Deut. Chem. Cessll. 66: l746~1750.1933

Cited by Daniel and Munsell. U.S.D.A.Miscellaneous
Publication 275,1937.

Heywang,B.,and Titus,H.W. “Alfalfa Leaf Meal as
a Source of Vitamin A for Growing Chickens“.

Jour. Agric. Res. §2,559-569,1937.

Krizenecky,J.
Casove Otaz. Zemed.,l936,§§,93-99
Nutritional Abstracts and Reviews,July 1937,p45.

Russe11,W.C. ”The effect of the Curing Process
upon the Vitamin A and D of Alfalfa”.
Jour. Biol. Chem. 85,289-297,1929.

19
7. Esselen,w.B.,Fellers.C.R.,and Isgur,B.
”Vitamins A,C,D,in Maize as Affected by Variety
and Stage of Growth". Jour. Nutrition 14,504,1937.

8. Fraps,G.S.,and Treichler,R.
”Effect of Storage on Vitamin A in Dried Foods“.
Indus. and Engin. Chem. 25,465-466,1933.

9. Kon,P.M.
'A Short Note on the Variation in Nutritional
Value of Dog Foods“. Vet.Rec. 46,632-634,1934.

10.8teenbock,H.,Nelson,E.M.,and Hart,E.B.
"The Incidence of an Ophthalmic Reaction in Dogs
Fed a Fat Solube Vitamine Deficient Diet”.
Amer. Jour. Physiol. 58,14,1921-1922.

11,8herman,H.C.,and Todhunter,E.N.
“The Determination of Vitamin A Values by a Method
of Single Feedings." Jour.Nutrition g,347-356,l934.

l2.Richards,M.B.,and Simpson,B.W.
IThe Curative Method of Vitamin A Assay“.
Biochcm. Jour. 28,1274,1934.

l3.Fraps,G.8.,Copeland,0.C.,and Treichler,R.
“The Vitamin A Requirements of Dairy Cows".
Texas Agric. Exper. Sta. Bull. 495,21pp,1934.

(I

14.

20
Fraps,G.S.,and Treichler,R.
“Vitamin A Content of Foods and Feeds".
Tex. Agr. Sta. Bull. 477,34pp,l938.

150 R106,P0Bo,and MWSOIl,H,E.

16.

“The Approximate Units of Vitamin A and Vitamin C
in Foods”. (N.Y. Assoc.for Improving the
Condition of the Poor.) 1931.

Taylor,M.,and Russell,W.
”The Stability of Carotene in Plant Tissues".
Jour. Nutrition_1§,l,l938.

 

 

 

 

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