the vitamin a, b(b1) and g(b2) contents of milk throughout the year

TH E VITAMIN A, B(B1) AND G(Bf) CONTENTS OF MILK THROUGHOUT THE YEAR 1

FLORENCE L. MAcLEOD, J E S S I E B. BRODIE AND E M I L Y R. MACLOON

The vitamin value of milk has frequently been shown to vary with the feed which the cow is receiving. Kennedy and Dutcher (1) in 1922 concluded from experiments on both stall- and pasture-fed cows that stall-fed cows will produce a milk rich in vitamins A and B provided their ration consists of a proper combination of grains and leafy foods.

Drummond and his associates (2), Luee (3), Chick and Roscoe (4), and Golding, Soames and Zilva (5), have shown that the vitamin A content of milk is raised by the feeding of cod-liver oil, fresh pasture or kale while the vitamin D content is higher when the cows are on pasture in the sun or when receiving a comparatively large quantity of cod-liver oil.

Milk has been found to be relatively richer in vitamin G(Bf) than in vitamin B(B1) by Sherman and Axtmayer (6), Hunt and Krauss (7) and Outhouse, Macy, Brekke and Graham (8).

Bechdel, Honeywell, Dutcher and Knutsen (9) reported experiments in 1928 which showed that the vitamin B complex may be synthesized by bac- teria in the rumen of the cow and, therefore, that the amount in the milk is not always dependent on the amount in the ration.

Our purpose in the experiments to be reported here, was to determine the vitamin A, B (B 1) and G (B~) values of milk from one dairy of stall-fed cows at all times of the year. In previous work from this laboratory (10) it has been shown that the vitamin C value of milk from the same dairy is high and remains constant throughout the year. The cows which furnished the milk received a ration containing an alfalfa hay of excellent quality, a good ensilage, and a concentrate.

VITAMIN A CONTENT

Series I

The first series of experiments on the vitamin A value of the milk was carried out during the year July, 1926, to July, 1927, using the method de- scribed by Sherman and 1Yiunsell (11). The diet (laboratory No. 379A) devoid of vitamins A and D but adequate in other respects had the following composition :--purified casein, 18 per cent; cornstarch, 67 per cent; dried brewery yeast, 10 per cent; Osborne and Mendel salt mixture, 4 per

Received for publicafton Msby 14, 1931. 1 This research wa~ aided by a ~rant by the Wal]~er-Gordon Laboratory Company. Contribution 1No. 681 from the DCl~art~ment of Chemistry, Columbia University,

New York City.

14

VITAMIN VALUE OF MILK THROUGHOUT THE YEAR 15

cen t ; a n d sod ium chlor ide, one p e r cent. I n th is ser ies the p r e - e x p e r i m e n -

ta l s tore of v i t a m i n D was d e p e n d e d u p o n to c a r r y t he an ima l s t h r o u g h

both t h e dep le t ion p e r i o d a n d the e x p e r i m e n t a l p e r i o d of c losely r e s t r i c t e d

growth .

Rats , 28 to 29 days old, which had been ra i sed on D i e t B of th is labora-

tory, con t a in ing whole mi lk powder one- th i rd by w e i g h t and whole whea t

two- th i rds by weight , were p laced on the die t descr ibed above and were con-

t i n u e d on i t un t i l t h e y ceased to grow. The an imal s w e r e then p laced in

sepa ra te cages wi th ra i sed screen bot toms and were g iven m e a s u r e d

a m o u n t s of milk. One a n i m a l f r o m eve ry l i t t e r was con t inued on t h e basal

diet w i th no mi lk as a n e g a t i v e control .

The an imal s of this series were fed amoun t s of milk, v a r y i n g in q u a n t i t y

f r o m 0.25 ee. to 1.0 ec. p e r day, six days in the week.

I t can be seen f r o m Table I t h a t a t all seasons of the yea r a u n i t ga in

in we igh t (a ga in of a p p r o x i m a t e l y 3 g rams pe r week fo r the e x p e r i m e n t a l

TABLE 1 Growth o f rats f ed a V i t a m i n A- f ree diet (Die t 379A) only or this diet plus graded

amounts o f m i l k at d i f f eren t sea~ons o f the year

A M O U N T O F SEASON M I L K FED N U M E E R O F AV. GAIN I N 8 ' R E M A R K S

PER DAY A N I M A L S W K S . AND P. E.

Summer ........... Fall ..................... Winter ............... Spring ...............

Summer ...........

Summer ............ ~'all ..................... Winter ............... Spring ..............

Summer ........... Fall ..................... Winter ............... Spring ...............

Fall ..................... Winter ...............

ec .

0 0 0 0

0.25

0.5 0.5 0.5 0.5

0.75 0.75 0.75 0.75

1.0 1.0

10 9

15 8

15

11 14 15 11

16 20 16 4

20 5

g r a m s .

25.2 _+ 1.6 26.3 ___ 1~2 27.2 +__ 1.7 27.4 + 2.2

34.6 ___ 2.1 33.2 _ 1.3 33.8 ___ 1.2 33.2 -+- 0.9

42.3 _ 1.3 38.4 _ 1.0

Av. survival 28.9 days Av. survival 25.8 days Av. survival 31.0 days Av. survival 28.3 days

Av. survival 39.7 days

Killed end exp. period Killed end exp. period Killed end exp. period Killed end exp. period

Killed end exp. period Killed end exp. period Killed end exp. period Killed end exp. period

Killed end exp. period Killed end exp. period

pe r iod ) was ob ta ined when 0.5 ee. of mi lk pe r day was f ed s ix days in the

week. No seasonal v a r i a t i o n in the v i t a m i n A e o n m n t of t he mi lk was

found , the gains fo r the e igh t week per iods d u r i n g the summer , fal l , w i n t e r

and s p r i n g being 25.2, 26.3, 27.2 a n d 27,4 grams, respec t ive ly .

16 F . L . MACLEOD~ J . B. BRODIE AND E. R. MACLOON

On autopsy thirty-five per cent of the animals receiving 0.5 cc. of milk showed signs of vitamin A deficiency such as pus in the tongue and sub- maxillary glands while 16 per cent receiving 0.75 cc. and 8 per cent receiving 1.0 cc. showed these signs.

Series I I

During 1928 to 1929 much of the work was repeated providing vitamin D in the basal diet in the form of irradiated commercial cholesterol. Half of the animals in each litter used were given the original basal diet while the other half received vitamin D in addition. No differences were found in the gains made in the experimental periods by the animals on the two diets. In this series, a unit gain in weight was made when 0.75 cc. of milk per day was fed; the fourteen animals which received an extra source of vitamin D gaining 25.5 grams and the same number receiving the basal diet without vitamin D, 24.1 grams. The milk, therefore, was a slightly poorer source of vitamin A in 1928 to 1929 than in 1926 to 1927.

From all the results obtained on the vitamin A value of the milk in both series it can be definitely concluded that animals receiving 0.5 to 0.75 cc. per day of the milk six days a week wil] gain about 3 grams a week for the eight weeks' experimental period at any time of the year. The milk contains, therefore, at least 600 units of vitamin A per pound or 1.3 to 2 units per gram. Dutcher, Ho~neywell and Dahle (13) found that 1 cc. of raw milk per day, as the only source of vitamin A, produced a gain of 3 grams a week in rats. The cows furnishing the milk received very little fresh pasture. Outhouse, Macy and co-workers (8) found, when feeding milk similar to that used in these experiments, that 3 cc. per day were required to give approximately normal growth. Supplee and Dow (14) reported that 1.65 cc. of milk per day gave a growth in rats of "2.16 to 3.75 grams per week," the first determination being made in' January and the second in April. The milk examined in the experiments reported here is, therefore, a.rich as well as a uniform source of vitamin A.

VITAMIN B~l ) CONTENT

The method here used for the measurement of the vitamin B (B1) values was that worked out by Chase (15). The experimental diet (laboratory No. 513) recommended by Chase (15) consisted of :--purified casein, 18 per cent; Osborne and Mendel salt mixture, 4 per cent; butter fat, 8 per cent; cod liver oil, 2 per cent; cornstarch, 53 per cent; and autoclaved ba- ker's yeast, 15 per cent: 2 Litters of young rats which had been raised to 28 or 29 days of age ~ Diet B of this laboratory ad libitum plus raw beef

2 T h i s ~ ' b a k e r ' s ~ yea s t was o f re la t iveiy low in i t ia l v i t amin B con ten t and was au toc laved for 6 hours a t 15 p o u n d s p re s su re a n d 120°C., in the presence o f 0.1 !~I p o t a s s i u m hydroxide .

V I T A M I N VALUE OF M I L K T H R O U G H O U T T H E YEAR 17

at a level of 30 grams per animal per week were given the diet devoid of v i tamin B and were continued on this diet unt i l they were s ta t ionary in weight. The animals were then placed in individual cages and fed milk in graded amounts. One animal f rom each l i t ter was continued on the basal diet as a negat ive control.

The quanti t ies of milk fed were 4, 8, 10 and 12 cc. per day, six days per week, with three to seven animals on each level dur ing every season. The 4, 8 and 12 cc. levels were fed at all seasons o~f the year, the 10 cc. level only in the winter and spring.

The var ia t ion in the gains made dur ing the year by animals roceiving the same quant i ty of milk is not grea t and it is a question whether the differences found f rom season to season are significant. The animals receiving 4 cc. of milk per day lost more weight in the summer than at any other t ime of the year. I n the fal l and winter, however, there were six cases of polyneur i t i s and three deaths among the animals recMving 4 cc. of milk. I n the spr ing none of the animals showed any signs of polyneur i t i s and their average loss in weight dur ing the 8-week period was only 5.5 grams as compared with losses of 18.7, 12.5 and 11.4 grams in the summer, fal l and winter, respectively. Judg ing f rom these results it might be concluded that the milk was richest in v i tamin B in the spring.

The average gains made by animals receiving 8 cc. of milk were 16.5, 29.4, 29.3 and 33.2 grams for the summer, fail, winter and spring, respectively. This rate of gain was found by Chase (15) to be most sa t i s fac tory in test ing for v i tamin B. The results obtained in the summer showed very li t t le var ia t ion and were definitely poorer than those obtained for the other seasons. The gains made by the animals in the spr ing were, sl ightly bet ter than those made in the fal l or winter.

The average gains made by the animals receiving 10 cc. of milk per day in the winter and spring were 39.7 and 46.5 grams, respectively, a s l ight ly larger gain, therefore, being made in the spring.

The animals receiving 12 cc. of milk per day made average gains of 51.7, 47.0, 58.2 and 55.5 grams in the summer, fall, winter and spring, respectively. A t this level of feeding there is a tendency for the milk to show an impro.vement in its v i tamin B content in the winter as well as in the spring.

There was li t t le var ia t ion in the controls, receiving the basal diet only, f rom season to season, the average lengths of survival for the summer, fall, winter and spr ing being 22.0, 21.2, 25.8 and 20.3 days, respectively.

In figure 1 the average results for the entire year are given. When the average results for the year are considered i t is found tha t

a uni t gain in weight, a gain of about 3 g rams a week fo.r the 8 weeks ' exper imenta l period, was secured whe~ 8 cc. of milk per day° 6 days in the

18 F. L. :I~ACLEOD~ J . B. BRODIE AND E. R. MACLOON

/ I ~ - (/z/eots)

~ 2o , e r a ts)

~ - / 0

- 3 0 ' " ' , t 0 co. ( 1 6 / e a t s ) "~ 74 Rats)

- 4 0 /0 20 3 0 4 0 5"0 6 0 70

D A Y S

Fro. 1. VITAMIN B (B~) CONTENT OF MILK

Average g a i n curves of r a t s f ed mi lk as sole source of v i t a m i n B d u r i n g the year Ju ly , 1929, to Ju ly , 1930. A m o u n t o f milk f ed per day is shown a t end o f each curve. Broken fine ind ica tes dea th of one or more ra t s .

week were fed. Slightly more milk than this would probably have been required in the summer and slightly less in the spring. Ten to fifteen times as much milk is required to give a uni t gain in vitamin B experiments as in vi tamin A experiments. Milk has, therefore, about 0.1 uni t of vitamin B(B1) per gram, or about 55 units per pound.

VITAMIN G(B2) CONTENT

The experiments on the vitamin G(B2) and vitamin B(B1) values of the milk were carried out simultaneously dur ing the year July, 1929, to

VITAlS[IN VALUE OF M I L K T H R O U G H O U T T H E YEAR 19

Ju ly , 1930. The method used for the determinat ion of v i tamin G ( B f ) was worked out in this labora tory by Bourquin (16). Rats, 28 ov 29 days of age, which had been raised on Diet B of this l abora to ry ad libitum, plus fresh raw beef to the extent of 30 grams per ra t per week were fed the following diet ( labora tory No. 554) : - -pur i f ied casein, 18 per cent ; Osborne and Mendel salt. mixture, 4 per cent ; bu t te r fat , 8 per cent; cod-liver oil, 2 per cent, and corn starch, 68 per cent, pa r t of which carr ied the 80 per cent alcoholic extract f rom 5(] grams of wheat for each 100 grams of diet. The animals were kept on this diet without any addit ional source of v i tamin G unt i l they were constant in weight. They were then placed in individual cages and fed milk in graded amounts in addit ion to the basal diet. One animal f rom each l i t ter was continued on the basal diet as a negative control.

The quanti t ies of milk fed were 1, 2, 4 and 6 cc. per day, six days a week. The 6 cc. level was not given in the summer but was fed in the other s e a s o n s .

The v i tamin G(Bf ) content of the milk was found to va ry litt le f rom season to season. The results obtained on the 2 cc. level in the summer were lower than those found for this level of feeding in the other seasons. The gains made by the animals receiving 1 cc. of milk per day were 4.3, 2.8, 7.8 and 10.0 grams for the summer, fall, winter and spring, respectively, and for the animals receiving 2 cc. the gains were 8.6, 18.6, 22.7 and 21.6 grams for the same seasons, respectively.

The gains made by the animals receiving 4 cc. of milk were of the order which has been found most sa t i s fac tory for determinat ions of this type. The animals receiving this level in the summer, fall, winter and spring gained 34.8, 37.0, 35.7 and 46.5 grams, respectively. The increase in the average gain made by these animals in the spr ing is p robably not significant, for at this same t ime the controls, receiving the basal diet only with no added source of v i tamin G(Bf ) made slight gains in weight dur ing the exper imenta l period. The negat ive control animals lost 10.0, 12.2 and 9.8 grams dur ing the summer, fal l and winter, respectively, and gained in the spr ing 6.2 grams.

In figllre 2 the average results for the entire year are given. These resul ts are near ly identical wi th those of Bourquin (16) who fed

equivalent amounts of skimmed milk powder. I n both sets of experiments, it was found tha t the gains made by the animals increase approx imate ly in

propor t ion to the levels of milk fed. In order to obtain a gain in weight of about 25 grams in the eight weeks '

exper imenta l period, i t would p robab ly be necessary to feed about 3 cc. of milk per day, or 4 to 6 t i m ~ as much as it required to l~oduce the same

20 F. L. MACLEOD~ J . B. BRODIE &ND E. R. MACLOON

6 0

co ,,40

'~ 4 0

t 3 0

I,. :~ 2 0

o

- / 0

-20 /0 20 30 4O SO aO 7O

DAY'S FIG. ~. VITAMIN G (B,) CONTENT OF MILK

Average ga in curves of r a t s fed milk as sole source of v i tamin G during the year July, 1929, to July , 1930. Amount of milk fed per day is shown a t end of each curve. Broken line indicates death of one or more animals.

gain in weight in vitamin A experiments. Milk, therefore, contains about 150 units of vitamin G(B,2) per pound, or 0.3 unit per gram, and is, therefore, a good source of this vitamin.

SUMMARY AND CONCLUSIONS

The vitamin A, B(Bi) and G(B2) values of raw milk have been deter- mined at all seasons of the year.

I t was found that the vitamin A value of the milk is comparatively constant from season to season and that 0.5 to 0.75 cc. of the milk per day fed six days in the week will give a unit gain in weight (a growth of 3 grams per week throughout the eight weeks' experimental period) at any time in the year. The milk contains, therefore, about 1.3 to 2 units of vitamin A per gram.

When 8 cc. of milk per day were fed 6 days in the week, as the only source of vitamin B(B1), an average gain in weight of 3 grams a week for the experimental period (unit .gain) was secured. The milk contains, therefore, about 0.1 unit of vitamin B (B1) per gram.

V I T A ] ~ I N V A L U E OF ~ I I L K T H R O U G H O U T T H E Y E A R 21

T h e v i t a m i n G ( B 2 ) v a l u e of t h e m i l k v a r i e d l i t t l e f r o m season to season.

W h e n 2 cc. of m i l k p e r d a y w e r e f e d as t h e o n l y s o u r c e o f v i t a m i n G ( B 2 )

t h e r e w a s a n a v e r a g e g a i n f o r t h e e i g h t w e e k s ' e x p e r i m e n t a l p e r i o d of 17.8

g r a m s a n d w h e n 4 cc. w e r e f e d t h e r e was a n a v e r a g e g a i n o f 38.5 g r a m s .

T o s e c u r e a u n i t g a i n i n w e i g h t a p p r o x i m a t e l y 3 cc. p e r d a y w o u l d h a v e

to be f e d . T h e m i l k c o n t a i n s , t h e r e f o r e , a b o u t 0.3 u n i t o f v i t a m i n G

p e r g r a m .

T h e r e s u l t s r e p o r t e d here , t o g e t h e r w i t h t hose p u b l i s h e d i n a n e a r l i e r

p a p e r (10 ) , i n d i c a t e t h a t a m i l k w h i c h is p r o d u c e d u n d e r e x c e l l e n t cond i -

t i ons o f s t a l l - f e e d i n g is a u n i f o r m a n d g o o d s o u r c e of v i t a m i n s A , B (B1) ,

C a n d G ( B 2 ) .

T h e a u t h o r s a r e i n d e b t e d to P r o f e s s o r I t . C. S h e r m a n f o r t h e a d v i c e

g i v e n b y h i m d u r i n g t h e c o u r s e of t h i s w o r k .

REFERENCES

(1) KENNEDY, C., AND DUTCHER, :R. A. The influence of the diet of the cow upon the quantity of vitamins A and B in the milk. J. Biol. Chem. 50: 339-359. 1922.

(2) DRUMMOND, J . C., COWARD, K. H. AND WATSON, A . F . Notes on the factors influ- encing the value of milk and butter as sources of vitamin A._ Biochem. J. 15: 540-552. 1921.

DRUMMOND, J. C.~ COWARD, K. H., GOI.,DING, J . MACKINTOSH, 5. AND ZILVA, S. S. Cod-liver oil in the winter feeding of milch cows. J . Agr. Sei. 13: 144-152. 1923.

(3) LUCE, E. /~. The influence of diet and sunlight upoa the growth-promoting and antirachitic properties of the milk afforded by a cow. Biochem. J . 18: 716-739. 1924.

LucE, E . M . Further observations on the influence of sunlight upon the growth- promoting and antirachitie properties of cow's milk. Biochem. J. 18: 1279- 1288. 1924.

(4) CHICK, ]-I., .~-~n ROSCOE, M. It . On the composite nature of the water-soinble B vitamin. Biochem. J . 21: 698-711. 1926.

(5) GOLDIN( 4 J., SOAMES, K. AND ZIhVA, S. S. The influence of the cow's diet on the fat-soluble vitamins of winter milk. Biochem. J. 20: 1306-1319. 1926.

(6) SHERMAN', H. C., AND AXTMAYER, 5. H. A quantitative study of the problem of the multiple nature of vitamin B. J. Biol. Chem. 75: 207-212. 1927.

(7) H~NT, C. H., A N~ K~AUSS, W. E. The relative antineurltic and anti-pellagric potency of cow's milk. J . Biol. Chem. 79: 733-738. 1928.

(8) OUTIIOUSI~, J., MAC~, I. G., BREKKE, "V., AND GRAHAM, A. A note on the vitamin A and B content of cow's milk. J. Biol. Chem. 73: 203-208. 1927.

(9) BECHDEL, S. L., HONEYWELL~ H. E., DUTCIIEI~, l~. A., AND KNUTSEN, M. H. Synthesis of vitamin B in the rumen of the cow. J . Biol. Chem. 80: 231-238. 1928.

(10) MACLEoo, F . L . Antiscorbutic vitamin content of milk of stall-fed cows throughout a year. J. A. M. A. 88: 1947-1949. 1927.

(11) SH~a~MA~, H. C., A~D MVNSSLI, H . E . The quantitative determination of vitamin A. J . Am. Chem. Soc. 47: 1639-164~. 1925.

22 F. L. MACLEOD~ J. B. BRODIE AND E. R. 1VIACLOON

(12) SHER~AI~, H. C., AND BU~IS, M. P. Factors affecting the accuracy of the quanti- tat ive determination of vitamin A. J . Biol. Chem. 78: 671-680. 1928.

(13) DUTCHF~, R. A., HON~'W~.L, H. E., AND D-~I~E, C.D. Vitamin A in evaporated milks made by vacuum and aeration methods. J . Biol. Chem. 75: 85-94. 1927.

(14) SUPPL~E, G. C., A~-~ DOw, O.D. Vitamin A potency of irradiated milk. J. Biol. Chem. 75: 227-239. 1927.

(15) CHASE, E. F. A quant i ta t ive study of the determination of the antineurit ic vi tami~ ( F or B1). Dissertation, Columbia University. 1928.

(16) BOURQUI~, A. Experiments on the quant i ta t ive determination of vi tamin G. Dis- sertation, Columbia University. 1929.

the vitamin a, b(b1) and g(b2) contents of milk throughout the year

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