HUMAN MILK STUDIES.

V. A QUANTITATIVE COMPARISON OF THE ANTIRICKETIC FACTOR IN HUMAN MILK AND COW’S MILK.*

BY JULIA OUTHOUSE, ICIE G. MACY, AND VIOLA BREKKE.

(From the Nutrition Research Laboratories of the Merrill-Palmer School and the Children’s Hospital of Michigan, Detroit.)

(Received for publication, March 17, 1928.)

The prevalence of rickets in babies, as shown by the recent investigation conducted by the United States Children’s Bureau (l), makes it of importance to know to what extent the milks generally recommended in infant feeding can justly be relied upon to supply the antiricketic factor. This is particularly pertinent when it is realized that rickets occurs at the period of development when milk constitutes the greater part, if not the whole, of the diet. It might be assumed that nature would provide an abundance of all essential growth-promoting factors in the pabulum for the young of all species; but clinical experience has demonstrated that under the present living conditions neither human milk nor cow’s milk can always be relied upon to protect babies against rickets.

With the advent of the study of experimental rickets in animals, various attempts have been made to evaluate the antiricketic potency of human milk and cow’s milk.

Steenbock and associates (2), using their ricketic ration, No. 2965, found that for cow’s milk the minimum daily requirement for healing of rickets in rats was 12 cc. Honeywell, Dutcher, and Dahle (3) corroborated these results, using the same ration in a preventive type of experiment. Lute (4), however, demonstrated that 2 to 5 cc. of milk from cows kept on pasture were potent in preventing rickets when fed to rats on the McCollum ration, No. 3143, whereas 15 to 20 cc. of milk were ineffective if the cow was kept in the dark on either dry or green food.

* A preliminary report of this investigation was presented before the Biological Chemistry Section of the American Chemical Society in Detroit, September, 1927.

129

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

Human Milk Studies. V

In studying human milk, Lesne and Vagliano (5) soncluded that this important nutrient contained no antiricketic properties. They fed, how- ever, a maximum of 5 per cent of the ether extract of breast milk to rats with the Sherman-Pappenheimer ricketogenic ration. Kennedy and Palmer (6), on the other hand, later demonstrated that a level of at least 8 per cent of human milk fat was necessary for the production of a distinct line test in rats made ricketic by the McCollum ration, No. 3143. Hess (7) has re- ported that whereas 25 cc. of human milk daily ordinarily failed to cure ricketic rats on the Sherman-Pappenheimer diet, this same amount could be made effective by treating the mother with ultra-violet light.

In a recent publication, Hess (8) has presented data on the antiricketic factor of these two milks when fed in a preventive type of experiment. He states that “when from 20 to 25 cc. (cow’s milk) were added to the standard ration, the development of rickets was prevented” and that “from 25 to 30 cc. of human milk had to be added to this ration to confer protection.”

It is impossible to explain the conflicting results of the studies reviewed above, since environmental factors surrounding the lactating individual and variations in experimental technique of feeding the milk play important rBles in the analysis of the results. The experiments report,ed here were started in March, 1926, for the purpose of making a more detailed comparison of the anti- ricketic properties of human milk1 and cow’s milk which have been produced under definite conditions and fed to rats with an experimental technique somewhat modified from the heretofore approved methods.

EXPERIMENTAL.

Technique.

It has been demonstrated repeatedly that in order to produce rickets in rats at least two conditions must exist; namely, the absence of the so called antiricketic factor, and the inclusion in the ration of a suitable salt mixture containing 4 or 5 parts of calcium to 1 part of phosphorus. It has likewise been shown (11-13) that the addition of more phosphorus to such rations results in the prevention or the arrest of the disease. Yet from a survey of the literature it would seem that this latter require- ment has not always been fully appreciated. Throughout the

1 The information obtained here on the antiricketic potency of human milk, together with the experiments published heretofore (9, lo), form the foundation for biological studies on the effect of diet upon the vitamin content of human milk.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

Outhouse, Macy, and Brekke 131

period of rickets development the ratio of Ca:P = 4 or 5 has been closely adhered to, but during the curative regime food substances rich in phosphorus or calcium have been superimposed on the ricketic diet without corresponding deductions being made in the salt composition of the basal ration. This correction is essential, particularly when large quantities of cow’s milk are fed. The addition of milk to a ricketogenic diet may result in an intake of calcium and phosphorus in a ratio that would tend to cause a healing of the bone lesion, even in the absence of the antiricketic factor. This criticism applies to any of the ricketogenic rations in general use. The primary consideration throughout this entire study, then, has been to maintain a ratio of Ca: P = 5 during the ricketogenic and curative periods, and to eliminate possible bone-calcifying agents other than the antiricketic.

Vigorous young rats, 21 to 23 days old, reared by mothers on the standard breeding ration2 were employed. They were placed in individual cages and on raised screens to prevent ingestion of feces and a consequent disturbance in the calcium: phosphorus ratio in the dietary. No attempt was made to keep the animals in absolute darkness; they were, however, placed in cages in a dark corner farthest from the windows, and at no time were exposed to the direct rays of the sun. The ricketic rations were fed ad libitum, accurate records of the weekly food intake being kept. Additions to the basal ration, i.e. milk or butter fat, were made daily in separate dishes. Fresh distilled water was available in the drinking bottles at all times except when the rats refused to drink the entire amount of milk allotted to them. The animals were held on the ricketogenic rations for a minimum period of time, to avoid the production of advanced pathological lesions. The occasional rat failing to grow on account of inadequate appetite has been discarded from the experiment.

For these experiments the Osborne, Mendel, and Park ration (14) was adopted, and somewhat modified to meet the require- ments of availability and low cost of food materials. The ad-

2 The breeding ration consisted of the Sherman diet of two-thirds whole wheat, one-third whole milk powder, and 1.3 per cent NaCl, together with daily supplements of fresh cabbage or lettuce, and fresh cow’s milk ad libitum.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

132 Human Milk Studies. V

vantage of this synthetic ration is that the mineral constituents3 are present in approximately the same proportion as in the Osborne and Mendel standard Salt Mixture IV with the exception of cal- cium and phosphorus, and that these two can be added separately in any amount and in any ratio desired. From the total calcium and phosphorus required, deduction has been made for the cal- cium and phosphorus content of the other constituents of the ration, and the balance added in the form of CaC03 and H3P04, previously mixed and dried. Butter fat was given daily so that the rats would not experience a shortage of vitamin A. It was assumed that approximately 1 per cent of butter fat would not prevent the production of rickets, since McCollum (15) found that only slight healing took place when 15 to 30 per cent was fed for 14 days. The modified ricketic ration has the following com- position:

HaPOd (85 per cent). . . . , . . 0.10 Salt Mixture xXx3.. . . . . . . 2.60

Calcium. . . . . . . . . . . 0.622 gm. “

Yeast.. . . . . . . . . . . . . . . . . . 6.00 Phosphorus.. . . . . . . . 0.125

Egg albumin4.. . . . . . . . . . . . 18 .OO Batio Ca.P =

5

Dextrin.................... 71.75) Butter fat.. . . . . . . . . . . 3 drops daily.

This standard ricketogenic ration has been used in studies only when the food substances under investigation contained but inappreciable amounts of calcium and phosphorus.

3 The authors desire to express their appreciation to Professors Osborne and Mendel for the privilege of using their ricketic salt mixture prior to its publication. The calcium- and phosphorus-free Salt Mixture XXX has the following composition:

MgCOs.. . . . . . . . . . . . . ;;80 Fe citrate-l+ HzO. . . . . :34 Na2C03.. . . . . . . . . . . . . . . 30.10 KI . . . . . . . . . . . . . . . . . . . . . 0.02 K&03.. . . . . . . . . . . 118.60 MnSOA.. . . . . . . . . . . . . . . . 0.079 HCl.. . . . . . . . . . . . . . 95.30 NaF . . . . . . . . . . . . . . . 0.248 H2SOd.. . . . . . . . . . . . 9.20 KzAlz(S01),. . . . . . . . . . 0.0245 Citric acid.HzO.. . . . . . . 30.60

In addition 622 mg. of calcium and 125 mg. of phosphorus were added to every 109 gm. of ration. These amounts have been found by Osborne and Mendel to be adequate for the production of rickets in rats.

4 Dried egg albumin was secured from T. M. Duche and Sons, 376-378 Greenwich Street, New York.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

TABL

E I.

Com

posit

ion

of

Ratio

ns

I an

d II

Fed

in

Conn

ectio

n wi

th

Diffe

rent

Am

ount

s oj

Hu

man

M

ilk

and

Cow’

s M

ilk.

Milk

.

Type

.

Hum

an.

Ca

per

cent

=

0.03

0.

P “

“ =

0.01

4.

Cow’

s. Ca

per

cent

=

0.11

0.

P “

“ =

0.09

7.

- _ 40

15

20

30

-

Perio

d.

Prem

ilk-fe

eding

. I

Milk

-feed

ing.

II

Prem

ilk-fe

eding

. I

Milk

-feed

ing.

II

Prem

ilk-fe

eding

. I

Milk

-feed

ing.

II

Prem

ilk-fe

eding

. I

Milk

-feed

ing.

II

Prem

ilk-fe

eding

. I

Milk

-feed

ing.

II

Cons

tituen

ts of

ratio

n. T

-l---4

- Pe

r P@

- Pe

r ce

nt

cent

ce

nt

1.90

0.

19

2.6

1.63

0.

00

2.6

-l-l-

2.30

0.

30

2.6

1.30

0.

00

2.6

--__

3.7

0.72

2.

6 2.

6 0.

00

2.6

----I-

5.

55

I 1.

27

I 2.

6 4.

00

0.00

2.

6 -l-

l---

6.50

4.

60

2. a.E!

PE

w Pe

r ce

nt 18

18

18

18

18

18

18

18

18

18

Per

cent

6 3 6 3

- _ _ _.

- -

6 3

. 3.

.$ *h J

1 t

*a

6 2”

-- ,“,“,‘

t dro

ps

71.1

3

74.8

0

-__ 70

.8

3 75

.1

0 ~-

69.0

3

73.8

0

-~ 66

.6

3 72

.4

0 -- 65

.4

3 71

.8

0

r

.-

.-

-

rota1

Ca

per

cent

0.77

0 0.

7701

0.92

5 0.

9251

1.49

1.

491

2.23

2.

23t

2.55

2.

55t

.- . _ ._

._

-

-- per

cent

“r

0.15

4 5

2 0.

154t

4-

5 .F

-- 0.18

5 5

5

0.18

5t

4-5

8 -_

_ P

0.29

8 5

L 0.

2981

4-

5 w

-- 0.44

5 5

3

0.44

51

45

-- 0.51

5

0.51

1 45

* Th

e dif

fere

nce

in

the

calci

um

and

phos

phor

us

cont

ent

of

the

25,3

0,

and

35 c

c.

leve

ls of

hu

man

m

ilk

was

so

sligh

t th

at

Ratio

ns

I an

d II

were

ca

lcula

ted

for

the

30 c

c.

serie

s an

d us

ed

in

the

25

and

35

cc.

serie

s as

wel

l. t

Appr

oxim

ately

. s w

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

134 Human Milk Studies. V

Variations of this standard ration have been used in succeeding studies in which different types and amounts of milk were fed. Each experiment was divided into a period of rickets development and a 7 day curative period of milk feeding. To avoid a marked change in the calcium and phosphorus intake of the rats during these two periods, it was necessary that for each proposed level of milk a separate ricketogenic ration should be prepared with an appropriate percentage of calcium and phosphorus. In addition to this, the basal food fed with milk during the curative period was so constructed that the sum total of the calcium and of the phosphorus eaten would still conform to the required ricketic ratio of 5:l. To this end, then, for every level of milk, two basal diets were prepared,5 Ration I, the ricketogenic ra- tion, and Ration II, fed only in connection with milk. These diets differed primarily in the percentage of calcium and phos- phorus actually incorporated in them (Table I). In the rations fed in connection with human milk, calcium and phosphorus were present in approximately optimal amounts; whereas the high phosphorus content of the cow’s milk necessitated the addition of abnormally large amounts of calcium to the ration.

During the milk feeding period, although theoretically calcu- lated to be constant and the same for all animals, the calcium and phosphorus ratio has varied somewhat on account of a differ- ence in the basal food intake of individual animals. In the analysis of the data secured in both the human milk and the cow’s milk series, therefore, all records showing an actual calcium and phosphorus intake not falling within the limits of the ricketic ratios of 5:l or 4:l have been discarded.

In the examination for ricketic changes the animals were

G The following method has been used in the calculation of these rations. Ration I (a) the calcium and phosphorus content of the proposed total milk intake (7 days) was determined; (b) from previous studies an estimation was made of the amount of basal food that would be eaten in connection with the different quantities of milk; (c) an equivalent percentage of phosphorus was incorporated in the ration; (d) calcium was added to the extent of 5 times the amount of phosphorus. Ration II (a) no phos- phorus was added, this being supplied largely in the milk; (b) the total calcium required in&Ration I minus the calcium supplied in the milk was added in the form of CaCO$. In both rations corrections were made for the content of calcium and phosphorus in the egg albumin and yeast.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

Outhouse, Macy, and Brekke 135

etherized and roentgenographed. Autopsies were performed immediately after, and notations were made on the condition of the costochondral junctions, the epiphyses of the long bones, and the thoracic and abdominal cavities. The bones of the hind legs were removed and both tibia were cut lengthwise, one-half of each being used at once for the silver nitrate “line test” of McCol-

Ration 0aCo3 - - - -- 1.55 per cent If@04 - - - - 0.10 3alt Mixture - 2.6 Yeast - - - - 6.0 Egg AlhInln - 18.0 Dextfin - - - 71.75 Butter fat - - 3 drops daily

/ 1 with Cod live? Oil / ~'- ' ( Ricketio Ration

RiCket IC Ration

CHART I. The standard ricketogenic ration. Composite growth curves and food intakes of rats on the standard ricketogenic ration with and without cod liver oil.

lum et al. (16) and the other portion preserved in formalin for histological examination. The femora were set aside for chemical analyses,6 determinations being made for each rat in order to study the individual variations of animals on the same ration.

6 Chemical analyses were made on the dry, fat-free femora, the muscle tissue having been previously removed by peeling the periosteum from the shaft. The bones, slit at each end, were dried to constant weight at loo”, and extracted first with hot 98 per cent alcohol for 24 hours, and then with anhydrous ether for the same length of time. They were then dried, weighed, and ashed in a muffle furnace at a temperature of 500-600”. The

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

Rat

No.

1028

1029

1030

1032

1033

TABL

E If.

Type

an

d De

gree

of

Bo

ne

Cond

ition

of

Rats

Fed

$1

Days

on

St

anda

rd

Rick

etic

Ratio

n wi

th

and

with

out

Cod

Liver

O

il.

d i R !lXU>

42

44

“ 42

49

“ 42

56

“ 42

43

“ 42

-

Cartil

age

band

.

Very

wi

de

and

irreg

- ul

ar.

Mod

erat

e wi

denin

g.

‘I ‘i

“ “

Very

wi

de

and

very

irr

egul

ar.

Line

test.

-

,

_-

I

-

3steo

id tis

sue.

Mod

erat

e am

ount

.

“ “

Smal

l am

ount

. “

I‘

Larg

e am

ount

.

-

( I /

.-

5 Te

ga-

tive.

Histo

logica

l ex

amina

tion.

Cartil

age

cells.

O&

aid

tissu

e.

Man

y pr

ong?

ex

tend

ing

into

m

eta-

ph

ysis

; ir-

re

gula

r ar

- ra

ngem

ent

“ ‘<

‘I I‘

“ “

6‘ “

Irreg

ular

fo

r, m

atio

n,

un,

calc

ified

.

“ “

I‘ “

Jnca

lcifi

ed

narro

w m

e-

taph

ysis

. rre

gula

r fo

r- m

atio

n,wi

dc

met

aphy

sis,

un

calc

ified

.

None

.

i-

X-ray

dia

g-

nosis

.

Seve

re

ricke

ts.

I‘ “

“ “

“ “

“ “

Bone

an

alysis

.

u”

PC

-- Per

Pm

cent

cen

t 0.

6 5.

C

- 4 ?i B - PW

cent

!8.1

$1.:

51.:

12.:

!8.:

-

1.8

5.2

25.7

1.6

5.:

23.7

2.2

5.:

28.2

0.3

4.7

27.3

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

1034

Aver

- ag

e..

1035

t-8

53

-4

10

36

1037

10

38

1039

10

40

Aver

- ag

e..

- -

L 10

-- - 10

--

- 9 15

17

17

13 8

-- - 13

-

-

521N

one.

l 42

IM

oder

ate

lSm

all

52 N

one.

42

M

oder

ate

Smal

l wi

denin

g.

wide

ning.

am

ount

. am

ount

.

---

--- 49

49

---

---

54

21

42

54

21

42

Narro

w,

Narro

w,

regu

- re

gu-

None

. No

ne.

lar.

lar.

54

21

42

54

21

42

“ “ I‘ I‘

“ “ 68

21

42

68

21

42

“ “

“ “ “ “

64

21

42

64

21

42

I‘ I‘ “ “

“ “ 54

21

42

54

21

42

“ “

“ “ “ “

44

21

42

44

21

42

“ “ “ “

“ “ ---

---

56

56

Nega

- tiv

e.

‘osi-

tiv

e.

‘I ‘I ‘I ‘I ‘I

Man

y pr

ongs

Irr

egul

ar

for-

exte

ndin

g m

atio

n,

un-

into

m

eta-

ca

lcifi

ed.

phys

is;

ir-

regu

lar

ar-

rang

emen

t.

Norm

al

ar-

None

. ra

ngem

ent.

I‘ ‘I

“ I‘

“ “

“ I‘

“ “

“ “

“ ‘I

“

None

.

Hea

vy

“ ‘I “ I‘ I‘

Seve

re

129.

8111

.21

5.

Seve

re

29.8

11.2

5.

ric

kets

. ric

kets

.

---

---

29.6

11.3

5.

29

.611

.3

5.

---

---

Appr

oach

- Ap

proa

ch-

41.2

15.

7 7.

41

.2 1

5.7

7.

ing

norm

al.

ing

norm

al.

‘I “

‘I “

37.5

14.

1 6.

37

.5 1

4.1

6.

“ “

“ “

39.2

14.7

6.

39

.214

.7

6.

“ ‘I

“ ‘I

40.6

15.3

7.

40

.615

.3

7.

“ “

“ “

43.6

16.9

7.

43

.616

.9

7.

“ “

“ “

39.3

14.3

6.

39

.314

.3

6.

---

---

40.2

15.

2 7.

40

.2 1

5.2

7.

29.6

27.9

14.7

14.5

16

.0

11.7

11

.6

15.5

14.0

-

* Th

is ra

tion

cont

ains

0.

622

per

cent

Ca

and

0.12

5 pe

r ce

nt

I’.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

138 Human IMilk Studies. V

Growth and Deportment of Rats on Ricketic Rations.

Each ricketogenic ration, No. I, irrespective of its calcium and phosphorus content, has allowed for continued growth and avid appetite in young rats. By the end of the 3rd week, however, the result of the deprivation of the antiricketic factor has manifested itself in a retarded rate of growth (see Chart I). Enlargement by the epiphyseal ends of the long bones has been markedly notice- able by the 10th day. Uncomplicated rickets has appeared as early as the 14th to the 21st day as judged by various methods in current use. Pathological changes, such as marked curving of the legs and spine, inability to stand, etc., have not been produced in this short experimental period. Xerophthalmia and respira- tory diseases have not been observed.

In Table II data are presented on the behavior of a typical group of rats receiving the standard ricketic ration with and with- out the addition of cod liver oil. The diagnosis of rickets has been confirmed by three different methods; i.e., histological, line test, and x-ray examinations. Additional evidence has been found in the percentages of calcium, phosphorus, and ash of the bones. These chemical analyses have been found to be of great value in determining the degree of calcification of bones, but should not be relied upon alone in the diagnosis of rickets in rats. As shown by Chick, Korenchevsky, and Roscoe (19), calcium and phosphorus determinations based on the ash content of bones are of no sig- nificance, these values remaining approximately constant, irre- spective of diet, sex, or age of the animal. Although complete metabolism studies were not possible, interesting data have been obtained on the ash content of feces? of rats on these dietaries. On the standard ricketic ration a fecal ash of approximately 28 per cent was excreted, whereas on the ricketic rations, containing four times as much calcium and phosphorus, 60 per cent ash was excreted.

weighed ash was dissolved in hydrochloric acid, transferred to volumetric flasks, neutralized with NH&H, and made up to volume. Aliquot portions were used for the determination of calcium by the Clark method (17) and for phosphorus according to the procedure of Fiske and Subbarow (18).

7 In the determination of fecal ash, the feces were extracted and ashed in the same manner as described for bone analysis.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

Outhouse, Macy, and Brekke 139

Human Milk and Cow’s Milk as Sources of the Antiricketic Factor.

Raw human milk was obtained fresh daily from the Detroit Bureau of Wet Nursing and was representative of the pooled milk from ten to sixteen women on the average American dietary. A survey indicated that these wet nurses had not taken codliver oil during the periods of lactation under investigation. Various samples of the milk contained, as an average, 30 mg. of Cal&m and 14 mg. of phosphorus per 100 cc. The fat content of the milk ranged between 2.7 and 3.5 per cent. The certified cow’s milk* was obtained from a herd of 450 animals fed a concentrate of grains, bone meal, and bran in addition to ensilage and alfalfa hay. The cows were kept in light, well ventilated stalls, but had no direct exposure to sunshine and little access to green pasture. The calcium and phosphorus contents were found to be respectively 100 and 97 mg. per 100 cc. The fat contents averaged 4 per cent.

As shown by the line test of the tibiae of the litter mate controls, the rats in the human milk series were ready for curative feeding after 21 days on the ricketogenic rations, No. I. At this time they were changed to rations, No. II, and given human milk in quantities of 25, 30, 35, and 40 cc. daily. Since rats do not eat human milk avidly, it was necessary in the latter series to con- dense the milk under partial vacuum to about 9 of the original volume. When this was done, the rats were able to consume all the milk given them. After 7 days, the animals were killed with ether and examined in detail. Pronounced beading of the ribs, enlargement of the joints, and softness of the bones were found. On treating the freshly cut tibiae with silver nitrate, it was demon- strated that no calcium phosphate had been deposited in the provisional zone of calcification at the base of the wide and irregu- lar bands of cartilage. This was in direct contrast to the picture presented by the animals receiving for the same length of time 5 drops of cod liver oil daily in addition to milk and Ration II. Here the cartilage band had become narrower and straighter and showed a heavy precipitation of calcium salts at its base; i.e., a positive line test. Analyses of the femora of rats receiving different amounts of milk showed no increase in the percentage of

8 The cow’s milk was obtained from the Walker-Gordon Laboratories of the Detroit Creamery Cornpan?.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

TABL

E III

.

Com

para

tive

Data

on

th

e An

tirick

etic

Prop

ertie

s of

Hum

an

and

Cow’

s M

ilk.*

I Lin

e tes

t. Cu

rativ

e pe

riod

(7

days

).

;i .bo $ 2 s g B

-- - w.

9 2 - 9 7

-

4 3

- -

- _-

3 a d s E & 8 3

ZI

- vm.

45

48

43

- 49

51

49

- 44

50

55

-

P x

- Pet

E ce

nt

4.5

Efl

F 4.

1 4.

4 z G

is

-

g 4.

8

4.6

-4

5.1

- 5.2

5.2

5.1

Tim

e on

at

ion.

dlVJS

35

28

28

21-2

1

21

21

21-2

1 21

21

10 9 10

11

10 9 11 9

-

30

29

- 34

31

- 30

24

-

25

25

30

30

- 40

40

-

Final

age.

Carti

lage

band

.

x-ra

y.

Calci

ficat

ion

3f

prov

ision

9 zo

ne.

Ash.

Ca

-- pet

per

cent

ce

nt

18.1

10

.1

17.7

9.

i 28

.2

9.E

-- 30.5

10.

8

29.7

10.

5 33

.5 I

1 .:

_-

32.8

11.f

32.9

Il.4

35

.3 1

0.:

l&2/

S

56

Wid

e,

irreg

ular

.

56

“ “

56

“ sl

ight

ly

ir-

regu

lar.

Neg

ativ

e.

‘I

Posi

tive.

42-4

9 W

ide,

irr

egul

ar.

49

“ “

49

“ re

gula

r.

Neg

ativ

e.

“ Po

sitiv

e.

4249

W

ide,

irr

egul

ar.

Neg

ativ

e.

49

“ “

I‘

49

“ re

gula

r. Po

sitiv

e.

Hum

an

milk

. R

icke

ts

seve

re,

ac-

tive.

“ “

Ric

kets

un

derg

oing

heal

ing.

Ric

kets

, m

oder

ate

to

seve

re.

“ “

Ric

kets

un

derg

oing

heal

ing.

Ric

kets

, m

oder

ate.

‘I

‘I

Ric

kets

un

derg

oing

heal

ing.

I

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

Cow’

s 32

2

27

53

Wid

e,

irreg

ular

. N

egat

ive.

R

icke

ts.

30.4

10.2

4.

8 m

ilk.

32

12

39

101

0 53

“

‘<

“ “

30.6

10

.6

4.Q

32

10

35

lO

i 5

53

Narro

w,

regu

lar.

Posi

tive.

No

rmal

bo

ne.

47.5

17

.3

8.5

__---

-~

-- ---

16

-28

8 43

37

49

Wid

e,

irreg

ular

. N

egat

ive.

30

.7

11.1

5.

0 16

-21

10

43

30

0 26

14

42

49

“ re

gula

r. Po

sitiv

e.

40.8

15

.1

7.1

16-2

1 8

41

30

5 26

8

4249

“

“ ‘I

39.8

15.

3 7.

3 2 “r

* R

esul

ts

pres

ente

d ar

e th

e av

erag

es

for

each

gro

up.

2 t

Milk

-fed

thro

ugho

ut

the

stud

y as

a p

roph

ylaxis

. .5

z ,Ei

Y E w”

2

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

142 Human Milk Studies. V

calcium, phosphorus, or total ash due to the addition of milk. The data in Table III show conclusively that there was no anti- ricketic factor present in 25,30, or 40 cc. of the human milk fed in these experiments.

On the high calcium and phosphorus ricke togenic rations of the cow’s milk series, it was found that rats could not be maintained in an unhealed ricketic condition beyond a period of 28 to 35 days. Since typical rickets was produced as early as the 14 th day, curative feeding was instituted at about this time. When 30 cc. of cow’s milk were given daily for a period of 7 days, very definite healing of the bone lesion was found in all cases (Table III). The degree of calcification appeared to be as great as that induced by cod liver oil for the same length of time. The antiricketic proper- ties of 5 cc. or of 10 cc., however, were so negligible that no bene- ficial effect was observed when these small amounts were fed daily to rats in a series of preventive experiments.g

In analyzing the results of these experiments it was observed that in some cases there was a lack of correlation of the x-ray and line test pictures with the ash content of the bones. The feeding of cod liver oil for 7 days to rats made ricketic on rations low in calcium and phosphorus, has resulted in the deposition of calcium phosphate at the epiphyseal line, as shown by the line test and x-ray pictures, without appreciably influencing the ash content of the bone. On the other hand, when calcium and phosphorus were present in large amounts in the food, the addition of cod liver oil for 7 days resulted not only in a positive line test but also in an increased percentage of ash in the femora. It is thus apparent that a level of food calcium and phosphorus of 0.622 and 0.125 per cent respectively, is too low to allow for the deposit,ion of calcium

g These experiments were conducted early in 1926 in cooperation with Dr. I. McQuarrie of the Henry Ford Hospital to determine whether or not the antiricketic potency of milk could be increased by irradiating cows with ultra-violet light. For this purpose four cows were kept in stalls under identical experimental conditions with the exception that while two of them had access to light only as it came through ordinary window glass, the other two were exposed to the rays of a quartz mercury vapor lamp at a distance of 36 inches for 1 hour daily. This was done for 3 months prior to starting feeding experiments with rats. In the small amounts of these milks fed (5 to 10 cc.) daily for 28 days to young rats on an appropriately constructed ricketic ration, no protection was afforded against rickets.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

Outhouse, Macy, and Brekke 143

salts in the shaft of the bone when cod liver oil is fed for a short period of 7 days. Whereas when 2.55 per cent of calcium and 0.51 per cent of phosphorus are present in the ration, cod liver oil feeding is attended by the deposition of calcium salts not only at the epiphyseal-diaphyseal junction but also in the shaft of the bone.

These experiments demonstrate that for rats human milk contains no demonstrable antiricketic factor, whereas cow’s milk does possess a rickets-healing substance. Since the Ca:P ratio of the dietary has been carefully controlled during the period of milk feeding, and since no appreciable change in the calcium and phosphorus level has occurred during the entire experimental period, it can be stated that the curative properties of cow’s milk are not due to the large percentage of calcium and phos- phorus present in the milk. It may be concluded, furthermore, that this factor in cow’s milk is analogous to the bone-calcifying properties of cod liver oil.

SUMMARY.

1. On a modified Osborne and Mendel synthetic ricketogenic ration, growth and appetite of rats have been satisfactory. Rickets has developed as early as 14 to 21 days.

2. By varying the calcium and phosphorus content of the ration, it has been possible to maintain an approximately constant ratio of Ca:P = 5 throughout the rickets-developing period and the curative period of milk feeding.

3. Data are presented which show that human milk fed in amounts of 25, 30, or 40 cc. daily contains no antiricketic factor.

4. Under the same carefully controlled conditions, 30 cc. of cow’s milk fed daily for 7 days induced marked healing of ricketic lesions in rats.

The authors wish to express their appreciation of the interest and cooperation of Dr. Lawrence Reynolds, director of the X-ray Department, at the Children’s Hospital of Michigan, who has made the roentgenological diagnoses for this study.

BIBLIOGRAPHY.

1. Eliot, M. M., J. Am. Med. Assn., 1925, lxxxv, 656. 2. Steenbock, H., Hart, E. B., Hoppert, C. A., and Black, A., J. Biol.

Chem., 1925, Ixvi, 4.41.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

144 Human Milk Studies. V

3. Honeywell, H. E., Dutcher, R. A., and Dahle, C. D., J. Biol. Chem., 1927, lxxiv, p. Ixxvii.

4. Lute, E., Biochem. J., 1924, xviii, 716. 5. Lesne, E., and Vagliano, Compt. rend. Sot. biol., 1924, xci, 143. 6. Kennedy, C., and Palmer, L. S., Proc. Sot. Ezp. Biol. and Med., 1925,

xxiii, 236. 7. Hess, A. F., Weinstock, M., and Sherman, E., J. Am. Med. Assn.,

1927, lxxxviii, 24. 8. Hess, A. F., and Weinstock, M., Am. J. Dis. .Child., 1927, xxxiv, 845. 9. Macy, I. G., Outhouse, J., Graham, A., andLong, M. L., J. Biol. Chem.,

1927, lxxiii, 175. 10. Macy, I. G., Outhouse, J., Graham, A., andLong, M. L., J. Biol. Chem.,

1927, lxxiii, 189. 11. Sherman, H. C., and Pappenheimer, A. M., Proc. Sot. Exp. Biol. and

Med., 1921, xviii, 193. 12. Shipley, P. G., Park, E. A., McCollum, E. V., and Simmonds, N.,

Proc. Sot. Exp. BioZ. and Med., 1921, xviii, 277. 13. Karelitz, S., and Shohl, A. T., J. BioZ. Chem., 1927, Ixxiii, 665. 14. Osborne, T. B., Mendel, L. B., and Park, E. A., Proc. Sot. Exp. Biol.

and Med., 1923-24, xxi, 87. 15. McCollum, E. V., and Simmonds, N., The newer knowledge of nutrition,

New York, 3rd edition, 1925, 404. 16. McCollum, E. V., Simmonds, N., Shipley, P. G., and Park, E. A.,

J. BioZ. Chem., 1922, li, 41. 17. Clark, G. W., J. BioZ. Chem., 1921, xlix, 487. 18. Fiske, C. H., and Subbarow, Y., J. BioZ. Chem., 1925, Ixvi, 375. 19. Chick, H., Korenchevsky, V., and Roscoe, M. H., Biochem. J., 1926,

xx, 622.

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from

BrekkeJulia Outhouse, Icie G. Macy and ViolaHUMAN MILK AND COW'S MILKTHE ANTIRICKETIC FACTOR IN

QUANTITATIVE COMPARISON OF HUMAN MILK STUDIES: V. A

1928, 78:129-144.J. Biol. Chem. 

  http://www.jbc.org/content/78/1/129.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  ml#ref-list-1

http://www.jbc.org/content/78/1/129.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be

by guest on July 8, 2020http://w

ww

.jbc.org/D

ownloaded from