THE COMPARATIVE COMPOSITION OF HUMAN MILK AND OF COW’S MILK.

BY EDWARD B. MElGS AND HOWARD L. MARSH.

(From the Robert Hare Ch.emical Laboratory of the University oj Pennsyl- vania and the Wistar Institute of Anatomy and Biology.)

(Received for publication. August 30, 1913.)

INTRODUCTIOR’.

The following article is an account of work done by Arthur V. Meigs, Howard L. Marsh, William H. Welker and W. L. Croll on the chemical analysis of human milk and of cow’s milk. The work was carried out in the Robert Hare Chemical Laboratory of the University of Pennsylvania and was to a large extent super- vised by John Marshall, the Director of that laboratory. The present account has been written by Edward B. Meigs, in collabo- ration with Howard L. Marsh, after the death of Arthur V. Meigs, on January 1, 1912.

The subject of milk analysis was taken up by Arthur V. M&gs more than thirty years ago with the idea of discovering how cow’s milk should be modified in order to make a proper food for very young infants. On the basis of analyses made in 1881- 1884, Meigs devised a food which he afterward used in his prac- tice with great success. He published an account of his early investigations in book form in 1885,l and has since published a number of smaller articles on the subject2 In 1908 he again began chemical work on the subject which he continued until his death in 1912. A short preliminary account of some of the results of this later work appeared in 1911;3 and since that time

1 Arthur V. Meigs: Milk Analysis and infant Feeding, Philadelphia, 1885.

2Arthur V. Meigs: Transactions of the College of Physicians of Phila- delphia, Third Series, viii, p. 139, 1885; Ibid., xxiv, p. 136, 1902; Archives of Pediatrics, December, 1889; Feeding in Early Infancy, Philadelphia, 1896.

3 Arthur V. Meigs and Howard L. Marsh: The Medical Record, December 30, 1911.

147

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148 Composition of Milk

the authors of this article have been endeavoring to coordinate the other results and to prepare them for publication.

LMeigs’ work on milk in 1881 consisted in an attempt to com- pare human milk and cow’s milk in their content of protein (or “ casein”), fat, lactose, water and salts. The chief outcome of the work was the conclusion that human milk contains less than half as much protein as cow’s milk and about 50 per cent more lactose. In other respects Meigs’ results were not in sharp dis- agreement with those of his predecessors, but his figures for pro- tein and lactose were quite different from those which were USU-

TABLE I.

The average composition of human milk and cow’s milk according to Meigs’ early analyses, and those of certain well-knotisn authors whose results were available in 1881. The various constituents are given as percentages of the whole milk.

w*TER PROTEIN FAT j IACTOSE ABH .4UTHOR m-7. -

I~ mdk i milk .Human Cow’s :Human C;$s Hz,” Cm”;; ‘H,urnan C;w$ Hz,? Cw& 1 mrlk

____~,_ -- -:-. ~

Lehmann*. j I 3.5 4.5 : ‘4 to 6 Gorup- 1

Besanezt.,88.908,84.28 ’ 3.924 4.351 2.6G6 6.47 4.364 4.34 0.138 0.63 I ’

Meigsi . . . . . . 87.163~87.780~ 1.046 3.022, 4.2531 3.7591 7.407 4,949, 0.101 0.488

* Lehmann: Physiological Chemistry, Cavendlsh Society translation, London, 1831, vol. 1, p. 383, vol. ii, p. 341.

t Gorup-Besanez: Lehrbuch der physioloyischen Chemie, Brsunschwelg, 1878, pp. 421 and 424: the figures for human milk are those quoted from Verno~s and Becquerel. and are averages from 89 analyses.

t Meigs: Milk Analysis and Infant Feeding, Phlladelphla, 1885. pp. 34 and 36.

ally given at that time. A comparison of Meigs’ figures with some of the best known figures which were available in 1881 is given in Table I.

Meigs was of course familiar with later analyses which had not at that time been quoted in text-books. In these analyses the protein of human milk was variously given at from 0.215 to

4 The word “casein” was, at that time, very generally used to designate all the proteins of milk; it now means, as is well known, a particular pro- tein body which constitutes about 80 per cent of the total protein of cow’s milk and a somewhat less proportion of the protein of human milk. In the subsequent discussion the words “casein” and ‘Lprotein” will be used in their modern senses.

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Edward B. Meigs and Howard L. Marsh I49

over 7 per cent, while the figures for lactose ranged from 1.921 to 8.805 per cent.

In the milk analyses which have been published since 1881, the figures given to represent the percentage of protein and lac- tose in cow’s milk have not varied widely from those published by Gorup-Besanez. But the percentages of these constituents in human milk have been quite variously given, as Table II shows; in the more modern analyses the results are quite near to those reached by Meigs in 1881.

It is generally agreed among physiological chemists at present that the fat, lactose, ash and total nitrogen of milk can be quan- titatively determined satisfactorily. The difficulties of milk anal-

TABLE II.

The average composition of human milk according to analyses published since 1881. The constituents are given as percentages of the whole milk.

) PROTEIN I WATER FAT i ‘*LOSE ASII i- ---!

Munk and UffeImann*. 89.2 j 2.1 3.4 j 5.0 0.2 Kiinigt 87.4 2.3 3.8 6.2 0.3 Heubnerj.. . . 1.03 4.07 ~ 7.03 0.21 Camerer and Siildner$ . 1.27 3.91 ’ 6.52 0.22

* Munk and Uffelmann: Em(ihrung des gesunden und kranken Menschen: Wien and Liepsig,

1887, p. 269.

t Quoted by Camerer and Siildner: Zeitscb. f. Bid., xxxiii, 18913, p. 43.

: Heubner: Bed klin. Wochenschr., 1894. Nos. 37 and 38.

5 Camerer and S6ldner: Zeitschr. j’. Bid., rxxvi, Table II, pp. 280 and 281, 1808. The fiwres

are average figures for milk between the 20th and 40th day post partum. The figure for protein

is obtained by multiplying the figure for total nitrogen glen by the authors by the factor 6.25.

ysis lie in the determination of the protein and of the unknown constituents. Meigs and his collaborators have, in the first place, determined the quantities of fat, lactose, ash and total nitrogen in a number of samples of human milk and cow’s milk; they then attacked the question of the protein and unknown constituents. It will be convenient to divide this article into two parts, in the first of which will be given the results on the easily determinable constituents of milk; and in the second, the work on the protein and unknown substances; this latter work was still in a very fragmentary state at the time of Meigs’ death. It will be well to begin with a description of the samples of milk used in the various studies.

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Composition of Milk

Each of the samples of human milk numbered 1, 2, 4, 6, 7, 8 and 10 was made up of milk from several women; each of the other samptes was com- posed of the milk from one woman. The composite samples of human milk came from women in maternity hospitals, and all were taken between the fourth and ninth days after delivery. The other samples of human milk may be described as follows:

No. 3. Colostrum-collected up to and including the fourth day after delivery; maternity hospital patient.

No. 5. Milk collected near the end of lactation, eighteen months or more post partum; woman in moderate circumstances. The flow of milk had almost ceased so that only very little could be obtained.

No. 9. Milk collected during the fifth month after delivery; woman in good circumstances.

The cow’s milk came part.ly from thoroughbred Guernsey cows and partly from grade cows; samples 1, 9, 13 and 20 are the former, the others, the latter. Each of the samples of cows’ milk except Nos. 1, 9, 13 and 20 was made up of milk from several cows, and the periods of lactation have not been accurately determined. All the samples may, however, be taken to represent milk from the middle period of lactation, from two to six months after delivery.

In preparing the work for publication, the data obtained from a few samples of milk have been left entirely out of consideration, either because they contained obvious analytical errors, or else because the samples were used for answering subsidiary questions which had no bearing on the com- position of milk.

PART I. STUDIES ON THE AMOUNTS OF ASH, LACTOSE, FAT AND

NITROGEN IN HUMAN MILK AND IN COW’S MILK.

Methods of experimentation.

The ash determinations were made by igniting the dry residues from the samples cautiously at low temperatures in platinum dishes.

The nitrogen determinations were made by the Kjeldahl method. The lactose was determined by Fehling’s method as follows: 25 cc. of

cow’s milk or 15 cc. of human milk were diluted to 400 cc. in a 500 cc. grad- uated flask. Fifteen cc. of r NaOH and 10 cc. of CuSO, of the strength used in a Fehling’s solution were added and the volume of the liquid in the flask was diluted to 500 cc. with water.

After this mixture had been well shaken, it was filtered through a dry filter into a dry flask. One hundred cc. of this solution were added to 50 cc. of hot Fehling’s solution and the mixture was boiled for six minutes. It was then quickly filtered through an alundum crucible and washed with about 600 cc. of boiling water. The resulting cuprous oxide in the crucible was dissolved with nitric acid and the solution poured into the be’aker which had been used in the reduction. The crucible was then thoroughly washed by passing hot water through it and this wash water was added to

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Edward B. Meigs and Howard L. Marsh 151

the nitric acid solution which had been poured from the crucible. This nitric acid solution of the cuprous oxide was evaporated on a water bath until free from nitric acid. It was then dissolved with acetic acid and water, 8 grams of zinc acetate were added and the liquid was transferred to a small glass-stoppered bottle, 4 grams of potassium iodide were added and the solution titrated against G sodium thiosulphate with starch as an indicator.

The amount of lactose corresponding to the amount of copper found was ascertained. by referring to a table given in Bulletin No. 107 (revised) of the U. S. Department of Agriculture, Bureau of Chemistry, pp. 48 and 49. The figures represent lactose plus one molecule of water of crystallization.

Fat was determined by a modification of the method recommended by Meigs in 1885.5 To 10 cc. of milk in a 100 cc. glass-stoppered cylinder were added 20 cc. of distilled water and 20 cc. of ethyl ether and the mixture shaken for five minutes. Twenty cc. of 95 per cent ethyl alcohol were then added and the whole again shaken for five minutes. The cylinder was then allowed to stand until its contents separated into two distinct layers. The upper layer was removed by the specially designed pipette shown in figure 1,5 cc. of ethyl ether were added in such a way as to wash down the sides of the cylinder, this was removed and added to the upper layer pre- viously removed, and the washing of the sides of the cylinder and of the top of the,lower layer of the mixture was repeated five times in order to remove all the fat. The upper layer from the mixture plus washings was then evaporated to dryneks on a water bath and the residue desiccated to constant weight over sulphuric acid at room temperature. The weight of the residue may be taken to be very nearly that of the fat contained in the milk.

The residue in question was in a number of cases treated with dry ether, and it was found that a minute portion of it usually failed to dissolve. This insoluble portion was, however, never as much as 3 per cent of the weight of the original residue; it amounted on the average to about 1 per cent. It was shown by Fehling’s and by the phenyl-hydrazine tests to con- sist largely of lactose.

The fat as obtained by Meigs’ method from two portions of a sample of cow’s milk (No. 14) was analyzed for nitrogen: 0.0094 per cent and 0.0064 per cent respectively of nitrogen was found. It is probable that a con- siderable portion of this comes from the ether-soluble lipoids.

In one sample of human milk (No. 8) the ash, as determined in the fat obtained by Meigs method, was found to be 0.0081 per cent of the weight of the whole milk.

In other experiments the fat as obtained by Meigs’ method in certain samples of milk was compared with the fat as obtained by the Soxhlet extraction of the dried solids. These results have already been published? in twelve determinations on human milk the fat as determined by Meigs’

6 Meigs: Milk Analysis and Infant Feeding, Philadelphia, 1885. 6 Hawk: Practical Physiological Chemistry, Philadelphia, 1912, p. 437.

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152 Composition of Milk

FIG. 1. PIPETTE ARRANGED TO REMOVE THE UPPER LAYER IN MEIGS’ METHOD OF FAT EXTRACTION.

The end of the tube A is placed at the surface of the division between the two layers of the mixture of milk treated with the water, alcohol and ether mixture and the upper layer is then forced out through it by forcing air into the space above the mixture through tube B.

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Edward B. Meigs and Howard L. Marsh ‘53

method averaged 0.017 per cent less than by the Soxhlet method; and in seven determinations on cow’s milk, 0.019 per cent less. It is by no means certain, however, that the figures for fat obtained bythe Soxhlet method in these experiments are more nearly correct than those obtained by Meigs’ method; for the prolonged treatment of the milk residue with ether in the Soxhlet apparatus is apt to dissolve substances other than fat, and thus give a fictitious additional weight to the fat extract.7

The result of this investigation has been that the product obtained by Meigs’ method of extracting the fat from milk contains 98 per cent or more of material soluble in dry ether. The results as obtained by Meigs’ method are practically the same as those obtained by the Soxhlet extrac- tion; and Meigs’ method is much quicker and subjects the milk to less treatment which is apt to produce changes in the protein and carbohydrate constituents of the fluid.

Meigs’ method of determining the fat in milk was used as a matter of routine in this investigation, and the figures for fat in the tables repre- sent the dry weight of the material obtained from the “upper layer” and washings in the procedure which has just been described. In some cases, however, the fat was determined by the Soxhlet extraction as well as by Meigs’ method. The figures obtained by the Soxhlet extraction are given in a footnote to Table III.

The results of the determinations of the ash, fat, lactose and total nitrogen of human milk and cow’s milk are shown in Table III.

These data agree with those of Camerer and S61dners and with most of the more recent work on milk analysis in showing the marked difference between human milk and cow’s milk in respect to their content of nitrogen and lactose. Human milk contains roughly 50 per cent more lactose than cow’s milk, and decidedly less than half as much nitrogen. The figures in Table III agree also, so far as they go, with the conclusion of Camerer and Sold- nerg that the lactose in human milk increases, while the nitrogen decreases with the progress of lactation.

7 Croll has published in detail his comparison of the results obtained by Meigs’ method of fat extraction and by the Soxhlet method in the Biochemical Bulletin for June, 1913.

* Camerer and Soldner: ZeitschT. f. Biol., xxxvi, Table II, pp. 280 and 281, 1898.

9 Ibid.

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TABL

E III

.

The

quan

titie

s of

ash

, fa

t, la

ctos

e an

d to

tal

nitro

gen

in

hum

an

milk

an

d co

w’s

milk

gi

ven

as

perc

enta

ges

of t

he

weig

ht

of t

he

(I;

whol

e m

ilk.

Dup

licat

e an

alys

es

were

ca

rried

ou

t on

ea

ch

sam

ple

of m

ilk

and

the

tabl

e gi

ves

the

figur

es

obta

ined

in

each

p

anal

ysis

and

the

aver

age.

Itum

an

milk

.

(Milk

N

o.

4 .

0.18

6 0.

189

0.18

7 2.

198

2.21

0 2.

204

6.78

3 6.

759

6.77

10.2

209/

0.22

740.

2242

2

5th

to

9th

day

post

M

ilkN

o.

6 .._

_.._

. 0.

191

0.17

1 0.

181

2.23

4 2.

217

2.22

5 6.

443

6.44

3 6.

4430

.276

6~0.

2765

0.27

65

Milk

N

o.

7 ,._

__.._

__

0.15

1 0.

154

0.15

2 2.

863

2.87

4 2.

868

6.37

9 6.

378

6.37

90.1

962!

0.19

510.

1956

$$

pa

rtum

. . .

. .

. M

ilkN

o.

8 .._

..,...

_ 0.

202

0.19

1 0.

196

2.03

7 2.

019

2.02

8 6.

504

6.50

4 6.

5040

.264

20.2

6020

.262

2 g

Milk

N

o.

10 ..

,,..,,

0.

249

0.25

6 0.

252

3.14

7 3.

134

3.14

0 6.

600

6.54

9 6.

5740

.286

00.2

8280

.284

4 z

Milk

N

o.

9, 5

th

mon

th

post

pa

rtum

. 0.

185

0.17

5 0.

180

4.08

1 4.

081

4.08

1 7.

067

7.09

4 7.

0800

.164

50.1

5740

.160

9 M

ilk

No.

5,

co

llect

ed

near

th

e en

d of

la

c-

z

tatio

n....

......

......

......

......

......

. 0.

165

0.16

2 0.

163

0.85

6 0.

850

0.85

3 7.

294

7.30

5 7.

2990

.196

50.1

9560

.196

0 0,

Cow’

s m

illc.

z E

‘No.

7

. . .

. . .

0.68

6 0.

676

0.68

1 4.

152

4.13

9 4.

145

4.59

9 4.

641

4.62

0~0.

5288

10.5

3210

.530

4 FF

N

o.

8 .._

. 0.

720

0.72

1 0.

720

4.17

6 4.

224

4.20

6 4.

632

4.60

1 4.

616~

0.55

M)0

.553

00,5

515

Sam

ples

of

m

ixed

m

ilk

from

N

o.

ll....

. 0.

719

0.66

0 0.

689

3.94

7 3.

936

3.94

1 4.

745

4.73

7 4.

741~

.560

0655

83~0

.559

1 gr

ade

cows

.

. N

o.

15..

0.69

2 0.

694

0.69

3 4.

383

4.38

4 4.

383

4.82

3 4.

823

4.82

30.5

042,

0.50

8310

.506

2 N

o.

16

0.72

5 0.

731

0.72

8 3.

785

3.80

4 3.

794

4.71

3 4.

713

4.71

3~0.

4981

!0.4

970!

0.49

75

,No.

19

.._

_ 0.

732

0.73

5 0.

733

3.21

5 3.

220

3.22

0 4.

490

4.47

4 4.

482’

0.52

690.

5191

’0.5

230

Sam

ples

of

m

ilk

from

si

ngle

N

o.

l*....

. 0.

728

0.65

4 0.

691

5.38

2 5.

409

5.39

5 4.

940

5.07

0 5.

005~

.569

20.5

555l

I.562

3 th

orou

ghbr

ed

Gue

rnse

y N

o.

9....

.. 0.

729

0.73

9 0.

734

4.69

5 4.

689

4.69

2 4.

805

4.81

9 4.

8120

.495

9nod

up.0

.495

9 co

ws

. .

No.

13

.. 0.

774

0.76

81

0.77

1 4.

605

4.61

5 4.

610,

4.

741

4.75

5 4.

748,

0.59

48~0

.594

9’0.

5948

---

---.

*The

fig

ures

ob

taine

d for

fa

t In

this

sim

ple

of m

ilk

by

the

Soxh

let

meth

od

of ex

tractI

on

were

I, 5.3

10;

II,

5.344

; av

erage

, 5.3

31.

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Edward B. Meigs and Howard L. Marsh ‘55

PART II. THE NATURE AND -4MOUNT OF THE PROTEIN AND UNKNOWN

SUBSTANCES IN HUMaN MILK AND IN COW’S MILK.

Meigs hoped in his later work to satisfactorily settle the quan- tities of protein in human milk and in cow’s milk, and perhaps even to determine the nature of some of the more important unknown substances, which are supposed to be present in the two secretions. Both these hopes were still unfulfilled at the time of his death, but the work has thrown some further light on the problem; and the results, so far as they go, will be given here.

Experiments which bear solely on the protein content of milk.

The casein and globulin were precipitated from several samples of human milk and cow’s milk by magnesium sulphate and the albumin was precipitated from the magnesium sulphate filtrates by acetic acid and heat.lO The nitrogen content was determined in each precipitate separately, and also, in a separate portion, for the whole milk. The results are given in Table IV.

In other samples of milk the colloids were completely precipi- tated from the whole milk according to Marshall’s11 aluminium hydroxide method. The nitrogen in the colloid precipitates, in the filtrates, and in separate portions of the whole milk was determined. The results are given in Table V.

No very definite conclusions regarding the usual protein con- tent of human milk can be drawn from this part of the work, because the samples of human milk used were so few and from so early a period of lactation. So far as the results go, however, they confirm the view that human milk contains less than half as much protein as cow’s milk. The results are interesting also in showing that, a large proportion of the nitrogen in early human milk exists in compounds which are not precipitable either by magnesium sulphate or by acetic acid and heat; and the results with Marshall’s reagent indicate that most of this ” non-precipi- table” nitrogen exists in non-colloidal bodies.

lo For details of these methods see Bulletin No. 107 (revised) of the U. S. Department of Bgriculture, 1910, p. 118.

I1 Marshall and Welker: Journ. Amer. Chem. Sot., xxxv, p. 820, 1913.

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TABL

E IV

.

The

tota

l ni

troge

n,

the

amou

nts

of n

itrog

en

cont

aine

d in

th

e ca

sein

an

d gl

obul

in

prec

ipita

tes

and

in

the

albu

min

pr

ecip

i- ta

tes,

an

d th

e no

n-pr

ecip

itabl

e*

nitro

gen

in

hum

an

milk

an

d co

w’s

milk

gi

ven

as

perc

enta

ges

of

the

weig

ht

of

the

whol

e m

ilk.

Avera

ge

Cow’

s m

ilk.

Sam

ple

No.

1

from

G

uern

sey

cow.

. .

. .

0.02

16

0.04

30

Sam

ples

of

m

ixed

m

ilk

from

0.

0259

gr

ade

cows

.

. 0.

0429

N

o.

5 0.

5251

0.50

19’0

.513

5~.4

3210

.413

’i0.4

2290

.039

40.S

~0.0

426

0.04

80

l Th

e fig

ures

for

the

“no

n-prec

ipltab

le”

nitrog

en

are

obtai

ned

by

subtr

actin

g the

su

m

of the

qu

antltl

es

of nit

rogen

ln

the

case

ln an

d glo

bulin

and

in

the

album

in pre

cipita

tes

from

the

tot

al nit

roge

n.

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TABL

E V.

The

tota

l ni

troge

n co

nten

ts

an.d

th

e am

ount

s of

ni

troge

n co

ntai

ned

in

the

prec

ipita

les

by

Mar

shal

l’s

met

hod

and

in

the.

fil-

trate

s th

eref

rom

in

hu

man

m

ilk

and

cow’

s m

ilk

give

n as

pe

rcen

tage

s of

th

e wh

ole

milk

. __

~ I

I /

Sam

ple

No.

3

(col

ostru

m).

. .

0.28

85

’

.I i

0.30

38

Sam

ple

No.

2

(ear

ly m

ilk).

. . .

.

0.21

81

0.21

91

~--

.--

~~_

.-~.

Sam

pIes

of

m

ixed

m

ilk

J N

o.

3..

\

No,

4,

.

No.

5.

.. --~

.-~

~-..

.-

0.51

21

0.51

49

0.53

55

0.50

29

0.51

29

0.52

51

0.52

51

0.50

19

Cow’

s 7

0.51

35

0.51

92

0.51

90

0.51

35

..--.

-.

ni lk

. -~__

0.47

91

0.50

35

0.52

13

0.47

32

0.48

39

0.48

15

0.50

08

0.50

22

0.49

83

0.50

98

0.47

64

0.47

48

0.03

80

0.04

06

0.01

90

0.03

50

0.03

85

0.03

82

0.03

69

0.03

88

0.02

26

0.02

08

0.03

40

0.03

45

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158 Composition of Milk

Experiments bearing on the amount and nature of the unknown material contained in milk as well as upon the protein content.

Camerer and Soldner subtracted the sum of the quantities of lactose, fat, ash and citric acidI in their samples of milk from the amount of total solids, and called the remainder so obtained the quantity of the ‘Lprotein plus unknown substances.” They found that the nitrogen in cow’s milk made up about 16 per cent of the quantity of ‘(protein plus unknown substances,” while that in human milk made up only from 11 to 12 per cent.13 They then precipitated the protein from milk by alcohol according to Munk’s method, and found that the dry ash-free protein of cow’s milk contained 14.54 per cent of nitrogen; while that from human milk contained 13.64 per cent.14 From all these results they con- cluded that human milk contains a considerable amount of un- known substances which contain little or no nitrogen.

Camerer and Sijldner found, as have other investigators, that alcohol does not precipitate the nitrogenous substances from milk so completely as does tannic acid; further, the filtrate from the alcohol precipitation gives a positive reaction with the biuret test and with Millon’s reagent. They have studied the non-fatty material which goes into solution in alcohol when the proteins are precipitated from milk by Munk’s method and find that it is a soft, sticky material very slightly soluble in water but highly soluble in dilute alkali. Dilute hydrochloric acid gives a white flocculent precipitate from the alkaline solutions. The substance contained about 13 per cent of nitrogen, 0.2 per cent of phosphorus and 1 per cent of ash. From the high nitrogen and phosphorus contents Camerer and Sijldner suspect that the material is altered casein.15

Meigs and his collaborators have gone over the ground covered by Camerer and Sijldner on the protein and unknown substances in milk: Their most important results are given in Tables VI and VII.

l2 Camerer and Sijldner: Zeitschr. f. Biol., xxxvi, p. 278, 1898. 13 Camerer and Siildner: ibid., xxxiii, p. 549 and pp. 562-565, 1896. I4 Camerer and Sijldner: ibid., xxxiii, p. 548, 1896; The alcohol precipi-

tate from the cow’s milk contained, on the average, 11.06 per cent of ash; that from the human milk, 6.29 per cent.

I5 Camerer and Siildner: ibid., xxxiii, p. 561, 1896.

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Edward B. Meigs and Howard L. Marsh 159

TABLE VI.

The quantities of total solids and of ‘(protein plus unknown substances” in human milk and cow’s milk given as percentages of the weight of the whole milk; and the percentages of nitrogen which may be supposed to be contained in the “protein plus unlznown substances.”

TOTdL 80LID8

Human milk.

5th to 9th day post partum.. .

11.73 11.70 12.35 11.82 11.75 12.63

lactation........................ 16.05

Cow’s milk.

iNo. 7 813.320’13.471~13.395~ 3.769 1 14.07

Samples of mixed No. 8.. ‘13.43713.484,13.460: 3.744 14.73

milk from grade No. ll,...,.. ~13.19513.188~13.191~..3.640

cows. . . . No. 15, ;13.343~13.313’13.325~

15.36

No. 16.. ~12.45912.503:12.481~ 3.249 15.58 3.066 16.23

Samples of milk from single thoroughbred Guernsey COWS.

+ The average percentage of nitrogen in the “protein plus unknown substances” in samples 4, 6, 7, 8, 9 and 10 of human milk is 12 per cent. Sample 5 is omitted from this calculation, as the milk came from very near the end of lact.vtion and q-as evidently abnormal. The average percentage of nitrogen in the “protein plus unknown substances” in the samples of cow’s milk is 15.18 per cent.

Table VI gives the amount of total solids and of ‘L protein plus unknown substances” in a number of samples of human milk and of cow’s milk. The figures for the “protein plus unknown sub- stances” were obtained in the same way as those of Camerer and Soldner. The average nitrogen contents of the “protein plus unknown substances” as calculated from our figures is 12 per cent in the case of human milk and 15.18 per cent in the case of cow’s milk.

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TABL

E VI

I.

The

dry

weig

ht

of

prot

ein

prec

ipita

ted

by

alco

hol

from

fa

t-fre

e hu

man

m

ilk

and

cow’

s m

ilk

given

as

per

cent

ages

of

Ih

e, w

eigh

t u

of t

he w

hole

m

ilk;

the

amou

nt

of n

itrog

en

cont

aine

d in

th

e al

coho

l pr

ecip

itate

gi

ven

as

perc

enta

ge

of

the

weig

ht

of

the

whol

e 0”

m

ilk;

and

the

perc

enta

ges

of

nitro

gen

cont

aine

d in

th

e dr

y al

coho

l pr

ecip

itate

s.

-. ._

_~~.

.

..-

.~

~~ ~_

-.__

1.06

4 1.

052

0.81

1 0.

869

Milk

N

o.

8.

1.29

2 1

.31.

5

per

cent

.. .

0.78

6 0.

793

1.44

0 1.

428

Prec

ipita

ted

by

QO

pe

r ce

nt

alco

hol

IMilk

/’

No ’

7,

0.99

1 0.

957

Prec

ipita

ted

by86

iM

ilk

No.

13

. pe

r ce

nt

,zlc

ohol

\M

illc

No.

IQ

. 3.

808

3.04

7

1.05

8 1

0.15

06

1.43

4 O

.lQ41

0.97

4 0.

1474

Cow’

s m

ilk. --

3.41

2 0.

4800

3

410

0.48

78

3.39

3 0.

4506

3.

811

0.54

64

3.03

0 0.

4276

0.15

30

0.15

18

0.14

68

0.14

66

0.16

66

0.16

66

0.10

24

0.10

36

0.19

70

0.10

55

0.14

71

0.14

72

0.47

40

0.47

70

0 40

48

0.4Q

13

0.4G

36

0.46

16

0.54

51

0.54

57

0.42

53

0.42

64

~._.

,.

14.1

5 14

.54

16.0

8 1G

.8Q

12

.00

12.6

7 13

.35

13 .Q

l 13

.45

13.8

0

14.8

7 15

.37

13.0

1 14

.03

14.2

3 14

GO

13

.62

13.5

9 14

33

14

.31

14.1

0 13

.96

..-

~~~

14.3

5 9

16.4

7 3 cb

12.7

8 0

13.1

3 13

.64

g $ 15

.11

0

~ ;

13.0

7 F?

14.4

1 13

.60

14.3

2 14

.06

.- l

The

figur

es

in

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col

umn

repr

esen

t th

e w

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the

as

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s.

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ash

in

the

alco

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prec

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~3s

dete

rmin

ed

by

Mar

sh

in

the

CAS

TS of

sam

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8.

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10 a

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7 of

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milk

, iln

d in

the

oas

es o

f sa

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9, 1

3 an

d 19

of

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’s m

ilk;

it ~~

59 f

ound

to

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.079

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and

11 p

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le pr

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ltate

In

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’s m

ilk.

In

the

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sam

ples

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an

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th

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h-Ire

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Is

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to

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04

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r ce

nt o

f th

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-con

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In t

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ples

of

cow

’s m

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89 p

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cent

. Se

e al

so C

amer

er

and

SB

ldne

r: Ze

ilsch

r. f.

B

iol.,

xx

x~il,

Tabl

e III

, p.

548

, 18

96.

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Edward B. Meigs and Howard L. Marsh 161

The dry weight of the material precipitated from samples of human milk and cow’s .milk by alcoho1 and the nitrogen content of this material are given in Table VII. The ash-free protein pre- cipitated from human milk by alcohol contained on the average 13.80 per cent of nitrogen; while that from cow’s milk contained 14.07 per cent.

The following is an account of the methods used by Meigs and his collaborators in this part of the investigation

Samples of milk were placed in platinum dishes (without sea sand) and were kept in a water oven at about 9P6 until ten hours after the visible fluid in them had disappeared. The residues were then placedin desic- cators over sulphuric acid at room temperature until they reached con- stant weight, and this figure was taken as the weight of the total solids. The lactose retains its water of crystallization under these conditions of drying.17

The protein precipitated from milk by alcohol was determined as fol- lows: The fat was removed from 10 cc. of milk by Meigs’ method,described on pages 151-153. After the removal of the fat had been completed the lower layer of the mixture (which contained the non-fatty constituents from 10 cc. of milk) was transferred to a 200 cc. beaker and evaporated on a water bath to a volume of about 10 cc.; 95 per cent alcohol was then added. The amount of alcohol added differed in different experiments; in some cases the mixture obtained by adding the alcohol contained 75 per cent of alcohol; in others, 86 per cent; and in still others, 90 per cent of alcohol. The mixture was aIlowed to stand five minutes and then filtered t,hrough a weighed porous alundum crucible by means of a suction pump and washed with 1 liter of boiling alcohol and subsequently with 500 cc. of ether. Before being washed, the protein was carefully broken up into small particles with a glass rod.

The contents of the crucible were dried first in a water oven at 93’ and then in a desiccator over sulphuric acid at room temperature unbil they reached constant weight. They were then weighed, and their content of nitrogen was afterwards determined.

The filtrates from the milk treated with alcohol as described above were studied in various ways. It was found that if these filtrates were evaporated to small volume and then treated with a considerable quantity of water, there was precipitated from them a material which resembled that described by Camerer and Soldner (see page 158). Marsh determined the solubilities of this materia1

16Temperatures are throughout this article expressed in terms of the centigrade scale.

I7 Camerer and Soldner: Zeitschr. f. Biol., xxxiii, pp. 538-.!&1, 18%.

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162 Composition of Milk

and the nitrogen content. He found that all of it was soluble in alcohol and only a portion in ether. The ether-insoluble portion of the material is insoluble in water and in acids; soluble in al- cohol, chloroform and alkalies. It gives a positive reaction to both Millon’s test and the biuret. It contains 13.8 per cent of nitrogen. On being ignited, it leaves a little ash which contains phosphorus. It can be obtained in larger quantities from human milk than from cow’s milk.

FIG. 2. CRYSTALS OF SUBSTANCE OF UNKNOWN COMPOSITION FROM HUMAN MILE.

It has been found possible to obtain two kinds of crystals from the alcohol filtrate of human milk treated as described on page 161. Of these one appears as long glistening prisms. These crystals are soft and represent, in all probability, a minute rem- nant of fat, which had not been removed by Meigs’ method of fat extraction. The appearance of the other kind of crystal is shown in Figure 2, which is reproduced from a photograph.

It was afterward found that the crystals shown in Figure 2 could most easily be obtained as follows:

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Edward B. Meigs and Howard L. Marsh ‘63

The fat was removed from samples of milk by hleigs’ method described on pages 151-153; the fat-free or nearly fat-free lower layer was then shaken up with ether; the mixture allowed to stand; and the upper layer, consisting of a mixture of alcohol and ether, was drawn off. This was evaporated over boiling water to a volume of about 20 cc. and then allowed to evaporate spontaneously. The crystals usually made their appearance before the supernatant fluid had entirely evaporated. The production of the crystals has, however, been rather capricious; no method has yet been devised by which they could be obtained with certainty.

We have submitted these crystals to Dr. A. P. Brown, Professor of Geology and Mineralogy in the University of PennsyIvania, for examination and have obtained the following report:

The crystals belong to the Tetragonal System. Their axial ratio is a : c = 1: 2.46. Form observed: The unit pyramid was the only form present. Angles: The plane angle of the face of the unit pyramid, between the

pole edges 111 - 111 A 111 - Ill = 35” 20’; also the edge angle over the pole, edges 111 - ill A 111 - nl = 44” 20’ (calculated 44” 6’).

Habit of crystals: The unit pyramid is alone developed; sometimes symmetrically, more often distorted by contact with the surface upon which it rests, giving the appearance at first sight of the unit pyramid combined with the basal pinacoid (001) and with the unit prism (llO), but these forms are not seen. No twins were observed, but parallel growths of the unit pyramids were fairly common. The crystals are brittle; no cleavage was observed.

The refractive index is high and the double refraction is very strong. Examined in polarized light, the extinction is straight in all ordinary positions. The greater refractive index is for light vibrat,ing parallel to the crystal axis, c; hence E>W, or the crystal is optically positive (-I-). On examining the crystal in the direction of the vertical axis, c, in con- vergent polarized light, the uniaxial interference figure is observed, thus confirming the tetragonal character.

As a check on the angles, the angle over the pole was measured, angle 111 ATT1 = 32” 40’ as against 32” 4’ calculated. The profile view of the crystal lying on the pole edge gave an edge angle over the pole of about 54”.

These crystals are fairly soluble in ether, somewhat soluble in 95 per cent alcohol, and nearly insoluble in water, though treat- ment with water alters them, rendering them opaque and in time destroying their crystalline form. They dissolve very slowly in acetone. They have been submitted to Lassaigne’s test for nitro- gen, and thereby shown to be free from nitrogen; but the appli-

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Composition of Milk

cation of the plumbic acetate tesP shows that they contain a considerable amount of sulphur.

They represent a substance, which, so far as we know, has not been heretofore described as a component of milk.

The immediately preceding paragraphs may be summed up by saying that the results of Meigs and his collaborators on the protein and unknown substances of milk are throughout in close agreement with those of Camerer and SBldner. Both sets of observers find that the total nitrogen in human milk makes up a decidedly smaller proportion of the “protein plus unknown sub- stances” than it does in the case of cow’s milk; both sets of ob- servers find that the materials precipitated from the two kinds of milk by alcohol have about the nitrogen content which is usual for protein; finally, both sets of observers obtained similar mate- rials from the alcohol filtrates. This agreement is made all the more striking by the fact that neither Meigs nor any of his col- laborators was familiar with the work of Camerer and Siildner at the time when their experiments were carried out. They ob- tained their results quite independently. We are inclined, there- fore, to regard it as established, or at least very highly probable, that human milk contains a considerable amount of unknown material, which has either a low nitrogen content or else none at all of that element.

What becomes of this material when milk is subjected to analy- sis? It has been shown above that unknown substances can be extracted from milk by means of alcohol, and we shall consider very briefly the question whether the material of the alcohol ex- tract is to any extent identical with the “z material,” the exist- ence of which in human milk can be inferred from the work of Camerer and Soldner and of Meigs and his collaborat,ors.

Camerer and Soldner advance the hypothesis that the mate- rial of the alcohol extract is “altered casein.” We think this improbable for the following reason. Cow’s milk contains about three times as much casein as human milk. If, therefore, alcohol alters casein so that a part of it becomes alcohol-soluble, more of this altered casein should appear in the alcohol filtrate from

I* Hawk: Practical Physiological Chemistry, 4th edition, Philadelphia, 1912, pp. 108, 109.

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Edward B. Meigs and Howard L. Marsh 165

cow’s milk than in that from human milk. But just the oppo- site is the case.

We have endeavored to get an approximate idea of the total amount of unknown material in the alcohol extract from cow’s milk and human milk. Two samples of human milk and three samples of cow’s milk were freed from fat by Meigs’ method, and the protein was precipitated from the fat-free residue by 86 per cent alcohol.

The filtrates from the 86 per cent alcohol protein precipita- tions were then evaporated to dryness and the weights of the ash-free residues were determined. These residues contained all the lactose from the milk plus the unknown alcohol-soluble mate- rial. In other portions of the same samples of milk the lactose was determined by the Fehling method-see Table III. Sub- tracting the amounts of lactose from those of the ash-free residues in the five samples of milk gives the following results:

Percentage of ash-free alcohol

r&he

Percentage of unknown alco-

hoi-soluble material

Human milk.. (p. 1;::. . . 7.952 - 6.574 = 1.378

N:: 16. .::: 7,575 5.104 - - 7.080 4.713 = = 0.495 0.391 Cow’s milk.. . No. 19.. . . . 5.068 - 4.482 = 0.586

No. 13.. . . . 4.935 - 4.748 = 0.187

The following is another method by which an approximate idea of the amount of unknown alcohol-soluble material in milk may be gained. In the samples of human milk Nos. 4, 5, 7, 8, 9 and 10 the weights of the material precipitated by alcohol have been determined (Table VII) and may be compared with the quan- tities of “protein plus unknown substances” found in Table VI. It will be seen that the quantities of the latter are always con- siderably larger than those of the former; the results obtained by subtracting the quantities of ash-free alcohol precipitates from those of the “protein plus unknown substances” are as follows: Milk No. 4, 0.853; No. 7, 0.610; No. 8, 0.916; No. 10, 0.981; Ko. 9, 0.485; No. 5, 0.331. The same data for the samples of cow’s milk, Nos. 7, 8, 9, 13 and 19 are given in Tables VI

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166 Composition of Milk

and VII; and the results of similar calculations are as follows: Milk No. 7, 0.357; No. 8, 0.334; No. 9, 0.281; No. 19, 0.076.1g

The figures which have just been given to represent the amounts of the unknown alcohol-soluble material in human milk at dif- ferent periods of lactation correspond very fairly with the figures for the amounts of the x material at the correspondingperiods of lactation as calculated from the data of Camerer and Sijldner and from those of Meigs and his collaborators. It seems prob- able, therefore, that the alcohol-soluble material is to a consid- erable extent identical with the 5 material. That it is not wholly identical with the x material is evident from general consider- ations as well as from its high nitrogen content. Milk contains such bodies as urea, ammonia,2O and purine bases21 which would not be precipitated by alcohol; and the nitrogen in these would account for a considerable part of the nitrogen in the alcohol- soluble material.

The known alcohol-soluble substances in milk.

Koch and W’oods22 find 0.036 to 0.049 per cent lecithin and 0.027 to 0.045 per cent kephalin in cow’s milk-O.072 to 0.086 per cent total ‘(le~ithans.“~~ For human milk the figures of these investigators are lecithin, 0.041 per cent; kephalin, 0.037 per cent; total lecithans, 0.078 per cent.

Raudnitz24 gives a review of previous determinations of cho- IesteroP in milk. Tolmatscheff found 0.0252 to 0.0385 per cent of this substance in human milk; Schmidt-Miilheim was able to demonstrate its presence in cow’s milk. Marsh determined the amount of cholesterol in cow’s milk according to the method of Ritter26 and found 0.021 per cent.

I9 In the case of sample No. 13 of cow’s milk there was some error in anal- ysis so that the weight of the material precipitated by alcohol appeared to be slightly greater than that of the “protein plus unknown substances.”

z0 Camerer and Siildner: Zeitschr. f. Biol., xxxvi, p. 299, 1898. 21 Raudnitz: Ergeb. d. Physiol., 1903, ii, p. 257. 22 Koch and Woods: This Journal, i, p. 211, 1906. 28 Jj y “lecithans” Koch and Woods mean the phosphorus-containing

lipoids. H Raudnitz: Ergeb. d. Physiol., 1903, ii, p. 264. *j Raudnitz uses the older term, “cholesterin.” 26 Ritter: Zeitschr. f. physiol. Chem., xxxiv, p. 430, 1901-02.

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Edward B. Meigs and Howard L. Marsh 167

The substances which have been spoken of above-lecithin, kephalin, cholesterol, urea, ammonia and the purine bases-would account for less than one-fifth of the unknown material in the alcohol extract of human milk.

It is realized that the data given above and bearing on the quantity of the material in the alcohol extract of milk are very incomplete. But they do indicate that human milk contains con- siderably more non-fatty, alcohol-soIuble material than cow’s milk; and it has been thought worth while to present them in view of recent interesting work on the importance of unknown substances in milk as accessory factors in diet. Stepp,27 for instance, has found that mice always die in a few weeks when given food mate- rials which have been fully extracted with alcohol and ether, and that the diet may be rendered again capable of sustaining life by the addition of the material from the alcohol-ether extract of skimmed milk. Stepp has been able to show that the mate- rial necessary to maintain life is not either fat, cholesterol, lecithin or salts.

GENERAL CONCLUSIONS.

Human milk differs from cow’s milk in three important ways. It contains considerably more lactose than cow’s milk, and more substances of unknown nature which contain little or no nitrogen; it contains very much less protein than cow’s milk. The com- posit,ion of milk varies more or less regularly with the progress of lactation so that average figures for its composition are not very satisfactory. The following, however, may be taken as the limit#s of normal variation of the constituents of the two kinds of milk from the beginning of the second month of lactation onward, the figures representing percentages of whole milk:

-- I I FAT LhCTOSE , PROTEIN

-1~~~ Human milk. .’ 2-4 ~ 6-7.5 ~ 0.7-1.5 Cow’s milk.. 2-4 ~ 3.5-5 1 2.5-4

Both kinds of miIk cont.ain substances which are important constituents of diet, which are soluble in alcohol and ether, which

27 Stepp: Zeitschr. f. Biol., lvii (9. F. xxxix), p. 135, 1911.

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168 Composition of Milk

contain little or no nitrogen, but of which the chemical nature is still unknown. These substances are most plentiful in early human milk and diminish in amount with the progress of lacta- tion. Early human milk contains about 1 per cent of these un- known substances; milk from the middle period of lactation about 0.5 per cent. Cow’s milk from the middle period of lactation contains about 0.3 per cent of the unknown substances.

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Edward B. Meigs and Howard L. MarshMILK

OF HUMAN MILK AND OF COW'S THE COMPARATIVE COMPOSITION

1913, 16:147-168.J. Biol. Chem. 

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