DISTRIBUTION

ACIDS
BY ELIOT
HOFFMAN,
(From

OF NITROGEN
IN HUMAN
AND
F.

the Research

AND PROTEIN
AMINO
IN COWS MILK*

BEACH,
SAMUEL
S. BERNSTEIN,
D. MAXWELL
TEAGUE,
AND ICIE
Laboratory
for

the Childrens

publication,

January

Fund

of Michigan,

D.
Detroit)

16, 1941)

The belief that human breast milk is superior to cows milk in

the nutrition of the human infant is held by many pediatricians.
The theory is based almost wholly upon clinical experience (1, 2).
Studies of the amino acid composition of the proteins of cows
milk have been numerous but little information
is available concerning the composition of human milk proteins.
The present
study of the constitution
of these proteins was undertaken,
first,
because the milk proteins which are elaborated for the young at
a time when requirements
for growth are at a maximum might
be expected to have the ideal pattern of amino acid distribution
for supporting rapid growth, and, second, because a comparative
study of the composition of cow and human milk proteins might
amplify our knowledge of their relative nutritive values.
While
amino acid composition
is not the only factor determining
the
biological value of proteins, its importance cannot be minimized.
EXPERIMENTAL

The distribution
of the casein, whey protein, and non-protein
nitrogen in the total nitrogen of milk samples was determined by
an adaptation of the methods of Rowland (3).1
* Preliminary
reports
of this investigation
were presented
before
the
meetings
of the American
Institute
of Nutrition,
at Toronto,
Spril
26,
1939, and the Society
for Pediatric
Research,
at Skytop,
Pennsylvania,
April
26-27, 1939.
1 Total
nitrogen
was determined
by the macro-Kjeldahl
method,
on 5
ml. of milk.
To determine
the non-casein
nitrogen
20 ml. of milk were
placed
in a 100 ml. volumetric
flask.
60 ml. of water
at 40 were added,
followed
by 2 ml. of 10 per cent acetic acid.
After
10 minutes,
2.0 ml. of
57

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(Received

of

OLIVE
G. MACY

Amino Acids of Milk

normal
sodium
acetate
were added.
The mixture
was alIowed
to cool to
room
temperature,
diluted
to volume,
and filtered.
A macro-Kjeldahl
determination
was made on 50 ml. of the filtrate
to ascertain
the amount
of
non-casein
nitrogen
present.
To determine
the non-protein
nitrogen
10 ml.
of milk were placed in a 50 ml. volumetric
flask and made to volume
with
15 per cent trichloroacetic
acid.
After
30 minutes
the mixture
was filtered
and the nitrogen
contents
of 10 ml. aliquots
of the filtrate
determined
by
the Van Slyke manometric
micromethod.
2 The milk was centrifuged
at high speed until
no further
separation
of
fat could
be obtained.
The fat separated
readily
from
cows milk
but,
frequently,
difficulty
in separating
was encountered
with
freshly
drawn
human
milk,
probably
owing
to the fine dispersion
of the fat globules.
Upon
being held in refrigeration
for 24 hours
the fat could
be removed
completely
enough
to permit
a sharp
precipitation
of casein.
1 liter
of
fat-free
milk was diluted
with an equal volume
of distilled
water and placed
in a 3 liter jar.
A mixture
of 1 part of normal
hydrochloric
acid and 2 parts
of normal
acetic acid was introduced
very slowly,
during
efficient
mechanical stirring,
through
a capillary
extending
below the surface
of the milk,
until
the isoelectric
point
of casein
was reached.
The precipitate
was
washed,
and then purified
once by dissolving
with 0.1 N sodium
hydroxide
(pH 7), as described
by Van Slyke and Baker
(4).
The casein was washed
with distilled
water,
then with 45 per cent alcohol,
finally
with 95 per cent
alcohol
and ethyl
ether,
and dried in a vacuum
oven at 70.

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The protein fractions of cows milk were prepared from three

lots of mixed milk obtained from the same herd of cows.
The
human milk samples were mixtures of milk obtained from mothers
producing large volumes of milk, known to be in the mature stage
of lactation.
No milk was taken earlier than 8 weeks or later
than 6 months post partum.
Casein fractions were prepared by the method of Van Slyke and
Baker (4) .2 The soluble protein fractions of the milk were prepared from the whey remaining after the precipitation
of casein.
The whey was placed in collodion sausage casing sacs with thymol
as a preservative
and dialyzed with running tap water until the
test for reducing sugar with Benedicts
reagent was negative.
About 16 to 18 hours were sufficient to complete the dialysis.
(It was found that protein preparations
made from whey which
was not dialyzed were contaminated with large amounts of carbohydrate.)
After dialysis the whey was mixed with a 33 per cent
by weight solution of trichloroacetic
acid in sufficient amount to
bring the resulting mixture to a concentration
of 8 per cent trichloroacetic acid. After 30 minutes the whey protein fraction

Beach, Bernstein,

Hoffman,

Teague, Macy

59

was centrifuged off and suspended in 95 per cent alcohol.

A half
volume of ether was added to the alcohol-whey
protein suspension
and the whey protein centrifuged off, washed with ethyl ether, and
transferred to a desiccating vacuum oven at 70 for drying.
TABLE

Chemical

Composition

of Milk

The values,
measured
in gm. per 100 ml., represent
averages
obtained
from the analyses
of three different
samples
of cows milk and five samples
of human
milk.

..............

..............
..............
..............
..............
..............
..............
..............
..............
..............

TABLE

Average

Nitrogen

Human

3.89
0.5176
0.780
0.1240
0.0122
0.1550
0.0497
0.0944
0.1050
0.0326

4.12
0.1616
0.210
0.0318
0.0038
0.0498
0.0113
0.0134
0.0322
0.0139

II

Distribution

in Milk

Cows

milk

Human

milk

-mg. per 100 ml.

No. of samples.
.................
Total
nitrogen.,
.................
Casein
I
...................
Whey
protein
nitrogen
...........
Non-protein

...........

3
517.6
404.6
88.8
24.2

nag. per 100 ml.

4
161.6
48.6
77.0
36.0

The milk proteins were analyzed for total nitrogen content by

the macro-Kjeldahl
procedure and for total sulfur as sulfate after
combustion
in the oxygen bomb.
The basic amino acids were
determined by the methods of Block (5) with nitranilic acid as the
precipitant
for histidine.
Correction factors were applied to the
arginine and lysine values to allow for solubility
losses in the
micro adaptation of the method.
These are described in the foot-

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Fat ...............
Total
nitrogen
.....
Ash. ...............
Calcium.
.....
..
Magnesium.
.....
Potassium.
......
Sodium.
.........
Phosphorus.
.....
Chlorine
.........
Sulfur.
..........

COW

Amino Acids of Milk

60

notes to Table III.3

Tyrosine and tryptophane
were determined
by the method of Folin and Marenzi (6). Cystine was determined
through its cuprous mercaptide by the method of Graff, Maculla,
III

TABLE

Amino

Acid

Composition

and Nitrogen

Content

of Milk

Protein

Casein

Whey

COW

0.27
0.23
0.20
0.06
0.13
0.07

cent

per

4.62
1.80
3.31
5.21
6.11
1.69
0.73
2.19
0.195
0.471
0.666
0.682

f
f
f
f
f
f
*

0.14
0.06
0.05
0.09
0.32
0.14
0.07

per cent

14.20
1.41
3.46
7.91
4.70
1.81
2.49
2.73
0.662
0.586
1.248
1.262

rt
f
f
f
f
f
f

0.05
0.15
0.08
0.30
0.11
0.10
0.02

cent

per

3.73
1.13
5.18
5.80
5.20
2.32
3.09
1.91
0.825
0.410

f
f
f
f
f
f
f

0.15
0.08
0.16
0.16
0.05
0.09
0.31

1.235
1.298

* Casein of cows milk represents

the average
obtained
on the analysis
of two preparations.
The other
protein
fractions
are averages
of three
preparations.
The plus-minus
figures
represent
the approximate
range of
values
among
the preparations.
No range
is given
for total
sulfur
or
methionine,
since these results were obtained
on mixed preparations,
owing
to insufficient
material.
t The arginine
values are corrected
for loss through
solubility
of arginine
silver
by the factor
3.6 mg. per 100 ml. proposed
by Gulewitsch
(10).
For
instance,
in the present
adaptation
of the Block method
a volume
of 325ml.
used (which
includes
mother
liquor
and washings)
gives a solubility
loss of
11.8 mg. of arginine.
$ The lysine
values
are corrected
for the constant
loss of 8.34 mg. of
lysine
mostly
through
the solubility
of lysine
phosphotungstate.
This
factor
was determined
by Tristram
(11).

and Graff (7) and methionine by the methods of Baernstein

with some modifications
introduced by Kassell and Brand

(8)
(9).

3 It is recognized
that small losses of histidine,
also, are incurred
in the
procedure.
Work
is in progress
on the estimation
of histidine
by the
nitranilic
acid procedure
and special
attention
is being given the determination of an accurate
correction
factor.

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f
f
f
f
f
f

protein

COW

per cent

Total
N . . . . . . . . . . 14.50
Histidine.
1.81
Argininet..
3.79
Lysine#.
6.20
Tyrosine.
6.01
Tryptophane
.
1.11
Cystine..
.
0.26
Methionine.......
3.10
Cystine
S..
0.069
Methionine
S.....
0.666
Cystine
S + methionine
S..
0.735
Total
S..
0.796

Fractions*

Teague, Macy

61

The fat content of the milk samples and their contents

mineral elements calcium, magnesium, sodium, potassium,
phorus, chlorine, and sulfur were also determined.

of the
phos-

Beach, Bernstein,

Hoffman,

Results
The lipid, nitrogen, and mineral ash constituents
of cow and
of
human milk samples are shown in Table I. The distribution
total nitrogen in cow and human milks is shown in Table II.
Differences in the distribution
are striking, human milk containing

of Milk

COW
Cm&

Nitrogen ..........

Histidine .........
Arginine ..........
Lysine............
Tyrosine ..........
.....
Tryptophane.
Cystine ...........
Methionine .......
Cystine S .........
Methionine
S.....
Cystine S + methionines
......

Whey
protein

9. per
100 ml.

7%x

TOtd
1 *zg. per
no. per
100 ml. 1F6.6 ml.

404.6
50.5
105.7
173.1
167.9
31.3
7.3
86.6
1.9
18.6

38.8
8.8
21.6
49.5
29.4
11.3
15.5
17.1
4.1
3.7

493.4
59.3
127.3
222.6
197.3
42.6
22.8
103.7
6.0
22.3

20.5

7.8

* 25.5 ml. of cows milk contain

human milk.

15.1
32.4
56.7
50.2
10.8
5.8
26.4
1.5
5.7

28.3
-

7.2
-

as much protein

Whey

Casein

1.25.6

Hllmitn
1protein

Total

mg. per
100 ml.

%S

ml. per
100 ml.

48.6
6.0
11.0
17.3
20.4
5.7
2.5
7.2
0.7
1.5

77.0
6.3
29.0
32.4
29.2
13.0
17.3
10.9
4.6
2.3

.25.6
12.3
40.0
49.7
49.6
18.7
19.8
18.1
5.3
3.8

6.9

9.1

2.2

nitrogen

as 100 ml. of

only 30 per cent as much total nitrogen and only 25 per cent as
much protein nitrogen as does cows milk. The mineral content
of human milk, shown in Table I, is correspondingly low. In the
two milks the whey protein occurs in approximately equal concentrations but the casein content of cows milk is 8 times as great
asthat of human milk. 82 per cent of the protein nitrogen of cows
milk was found to be in the form of casein, while in human milk
only 39 per cent was contained in the casein fraction, results
which are similar to those found in the literature.

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TABLE
IV
Amino Acid Composition

62

Amino Acids of Milk

DISCUSSION

To obtain a comparison of the relative amino acid composition

of the mixed proteins of cow and human milks the data must be
calculated on the basis of equal amounts of protein nitrogen.
For this reason Table IV has a column showing the amino acid
composition of 25.5 ml. of cows milk, which contain a quantity
of protein equal to that in 100 ml. of human milk.
In spite of the
differences between the two milks, in the distribution
of protein
nitrogen between casein and whey proteins, there is a surprising
similarity in contour of the amino acid composition of the protein
mixtures, with respect to histidine, arginine, lysine, tyrosine, and
tryptophane.
It is interesting to speculate whether the amino
acid pattern of whole milk protein is ideal for growth purposes
and therefore is repeated in the two species.
The similarity
in
composition of the milk protein mixtures of the two species is not
carried out with respect to cystine and methionine although the
amounts of sulfur furnished by the proteins of the two milks are
approximately
equivalent.

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The amino acid composition of the casein and whey protein

fractions of cow and human milks is shown in Table III.
The
differences in amino acid composition
are not great among the
protein fractions of the two milks.
The casein fraction of human
milk has a significantly
higher tryptophane
content and is higher
in cystine and lower in methionine than cow casein.
The whey
protein of human milks is significantly
higher in arginine and
cystine and lower in lysine and methionine than cow whey protein.
While these data differ somewhat from those of Plimmer and
Lowndes
(12) in minor details, the results are similar.
The
analyses of cows milk casein for basic amino acids agree with
values obtained by Vickery and White (13).
The quantities of each of the seven amino acids and nitrogen in
the mixed proteins of 100 ml. of each of the two kinds of milk
have been calculated from the data in Tables II and III and are
presented in Table IV.
It is apparent that all of the amino acids
with the exception of cystine occur in much higher amounts in
cows milk.
Approximately
equal quantities of cystine occur in
100 ml. of each of the two kinds of milk.

Beach, Bernstein,

Hoffman,

Teague, Macy

63

SUMMARY

BIBLIOGRAPHY

1. Grulee,
C. G., Sanford,
H. N., and Herron,
P. H., J. Am. Med. Assn.,
103, 735 (1934).
2. Grulee,
C. G., Sanford,
H. N., and Schwartz,
H., J. Am. Med. Assn.,
104, 1986 (1935).
3. Rowland,
S. J., J. Dairy
Research,
9, 42 (1938).
4. Van Slyke,
L. L., and Baker,
J. C., J. Bid.
Chem., 36, 127 (1918).
5. Block,
R. J., J. Biol. Chem., 106,457
(1934); 133, 67 (1940).
6. Folin,
O., and Mare+
A. D., J. Biol.
Chem., 83, 89 (1929).
7. Graff,
S., Maculla,
E., and Graff,
A. M., J. Biol. Chem., 121, 81 (1937).
8. Baernstein,
H. D., J. Biol. Chem., 1X,25
(1936).
9. Kassell,
B., and Brand,
E., J. BioZ. Chem.,
126, 145 (1938).
10. Gulewitsch,
W., 2. physiol.
Chem.,
27, 178 (1899).
11. Tristram,
G. R., Biochem.
J., 33, 1271 (1939).
12. Plimmer,
R. H. A., and Lowndes,
J., Biochem.
J.. 31, 1751 (1937).
13. Vickery,
H. B., and White,
A., J. BioZ. Chem . 103, 413 (1933).

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1. Samples of human and cows milk were examined for their

lipid and mineral content and the distribution
of total nitrogen
in casein, whey protein, and non-protein nitrogen.
2. Samples of casein and whey protein were prepared from cow
and human milks and analyzed for seven amino acids: histidine,
arginine, lysine, tyrosine, tryptophane,
cystine, and methionine.
3. In the proteins of cows milk the preponderance of sulfur is
in the form of methionine, with very little in the form of cystine,
while in the proteins of human milk the sulfur is about equally
divided between cystine and methionine.
4. The amounts of the seven amino acids contained in the
proteins of 100 ml. of human and cows milk, respectively,
were
calculated to be 12 and 59 mg. of histidine, 40 and 127 mg. of
arginine, 50 and 223 mg. of lysine, 50 and 197 mg. of tyrosine,
19 and 43 mg. of tryptophane,
20 and 23 mg. of cystine, and 18
and 104 mg. of methionine.

DISTRIBUTION OF NITROGEN AND

PROTEIN AMINO ACIDS IN HUMAN
AND IN COW'S MILK
Eliot F. Beach, Samuel S. Bernstein, Olive D.
Hoffman, D. Maxwell Teague and Icie G.
Macy
J. Biol. Chem. 1941, 139:57-63.

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