PHOSPHORUS IN MILK
I. THE PHOSPHORUS PARTITION IN THE MILK OF COW, GOAT, SHEEP AND
HUMAN BEINGS
By K. P. BASU and K. P. MUKHERJEE, Biochemical Laboratory, Dacca University
(Received for publication on 19 January 1943)
THE importance of phosphorus in nutrition is
well known. Investigators have, therefore, from
time to time examined the nature of phosphorus
compounds in cow's milk which is the most im-
portant food for infants and growing children.
References to earlier investigations are given by
Graham and Kay [1933]. In India some work on
the compounds of phosphorus in buffalo milk has
been done by Acharya and Devadatta [1940, 1941].
Lack of a rapid & accurate technique has hampered
work in this line.
For removing proteins & fats from milk, we
have, following the procedure of Sanders [1931],
added to it trichloracetic acid (upto 8 per cent
strength). This procedure was also adopted by
Graham and Kay [1933] in their investigations on
cow's milk. Although these investigators have
confirmed the presence of organic phosphorus
compounds, they could not determine their na-
ture. Attempts have been made by Lang and
Miethke [1932], Hochheimer [1932-33] and
Bomskov [1932] to partition the phosphoric
esters into fractions on the lines used by Lohman
[1928] for the partition of the organic phosphorus
compounds in muscle, but the findings of the
investigators are by no means in agreement.
We investigated the phosphorus distribution of
milks of different animals—cow, goat, sheep,
buffalo and also of human beings—in different
fractions. The experimental method was similar
to that applied by Graham and Kay, with some
modifications, and special attention was paid to
the trichloracetic acid soluble fraction which was
further worked up according to the method adopt-
ed by Needham [1937] for his studies on the
nature of ester phosphorus in the chick embryo.
Graham and Kay [1933] in their experiments
determined :—
(1) the total phosphorus in milk,
(2) the trichloracetic acid soluble phosphorus,
in which fraction the following estima-
tions were made :—
(a) the total phosphorus,
(b) the inorganic phosphorus,
(c) the ester phosphorus as the difference
of (a) & (b).
(3) total acid-insoluble P as the difference
between the total P and the total acid-
quantitative soluble P. This equals the
sum of lipin P [3 (a)] and casein P [3 (b)].
3(a) the lipin P by ether-alcohol extraction
of the original milk.
3(b) the casein P as the difference between
3 and 3(a).
The fraction (2) was further investigated by us, as
indicated in the experimental part, according to
the technique of Needham. It was fractionated
into barium insoluble and soluble fractions. The
barium insoluble fraction was worked up for the
detection and estimation of adenyl pyrophosphate
and hexose-diphosphate phosphorus and the bari-
um soluble fraction for labile phosphoric ester and
hexose monophosphate.
EXPERIMENTAL
Instead of using, as was done by Graham and Kay,
the trichloracetic acid at the ordinary temperature
we have first cooled the trichloracetic acid solution
to 4ºC. as the heat generated due to the addition
of trichloracetic acid to the diluted milk might
cause the hydrolysis of ester phosphorus and possi-
bly also of the precipitated lipin P. The trichlor-
acetic acid extract was then separated into (1) Ba-
precipitable and (2) the filtrate fractions according
to the technique of Needham [1937]. The two
fractions were further worked up as indicated
below. The colorimetric method of Briggs [1924]
was used for estimating inorganic phosphate.
Ten c.c. sample of milk was pipetted into a 100
c.c. measuring flask, 50 c.c. of distilled water were
added and 25 per cent trichloracetic acid solution
(cold) were added upto the mark. The flask was
now shaken thoroughly and allowed to stand for
5 minutes. The liquid was now filtered and 20 c.c.
of this filtered solution were taken into a centri-
fuge tube capable of holding 100 c.c. The liquid in
the centrifuge tube was neutralized and the pH
was adjusted to 7.8—8.0. 1.5 c.c. of 25 per cent
barium acetate were added to the neutralized
solution. After standing some 20 minutes in the
refrigerator the precipitate was centrifuged and
washed twice in the centrifuge with 1 per cent
barium acetate. The clear supernatant fluid was
separated from the precipitate very carefully
and poured into another centrifuge tube and
excess barium was precipitated by about 1.5 c.c. of
saturated sodium sulphate solution. The solution
after treatment with Na 2 So 4 solution was filtered
through a quantitative filter paper and the filtrate
was quantitatively collected in a 50 c.c. measuring
flask. The volume of the filtrate was adjusted upto
the mark. Aliquots of the filtrate after removal
of excess of barium acetate with sodium sulphate
were used for the determination of—
(1) Total barium-nonprecipitable phosphorus.
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