232             Phosphorus in Milk, I

(2) Labile phosphoric ester—that fraction spon-
taneously hydrolysing on standing with the molyb-
date reagent for half an hour at room temperature.

(3) Hexose-monophosphate phosphorus that
would appear in this portion as the difference
between the total barium-nonprecipitable phos-
phorus and the labile phosphoric ester phosphorus.

The original barium precipitate (washed) was
dissolved in 5 to 8 drops of N HCl and barium
was removed with saturated sodium sulphate solu-
tion. The solution after treatment with sodium
sulphate solution was filtered and made up to
50 c.c. Aliquots of this filtrate were used for the
determination of:—

(1) total barium-precipitable phosphorus,

(2) inorganic phosphorus,

(3) inorganic phosphorus after fractional hy-

drolysis in the boiling water-bath with
normal hydrochloric acid:
(a) the 7 min. value giving the adenyl-

pyrophosphate phosphorus,
(b) the difference between the 7 and 100
min. value giving the hexose-diphos-
phate phosphorus.
Before determination of phosphorus in the frac-
tions (a) and (b) the solution was neutralized with
normal caustic soda. Our attention has been
drawn to a study of phosphorus distribution in
ass's milk by Anantakrishnan [1941]. The author
also carried out deproteinization under ice-cold
conditions and the acid soluble phosphorus was
fractionated into barium insoluble and soluble
fractions. No reference is, however, made to
Needham's work.

Working with cow's milk—10 c.c. samples being
used for extraction with trichloracetic acid—
adenylpyrophosphate, hexose-diphosphate and
hexose-monophosphate were found to be practi-
cally absent. The whole of the Ba-nonprecipita
ble phosphorus was found to consist of labile
phosphoric ester. The inorganic phosphorus in
the trichloracetic acid filtrate was wholly precipit-
ated by barium. For the detection of hexose di-
phosphate in cow's milk the entire 10 c.c. portion
of the milk was also worked up—the trichloracetic
acid extract was brought to pH 8, precipitated with
25 per cent barium acetate solution, the Ba-preci-
pitate dissolved in very dil. HCl and the barium
removed with sat. Na₂ SO₄. Treated with phenyl
hydrazine the filtrate gave only a minute amount
of precipitate showing that only traces of hexose
diphosphate are present in cow's milk.

Some properties of the labile phosphoric ester
were studied and the following results were
obtained:—

1. When alcohol was added at room temperature
to an aqueous solution of the barium salt an
amorphous precipitate was formed.

2. With AgNO₃ alone, in neutral or slightly acid
solution no precipitate appeared but a precipitate
(colourless at first then darkening when exposed
to light) was obtained with silver nitrate and
barium hydroxide.

3. A few c.c. of the filtrate containing labile
phosphate were taken, copper acetate was added
and then sufficient sodium hydroxide to make the
mixture very slightly alkaline. The precipitate
was filtered off. The precipitate was extracted
with a few c.c. of 05 N NaOH. The extract gave
the test for the presence of labile phosphate.

The above tests suggest that the labile phospho-
ric ester is likely to be creatine phosphate.
The results are indicated in Tables I—V.

                                                                    TABLE I

                                            Phosphorus distribution in cow's milk
                                                P given as mg. P per 100 c.c. milk

            In the mean values the figures within brackets represent the various phosphorus fractions
                                        expressed as percentages of total phosphorus.