242
Phosphatase in Faeces
After 24 hours, the suspension was again shaken. A portion of it was
centrifuged for half an hour and the centrifugate was carefully filtered through
a Whatman filter No. 42 (7 cm.). The process of filtration was slow, about
5 ml. being collected in 15 to 20 minutes. A clear but slightly coloured filtrate
was obtained and the phosphatase determination in the filtrate was made by
the Bodansky [1932-33] method. The procedure may be briefly described as
follows :
In a 30 ml. test tube, 10 ml. of buffered substrate was placed. The subs-
trate is a 0.5 per cent solution of sodium-B-glycerophosphate adjusted to
pH 8.6 with the buffer salt monosodium diethylbarbiturate. The substrate
was brought to 37° C. by placing the tube for a few minutes in the incubator
bath and then 1 ml. of the faecal extract was added to it. The tube was then
returned immediately to the incubator and left there for exactly an hour. At
the end of this time, the tube was immersed in an iced bath and 9 ml. of 10
per cent trichloracetic acid was added to its contents and thoroughly mixed.
The solution in the tube was filtered through a Whatman filter No. 42 (7 cm.)
and the inorganic phosphorus in the filtrate was estimated by the method
described by Bodansky [1932-33].
Simultaneously with the incubated sample, a blank was run to estimate the
inorganic phosphorus in 1 ml. of the faecal extract. The difference in the
inorganic phosphorus content between the incubated sample and the blank
gave the amount of sodium-B-glycerophosphate hydrolysed by enzyme in
1 ml. extract.
Usually in 1 ml. of the extract of normal faeces, the inorganic phosphorus
content was negligible and its estimation in the blank was unnecessary. In
the extract of faeces voided during the period of starvation, however, a signifi-
cant amount of inorganic phosphorus was found. It was considered safe to
test the presence of inorganic phosphorus in the faecal extract before running
the blank. The filtrates collected for estimating inorganic phosphorus, both
from incubated and blank samples, were pale yellow in colour. This however,
did not seriously interfere with the reading of the final blue colour of the solu-
tion in a Leitz (Wetzlar) colorimeter, fitted with an appropriate colour filter.
To read the blue colour correctly in an ordinary colorimeter, it was found
necessary to eliminate the last trace of colouring matter from the trichloracetic
acid filtrate. The elimination was carried out by ultrafiltering the filtrate
from a semi-permeable collodion membrane by external pressure. The techni-
que of ultrafiltration adopted was that described by Duckworth [1935]. A
sufficient quantity of crystal clear ultrafiltrate could be collected in half an
hour. During this interval, no hydrolysis of the ester phosphorus in the dilute
acid medium was detected, nor did the ultrafiltration appreciably affect the
distribution of the phosphate ion on either side of the membrane.
Experiment 1.—The faecal phosphatase activity in normal metabolism
For a proper understanding of any physiological activity, it is essential
at the outset to study its trend under normal, standard conditions. With this
in view, an attempt was made in this experiment to estimate the units of faecal
phosphatase excreted and their range of variation from day to day when the
experimental animals were fed a ration which ensured, as tested by an actual