SUGAR OF BLOOD. 101
alcohol was found to be the best extraction medium; this strength
extracts all the sugar likely to be present and is sufficiently strong to
coagulate all the remaining proteins. Until a neutral sample of sodium
tungstate was obtained care was taken to adjust the reaction in the way
recommended by Folin(6). Neutral samples were finally obtained from
Messrs Harrington.
The method finally adopted is as follows: The blood is precipitated
according to the method of Folin and Wu. Filtration is effected by
filtering in succession through two funnels. In the upper one is placed
a coarsely grained paper, in the lower a Whatman paper No. 40. The
double filtration removes all of the precipitate which it is possible to
remove by filtration at this stage. The filtrate is concentrated almost to
dryness in vacuo, not allowing the temperature to rise above 40° C.
Caprylic alcohol is used to prevent foaming. A volume of 85 p.c. alcohol
equal to the original volume of blood is then added to the distillation
flask, and an immediate precipitation of the remaining proteins and
tungstate takes place. The alcohol is allowed to stand in contact with the
residue thus formed at 40° C. for 20 minutes with occasional shaking, so
as to ensure complete extraction of the sugar from the solid mass. The
alcohol is filtered into a small distillation flask and a further quantity of
alcohol equal to half the original amount is added to the first flask in
order to complete the extraction. This is allowed to stand for ten minutes
with shaking as before. The combined filtrates are concentrated in vacuo
at 40° C. until almost dry. The residue is taken up in a small quantity of
water, the amount depending upon the original volume of blood used,
and then filtered (usually 20 to 30 c.c.). The filtrate is perfectly clear
and suitable for polarimetric determinations. A one decimetre tube is
usually employed owing to the amount of fluid available being small,
and a considerable amount being required for the copper reducing
estimation.
The final filtrate was tested by the protein and biuret colour reactions;
negative results were obtained. A determination of the nitrogen showed
that it was no greater than could be accounted for by the extraction by
the alcohol of urea and similar substances. In order that an accurate
comparison may be made between the polarimetric and copper reducing
values of the blood sugar, it is necessary that every trace of protein be
removed; if this condition be not obtained, not only are the optical
properties of the solution liable to be affected to an unknown and variable
extent in each experiment, but in determining the copper reducing value
of the sugar, there is a danger of traces of protein giving rise to substances