ESTIMATION OF DIASTASE IN BLOOD. 69 



leaden and to pale blue, the final disappearance of which marks the completion of 

 titration ; in some cases when the blue colour is very faint the end-point may 

 be rendered more distinct by the addition of a drop of 3 per cent, starch 

 solution. The amount of sodium thiosulphate used is noted and its exact 

 glucose equivalent ascertained from the glucose-thiosulphate table, or very 

 much better, from a plotted graph. The second flask is treated in exactly the 

 same manner except that starch must be added to complete the titration. 



Calculation. 



The result is calculated as the percentage of soluble starch transformed to 

 sugar (calculated as glucose) by the 0"2 c.c. blood employed. The amount of 

 starch used is 1 mg., and the difference between the sugar contents, measured 

 in fractions of a milligramme, of the two samples of 0'2 c.c. blood will be 

 equivalent to the amount of starch reduced to sugar. 



Suppose that, reading from the glucose-thiosulphate graph, the sugar con- 

 tent of the control preparation is 0'164 mg., while for the starch preparation it 

 is 0"259 mg. Since the amount of filtrate used corresponds to -f of 0*2 c.c. blood, 

 that amount of blood would contain 0'205mg. sugar in the one case and 0"323 mg. 

 sugar in the other. The difference, 0'118, is equivalent to the amount of starch 

 transformed to sugar by incubation with 0"2 c.c. blood, so that the diastatic 

 index in this case is 11'8. Allowance should, of course, be made for any slight 

 reducing action of the soluble starch. During the course of this investigation 

 the starch solutions were repeatedly tested for any such action, and in no case 

 was it found necessary to make correction. 



In view of the fact that glycolysis does not occur on incubation of 0"2 c.c. 

 blood under the conditions of the experiment, there appears to be no good 

 reason for running a control. The half hour during which the starch prepara- 

 tion is incubating may be very conveniently occupied in making a direct 

 estimation of freshly-drawn blood. 



DETAILS OF METHOD AND EESULTS. 



Accuracy. — It may seem superfluous to call attention to the need for 

 accuracy in this estimation, but in view of the fact that determinations are 

 made in milligrammes and fractions of milligrammes, perhaps a few points 

 which lend themselves to precision are worthy of note. Cleanliness and 

 attention to aseptic precautions should be maintained throughout. Burettes, 

 flasks and pipettes should be thoroughly cleaned. Antiformin or a solution of 

 potassium bichromate in sulphuric acid is a very useful help. Standardised 

 burettes and pipettes should be used. During the process of the boiling of the 

 blood filtrate and the copper solution, the manometer should be carefully 

 watched for any change in gas pressure and any necessary adjustment made. 



Preparation of the starch solution. — Lintner's soluble starch was used 

 throughout this investigation. The solvent was physiological salt solution 

 prepared with water, double (glass) distilled and practically neutral to rosolic 

 acid. The presence of the salt solution prevents haemolysis, and tends to 

 accelerate the action of diastase. A litre of 0"9 per cent. NaCl is prepared 

 with re-distilled water, and 0"2 grm. of soluble starch weighed out and 

 suspended in about 5 c.c. of the saline solution. Sufficient saline to make 

 200 c.c. is measured out and heated in a large flask almost to boiling-point. 



