146 DIASTATIC ACTIVITY IN BLOOD AND URINE. 



from the incubator, and their contents poured into a series of larger test-tubes 

 about three parts full of cold tap-water. One or two drops of a N/10 solution 

 of iodine were added to the series of tubes (1-10) until a tube was obtained 

 where the blue or violet tint was not perceptible. The amount of diastase in 

 this tube was sufficient to digest all the starch present. The diastatic activity 

 of the urine was found empirically by dividing the number of c.c. of the starch 

 solution digested by the amount of urine in c.c, the number obtained often 

 being called Wohlgemuth units. With the above technique the range of 

 diastatic activities would be 20, 18, 16, 14, 12, 10, 8, 6, 4, 2 — an even series 

 of numbers not given by simple pipettings in previous methods. The range of 

 diastatic activities could be easily increased by the use of stronger solutions 

 of starch. For example, the use of a 0*4 per cent, solution of starch would 

 give a range of diastatic activities from 40, 38, 36, . . . 24, 22, but the 

 necessity for this was not common when the specimen of urine for examina- 

 tion was passed in the second two-hourly interval after the first meal of the 

 day. This period was chosen because it appeared to be a convenient time to 

 examine both the blood and urine, and because most of the diuresis due to the 

 intake of fluid with the meal had ceased. 



It was shown by Michaelis and Peckstein (1914) that the pH at which 

 diastase was most active varied with the salt content of the medium. Stafford 

 and Addis (1924) pointed out that in the technique of Dodds {loc. cit.) and 

 Sladden (1922) there seemed to be a danger of a disturbing variation in the 

 chloride and phosphate concentration respectively. This difficulty has been 

 got over by the above modification, in which there was always in each tube a 

 sufficiency of chloride and phosphate, and in which the urine with the enzyme 

 was diluted equally in all the tubes, although the concentration of the substrate 

 varied. This, however, would not prevent a reasonably accurate study of the 

 diastatic activity of different urines. 



In the present investigation the above modification was adopted, and was 

 used before the publication of the second and third papers referred to in the 

 preceding paragraph. Sladden {loc. cit.) considered that the addition of 

 phosphate buffer solutions tended to obscure the final readings. But in the 

 present inquiry no difficulty in reading the end-point was observed if small 

 tubes (4 c.c.) were used for incubation, and if these were emptied into larger 

 tubes (20-30 c.c.) containing cold water before the addition of iodine. 



Blood. — For the examination of the diastase in the blood, the method 

 described by G. Matthew Fyfe (1923) was employed with one or two small 

 modifications. 



Into one of two 100 c.c. Erlenmeyer flasks, 1'8 c.c. of Sorensen's buffer 

 solutions and 1 c.c. of 0*1 per cent, solution of Lintner's soluble starch in 0*9 

 per cent. NaCl are pipetted, and into the other 23*8 c.c. of a 15 per cent, solution 

 of sodium sulphate acidified by the addition of glacial acetic acid to the extent 

 of 01 per cent. Into each flask is introduced 0*2 c.c. blood by means of two 

 special pipettes which are thoroughly rinsed out in the clear fluid. The flask 

 containing starch is placed in a water-bath in an incubator for 30 minutes at 

 37° C. At the end of this period 21 c.c. of the acid sodium sulphate solution 

 are added immediately. The amount of sugar is estimated in both flasks by 

 MacLean's method (1919). 



