Measurement of Amylase Activity*

The Nelson photoelectric method for the determination of glucose in biological fluids (1) has proved to be the method of choice in this laboratory and in many others, owing to its reproducibility, stability of color, and excellent agreement with the Beer-Lambert law. Nelson, however, states that the method is unsuitable for the determination of diastatic activity. Since it is convenient and economical for a laboratory running large numbers of glucose and amylase determinations to use the same reagents for reduction and color development, it seemed advisable to attempt to modify the amylase procedure to this end. The difficulty in using the substrate of the Somogyi amylase method (2) with the Nelson reagents arises from the clouding of the final colored solution by the presence of unhydrolyzed starch. It was reasoned that, if a smaller amount of starch were present, the final solution might be clear. This was realized by diluting the Somogyi substrate after incubation and running the diluted solution through the Nelson procedure. Thus, while the original relative concentrations of substrate and serum which Somogyi found to be optimum were preserved, the final colored solutions were clear in all cases, including the starch blank, and were suitable for photoelectric reading. This final concentration of starch may be attained in a slightly different manner by using only 0.3 per cent starch and 0.2 ml. of serum and omitting the 1:5 dilution after incubation. It is interesting to note in this regard that “micromethods” in current use (2) require the employment of 1 ml. of serum.