A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase

A continuous spectrophotometric method for measuring serum alkaline phosphatase activity is described. The effects of temperature, pH, substrate concentration, type and molarity of the buffer, sample size, cofactors, and inhibitors on the enzymatic hydrolysis of p-nitrophenyl phosphate were studied. The optimal conditions for assay of serum alkaline phosphatase at 300 were found to be 0.75 M 2-amino-2-methyl-1-propanol buffer, pH3o0 10.15, 4 mmole substrate, and 100 or less sample size. Studies of the factors affecting analytical precision-i.e., control of reaction temperature, of reagent manufacture, and of standardization-are discussed. The precision of this method was 2.3 % (relative standard deviation) on 10 within-day replicates and 5.0 % on day-to-day replicates spread over a 5-week period. The range of activity for 258 apparently healthy adult blood donors was 6-110 mU./ml. (International milliunits per milliliter), with a mean of 49 and a standard deviation of 14. TI HE MEASUREMENT of serum alkaline phosphatase * activity was one of tile first enzynie determinations to gain widespread acceptance ill clinical medicine. Today, in spite of the interest in ‘‘the newer enzyme tests, ” alkaline phosphatase continues to be one of the most useful determinations performed in the clinical chemistry laboratory. Over the years, man new phosphatase methods and modifications (2-6) have been introduced to shorten the assay time, to increase the sensitivity, or to better tile analytical precision and accuracy over the original Martlalld and Robison procedure (7). The present study was initiated because further improvement in each of these areas appeared to be possible by utiliZillg a continuous spectrophotometric technic