A Continuous Spectrophotometric Method for Measuring the Activity of Serum Alkaline Phosphatase

TLDR

The study presents a continuous spectrophotometric assay for measuring serum alkaline phosphatase activity. The assay was optimized by systematically varying temperature, pH, substrate concentration, buffer composition, sample volume, cofactors, and inhibitors, and by controlling reaction temperature, reagent quality, and standardization. Under optimal conditions (30 °C, 0.75 M 2‑amino‑2‑methyl‑1‑propanol buffer, pH 10.15, 4 mM substrate, 100 µl sample), the assay achieved 2.3 % intra‑day and 5.0 % inter‑day precision, with a healthy donor activity range of 6–110 mU ml⁻¹ (mean 49 mU ml⁻¹).

Abstract

Abstract 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 30° were found to be 0.75 M 2-amino-2-methyl-1-propanol buffer, pH30° 10.15, 4 mmole substrate, and 100 µl. 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.