Abstract
Procedure for the determination of alkaline phosphatase activity in human serum by adaptation of the histochemical method for demonstrating phosphatase activity with alpha naphthyl phosphate as the substrate was described.
The alpha naphthol produced by enzymatic hydrolysis, was converted to a purple azo dye by coupling with Fast blue B salt. Although the azo dye was an insoluble product, it was dissolved with sodium lauryl sulfate solution, and then its optical density was measured colorimetrically.
It may be useful in the diagnosis of liver and bone diseases to determine on serum alkaline phosphatase by this method combined with the zymogram method. The procedure that is adopted are outlined as follows; Determinations are made in duplicates. With a 0.05 ml pipette. serum is transferred into each of two test tubes, which contain 3.0 ml of substrate buffer solution (pH 10.4). Two tubes containing 3.0 ml of the substrate buffer solu- tion without serum are used as a blank.(see to table 1)
These test tubes with or without serum are incubated at 37°C for 30 minutes. 1.0m1 of inhibitory solution is then added to each test tube which is alkalic condition. Then 0.05 ml of serum is added to each tube without serum. And 1.0ml of Fast blue B salt solution is added to all test tubes. The solution is prepared by dissolving 40mg of Fast blue B salt and 5g of sodium lauryl sulfate in 100m1 of citrate buffer (pH 4-8), immediately before use. Each Purple pigment solution obtained from the above reaction, is transferred into a colorimeter tube. Color density is then determined with a photoelectric colorimeter through a 540mμ filter.
