Ammonium thymolphthalein monophosphate as a new substrate for alkaline and acid phosphatase determinations in serum.

A new substrate, ammonium thymolphthalein monophosphate, is proposed for use in alkaline and acid phosphatase determinations. The ammonium salt shares the advantages of the sodium salt, but is a more stable substrate and results in more stable working reagents. The substrate is buffered at ph 10.1 in diethanolamine for determining alkaline phosphatase activity in 5 mm, and at pH 5.9 in citrate buffer for determining acid phosphatase activity in 30 mm. The substrate is relatively specific for bone and liver alkaline phosphatase and prostatic acid phosphatase. Coefficients of variation were 2.5% and 6.6% for alkaline and acid phosphatase, respectively. Numerous substrates have been proposed for alkaline and acid phosphatase determinations (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1 and EC 3.1.3.2, respectively) and, of these, p-nitrophenyl phosphate (1), phenolphthalein monophosphate (2), a-naphthyl phosphate (3), and thymolphthalein monophosphate (4) are commonly used. p-Nitrophenyl phosphate is unstable, even at freezer temperature (5), and the absorption of p-nitrophenol at 404 nm is very close to that of bilirubin. Phenolphthalein monophosphate is also relatively unstable (2), and the absorption maximum of phenolphthalein at 550 nm is close to that of hemoglobin. Likewise, the coupled product of a-naphthol and tetrazotized o-dianisidine absorbs at 530 nm, close to the Amax of hemoglobin. Thymolphthalein, however, has an absorption maximum at 590 nm and, consequently, bilirubin and hemoglobin pose virtually no problem in phosphatase assay with thymolphthalein monophosphate. Thymolphthalein monophosphate is currently commercially available as the magnesium salt (Eastman Organic Chemicals, Rochester, N. Y. 14650), the sodium salt (Worthington Biochemical Corp., Freehold, N. J. 07728), and the ammonium salt (Kiess Instruments, Inc., Miami, Fla. 33156). Magnesium thymolphthalein monophosphate is very poorly soluble and requires the addition of citrate or other chelating compounds, a fundamentally undesirable approach for alkaline phosphatase determinations. Sodium thymolphthalein monophosphate is highly soluble, but is stable only under refrigeration,