Problem Clinical chemistry determination of serum alkaline phosphatase activity

The activity of alkaline phosphatase in serum is elevated above normal in a variety of different bone diseases, as well as biliary obstruction and some other liver diseases. Measurement of alkaline phosphatase activity in serum can thus give useful information about these conditions. It is especially useful for diagnosis of preclinical rickets and osteomalacia (section 11.3.4).

According to the literature, the range of alkaline phosphatase activity in serum from healthy adults is (mean ± sd) 75 ± 12 units/L. The reference range (that which

Table 2.6 The absorbance of p-nitrophenylate atpH 12

p-Nitrophenylate (mmol/L)

Absorbance at 405 nm











is considered 'normal') is ± 2 sd around the mean; values outside this range are considered abnormal.

The activity of alkaline phosphatase is measured by hydrolysis of ^-nitrophenyl phosphate to yield ^-nitrophenol and free phosphate. At alkaline pH, ^-nitrophenol dissociates; the ^-nitrophenylate ion has a strong yellow colour, with maximum absorbance at 405 nm.

The activity of alkaline phosphatase in a serum sample was determined by incubating 0.1 mL of serum with 0.2 mL of^-nitrophenyl phosphate at 14 mmol/L in 2.7 mL of buffer at pH 10.4 at 30 °C for 10 min. The reaction was stopped by addition of 3 mL of 0.2 mol/L sodium hydroxide, which both denatures the enzyme and also raises the pH of the incubation mixture to 12. The absorbance of the final reaction mixture was then determined at 405 nm, using a cuvette with a 1-cm light path. The result was a reading of 0.95 absorbance units.

Table 2.6 shows the absorbance at 405 nm of^-nitrophenylate in buffer at pH 12, using a cuvette with a 1-cm light path.

Was the activity of alkaline phosphatase in the serum sample within the reference range?


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