Veterinary Clinical Biochemistry and Laboratory Diagnosis-II

Estimation of Alkaline Phosphatase

ESTIMATION OF SERUM ALKALINE PHOSPHATASE (p-NPP method – Kinetic mode)

Principle:

• Alkaline phosphatase (ALP) hydrolyzes p-nitrophenyl phosphate (p-NPP) into p-nitrophenol and inorganic phosphate at pH 10.0. The p-nitrophenol is a yellow compound, which absorbs light at 405 nm. The rate of increase in absorbance at 405 nm is proportional to alkaline phosphatase activity in specimen.

                                                                          _ALP

• p-nitrophenyl phosphate + H₂O → p-nitrophenol + inorganic phosphate

Reagents :

• Reagent 1 (Diethonalamine buffer, Magnesium chloride)
• Reagent 2 (p-nitrophenyl phosphate)

Preparation of working reagent:

• Mix four Volumes of Reagent 1 with one volume of Reagent 2. This working reagent is stable for 30 days at 2 – 8 ⁰ C.

Procedure:

• Mark one clean test tube as (T):

S.No	Reagents	T
1.	Working Reagent	1000 m l
2.	Serum	20 m l
3.	Distilled Water	2000 m l

• Mix well and incubate for 1 minute at 37^oC. Read the change in absorbance per minute (Δ OD / min.) during 3 minutes at 405 nm, setting zero with D.W.

Calculation:

• Serum ALP activity (U/L) = (Δ OD / min.) x 2750 x 3

Result:

• The ALP activity of the given serum = U/L.

Clinical Significance:

• Serum ALP estimation is useful in diagnosis of hepatobiliary diseases and also diseases associated with increased osteoblastic activity. It is elevated in osteomalacia and rickets. Very high levels are found in patients with bone cancer.
• Elevated levels are also found in intrahepatic obstruction due to stone or spasm and also in extrahepatic obstruction (cholestasis) where the levels are still higher.