Determination of Serum Alkaline Phosphatase

In this determination of serum alkaline phosphatase post we have briefly explained about determination of serum alkaline phosphatase practical principle, requirements, procedure, and calculation.

Determination of Serum Alkaline Phosphatase

Alkaline phosphatase (ALP) is an enzyme that is present in many parts of the body, but it is primarily found in the liver, bones, intestine, and kidneys. Alkaline phosphatase testing measures the amount of this enzyme in the blood. Abnormal levels of ALP can be caused by liver problems and other types of health problems.


Determination of serum alkaline phosphatase used was that of King and Armstrong in which disodium phenylphosphate is hydrolyzed with the liberation of phenol and inorganic phosphate. The liberated phenol is measured at 700nm with Folin-Ciocalteau reagent.

Materials Required



2. Incubator

3. Spectrophotometer

4. Test tubes

5. Test tubes stand


1. Chloroform

2. Citric Acid

3. Disodium Phenylphosphate

4. Phenol

5. Sodium Carbonate Solution

6. Sodium Citrate


1. Sodium carbonate-Sodium bicarbonate buffer, 100mmol/L: Dissolved 6.36g anhydrous sodium carbonate and 3.36g sodium bicarbonate in water and made to a litre.

2. Disodium phenyl phosphate, 100mmol/L: Dissolved 2.18g of disodium phenylphosphate in distilled water and heated to boil, cooled and made to a litre. Added 1.0ml of chloroform and stored in the refrigerator.

3. Buffered substrate: Prepared by mixing equal volume of the above two solution. This has a pH of 10.

4. Folin-Ciocalteau reagent: Prepared by mixing one volume of reagent and two volumes of water.

5. Sodium carbonate solution, 15%: Dissolved 15g of anhydrous sodium carbonate in 100ml water.

6. Standard phenol solution, 1g/L: Dissolved 1g pure crystalline phenol in 100mmol/L HCl and made to a litre with acid.

7. Working standard solution: Added 100ml diluted phenol reagent to 5.0ml of stock standard and diluted to 500ml with water. This contains 10μg of phenol/ml.


Homogenize 1 g fresh tissue in 10 ml of ice-cold 50 mM citrate buffer (pH 5.3) in a pre-chilled pestle and mortar. Filter through four layers of cheese cloth. Centrifuge the filtrate at 10,000 g for 10 min. Use the supernatant as enzyme source.


1. Pipetted out 4.0ml of buffered substrate into a test tube and incubated at 37˚C for 5 minutes. Added 0.2ml of sample and incubated further for exact 15 minutes.

2. Removed and immediately added 1.8ml of diluted phenol reagent. At the same time, set up a control containing 4.0ml buffered substrate and 0.2ml of sample to which 1.8ml of phenol reagent was added immediately.

3. Mixed well and centrifuged. To 4.0ml of supernatant added 2.0ml of 15% sodium carbonate.

4. Took 4.0ml of working standard solution and for blank taken 3.2ml water and 0.8ml of phenol reagent.

5. Then added 2.0ml of sodium carbonate. Incubated all the tubes at 37˚C for 15 minutes. Read the color developed at 700nm.

A standard graph was drawn by plotting the concentration on x axis and the optical density on y axis. From this, the concentration of acid phosphatase in the given sample solution was calculated.


The activity of serum alkaline phosphatase was found to be ———— μmoles of phenol liberated per litre.

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