Determination of Serum Acid Phosphatase

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

Serum Acid Phosphatase Test

Acid phosphatase, An enzyme that acts to liberate phosphate under acidic conditions and is made in the liver, spleen, bone marrow, and prostate gland. Abnormally high serum levels of acid phosphatase may indicate infection, injury, or cancer of the prostate.


Serum acid phosphatase test method 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.



  1. Centrifuge
  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. Citrate buffer: 0.1M, pH 5. A: Citric acid (21.01g in 100ml) B: Sodium citrate (29.41g in 100ml)

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 solutions. This has a pH of 5.0.

4. Folin-Ciocalteau reagent: Prepared by mixing one volume of reagent and two volumes of water. Sodium carbonate solution, 15%: Dissolved 15g of anhydrous sodium carbonate in 100ml water.

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

6. Working standard solution: Diluted 10ml of stock standard to 100ml with water. This contains 100μg 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.

2. Added 0.2ml of sample and incubated further for exact 60 minutes. Removed and immediately added 1.8ml of diluted phenol reagent.

3. 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. Mixed and centrifuged.

4. To 4.0ml of supernatant added 2.0ml of 15% sodium carbonate. Took 4.0 ml of working standard solution and for blank taken 3.2 ml water and 0.8ml of phenol reagent.

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

6. 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.


Specific activity is expressed as m moles p-nitro-phenol released per min per mg protein. The activity of serum acid phosphatase was found to be ————– μmoles of phenol liberated per litre.

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