Estimation of alkaline phosphatase in serum
Introduction
Phosphatase are enzymes which catalyze the splitting of phosphoric acid from certain mono phosphoric esters such as (glycerophosphates and phenyl phosphates)
Alkaline phosphatase with a maximum activity at about PH 10
Principle
Alkaline phosphatase in serum acts on the substrate disodium phenyl phosphate buffered at PH 10 at 37ᵒ to liberate phenol
This phenol condenses with 4- amino antipyrine in the presence of alkaline oxidizing agent potassium ferricyanide to give red colored complex which is measured at 520 nm
Normal range
Alkaline phosphatase : 21 – 92 IU / L or 3 – 13 KA units
Note : one IU of ALP activity = 7.13 KA units
Reagents
Buffered substrate PH 10
Phenol std
Chromogen reagent 4 – amino antipyrine 0.6 % , potassium ferricyanide 2.4 % Procedure
| Reagent | Blank | Standard | Test |
| Buffered substrate PH 10 | 0.5 ml | 0.5 ml | 0.5 ml |
| D / W | 1.55 ml | 1.55 ml | 1.55 ml |
| Mix well and incubate at 37ᵒ for 15 min | |||
| Serum | – | – | 50µ |
| Phenol STD | – | 50µ | – |
| Mix well and incubate at 37ᵒ for 15 min | |||
| Colored reagent | 1 ml | 1 ml | 1 ml |
| Mix well and read at 520 nm |
Calculation
ALP activity (IU/L) = Serum ALP levels are increased in:
Liver diseases: enzyme is produced by the hepatocytes canaliculi and hence marked increase is seen in condition of obstruction of bile flow
Extrahepatic biliary obstruction due to : stones in bile duct , cancer of head of pancreas
Intrahepatic biliary obstruction due to : viral / drug induced hepatitis or malignancy
:Bone diseases: when osteoblastic activity in bone formation is take place like in
Rickets : values of ALP more than 100 KA units can be seen
Osteomalacia : less rise than in rickets
Paget’s disease : very high values seen
Others: fanconi’s syndrome, hyperparathyroidism
:Serum ALP levels are decreased in
Severe anemia
Malnutrition