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
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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 %
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Procedure
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
|
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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
|
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Calculation
- ALP activity (IU/L) =
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Serum ALP levels are increased in:
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