What is Alkaline Phosphatase Enzymes?
ALP is a hydrolase enzyme responsible for
removing phosphate groups from many types of
molecules, including nucleotides and proteins.
In humans, alkaline phosphatase is present in all
tissues throughout the entire body, but is
particularly concentrated in liver, bile duct, kidney,
bone.
1- Hepatobiliary Disease: bile duct obstruction
2- Bone Diseases:
Paget’s Disease:
 This is bone disease in which there is marked
proliferation of blood vessels within the bone which
actually destroys the bone.
Bone Tumors:
 primary tumors of bone (i.e., cancer of the bone itself)
which destroy bone without causing new bone formation
are called osteolytic (lysis = dissolve) tumors.



Osteomalacia : (malacia = softening),
this is a bone disease in which there is an
abundant amount of protein matrix but an
inadequate
amount
of
calcium
hydroxyphosphate(hydroxyapatite) deposited
in the matrix.
In children it is known as rickets.
In such children the ALP level is usually
several times higher the normal value for their
age group. In adults osteomalacia is usually
due to the diseases of small bowel, such as
sprue, which lead to failure to absorb sufficient
calcium.
Bone Fractures: ALP may be slightly elevated
as the fracture heals.
Paget's disease
Rickets:
Before treatment
(Bowleg deformity)
and 2 years after
treatment with
calcium
6
Requirements
Requirements:
Automatic pipettes
Tips
Cuvettes
 Spectrophotometer
 Reagent
Sample:
Serum or heparin plasma
Working Reagent
Reagent 1
Reagent 2
Buffer Solution:
Diethanolamine buffer, pH 10.35
1.25 mol/L
Magnesium Chloride
0.625 mmol/L
Substrate:
p-Nitrophenyl-phosphate
55 mmol/L
Reagent preparations and Stability:
Pipette 2 ml from reagent 2 (SUB) into one bottle of buffer solution. Mix
throughly. The working reagent is stable for 4 weeks at 2-8o C, for 5 weeks
at 15-25o C.
Preparation of Cuvettes
Procedure
Blank
(only air or distilled water)
Sample
(Reagent+Serum)
Principle :
ALP catalyzes the hydrolysis of 4nitrophenyl phosphate, forming phosphate
and free 4-nitrophenol, which in dilute acid
solutions is colorless, as follows:
Under alkaline conditions, 4-nitrophenol is
converted to the 4-nitrophenoxide ion,
which is an intense yellow color.
P-Nitrophenylphosphate + H2O
ALP
acid media
Phosphate + p-nitrophenol (colorless)
alkaline media
4-Nitrophenoxide (yellow)
Sample #1 Sample # 2
ALP reagent (ml)ِِِ
2.0
2.0
Sample (s) (µl)
20
20
Mix and wait 1 minute.
Zero the spectrophotometer at 405 nm against air or with
distilled water.
Measure the absorbance increase every minute for 1-3
minutes.
Record your results for further calculations.
Calculate ΔA / min.
ALP activity (U/L)
=
ΔA / min.X 2757
25 0C
U/L
Children
Up to 15
Children
Up to 17
women
Men
Up to 400
30 0C
U/L
37 0C
U/L
Up to 488 Up to 644
Up to 300
Up to 366
Up to 483
40 –190
49-232
64 – 306
50 – 190
61 – 232
80 – 306