Al-Muthana University

Clinical Chemistry Lab.

College of Medicine

Al-Muthana University
College of Medicine
Laboratory of Clinical Biochemistry

Number of Experiment: -5-

Name of Experiment: Determination of alkaline

phosphatase (ALP)

The purpose of Experiment:

Name:

Class:

Group:

The date of experiment work:

MSc. Ahmad Al Sarraf

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Al-Muthana University
Clinical Chemistry Lab.
College of Medicine

Principle of The Method

Kinetic photometric test, according to the International Federation of
Clinical Chemistry and Laboratory Medicines (IFCC).
Alkaline phosphatase (ALP) catalyses the transfer of the phosphate
group from
p-nitrophenylphosphate to 2-amino-2-methyl-1propanol (AMP), liberating p-nitrophenol according to the following
reaction:
p-nitrophenylphosphate + AMP
Phosphate

ALP

p-Nitrophenol +

The rate of p-Nitrophenol formation, measured photometrically, is

proportional to the catalytic concentration of alkaline phosphatase
present in the sample.
Clinical Significance
Alkaline phosphatase is an enzyme present in almost all weaves of the
organism, being particularly high in bone, liver, placenta, intestine and
kidney. Both increases and decreases of plasma ALP are of importance
clinically. Causes of increased plasma ALP: Pagets disease of bone,
obstructive liver disease, hepatitis, hepatotoxicity caused by drugs or
osteomalacia.
Causes of decreased plasma ALP: Cretinism and
vitamin C deficiency. Clinical diagnosis should not be made on a single
test result; it should integrate clinical and other laboratory data.

Reference Value
Serum
Type

Normal Value
Adult
3-13 KAU/dL
Children 6-25 KAU/Dl

ALP

MSc. Ahmad Al Sarraf

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Al-Muthana University
Clinical Chemistry Lab.
College of Medicine

Reagents
R1 (Buffer)

2-Amino-2-methyl-1-propanol
0,35
mol/L
Zinc sulfate
1
mmol/L
Magnesium acetate
2
mmol/L
Nhydroxyethylethylenediaminetriac
etic acid
2 mmol/L
(EDTA)
p-Nitrophenylphosphate
(pNPP)10mmol/L

R2 (Subtrate)

PREPARATION
Working reagent (WR):
Mix: 4 vol. (R1) Buffer + 1 vol. (R2) Substrate
Stability: 21 days at 2-8C or 5 days at room temperature (15-25C).

Procedure
1. Assay condition
Wave length 405 nm
Cuvette 1 cm
Temperature 15-25 C
2. Adjust the instrument to zero with distilled water
3. Pipette into a cuvette
WR (mL)
Sample (L)
MSc. Ahmad Al Sarraf

1.0
20
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Al-Muthana University
Clinical Chemistry Lab.
College of Medicine
4. Mix and incubate exactly for 1 min. at 15-25 C
5. Read initial absorbance (A) of the sample, start the
stopwatch and
read absorbances at 1 minute intervals thereafter for 3
minutes.
6. Calculate the difference between absorbances and the
average absorbance differences per minute (A/min).

Calculations

ALP Activity (U/L) = A/min x 2764

Units:
One international unit (IU) is the amount of enzyme that transforms 1
mol of substrate per minute, in standard conditions. The
concentration is expressed in units per litre of sample (U/L).

Discussions

Q1/

Q2/
MSc. Ahmad Al Sarraf

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Al-Muthana University
Clinical Chemistry Lab.
College of Medicine

Q3/

Q4/

Q5/

MSc. Ahmad Al Sarraf

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