Alkaline Phosphatase Assay in Serum

Alkaline Phosphatase Assay in Serum

Clinical enzymology

 Various diseases are diagnosed by determining enzyme

activity in body fluids and tissue extracts.

 Blood

 Urine

 Body fluids

 Tissues

Clinical enzymology-materials

 In normal conditions,

 Enzyme activity is stable in healthy people.

 The activities of some enzymes may show temporary increases depending on the conditions.

 Such as temporary increase of creatine kinase after severe muscle exercises or temporary increase of intestinal

isozyme of alkaline phosphatase after eating.

Increase in plasma enzyme activity

 Necrosis or severe destruction of cells

 When cell regeneration is accelerated (osteoblastic activity)

 Increased enzyme concentration in the cell

 Channel blockage  

Phosphatases

 They react with organic phosphate esters.

 They hydrolyze phosphoester bonds.

 They release the inorganic phosphate ion.

 They are named according to the pH where they show optimum

effectiveness. (Acid / alkaline) 

Alkaline phosphatases (ALPs)

 Maximum activity between pH 9-10.5    

 These enzymes are found in the liver, bones,

placenta and intestines.

The conditions which cause a rise in alkaline phosphatase activity

 Bone diseases (Paget's disease, hyperparathyroidism, rickets, osteomalacia)

 Liver diseases

 Cancer

 Pregnancy

 Fatty food intake

The conditions which cause a decrease in alkaline phosphatase activity

 Hypophosphatasia is a genetic condition that

causes abnormal development of the bones and teeth.

 Hypophosphatasia excess amount of phosphoryl

ethanolamine in urine

The determination of alkaline phosphatases activity in serum (King Armstrong Method)

 Principle: Disodium phenylphosphate is hydrolyzed by the enzyme phosphatase and the released phenol is detected.

 1 King Armstrong Unit for alkaline phosphatase:

 It is the amount of phosphatase that releases 1 mg phenol from

the specific substrate (pH 10) at 37 °C in 15 minutes.

Experimental Procedure-1

1.tube (Control tube) 2.tube (Sample tube)

Buffered alcaline substrate (10 ml)

Incubation in water bath at 37⁰C

Take the sample tube out of the water bath, add 1 ml serum and mix well.

Then put the tube in water bath, again.

Incubation for 15 minutes (Take both tubes out of the water bath)

Experimental procedure-2

1.tube (Control tube) 2.tube (Sample tube) +1 ml serum -

+5 ml diluted phenol reagent +5 ml diluted phenol reagent

Experimental procedure-3

3.Tube 4.tube (standart) (blank)

1 ml diluted phenol standart 2 ml diluted phenol reagent 3,2 ml distilled water 4,2 ml distilled water

2 ml diluted phenol reagent

Experimental procedure-4

SAMPLE CONTROL STANDART BLANK

Take 2ml from each prepared tubes +8 ml %6 Na

CO

Optical density measurement at 660 nm

Calculation

OD sample - OD control K.A.U = --- x 25 OD standard

The King-Armstrong unit (K.A.U) of alkaline phosphatase is defined as the

amount of phosphatase that releases 1 mg of phenol from substrate (pH 10) at 37°C in 15 minutes.

Normal values for alkalen phosphatase in King-Armstrong method: 4.5-

9.5 K.A.U

References

 Ankara University Faculty of Pharmacy Biochemistry Practice Book-2004

 Practical Biochemistry (2015). Aljebory, A., And Alsalman, A.

 A Laboratory Text Book of Biochemistry, Molecular Biology and Microbiology (2014)

 Lehninger Principles of Biochemistry- 5th Edition (2008)

 King, E. J., Abul-Fadl, M. A. M., & Walker, P. G. (1951). King-Armstrong

Phosphatase Estimation by the Determination of Liberated Phosphate. Journal of

Clinical Pathology, 4(1), 85–91.doi:10.1136/jcp.4.1.85