Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

Phenyl phosphate!

ALP,pH 10

PhenolþPhosphate

Phenolþ4-aminoantipyrine!

Potassium ferricyanide

Orangeredcomplex

26.4 Reagents........................................

1.Disodium phenyl phosphate (0.1 M): Dissolve 2.18 g of disodium phenyl
phosphate in distilled water, and makefinal volume to 100 ml. Store at 4C.
2.Na 2 CO 3 -NaHCO 3 buffer (pH 10.0): Prepare 100 ml 0.1 M Na 2 CO 3 -NaHCO 3
buffer, (pH 10) by adding 51 ml 0.1 M Na 2 CO 3 to 49 ml 0.1 M NaHCO 3 , and
adjust pH if required. Also prepare 0.5 N bicarbonate buffer separately.
3.0.5 N NaOH.
4.4-aminoantipyrine (0.6%) aqueous solution. Store at 4C.
5.Potassium ferricyanide (2.4%). Store at 4C.
6.Stock phenol standard (100 mg %) in 0.1 N NaOH. Store at 4C.
7.Working phenol standard (10 mg %).Prepare in distilled water.

28.5 Procedure

1. Take a set of test tubes in duplicate and mark them as blank, standard (S 1 to S 6 ),
   control (C), and test (T).


2. Add working phenol standard according to table in tubes S 1 to S6.Add 1 ml each
   of carbonate-bicarbonate buffer and substrate in all tubes. Incubate for 5 min.


3. Then add 0.1 ml serum in test sample and add 1 ml distilled water in blank.
   Incubate for 15 min.


4. Then add 1 ml NaOH to stop reaction followed by 0.1 ml serum in control tube,
   1 ml of bicarbonate, 1 ml 4-aminoantipyrine, and 1 ml potassium ferricyanide.

Reagents Blank S 1 S 2 S 3 S 4 S 5 S 6 TC

Working phenol standard (ml) – 0.1 0.2 0.4 0.6 0.8 1 ––

Amount of phenol in added

volume (μg)

• 1246810 ––

Carbonate/bicarbonate buffer (ml) 1 0.9 0.8 0.6 0.4 0.2 – 0.9 0.9

Disodium phenyl phosphate (ml) – 11111111

Incubate at 37C for 5 min

Serum (ml) –––––––0.1 –

Distilled water (ml) 1 ––––––––

Mix well and incubate at 37C for 15 min

0.5 N NaOH (ml) 1 11111111

Serum (ml) ––––––––0.1

0.5 N bicarbonate buffer (ml) 1 11111111

4-aminoantipyrine (ml) 1 11111111

(continued)

108 26 To Estimate the Activity of Alkaline Phosphatase in Serum