1. Phosphatases
Lab # 7

2. Phosphatases Description Types of phosphatases They are hydrolases
They catalyze the splitting of phosphoric acid from mono-phosphate esters
Types of phosphatases
1- Alkaline phosphatase → max. activity at pH10
2- Acid phosphatase → max. activity at pH5

3. Alkaline Phosphatase (ALP)
Tissues sources
Highest conc. in bone, liver, intestine, kidney, breasts e.t.c.
N.B:
ALP is activated by Mg+2 , Mn+2, Co+2
ALP is inactivated by Zn+2, Cu+2, Hg+2, EDTA

4. Mix, incubate1min at 37°C then measure ΔA at 405 nm ≠ air
Determination of ALP
Principle
Nitrophenyl phosphate ALP nitrophenol + phosphate (pH9.8 in diethanolamine buffer)
Procedure
Add 1 ml of
Working reagent
Add 0.2 ml serum
37°C
Mix, incubate1min at 37°C then measure ΔA at 405 nm ≠ air

5. Determination of ALP: Cont.
Calculation
∆ A /min=( (A1 – A0) + (A2 – A1) + (A3 – A2) ) / 3
ALP activity (U/L) = ∆ A /min × 3300
Normal level
Adult: 100 – 290 IU/L
Children: 180 – 1200 IU/L

6. Determination of ALP: Cont.
Clinical significance
↑ ALP
1- Hepatobiliary diseases (mainly in obstructive conditions)
2- Bone diseases e.g. Paget’s disease & rickets
4- Drugs e.g. sulfonamide
↓ ALP
1- Anemia
2- Scurvy
3- Cretinism

7. Acid Phosphatse (ACP) Occurrenc N.B: 1- Prostatic ACP → in prostate
2- Nonprostatic → in RBCs, WBCs & platelets
N.B:
Maximum activity → at pH5.6

8. Determination of ACP Principle
1 naphthylphosphate + H2O ACP phosphate + 1-naphthol
pH6.7
1-naphthol + 4-chloromethylphenyl diazonium azo dye

9. Determination of ACP
Procedure

10. Determination of ACP Calculation Normal level
∆ A /min=( (A1 – A0) + (A2 – A1) + (A3 – A2) ) / 3
TACP activity (U/L) = ∆ A /min × 750
NPACP activity (U/L) = ∆ A /min × 750
PACP activity (U/L) = TACP - NPACP
Normal level
Total ACP: 5.4 U/L
Prostatic ACP: 1.7 U/L

11. Determination of ACP Clinical significance:
High level of ACP indicates prostate cancer