Summary

Acid phosphatase (ACP) is an enzyme of the hydrolase

class of enzymes and acts in an acidic medium. It is widely

distributed and found in high concentrations in the liver,

RBCs and the prostate. Increased levels of the prostatic

fraction are associated with prostatic carcinomas. Increased

levels of the non-prostatic fraction are associated with liver

diseases, hyperparathyroidism, and Paget’s disease.

Principle

ACP at an acidic pH hydrolyzes α naphthyl phosphate to

form α naphthol and inorganic phosphate. The α naphthol

formed is coupled with fast red TR salt to form a diazo dye

complex. The rate of formation of this complex is measured

as an increase in absorbance which is proportional to the

ACP activity in the sample. Tartrate inhibits prostatic ACP

and the testing in its presence is done to find the nonprostatic ACP. The difference between the activities of

the total and non-prostatic ACP gives the activity of the

prostatic ACP.

 ACP

α Naphthyl phosphate + H2O αNaphthol + Phosphate

α Naphthol + Fast Red TR Salt Diazo dye complex

Normal Reference Values

Serum (male) : Up to 4.2 U/L at 30°C/up to 4.7 U/L at 37°C

 (female) : Up to 3.0 U/L at 30°C/up to 3.7 U/L at 37°C

Prostatic ACP : Up to 1.5 U/L at 30°C/up to 1.6 U/L at 37°C

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 10 × 2 mL 30 × 2 mL

L1 : Buffer reagent 25 mL 75 mL

T1 : Substrate tablets 10 Nos 30 Nos

L2 : Tartrate reagent 2 mL 2 mL

L3 : Acetate buffer 2 mL 2 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents L2 and L3 are ready to use.

The Buffer (L1) when retrieved from 2–8°C may appear

turbid. However, the turbidity clears up on attaining RT.

In case, the turbidity persists a little warming of the Buffer

to 30/37°C may be required.

Working reagent: Dissolve 1 Substrate tablet (T1) in

2.2 mL of Buffer reagent (L1). Allow the tablet to hydrate

for around 5 minutes and then shake to dissolve. This

working reagent is stable for at least 3 days when stored at

2–8°C. The working reagent may be used for the total ACP

assay or the non-prostatic ACP assay as required.

Contd...

Enzymology 529

Sample Material

Serum. Free from hemolysis.

ACP, especially the prostatic fraction, is unstable in a

collected sample, hence the serum should be separated from

the clot, as soon as possible, and assayed. In case of a delay

in testing the serum should be acidified to a pH of 5.0 with

0.02 mL acetate buffer (5M) provided for each mL of serum.

Procedure

Wavelength/filter : 405 nm

Temperature : 30/37°C

Light path : 1 cm

Total ACP Assay

Pipette into a clean dry test tube labeled as Test (T):

Addition

Sequence

(T)

(mL)

Working reagent 1.0

Sample 0.1

Mix well and read the initial absorbance A0 after 5

minutes and repeat the absorbance reading after every 1,

2, and 3 minutes. Calculate the mean absorbance change

per minute (∆A/min).

Non-prostatic ACP Assay : (Tartrate Inhibited)

Pipette into a clean dry test tube labeled as Test (T):

Addition

Sequence

(T)

(mL)

Working reagent 1.0

Tartrate reagent 0.02

Incubate at the assay temperature for 1 minute and add

Sample 0.1

Mix well and read the initial absorbance A0 after

5 minutes and repeat the absorbance reading after every 1,

2, and 3 minutes. Calculate the mean absorbance change

per minute (∆A/min).

Calculations

ACP activity in U/L = ∆A/min × 750

Prostatic ACP activity in U/L = Total ACP–non-prostatic ACP.

Linearity

The procedure is linear up to 75 U/L at 37°C. If the

absorbance change (∆A/min) exceeds 0.100, dilute the

sample 1 + 4 with normal saline (NaCI 0.9%) and repeat

the assay (Results × 5).

Notes

Samples having a high activity show a very high initial

absorbance. If this is suspected then dilute the sample and

repeat the assay.

The working reagent should have an absorbance below

0.800 against distilled water at 405 nm. Discard the reagent

if the absorbance is above 0.800. It has been seen that in a

collected sample ACP, especially the prostatic form, may

lose around 50% of its activity in an hour at RT.

System Parameters

Reaction : Kinetic Interval : 60

Wavelength : 405 nm Sample volume : 0.1 mL

Zero setting : Distilled water Reagent volume : 1.00 mL

Incubation

temperature

: 30/37°C Standard :

Incubation

time

: — Factor : 750

Delay time : 300 sec Read scope : Increasing

Read time : 180 sec Linearity : 75 U/L

No. of read : Units : U/L

Clinical Relevance

1. A significantly elevated value nearly always indicative

of metastatic cancer of the prostate. If the tumor is

successfully treated, this enzyme level will drop within

3 to 4 days after surgery or 3 to 4 weeks after estrogen

administration.

2. Moderately increased values also occur in the absence

of prostate disease in:

a. Paget’s disease

b. Gaucher’s disease

c. Hyperparathyroidism

d. Multiple myeloma

e. Any cancer that has metastasized to the bone

f. Hepatitis

g. Obstructive jaundice

h. Acute renal impairment

i. Sickle cell crisis/hemolytic anemia

j. Excessive destruction of platelets.

3. Levels are reported to be elevated in the bone marrow

of patients with prostatic cancer metastatic to the

bone.

Interfering Factors

1. Drugs that may cause increased levels include:

a. Androgens in females

b. Clofibrate.

2. Drugs that may cause decreased levels include:

a. Fluorides

b. Oxalates

c. Phosphates.

Serum Alkaline Phosphatase and Acid Phosphatase

The following table gives the differences between the two: