The document presents information on estimating alkaline phosphatase levels in serum, including that alkaline phosphatase is an enzyme responsible for dephosphorylation, it is commonly tested to evaluate liver and bone function, and the test involves combining serum with reagents to produce a color change that corresponds to alkaline phosphatase levels.

1. Presentation on
Estimation of Alkaline Phosphatase
in Serum
Presented by:
Dr. Monika Jangid,
Assistant Professor,
Dept. of Chemistry , Pacific University, Udaipur

2. Description
Why to be done
Normal and Abnormal values
Symptoms
How to be done
Applied method
Application

3.  Alkaline phosphatase (ALP, ALKP) is a hydrolase enzyme
responsible for removing phosphate groups from many types
of molecules, including nucleotides proteins and alkaloids.
 The process of removing the phosphate group is called
dephosphorylation. As the name suggests, alkaline
phosphatases are most effective in an alkaline environment.
 It is sometimes used synonymously as basic phosphatase.

4.  Medical testing of Alkaline Phosphatases is concerned with a group of
enzymes found primarily the liver (isoenzyme ALP-1) and bone
(isoenzyme ALP-2).
 Small amounts are also produced by cells lining the intestines
(isoenzyme ALP-3), the placenta, and the kidney (in the proximal
convoluted tubules).
 Since the mucosal cells that line the bile system of the liver are the
source of alkaline phosphatase, the free flow of bile through the liver
and down into the biliary tract and gallbladder are responsible for
maintaining the proper level of this enzyme in the blood.
 When the liver, bile ducts or gallbladder system are not functioning
properly or are blocked, this enzyme is not excreted through the bile
and alkaline phosphatase is released into the blood stream.

5.  The optimal range for alkaline phosphatase depends on age. Children
will have a much higher alkaline phosphatase than a full grown adult
because his/her osteoblasts are laying down bone very rapidly. The normal
range for serum ALP level is 20 to 140 IU/L, but this can vary from
laboratory to laboratory. The normal range runs higher in children and
decreases with age.
Abnormal Level-
Increased serum Alkaline Phosphatase causes Congestion or obstruction
of the biliary tract Paget's Disease, Hyperthyroidism, Rickets-Vitamin D
deficiency.
 Decreased serum alkaline phosphatase causes Zinc deficiency,
Hypothyroidism, Vitamin C deficiency/Scurvy, Folic acid deficiency.
Excess Vitamin D intake, Low phosphorus levels(hypophosphatasia).

7.  Signs and Symptoms of liver involvement may include:
 Weakness, fatigue
 Loss of appetite
 Nausea, vomiting
 Abdominal swelling and/or pain
 Jaundice
 Dark urine, light colored stool
 Itching
 Some examples of the signs and symptoms suggesting a bone
disorder include:
 Bone and/or joint pain
 Increased frequency of fractures
 Deformed bones

8.  Alkaline phosphatase is measured by combining the person's
serum with specific substances with which alkaline
phosphatase is known to react. The end product of this
reaction is measured and from that measurement, the amount
of alkaline phosphatase in the person's serum is determined.
 It is done in the pathology lab by kit method.
 One of the method is kind and king’s method to estimate this
in the lab.

9. Principle
 ALP at an alkaline pH hydrolyses disodium Phenylphosphate to form
phenol. The Phenol formed reacts with 4-Aminoantipyrine in the
presence of Potassium Ferricyanide, as an oxidising agent, to form a
red coloured complex. The intensity of the colour formed is directly
proportional to the activity of ALP present in the sample.
 Reaction

10. Reagents:
• Reagent 1: Buffered substrate, pH 10 , Reagent 2: Chromogen reagent, Reagent 3: Phenol substrate
• We have taken two serum samples for this. One is of 21 years and another is of 34 years.
Reagents Blank Standard Control Test
Working
buffer
substrate
0.5 0.5 0.5 0.5
Distilled
water
1.5 1.5 1.5 1.5
Mix well and incubate at 37 0C for 3 mins.
Pipetting chart
To be continue ...

11. Serum - - - 0.05
phenol
substrate
- - 0.05 -
After that:
Mix well and incubate at 370 C for 15 mins.
Chromogen
reagent
1.0 1.0 1.0 1.0
Serum - - 0.05 -
Mix well and measure the Optimum Density against purified
water at 520 nm.
To be continue ...

12. Absorbance at 520 nm
O.D. of Blank - ………………………..
O.D. of STD - …………………………
O.D. of Test - ……………………........
Calculation:
ALP activity (IU/L)=
Result:
Interpretation:
O.D. of Test- O.D. of Blank
O.D. of STD- O.D. of Blank
X concentrate
STD
To be continue ...

13.  Alkaline phosphatase has become a useful tool in molecular biology
laboratories, since DNA normally possesses phosphate groups on the
5' end. Removing these phosphates prevents the DNA from ligating
there by keeping DNA molecules linear until the next step of the
process for which they are being prepared.
 Removal of the phosphate groups allows radiolabeling (replacement
by radioactive phosphate groups) in order to measure the presence of
the labeled DNA.
 Undifferentiated pluripotent stem cells have elevated levels of alkaline
phosphatase on their cell membrane, therefore alkaline phosphatase
staining is used to detect these cells and to test pluripotency (i.e.
embryonic stem cells or embryonal carcinoma cells) .