Laboratory

Alpha-amylase:- Range, CNP and PNP Method of Measurement

Alpha-amylase can be measured by the CNP method or PNP method using a semi-auto biochemistry analyzer.

Alpha-amylase is mainly present in the salivary gland and the pancreas. The alpha-amylase in the salivary gland is known as ptyalin while that of the pancreas is known as the pancreatic amylase. Both of them are responsible for the breakdown of carbohydrates into sugar.  Ptyalin acts from the mouth to the large intestine. Finally, from the small intestine, the remaining portion of carbohydrate converts into sugar by the pancreatic amylase.

In case of any injury to the pancreas or the salivary gland, the enzyme leaks to the bloodstream. This causes an increase in the value of alpha-amylase in the blood and urine.

Alpha-amylase Normal Range

The normal range of alpha-amylase depends upon numerous factors. For example, the method of measurement or the type of reagent used can alter the range in the laboratory. Similarly, the temperature of the sample, habitat, geographical location, etc, of the people also alters the level of the enzyme in the blood or urine. Thus, the table shown below is an approximate value of the alpha-amylase.

  250C 300C 370C
Serum < 120 U/L <160 U/L <220 U/L
24 hours urine 450 U/L 650 U/L 900 U/L

Since alpha-amylase is a major secretion of the pancreas and the salivary gland, any defect in these two organs can alter the level of the enzyme in the blood. The increased level of alpha-amylase may indicate the problem of acute pancreatitis, penetrating peptic ulcers, etc. Similarly, a decrease in the enzyme may indicate the problem of chronic pancreatic disorders. In both cases, you are recommended to visit the doctors.

CNP Method for Alpha-amylase Measurement

1. Principle Reaction

Alpha-amylase acts as a catalyst during the hydrolysis of 2 -chloro -4 nitrophenol salt to chloro nitrophenol(CNP). The intensity of the color of the formed CNP is directly proportional to the activity of the alpha-amylase enzyme.

2. Sample Preparation

The table shown below is valid for Tulip and Accurex companies. So, if you have a reagent of any other company then there can be a minor variation in the procedure.

First of all, make a working solution out of the given two reagents. The ratio of their composition will be given in the literature. But, if you have only one reagent besides the calibrator, then that will act as a working solution.

Now, mix the sample volume with the required volume of the working solution. Then feed the solution in a semi-auto biochemistry analyzer.

If the concentration of alpha-amylase in the sample is very high (out of range/ non-linear curve) then dilute the solution with 0.9% NaCl or distilled water at the ratio of 1:y. Finally, when you get the result, multiply it with the dilution factor (1+ y).

Always remember that the total volume of sample and working solution should be more than the sipping volume set in the instrument. Else, the machine will suck up air and will give you the fault reading.

3. Programming of Alpha-amylase for a Semi-auto Biochemistry Analyzer (CNP Method)

Alpha amylase, CNP method
CNP Method

PNP Method for Alpha-amylase Measurement

1. Principle Reaction

Different oligosaccharides are cleaved under the catalytic action of alpha-amylase. Then the formed fragments are hydrolyzed to p-nitrophenol (PNP) and glucose by alpha-glucosidase.

The color intensity of the p-nitrophenol (PNP) formed is directly proportional to the alpha-amylase activity.

2. Sample Preparation

The table shown below is valid for Analyticon and Diasys Companies.  If you have a reagent of any other company then there can be a minor variation in the procedure.

3. Programming of Alpha-amylase in a Semi-auto Biochemistry Analyzer (PNP Method)

alpha-amylasea, PNP method
PNP Method

Handling of Alpha-amylase Reagent

  • Wear an apron and surgical gloves before carrying out the measurement.
  • Look for the expiry date of the reagents during purchase and measurement time. Suppliers tend to give you reagent kits with a low expiry interval.
  • Once you open the reagent bottles, the lifespan will decrease. So, look in your reagent kit literature for that.
  • Bring the reagents and samples to room temperature before you can carry out any measurement.
  • Always store reagents in the refrigerator when not in use.

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