How to Test the Concentration of Amylase Enzymes

Amylase enzymes are produced in the pancreas and in the saliva glands and help digest and process carbohydrates. When the pancreas becomes diseased, amylase is released into the blood stream. When a patient is experiencing digestive tract problems or there is disease of the pancreas, a doctor will order a blood test to establish the level of amylase in the blood. The monitoring of amylase enzyme levels will yield much information about the progression of the disease and the effects of treatment.

Things You'll Need

• UV/Vis spectrophotometer
• 15 large test tubes
• Test tube rack
• 1 mg/ml starch solution
• De-ionized water
• KI/iodine reagent
• Pipettes
• Dropper
• Tissue
• Cuvettes
• Graph paper
• Blood sample
• Centrifuge
• Stop watch

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Instructions

Preparation of a Starch Standard Curve

• 1

  Turn on the spectrophotometer and set it to a wavelength of 620 nm. Allow the instrument to warm up for a minimum of 10 minutes so that the response will be stable during the experiment.

• 2

  Set out six test tubes, label them 1-6 and place them in a test tube rack. Arrange the solutions of starch, de-ionized water and KI/iodine solution on the bench top.

• 3

  Fill tube 1 with 3 ml of de-ionized water using a pipette; this will serve as the blank. Fill tubes 2-6 with 3.0 ml, 2.9 ml, 2.7 ml, 2.4 ml and 2.1 ml of de-ionized water respectively. Add starch solution to each of the tubes 2-6 in the following order: tube 2 gets 0 ml, tube 3 gets 0.1 ml, tube 4 gets 0.3 ml, tube 5 gets 0.6 ml and tube 6 gets 0.9 ml.

• 4

  Add one drop of KI/iodine solution to each of the six tubes. Carefully mix each tube thoroughly before recording their absorbance in the spectrophotometer.

• 5

  Fill the cuvette with the blank solution, clean the outside of the cuvette and place it in the blank hole of the spectrophotometer. Adjust the absorbance to zero. All other solutions will yield a positive absorbance.

• 6

  Fill the sample cuvette with each of the other five solutions in turn. Wipe the outside of the cuvette before each measurement. Record the absorbance value for each tube. Rinse the cuvette with the new solution at least once before filling for the test.

• 7

  Make a graph that plots the absorbance readings against starch concentration. This is the standard curve that allows you to determine the concentration of a solution that has a given absorbance reading.

Determining Amylase Concentration

• 8

  Clean all the test tubes from the first part of the test. Retain the blank tube for use later.

• 9

  Obtain a blood sample from the patient. Place the sample in a centrifuge and separate the red blood cells from the blood plasma.

• 10

  Fill a test tube with 9 ml of de-ionized water and 1 ml of the blood plasma. This is a 1:10 dilution of the plasma. Mix the tube thoroughly and pipet 1 ml of the solution into a second test tube. Label the two tubes with their contents.

• 11

  Prepare to perform a timed test. Have a stop watch handy and set up seven test tubes with cuvettes in the test tube rack. You will take readings every two minutes once the test starts. Record the exact time and absorbance reading for each of the seven data points. Fill each of the seven test tubes with 2 ml of de-ionized water.

• 12

  Add 9 ml of the starch solution to the test tube that has the 1 ml of 1:10 dilution of plasma. As soon as you begin adding the starch solution, start the stop watch. Mix the test tube well and pipet 1 ml of the starch/plasma solution into the first of seven test tubes. Record the time. Add 1 drop of KI/iodine solution to the test tube and mix.

• 13

  Transfer to the cuvette, wipe the outside and record the absorbance. Repeat the process with the remaining six test tubes at two minute intervals. At the conclusion of the timed test you should have readings for the actual time and absorbance for the time spans of 0, 2, 4, 6, 8, 10 and 12 minutes.

• 14

  Use the standard curve to find the concentration of starch for each of the seven data points. These points will be decreasing with time. The amylase digests the starch and over time consumes all the starch available.

• 15

  Graph the data points for the timed test as starch concentration versus time. The slope of this curve indicates the amount of amylase in the plasma sample.