2.21: Amylase (Modified Caraway Method)

RELATED READING: Chapter 29. See Methods on CD-ROM for Amylase.

OBJECTIVES

Upon completion of this exercise, appropriate discussion and related reading, the student will be able to:

1. Describe the physiological action of amylase.
2. List four types of amylase methodologies.
3. Perform a serum amylase determination.
4. Identify the unique color of the iodide-starch complex.

PRINCIPLE

The iodometric (amyloclastic) method is based on the ability of iodide to form a vivid blue color in combination with starch. The starch is hydrolyzed by amylase in the sample to liberate smaller molecules such as dextran, maltose, and some glucose molecules. The smaller sugar molecules do not form a complex with iodide and do not give a blue color. As more starch is broken down, the intensity of the blue color decreases. Substrate (starch) and sample (amylase) are mixed together and incubated for a fixed time. Iodide color solution is added and the starch/iodide color complex is formed. A spectrophotometric measurement is made to determine color intensity (absorbance). The lower the final absorbance (the greater the difference between the blank and test solutions), the higher the serum amylase activity.

MATERIALS

• 13 x 100 mm test tubes
• Heating block or Water bath
• 16 x 100 mm test tubes
• Spectrophotometer
• 19 x 150 mm test tubes
• Normal control
• Starch Substrate
• Serum samples
• Iodide Reagent
• Pipets
• Distilled Water
• 0.9% saline

PROCEDURE

1. Place approximately 4 mL of starch substrate into a labeled 16 x 100 mm test tube. Place the tube in a 37°C heat block or water-bath for a minimum of 5 minutes to prewarm. Continue to warm at 37°C until needed for step 7.
2. Label two 19 x 150 mm test tubes for each control and sample to be tested. Label one of each pair of tubes “blank” and the other “test”.
3. Pipet 21.0 mL of distilled water into each of the 19 x 150 mm test tubes (“blank” and “test”). Set these tubes aside for future use.
4. Label two 13 x 100 mm test tubes for each control and sample to be tested.
5. Pipet 1.0 mL of 0.9% saline into each 13 x 100 mm tube.
6. Pipet 30 \(\mu\)L of the appropriate control or sample into each 13 x 100 mm tube labeled “test”. Mix each by inversion and incubate all 13 x 100 mm tubes (“test” and “blank”) at 37°C for 5 minutes to prewam.
7. Following the 5 minute prewarming, while continuing to incubate, add 0.5 mL of
   warmed starch substrate (from step 1) to all 13 x 100 mm tubes (“blank” and “test”). Incubate each tube for exactly 7 minutes.

Note

You may find it desirable to add the starch substrate to the tubes at 30 second intervals. This will allow for more exact timing.

8. After exactly 7 minutes of incubation, add 2.0 mL of iodide solution to each tube.
9. Add 30 \(\mu\)L of appropriate control or sample to each “blank” tube.
10. Transfer the contents of each 13 x 100 mm tube to the corresponding 19 x 150
    mm test tube from step 3. Mix by inversion.
11. Measure the absorbance of the mixture in each tube (“test” and “blank”) at 660
    nm against a distilled water blank. Record the absorbance on the data sheet.
12. Calculate the amylase value for each material tested using the formula shown
    below: $$\frac{A_{blank} - A_{test}}{A_{blank}} \times 5000 = \text{Amylase units/L}$$Record the values on the data sheet.

DATA SHEET, EXERCISE #21

NAME: ___________ DATE: ___________

RESULTS

	Absorbance 660 nm		Amylase units/L
	Blank	Test
Normal control
Sample #
Sample #

DISCUSSION QUESTION

1. Is the blank used in this experiment a reagent or sample blank?
2. Why is it necessary to use a “blank” for each sample?
3. Why is it necessary to know the method used to determine an amylase value to discuss a reference range?