Serum amylase test is important for calculation of hydrolytic enzyme which hydrolyses starch into maltose. It present in saliva and pancreatic juice where it is secreted by parotid glands and pancreas respectively.
Small amounts of leak into circulation due to where and tear of cells in these glands. The circulating enzyme is excreted by the kidneys into urine. Therefore, only a small amount of amalyse in present in serum nonorml.
Serum amylase test principle
Serum is incubated with starch substrate. The amylase in the serum hydrolyses the starch to simpler units with a resulting increase in reducing groups. In the method presented here iodine is added which reacts with the starch molecules not hydrolysed by the amylase.
The iodine-starch complex is blue in colour and is measured in the spectrophotometer. The degree of loss in colour is proportional to the amount of starch hydrolysed and hence to the activity of the amylase in the serum.
A substrate control is carried though the procedure to give a reference value for the amount of starch substrate present before hydrolysis.
Serum amylase test procedure
- Pipette 5 ml of substrate into two 50 ml volumetric flasks.
- Place the ‘test’ flask into a 34°C water bath for 5 minute to warm the contents.
- Using a pipette that deviloers between two marks add 0,1 ml of serum to the ‘test’ flask and mix.
- Do not use blow out pipettes as the smallest amount of saliva can give a large error.
- Time the addition of serum using a stop watch.
- After exactly 7.5 minutes add 5 ml or working iodine solution, mix and immediately remove from the water bath.
- Similarly add 5 ml of the working iodine solution to the flask containing the ‘substrate control, which has not been incubated.
- Dilute the contents of both flasks to the 50 ml mark with distilled water and mix the flasks well.
- Read the absorbance of both against water using the large (19 mm) cuvettes at 660 nm.
Serum amylase enzyme calculation
Absorbance of substrate control-absorbance of test x 800 Absorbance of control units of amylase activity per 100 ml of serum.:
In the result is greater than 400 units per 100 ml, repeat the test using 0.1 ml of a 1 in 5 dilution of the serum or 0.02 ml of undiluted serum.
An amylase units is defined as the activity that catalyses the conversion of 10 mg of starch substrate to non-iodine reacting product in 30 minutes under the condition of the assed
(a) Since the test is incubated for 7.5 minutes multiply by ‘4’ to estimate for 30 minutes.
(b) Since the 5 ml of starch substrate solution added only contain 2 mg of starch devide by ‘5’ to estimate the activity that would be indicated if 10 mg of starch were present, the activity would be less if there was more substrate to work up, thus the need to devide.
(c) Only 0.1 ml or serum is used in the assay but the activity is reported per 100 ml. thus 0.1 must be multiplied by 1000 to give 100 i.c. 4/5 x 1000-800
Source of Errors in Sr. amylase enzyme calculation
- Saliva: Saliva has a very high content of amylase. Even the slightest contamination of the test with saliva will give large errors.
- Substrat: Contamination with saliva is common cause of substrate unsuitability.
- Enzyme: The precautions which apply to enzyme assays in general with regard to time, pH, temperature, substrate concentration etc. apply to this assay for amylase activity.
- Precipetate: The final blue colour is really due to a fine suspension of blue particles in water rather than a true solution. Care must be taken upon dilution to 50 ml to mix well to prevent a precipitate forming, which would distort its absorbtion from closely obeying beer’s Law, as it can under optimal conditions,
- Urine : Amylase in urine can be measured exactly the same as in serum, though a 24 hour’s