Dr. Samah Kotb Nasr Eldeen

Quantitative determination of serum amylase activity

Amylase is an enzyme that changes complex sugars (starches) into simple sugars during digestion. Levels of amylase in the blood can be used to help diagnose and monitor diseases, such as diseases of the pancreas and salivary glands, or to determine whether the intestines have been damaged.

There are two kinds of amylase enzymes: Alpha-amylase (ptyalin), which is produced by the salivary glands. This enzyme begins starch digestion in the mouth and continues to work in the stomach. Pancreatic amylase, which is secreted by the pancreas into the small intestine. This enzyme continues the starch digestion process.

An amylase test measures the amount of this enzyme in a sample of blood taken from a vein or in a sample of urine.

Normally, only low levels of amylase are found in the blood or urine. But if the pancreas or salivary glands become damaged or blocked, more amylase is usually released into the blood and urine. In the blood, amylase levels rise for only a short time. In the urine, amylase may remain high for several days.

Salivary glands produce saliva, which moistens the mouth to help a person chew and swallow food. Saliva also contains substances (enzymes) that begin the breakdown of food.

There are four pairs of salivary glands: The parotid glands are located between the ear and the jaw. The submandibular glands are located under the jaw.

The sublingual glands are located on the floor of the mouth under the tongue. The buccal glands are not shown in this picture. They are located in the mucous membrane lining the cheeks and mouth. These glands produce only a small amount of saliva.

Salivary Glands

Why It Is Done A test for amylase is done to: Find pancreatitis and other pancreatic diseases. See if the treatment for pancreatitis and other pancreatic diseases is working. Check swelling and inflammation of the salivary glands.

How To Prepare To prepare for an amylase test: For a blood test for amylase, do not eat or drink anything except water for at least 2 hours before having the test. For a 24-hour urine test for amylase, be sure to drink enough fluids during the test to prevent dehydration.

Many medicines may affect the results of this test. Be sure to tell your doctor about all the nonprescription and prescription medicines you take.

How It Is Done Blood test Wrap an elastic band around your upper arm to stop the flow of blood. This makes the veins below the band larger so it is easier to put the needle in the vein.

Clean the needle site with alcohol. Put the needle in the vein. Attach a tube to the needle to fill it with blood.

Remove the band from your arm when enough blood is collected. Put a gauze pad or cotton ball over the needle site as the needle is removed.

Urine test Amylase can be measured in a 24-hour. A 24-hour urine sample is all of the urine you produce over a 24-hour period.

You start collecting your urine in the morning. When you first get up, empty your bladder but do not save this urine. Write down the time that you urinated to mark the beginning of your 24-hour collection period.

For the next 24 hours, collect all your urine. Your doctor or lab will usually provide you with a large container that holds about (4 L). The container has a small amount of preservative in it. Urinate into a small, clean container and then pour the urine into the large container. Do not touch the inside of the container with your fingers.

Keep the large container in the refrigerator for the 24 hours. Empty your bladder for the final time at or just before the end of the 24-hour period. Add this urine to the large container and record the time.

Do not get toilet paper, pubic hair, stool (feces), menstrual blood, or other foreign matter in the urine sample.

Normal Amylase in blood for adults and younger: 25–125 units per liter (U/L).units per liter (U/L) Adults older than age 60: 24 –151 U/L24 –151 U/L

Amylase in urine 24-hour urine sample: 24–408 U Amylase/creatinine clearance ratiocreatinine Normal: 1%–4% or 0.01–0.04 clearance fraction

Blockage of, or severe damage to, the intestines. A stomach ulcer that has caused a hole in the stomach wall.

Gallstones that are causing pancreatitis. Diabetic ketoacidosis. A ruptured ectopic pregnancy. Kidney failure.

Appendicitis or peritonitis. Macroamylasemia, an uncommon and harmless condition in which amylase is bound to a protein in the blood.

Lipase is an enzyme produced only by the pancreas. A lipase test may be done at the same time as an amylase test when pancreatitis is suspected. Lipase Occasionally, a test that compares urine amylase with creatinine (a renal clearance ratio) may be done to help diagnose pancreatitis.creatinine

Reagents

Item Quantity Amylase Assay Buffer 55 ml Amylase Substrate Mix 5 ml Amylase Positive Control (Lyophilized) 1 vial Nitrophenol standard (2mM) 150 µl * Store reagents at -20°C.

Procedure Warm the assay buffer to room temperature before use. Briefly centrifuge vials before opening. Keep samples and Amylase Positive Control on ice during the assay.

Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker

Assay Protocol Sample Preparation: 1- Dissolve Amylase Positive Control into 50 μl Assay Buffer, and store at -20°C. For serum or urine samples: Serum and urine samples can be tested directly: add 5 μl samples or 5 μl Amylase Positive Control into each well, and adjust volume to 50 μl with distalled H 2 O.

2. Standard Curve Preparation: Add 0, 4, 8, 12, 16, 20 μl /well nitrophenol standard. Bring the total volume to 50 μl with dH 2 O.

3. Reaction Mix: Prepare enough reaction mix for samples, standard and positive control. For each reaction: Assay Buffer 50 μl Substrate Mix 50 μl

4. Add 100 μl of the reaction mix into each reaction and mix. 5. Measure immediately (T0) at OD 405 nm to get ODT0. Incubate the reaction at 25°C for various times (T1) and measure OD 405 nm to get ODT1.

Data Analysis Plot the Nitrophenol standard curve. Apply the ΔOD (ΔOD = ODT1 – ODT0) to the Nitrophenol standard curve to get B nmol of Nitrophenol generated by amylase between T0 and T1.