Quantitative determination of serum amylase activity
Slide 3
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.
Slide 4
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.
Slide 5
An amylase test measures the amount of this enzyme in a sample
of blood taken from a vein or in a sample of urine.
Slide 6
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.
Slide 7
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.
Slide 8
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.
Slide 9
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.
Slide 10
Salivary Glands
Slide 11
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.
Slide 12
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.
Slide 13
Many medicines may affect the results of this test. Be sure to
tell your doctor about all the nonprescription and prescription
medicines you take.
Slide 14
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.
Slide 15
Clean the needle site with alcohol. Put the needle in the vein.
Attach a tube to the needle to fill it with blood.
Slide 16
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.
Slide 17
Slide 18
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.
Slide 19
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.
Slide 20
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.
Slide 21
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.
Slide 22
Do not get toilet paper, pubic hair, stool (feces), menstrual
blood, or other foreign matter in the urine sample.
Slide 23
Normal Amylase in blood for adults and younger: 25125 units per
liter (U/L).units per liter (U/L) Adults older than age 60: 24 151
U/L24 151 U/L
Slide 24
Amylase in urine 24-hour urine sample: 24408 U
Amylase/creatinine clearance ratiocreatinine Normal: 1%4% or
0.010.04 clearance fraction
Slide 25
Slide 26
Blockage of, or severe damage to, the intestines. A stomach
ulcer that has caused a hole in the stomach wall.
Slide 27
Gallstones that are causing pancreatitis. Diabetic
ketoacidosis. A ruptured ectopic pregnancy. Kidney failure.
Slide 28
Appendicitis or peritonitis. Macroamylasemia, an uncommon and
harmless condition in which amylase is bound to a protein in the
blood.
Slide 29
Slide 30
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
Slide 31
Reagents
Slide 32
Item Quantity Amylase Assay Buffer 55 ml Amylase Substrate Mix
5 ml Amylase Positive Control (Lyophilized) 1 vial Nitrophenol
standard (2mM) 150 l * Store reagents at -20C.
Slide 33
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.
Slide 34
Additional Materials Required Microcentrifuge Pipettes and
pipette tips Fluorescent or colorimetric microplate reader 96 well
plate Orbital shaker
Slide 35
Assay Protocol Sample Preparation: 1- Dissolve Amylase Positive
Control into 50 l Assay Buffer, and store at -20C. For serum or
urine samples: Serum and urine samples can be tested directly: add
5 l samples or 5 l Amylase Positive Control into each well, and
adjust volume to 50 l with distalled H 2 O.
Slide 36
2. Standard Curve Preparation: Add 0, 4, 8, 12, 16, 20 l /well
nitrophenol standard. Bring the total volume to 50 l with dH 2
O.
Slide 37
3. Reaction Mix: Prepare enough reaction mix for samples,
standard and positive control. For each reaction: Assay Buffer 50 l
Substrate Mix 50 l
Slide 38
4. Add 100 l of the reaction mix into each reaction and mix. 5.
Measure immediately (T0) at OD 405 nm to get ODT0. Incubate the
reaction at 25C for various times (T1) and measure OD 405 nm to get
ODT1.
Slide 39
Data Analysis Plot the Nitrophenol standard curve. Apply the OD
(OD = ODT1 ODT0) to the Nitrophenol standard curve to get B nmol of
Nitrophenol generated by amylase between T0 and T1.