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Contents1 Amylase
2 CK-Total( Creatine Phosphokinase Total)
3 Triglyceride
4 Urea
5Separation of Hemoglobin and Riboflavin by Gel
Filtration Chromatography
6Cellulose Acetate Paper Electrophoresis of
Serum Proteins
7 Uric acid
8 ALT transaminase
9 DNA gel eletrophoresis
10 Bilirubin
11 Final practical exam
Final practical grade
• Final practical grade=– Daily performance(30%)– Final opera(35%)– Final writing(35%)– (-The times of absent)
• 1/3 absent, can not take part in the final exam.
Amylase (AMS)• Amylases are a group of hydrolases
that split complex carbohydrates into maltose and some residual glucose.
• α-Amylase – the major form of amylase found in humans and
other mammals – calcium metalloenzymes– catalyzes the endohydrolysis of 1,4-α-D-glucosidi
c linkages in polysaccharides
Salivary and Pancreatic Amylase• In the body, the highest concentrations of amylase
are found in the saliva and pancreas. – Salivary amylase (ptyalin, S-type): hydrolysis of starches
while the food is in the mouth and oesophagus. – Pancreatic amylase (P-type): synthesized by the acinar cel
ls and then secreted into the intestinal tract by way of pancreatic duct system.
• Small amounts of amylase are normally present in the blood.
• However, increased amounts may be released into the blood when the pancreas is injured, inflamed, or blocked.
Pancreas• Functions:
– produces insulin and glucagon: control the use of sugar in the body.
– secretes enzymes: digest other proteins, sugars, and fats.
• These digestive juices are carried to the small intestine by the biliary system.
• When the pancreas becomes inflamed, its powerful digestive enzymes leak out and begin to attack the pancreas itself.
Acute and chronic pancreatitis• Acute pancreatitis occurs when the pancreas
suddenly becomes inflamed but then gets better.
• Chronic pancreatitis is persistent inflammation of the pancreas or a combination of persistent inflammation with repeated attacks of acute pancreatitis.
• Causes: alcohol abuse or gallstones.
• Symptoms of a pancreatic disorder: abdominal pain, fever, loss of appetite, or nausea.
Blood amylase • To diagnose and monitor pancreatitis or
other pancreatic diseases– In acute pancreatitis, a transient rise in
serum amylase activity occurs within 2-12 hours of the onset (often to 4-6 times higher than the highest reference value), levels return to normal by 3-4 day.
– In chronic pancreatitis, amylase levels initially will be moderately elevated but often decrease over time with progressive pancreas damage.
– Amylase levels may also be significantly increased: pancreatic duct obstruction, cancer of the pancreas, and gallbladder attacks
Urine amylase
• Its level will mirror blood amylase concentrations, but both the rise and fall occur later.
Blood amylase test(Starch iodine method)
• Principle– In solution iodine reacts with starch to give
an intense blue-violet complex.
– Amylase hydrolyses starch forming maltose and other fragments which do not react with iodine.
α-AMS
Starch Glucose + Maltose + DextrinIodine
Blue
+
Iodine+
×
• Principle– After incubation of serum with buffered
starch solution, the amount of starch remaining is assayed by measuring the absorbance at 630nm after the addition of iodine.
Specimens & Materials• Specimen: serum • Working reagent:
– buffered starch substrate (0.8mg/ml)
• Color reagent: iodine solution – Function: stop the reaction, reveal color
• Water bath• Test tubes• Pipettes• Spectrophotometer
Method
Test Control
Working reagent 0.5ml 0.5ml
Serum 0.02ml -
Mix, incubate for exactly 6 mins at 37C
Color reagent 3ml 3ml
Serum - 0.02ml
•Mix, measure the absorbance of test and control (AT, AC), setting zero with dH2O, λ=630nm.
Calculation
• Expected normal value: 20-240 U/L
• One Unit= the activity of the enzyme in 100ml serum that hydrolyze 5mg starch for 15min at 37C.
100002.0
100
6
15
5
5.08.0
( ml)( min)( mg)
1000C
TC
A
AA• Amylase activity (U/L)=
Micropipette
•Never try to measure a volume that the micropipettor cannot measure.• Micropipettes have 3 positions:
1. Rest position2. First stop3. Second stop
Step-wise Operation of the Automatic Step-wise Operation of the Automatic PipettePipette
• Set volume. Set volume. Notice:Notice:Do not adjust the micropipette volume above or below that recommended(!!!).
• Attach disposable tip.Attach disposable tip.• Depress the plunger to Depress the plunger to 11stst stop stop..• Immerse tip in sample and Draw up sample.Immerse tip in sample and Draw up sample.• Withdraw the tip.Withdraw the tip.• Dispense the sample by pushing the plunger tDispense the sample by pushing the plunger t
o the o the 22ndnd stop stop..• Withdraw the pipette and release the plunger.Withdraw the pipette and release the plunger.• Discard the tip.Discard the tip.
Spectrophotometry
1. Power switch2.Wavelength select
ion3.“Mode”4. “100%T/ 0A”5.“ 0% T” 6. Cuvette holder(sample compartment)7. Pole
sample compartment
Pole
1 、 distinguish transparence and opaque 2 、 control solution at 2/3 volume 3 、 sop up water with paper 4 、 after test, cleanout , upend it
Cuvette
1. Switch on, allow 20 min for warm up before use.2. Adjust wave length of maximal absorption.3. Prepare test, blank and standard sample. sop up liq
uid with paper, Place them in the cuvette holder.(Notice: put the blank in position 1, Make sure the cuvette is ali
gned with the light source.)4. To “Blank”, Mode “A or T” , press“100%T / 0A”,
Set A=0 or T=100. 5. Pull the pole once time. 6. Change mode to “T”, press“0%T”, Set T =0 7. Change mode to “A”. 8. pull the pole second time, record A1; third time, rec
ord A2; forth time ,record A3.
Operating steps of spectrophotometry
Blank
Standard
Test1
Test2
Blank
Standard
Test1
Test2
Blank
Standard
Test1
Test2
Blank
Standard
Test1
Test2Blank
Standard
Test1
Test2
T handle 0 set T 100% : Blank
T handle 1 set T 0% : A=1.-----
A handle2 、 3 、 4 assay A : Standard 、 test1 、 2
Rest state : handle 1 A=1.-----
( 3 ) Determine
Mode: T 0% ~ 100% A 0 ~ 1