Contents | | | | | - CK-Total( Creatine Phosphokinase Total)
| | | | | | | | | - Separation of Hemoglobin and Riboflavin by Gel Filtration Chromatography
| | | - Cellulose Acetate Paper Electrophoresis of Serum Proteins
| | | | | | | | | | | | | | | | 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.
- Assay the Activity of Amylase (AMS) in Serum (Starch iodine method)
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-glucosidic 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 cells 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 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.
- Starch Glucose + Maltose + Dextrin
Principle - 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
- Water bath
- Test tubes
- Pipettes
- Spectrophotometer
Method | | | | | | | | | | | | - Mix, incubate for exactly 6 mins at 37C
| | | | | | | | | | | - 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.
Micropipette - Never try to measure a volume that the micropipettor cannot measure.
- Micropipettes have 3 positions:
- 1. Rest position 2. First stop 3. Second stop
Step-wise Operation of the Automatic Pipette - Set volume. Notice:Do not adjust the micropipette volume above or below that recommended(!!!).
- Attach disposable tip.
- Depress the plunger to 1st stop.
- Immerse tip in sample and Draw up sample.
- Withdraw the tip.
- Dispense the sample by pushing the plunger to the 2nd stop.
- Withdraw the pipette and release the plunger.
- Discard the tip.
Spectrophotometry - 1．Power switch
- 2．Wavelength selection
- 3．“Mode”
- 4. “100%T／0A”
- 5．“0％T”
- 6．Cuvette holder
- (sample compartment)
- 7．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
- Switch on, allow 20 min for warm up before use.
- Adjust wave length of maximal absorption.
- Prepare test, blank and standard sample. sop up liquid with paper, Place them in the cuvette holder.
- (Notice: put the blank in position 1, Make sure the cuvette is aligned with the light source.)
- To “Blank”, Mode “A or T” , press“100%T／0A”, Set A=0 or T=100.
- Pull the pole once time.
- Change mode to “T”, press“0%T”, Set T =0
- Change mode to “A”.
- pull the pole second time, record A1; third time, record A2; forth time ,record A3.
- Operating steps of spectrophotometry
- How to operate Spectrophotometer ?
- （1） Turn on ，set wavelength ，warm-up for 20min
- （2） Respectively transfer sample solutions to cuvettes
- Blank, Standard, Test
- Height: 2/3~4/5
- Hold the rough face ，
- keep the smooth face tidy.
- Put cuvettes into the
- cuvette holder in the
- proper order .
- 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.-----
- Mode:
- T 0% ~ 100%
- A 0 ~ 1
Next experiment - CK-Total(Creatine Phosphokinase Total)
- (p23)
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