Biochemistry Practice Dept of biochemistry Contents



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  • Dept.of biochemistry

Contents

  • 1
  • Amylase
  • 2
  • CK-Total( Creatine Phosphokinase Total)
  • 3
  • Triglyceride
  • 4
  • Urea
  • 5
  • Separation of Hemoglobin and Riboflavin by Gel Filtration Chromatography
  • 6
  • Cellulose 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.

  • 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.
  • α-AMS
  • Starch Glucose + Maltose + Dextrin
  • Iodine
  • Blue
  • +
  • Iodine
  • +
  • ×

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

  • Test
  • Control
  • Working reagent
  • 0.5ml
  • 0.5ml
  • Serum
  • 0.02ml
  • -
  • Mix, incubate for exactly 6 mins at 37C
  • 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 37C.
  • (ml)
  • (min)
  • (mg)
  • Amylase activity (U/L)=

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
  • 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
  • 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 .
  • Blank
  • Test1
  • Test2
  • cuvette holder
  • Blank
  • Standard
  • Test1
  • Test2
  • Blank
  • Standard
  • Test1
  • Test2
  • Blank
  • Standard
  • Test1
  • Test2
  • Blank
  • Standard
  • Test1
  • Test2
  • Blank
  • 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

Next experiment

  • CK-Total(Creatine Phosphokinase Total)
  • (p23)


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