Biochemistry Practice

Dept.of biochemistry

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.

Assay the Activity of

Amylase (AMS) in Serum

(Starch iodine method)

Human alpha-amylase

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

Next experiment

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