Biochemistry Practice Dept.of biochemistry

Contents 1Amylase 2CK-Total( Creatine Phosphokinase Total) 3Triglyceride 4Urea 5 Separation of Hemoglobin and Riboflavin by Gel Filtration Chromatography 6 Cellulose Acetate Paper Electrophoresis of Serum Proteins 7Uric acid 8ALT transaminase 9DNA gel eletrophoresis 10Bilirubin 11Final 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- 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 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 + Dextrin Iodine 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 TestControl Working reagent0.5ml Serum0.02ml- Mix, incubate for exactly 6 mins at 37  C Color reagent3ml Serum-0.02ml Mix, measure the absorbance of test and control (A T, A C ), setting zero with dH 2 O, λ=630nm.

Calculation Expected normal value: 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 Step-wise Operation of the Automatic Pipette Set volume. Notice:Set volume. Notice: Do not adjust the micropipette volume above or below that recommended(!!!). Attach disposable tip.Attach disposable tip. Depress the plunger to 1 st stop.Depress the plunger to 1 st 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 to the 2 nd stop.Dispense the sample by pushing the plunger to the 2 nd stop. Withdraw the pipette and release the plunger.Withdraw the pipette and release the plunger. Discard the tip.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

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 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. ) 4.To “Blank”, Mode “A or T”, press“100%T ／ 0A”, Set A=0 or T= 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, 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 Standard 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= A handle2 、 3 、 4 assay A ： Standard 、 test1 、 2 Rest state ： handle 1 A= （ 3 ） Determine Mode: T 0% ~ 100% A 0 ~ 1

Next experiment CK-Total(Creatine Phosphokinase Total) (p23)