biochemistry experiment
DESCRIPTION
experiment _ mesasurement of blood glucoseTRANSCRIPT
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(GOD-POD method )
Experiment 6
Measurement of blood glucose
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Aim:
1. Learn the principle of GOD-POD method
2. Understand it's clinical significance
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What is blood glucose ?
The main sugar that the body makes from the food in the diet.
Glucose is carried through the bloodstream to provide energy to all cells in the body.
Cells cannot use glucose without the help of insulin.
The body produces it from protein, fat and, in largest part, carbohydrate.
Ingested glucose is absorbed directly into the blood from the intestine and results in a rapid increase in blood glucose.
Generally speaking, fasting blood glucose level is constant for healthy people
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Serum - serum glucose values decrease rapidly in samples that have not been separated from the cellular constituents of blood. Glucose values decrease by 10% per hour if serum is left in contact with cells. Note that the decrease in glucose is enhanced in patients with increased leukocyte or platelet counts, even if collected into fluoride oxalate tubes.
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Why do we choose Glucose
oxidase-peroxidase method
(GOD-POD) to measure blood
glucose ?
GOD-POD
High accuracy,High sensitivity ,
Good stability
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Principle
Glucose ﹢ O2 H2O2
Gluconic acid
﹢( GOD )
H2O2 ﹢4-NH3-
Antipyrine H2OQuinonimine﹢
( POD )﹢ Phenol
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The absorption peak of quinonimine is 500nm
The glucose concentration in the sample can be calculated using the standard comparison method
Generation amount of quinone imine is proportional to the content of glucose
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Equipment
pipet : 2mL ( ×1 )
micropipette : 20μl
Water bath
centrifuge , centrifuge tube 10ml ( ×2 )
Test tube : 1.5×5cm ( ×3 )
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Reagents
Glucose measurement kit
The glucose standard solution (5.55mmol/L)
Sample : fresh serum
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Operation Steps
1. Sample processing
Draw blood 3-5 mL
37℃ water bath it for 15min
Centrifuge the blood for 10min (3000rpm)
Extract the supernatant
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2. Use 3 small test tubes and add reagent according to the table below
NO.
Reagent
Blank tube
Standard tube
Sample tube
dd-Water (μL ) 10 —— ——
Standard glucose (μL )
—— 10 ——
Serum samples (μL )
—— —— 10
Enzyme solution ( mL )
1.5 1.5 1.5
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3. After mix the reagents in each tube, 37℃ water bath for 25min , then use 0.5cm cuvette to measure their UV absorption value At 500nm wavelength
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Calculation
Write down the data and then calculate them by Lambert-Beer law
Csample =Asample
Astandard
× Cstandard
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Cstandard = 5.55 mmol/L
range of normal value : 3.89 ~ 6.11mmol/L
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Clinical significance
1. Hyperglycemia
Glucose concentration >7.0mmol / L
e.g. diabetes
2. Hypoglycemia
Glucose concentration <2.8mmol / L
Effects can range from mild dysphoria to more serious issues such as seizures, unconsciousness, and (rarely) permanent brain damage or death.
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Question
Why human can maintain normal serum glucose at a certain level?
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Blood Glucose Meter
Lancing the finger
Apply blood to strip
1-800-DIABETES www.diabetes.org
Applying Blood to Strip
17
Drop, not smear
Cover ALL of test strip window
Some strips wick blood onto the
strip
8. Follow instructions included with the meter when applying blood to strip
1-800-DIABETES www.diabetes.org
Lancing the Finger
7. Hold the lancet device to the side of the finger and press the button to stick the finger.
• Alterative site (per DMMP) the school nurse and/or parent/guardian will give further instructions which sites are appropriate
• Note: In the case of suspected hypoglycemia, only the finger should be used for blood glucose sampling
16
People who have diabetes should be testing their blood glucose regularly at home.
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Glucose + GO(FAD) + 2 H+ - -> gluconolactone + GO(FADH2)
GO(FADH2) + 2 Fc+ --> GO(FAD) + 2 Fc + 2 H+
Fc --> Fc+ + 2 e- (at electrode)
Mechanism for catalytic oxidation of glucoseWith Glucose oxidase (GO) and Fc mediator
Signal can also be measured by amperometry:Hold const. E where oxidation occurs, measure I vs
time
Fc = ferrocene carboxylateGluconic acid/gluconolactone ferrocene
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Glucose biosensor test strips
MeterRead glucose
Dry coating of GO + Fc
Patient adds drop of blood,then inserts slide into meter
Output: amperometry
I
t
Patient reads glucose level on meter
e’selectrodes