Presentation on theme: "DETERMINATION OF BLOOD GLUCOSE CONCENTRATION"— Presentation transcript:
1 DETERMINATION OF BLOOD GLUCOSE CONCENTRATION SITI ANNISA DEVI TRUSDABIOCHEMISTRY DEPARTMENT
2 INTRODUCTIONNeeded for supporting the D/ of some diseases, including DM.When to measure: fasting state or 2 hours after taking a meal.Sometimes at a present time detecting a present increased or decreased of blood glucose level (hyperglycemia or hypoglycemia).
3 Two Methods of Measuring Blood Glucose Level Reduction method, which is based on the ability of glucose to reduce Cu++ to Cu+ less sensitive, substances that could reduce Cu++ : fructose, galactose,vitamin C, creatinin, uric acid, glutathion, etc.Enzymatic method (more specific and precise result) : Glucose is oxidized by glucose oxidase gluconic acid + H2O2 red dye.
7 To make the GOD color reagent : dissolve the content of one bottle of GOD enzyme with its solvent available. This solution is stable for 30 days at 2 – 8 oC temperature.The absorbancy of the blank’s reagent must be about 0.0 – 0.4 AU if it is read at 505 nm wavelength compared with aquadest.For every series of measurement, only one standard and one blank are needed.
8 Procedure :1. Centrifuge 3 ml EDTA blood, 2000 rpm for 10’. The plasma will be separated from the blood cells. Use plasma for sample2. Pipette into each of the three reaction tubes according to the following table :
10 3. Mix the content of each tube well, then incubate them at 37oC for 10 minutes or let stand at room temperature for 25 minutes. Avoid direct sunlight4. Using cuvette tube, read the sample’s and standard’s absorbancy against the blank at 505 nm.
12 Notes :By this method the blood glucose level can be measured linearly up to 600 mg/dl. If the blood glucose level is higher than 600 mg/dl, dilute the plasma three times by adding 2 volumes of aquadest, then repeat the procedure. Multiply the result three times.The result is not influenced by blood creatinin, fructose, galactose, uric acid, glutathion, ascorbic acid or bilirubin as long as these substances’s concentration are at normal range.Bilirubin concentration up to 10 mg/dl does not influence the result, but a high dose of oral ascorbic acid (vitamin C) could decrease the result.The color produced will be stable for 2 hours.
15 IDENTIFICATION OF GLUCOSE IN URINE Urine Reduction Test (Benedict’s Test) :Principle :Glucose reduces the alkaline copper solution, Cu++ Cu+ and is precipitated as Cu2O, a red brick color substance.Depend on concentration of Cu2O produced, the shaked solution will produce different color that can be used to estimate the glucose concentration
17 Materials and equipments : Urine of a diabetic patient as sampleBenedict’s qualitative solution (semi qualitative) :- 17 gr CuSO4- 100 gr NaCO3 anhydride- 170 gr tri – Na- Citrate 2 H2ODissolved in 1000 ml of aquadestTest tubesWaterbath 100 0C/ Bunsen burner
18 Procedure :Mix 3 ml Benedict’s solution with 3 drops of urine in a test tube, heat it in 100oC waterbath or just boil it for a moment, note the color produced after shaking the tube.Repeat the same procedure with the urine diluted 2, 4 and 8 times.
20 Note :The positive result of Benedict’s test could be given by many reductors, such as :- Sugars : glucose, pentose, lactose, galactose.- Drugs : antipyrine, pyramidon, PAS, xanthonin.- High concentration of normally urine substances : indican, uric acid, creatinin.- Preservative agents :formalin, CHCl3.
21 To read the result, shake the tube and then note the solution’s color produced : - Blue : (-) → means there is no glucose in the sample urine- Green : (+) → 0.5 – 1 gr % of glucose- Yellowish green : (++) → 1 – 1.5 gr % of glucose- Yellow : (+++) → 2 – 3.5 gr % glucose- Red brick color : (++++) → 4 gr % of glucose