Practical 2: Determination of Serum Glucose PDF

UNIVERSITY OF KWAZULU-NATAL (WESTVILLE CAMPUS) DISCIPLINE OF BIOCHEMISTRY BIOC202: BIOENERGETICS AND INTEGRATED METABOLISM PRACTICAL 2: DETERMINATION OF SERUM GLUCOSE NELSON-SOMOGYI & GLUCOSE OXIDASE METHODS ...

UNIVERSITY OF KWAZULU-NATAL (WESTVILLE CAMPUS) DISCIPLINE OF BIOCHEMISTRY BIOC202: BIOENERGETICS AND INTEGRATED METABOLISM PRACTICAL 2: DETERMINATION OF SERUM GLUCOSE NELSON-SOMOGYI & GLUCOSE OXIDASE METHODS  2 PRACTICAL 2: DETERMINATION OF SERUM GLUCOSE (NELSON-SOMOGYI AND GLUCOSE OXIDASE METHODS) Background Since the introduction of insulin for the treatment of diabetes mellitus, the determination of blood 'sugar' has been one of the tests most frequently carried out in the biochemical laboratory. Normally during a ‘fast’ the liver can regulate with great precision and rapidity, the amount of glucose secreted into the blood to match exactly the amount of glucose being removed. This ensures a constant blood sugar level during fasting. A fasting person can suddenly leap out a chair, and run rapidly with only minor effects on the blood glucose levels, because of the speed with which the liver can increase or decrease glucose output. By contrast, in pathological or diseased states, the blood glucose does not maintain this constant concentration. It may become elevated (hyperglycaemia) or depressed (hypoglycaemia). Hyperglycaemia, most commonly results from a deficiency in either the amount or the effectiveness of insulin, which is the basis of the condition known as diabetes mellitus. In this disease, the elevation of blood glucose is usually high enough to exceed the renal (kidney) threshold, so that sugar appears in the urine (glucosuria). Two forms of diabetes mellitus are recognized:- Type I - Juvenile onset type - because it often manifests itself in children and young adults. Here there is a marked failure to release insulin from the pancreas. The only effective treatment, is the administration of insulin and is thus often referred to as insulin dependent diabetes mellitus (IDDM). Type II - Maturity onset type - because it often first manifests itself in the adult, especially if obese. In this type of diabetes, the body becomes resistant to insulin or the pancreas is unable to produce enough insulin. In most cases, treatment initially takes the form of dietary recommendations and the use of drugs to stimulate release of insulin. Thus it is often referred to as non-insulin-dependent diabetes mellitus (NIDDM). Ultimately however, the administration of insulin by injection maybe necessary. Glucose is essential as an energy source for survival of mammalian tissues. It is normally transported in the blood to different tissues of the body. Its concentration in the blood varies between narrow limits. However, in certain disease conditions the level may rise (Hyperglycemia) such as obtained in diabetes mellitus, or alternatively fall (hypoglycemia) due to over-dosage with insulin in the treatment of diabetes. Insulin-secreting tumours of the pancreas, hypothyroidism, hypopituitarism and hypoadrenalism (Addison's disease) are all known to produce hypoglycemia. Finally, liver malfunction may affect the level of glucose in the blood. Hence, it is seen that accurate determination of blood glucose is useful in diagnosis of body malfunction.  3 NELSON SOMOGYI METHOD: Test Principle This is an alternative test for the determination of reducing sugars. The sugar is heated with an alkaline solution of copper tartrate and cuprous oxide is produced. Copper reagents are highly specific for reducing sugars and the most utilized method is the alkaline-copper technique developed over many years by Somogyi. The principle is similar to the principle of Benedicts test:- When CuSO4 solution is added to a sugar in the presence of alkaline Hardings solution, glucose is oxidised to produce gluconic acid via the 1-2 endiol intermediate. The cupric ion (Cu2+) is reduced to cuprous ions (Cu+) which can be determined colorimeterically (sphectrophotometrically). Cuprous ions from cuprous oxide (Cu2O) are allowed to react with Nelson's reagent containing Arseno-molybdenate to form molybdenum blue. The intense greenish-blue colour is then measured in a spectrophotometer (colorimeter) set at a wavelength of 650 nm. Sodium sulphate is included in the reaction mixture to minimize entry of atmospheric oxygen into the solution, which would cause re-oxidation of cuprous oxide. The calibration curve obtained depends to some extent on the sugar being estimated, so that the method is not suitable for the determination of a complex mixture of reducing sugars. Protein must be removed before proceeding with the determination and this is carried out using sodium tungstate (Na2WO4) and H2SO4 as protein precipitants to give a neutral protein-free solution. Tungstate solution contains heavy metal which denatures proteins and the H2SO4 then precipitates them. O HC OH C H C OH HC OH Alkaline (OH-) HO CH Harding's reagent HO CH HC OH HC OH HC OH HC OH CH2 OH OXIDATION 1-2 Endiol intermediate CH2 OH 2 Cu2+ (cupric ion) D-Glucose REDUCTION H2O OH- 2 Cu+ Cu2O Cu2 (OH) 2 O (cuprous ion) C OH Na2SO4 added to prevent O2 Arseno-molybdenate (Nelson's reagent) entry thus preventing HC OH reoxidation of Cu2O HO CH HC OH Molybdenum blue (green-blue) HC OH CH2 OH Colorimetric analysis at 650 nm Gluconic acid  4 Reagents: Section A - Protein precipitation and removal from serum 1. Isotonic Sodium Sulphate 2. Sodium Tungstate solution 3. 2/3 N H2SO4 Sulphuric Acid Section B - Determination of blood glucose 1. Copper Sulphate Solution (CuSO4) 2. Hardings solution (Alkaline anticoagulant medium) 3. Nelson's solution (contains Arseno-molybdenate) 4 CuSO4 - Na2SO4 solution 5. Stock Standard Glucose Solution 100mg/100ml a. Low Standard Glucose Solution : Working Standard (WS) 1 Dilute 5.0ml of stock Standard Solution to 200ml with CuSO4 - NaSO4 solution. Two millilitres of this solution are equivalent to a glucose concentration of 100mg/100ml of blood. b. High Standard of Glucose Solution : Working Standard (WS) 2 Dilute 10.0ml of stock Standard solution to 200ml with CuSO4-NaSO4 solution. This is equivalent to a glucose concentration of 200mg/100ml.  5 Method A. Protein precipitation and removal from serum 1. To 3.6ml of Isotonic Na2SO4 in a centrifuge tube, add 0.2ml serum. Mix. 2. Add 0.1ml Na2WO4 (Sodium Tungstate) solution. Mix. 3. Add 0.1ml 2/3N H2SO4. Mix. 4. Centrifuge (2000-3000 rpm) for 5 minutes. This precipitates out the protein. 5. Collect the supernatant that is now free of protein. *MOODLE TASK: Perform Micropipette interactive *MOODLE TASK: Perform Micro-pipetting simulation *MOODLE TASK: Perform LabSim-Centrifugation  6 B. Determination of blood glucose 1. Label 4 test tubes B, S1, S2 and T (Blank, Standards 1 and 2 and Test) and proceed as follows:- TEST TUBES SOLUTIONS (ml) TEST STANDARD 1 STANDARD 2 BLANK 100mg/100ml 200mg/100ml 0mg/100ml Harding solution 2.0 2.0 2.0 2.0 CuSO4 solution 1.0 Supernatant 1.0 Working Standard 1 2.0 Working Standard 2 2.0 CuSO4 - Na2SO4 2.0 2. Mix and place in a boiling water-bath for 10 minutes. Allow the test tubes to cool. 3. Add 2ml Nelson's arseno-molybdenate solution to each tube. (Toxic). 4. Dilute to 25ml in a volumetric flask with distilled water. 5. Let the tubes stand for 5 minutes. 6. Read absorbance in a spectrophotometer set at a wavelength of 650nm.  7 *MOODLE TASK: Perform LabSim - Spectrophotometer parts and theory *MOODLE TASK: Perform LabSim - How to Calibrate a Spectrophotometer RESULTS: Sample Absorbance reading (650 nm) Standard 1 0.241 Standard 2 0.487 Test 0.167 CALCULATE THE AMOUNT OF GLUCOSE IN THE SERUM SAMPLE (This will be one of the questions in the practical report quiz so do this calculation in advance if possible):- Determination of glucose levels in samples as follows. Conc. of sugar = reading of Test x conc. of standard (mg/100ml) (mg/100ml) reading of Standard Since 2 standards were used, calculate the glucose level using each of the two standards. 2ml of WS1 are equivalent to a glucose concentration of 100mg/100ml 2ml of WS2 are equivalent to a glucose concentration of 200mg/100ml N.B. : Blood Glucose 65-105mg/100ml Normal range CSF Glucose 40-70mg/100ml for humans  8 GLUCOSE OXIDASE METHOD: Test Principle For quantitative determination of glucose we will use an enzymic reaction in which the substrate is D-glucose and the catalyst is D-glucose oxidase. This method is based on that of Saifer Gerstenfeld and Marks. This enzyme is strongly specific for D-Glucose only and has been isolated from Penicillium notatum. The enzyme catalyses the oxidation of D-Glucose first to D-Gluco 1.5-lactone. Lactone is hydrolysed to Gluconic acid whilst the enzyme reduces oxygen to the level of hydrogen peroxide. The above reaction is coupled with a chromogenic reaction in which hydrogen peroxide (H2O2) reacts with an oxygen acceptor o-dianisidine in the presence of the enzyme peroxidase to give a brown colour which can be measured in a spectrophotometer (colorimeter) set at a wavelength of 500 nm. Peroxidase originates from plants, turnip or horseradish. This reaction is used in glucometers for rapid determination of blood glucose levels. * MOODLE TASK: Watch video on ‘Detecting Blood Glucose using a Glucometer’. Please note that time points 3:39 to 5:54 minutes explains this application.  9 Reagents 1. Zinc sulphate (ZnSO4.7H2O) 5%. 2. Sodium hydroxide, 0.3M. 3. o-Dianisidine hydrochloride, 0.5% in distilled water. Lasts for at least 2 weeks if refrigerated. 4. Glucose oxidase (obtainable in the form of a pale amber liquid, 660 units/ml. Stable almost indefinitely if refrigerated. 5. Acetate buffer, 0.1M, pH5. To 7 volume of 0.1M-sodium acetate add 3 volumes of 0.1M- acetic acid. 6. Peroxidase. Prepare a solution containing 1 mg per 5ml of acetate buffer. Lasts for several months if refrigerated. 7. Glucose oxidase reagent. To about 80 ml of acetate buffer add 0.5 ml of glucose oxidase, 5ml of peroxidase solution, and 1 ml of 0.5% o-dianisidine-HCl. Make up to 100ml with buffer and store in the refrigerator. It maintains its activity for at least 2 weeks. 8. Standard glucose solution. Prepare a stock glucose solution by dissolving 100 mg of glucose in 100 ml of 0.1% benzoic acid. This standard solution is equivalent to 100 mg glucose/100 ml. The glucose solutions are stable indefinitely at room temperature. 9. Normal sulphuric acid (IN). NOTE : TAKE SPECIAL CARE/ WARNING!!! ♦ Do not pipette or spill glucose oxidase-contains γ-dianisidine hydrochloride which is a potential carcinogen. ♦ Standard glucose solutions contain Benzoic Acid a skin irritant if you should spill any on your hands, flush well with water. ♦ Zinc Sulphate can cause irritation to skin. If spilt flush exposed area well with water.  10 Method A. Protein precipitation and removal from serum 1. To 2,8 ml of distilled water in a centrifuge tube add 0,2 ml of serum. 2. Then add 1,0 ml of 5% zinc sulphate and 1,0 ml of 0.3M NaOH. 3. Mix, stand for 5 minutes and centrifuge at (3000 r.p.m.) for 10 minutes. 4. Carefully pour the supernatant into a clean test tube. Please note that when preparing the protein-free supernatant, the blood is diluted 25 times and thus during calculation, the answer must be multiplied by 25 in order to get the final concentration of glucose. B. Determination of blood glucose 1. To 1 ml of distilled water (blank tube) (B), 1 ml of blood supernatant (Test, T), and to 1 ml of glucose standard (Std) solution in separate test tubes add 5 ml of the glucose oxidase reagent. Mix well, 2. Incubate in a water bath at 37°C for 10 minutes. 3. At the end of this time remove the tubes from the water bath and to each tube add 1 ml of 1N-H2SO4 and mix. This stops the enzyme action, eliminates any turbidity and stabilizes the amber colour. 4. Read against the distilled water blank in a colorimeter set at a wavelength of 500 nm. 11  12 RESULTS: Sample Absorbance reading (500 nm) Standard 0.965 Test 0.044 CALCULATE THE AMOUNT OF GLUCOSE IN THE SERUM SAMPLE (This will be one of the questions in the practical report quiz so do this calculation in advance):- Determination of glucose levels in samples as follows. Conc. of sugar = reading of Test x conc. of standard (mg/100ml) (mg/100ml) reading of Standard N.B. : Blood Glucose 65-105mg/100ml Normal range CSF Glucose 40-70mg/100ml for humans *MOODLE TASK: Watch YouTube video on Determination of Blood Glucose https://www.youtube.com/watch?v=bAhhIMD3Af0 *MOODLE TASK: Complete the Quiz titled ‘Practical 2 Report‘ between 14:00 and 17:00 on Thursday 2 September.