Glucose, glucose oxidase/peroxidase method, photometry, diabetes mellitus.

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1 Quantitative Determination TEAS Related topics Glucose, glucose oxidase/peroxidase method, photometry, diabetes mellitus. Principle Blood sugar is the key energy provider for our cells. The primary hormone in blood sugar regulation is insulin. The glucose oxidase/peroxidase method for the determination of blood sugar introduced here is also applied in multiple test strips for the determination of glucose in the urine. In this method, the glucose oxidase (GOD) is used to oxidize glucose by the oxygen in the air to gluconic acid. The H 2 O 2 generated in this process reacts, by action of peroxidase (POD), with a colour indicator and is converted into a dye which is detectable by photometry. Equipment Student Basic Set 1 Microliterpipette dig µl Microliterpipette dig µl Digital stop watch, 24 h, 1/100 s & 1 s Beaker, 100 ml, low form, plastic Lab. pencil, waterproof Semi-micro cuvettes 1.5ml, 100pcs Cuvette rack, PE, 16 places Pipette tips, µl, racked Pipette tips, µl, racked Disposable gloves, 100pcs,medium Disposable stirring spatula, plastic, pcs Lab Equipment 1 Filter photometer 400 to 700 nm Chemicals 1 Water, distilled, 5 l Required additionally Glucose reagent incl. Standard sample Control TruLab P and N Note: The reagents and control samples used for this experiment are listed in the Appendix. Please note! Always observe the preliminary remarks when performing this experiment. Fig.1: P P PHYWE Systeme GmbH & Co. KG All rights reserved 1

2 TEAS Quantitative Determination Tasks 1. Determine the glucose concentration in the blood of a patient and in a control sample. 2. Calculate the deviation of the individual measured value of the control sample. Set-up and procedure Preparation of reagent - The reagent is ready for use - Bring reagent to room temperature, follow the instructions in the package leaflet. Material for the examination Please refer to Chapter Preanalytics. - Serum, NaF-plasma, 24-hour urine, liquor - The standard solution (from package with reagent) is ready to use = 100 [mg/dl] (according to package Fig. 2: leaflet) - TruLab P or N are used as control samples. They are lyophilised. Dissolve in distilled water, following instructions in the package leaflet (refer to Chapter with preliminary remarks). Note A number of aspects have to be taken into consideration in preanalytics. A glucose concentration gradient exists between arterial and venous blood as well as between plasma and erythrocytes. In healthy subjects, this arterial-venous difference amounts to approx mg/dl and may rise to up to mg/dl postprandially. Immediately after taking a blood sample, the glycolytic effect of erythrocytic enzymes initiates a loss of glucose. This can be reduced by adding glycolysis inhibitors like sodium fluoride, for example. When no NaF-tubes are available when the blood sample is taken, the serum has to be separated from the glucose consuming cells within 60 minutes and glucose determination has to be carried out immediately. Task 1: Determine the glucose concentration in a sample taken from a patient and the control sample In accordance with the pipetting scheme, 10 µl each of the sample to be analysed (standard, control or patient s sample) are mixed with 1000 µl of the reagent and incubated for 10 min. Subsequently, the extinction is measured. To obtain the blank value for the reagent, the reagent is pipetted with 10 µl of distilled water instead of sample. Pipetting scheme 1 st cuvette Reagent blank value (LW) 2nd 4th cuvette Sample (std. control, patient) Sample (std. control, - 10 µl patient) Aqua dest. 10 µl - Reagent 1000 µl 1000 µl Figure 3: Measuring the blank value 2 PHYWE Systeme GmbH & Co. KG All rights reserved P

3 Quantitative Determination TEAS Measurement The temperature should be C. Initially, the reagent blank value has to be set. - Switch on device: on/off - Set wave length: 550 nm - Insert blank value cuvette (LW) into measuring point Press button R The remaining samples (standard, control and patient s sample) are determined as follows: - Insert cuvette into measuring point Press button T - Read extinction When finished - Switch off device: on/off Theory Blood sugar is the key energy supplier for our cells. We take up sugar from our food and burn it, mostly producing CO 2 and water. This generates energy, which is required for numerous processes in the cells. Excess sugar molecules are stored as glycogen in liver and muscles. If we do not consume enough carbohydrates for a prolonged period of time, fat and protein will be degraded to produce energy (gluconeogenesis) The primary hormone of blood sugar regulation is insulin. Insulin has a blood glucose reducing effect. Main purpose of the determination of blood glucose is the diagnoses of diabetes. Increased fasting blood glucose levels are an indication for diabetes. The aim of further diagnostics is a differentiation between type I and type II diabetes. Whereas type I diabetes has its roots in an insulin deficiency, normal or even increased insulin levels are observed in type II diabetes. The reason for type II is a disorder of the insulin receptors. Type I basically necessitates an insulin substitution therapy, whilst type II can be treated by dietary measures or medication, depending on its severity. Principle of the test (refer to package leaflet of reagent) In the glucose oxidase/peroxidase method (GOD/POD) introduced here, glucose oxidase (GOD) is used to oxidize glucose by the oxygen in the air to gluconolactone. GOD Glucose + O 2 Gluconic acid + H 2 O 2 Under the influence of the peroxidase (POD), the H 2 O 2 generated during this process reacts with a colour indicator F-H 2 (4-aminoantipyrine and phenol), forming the dye F (chinonimin) and water. POD 2H 2 O 2 + F-H 2 F (Chinonimin) + 4 H 2 O The measurable colour intensity is proportional to the glucose concentration. This method is applied as well on the multiple test strip for the semi-quantitative determination of glucose in the urine. P PHYWE Systeme GmbH & Co. KG All rights reserved 3

4 TEAS Quantitative Determination Evaluation Task 1: Calculation of the concentration The glucose concentration can be determined directly from the extinction of the samples, in comparison with the standard. Glucose[mg/dl] = E sample E standard [standard] The standard is shown in mg/dl. Subsequently, the concentration may be converted into SI-units (mmol/l). Amongst others, the conversion factor includes the molar mass of the glucose. Glucose [mg/dl] x 0,05551 = Glucose [mmol/l] Example for the calculation Extinction Standard (Std): 0.26 Extinction Control (Ko): 0.24 c Control mg dl = E Sample E Standard c Standard = mg dl = 92.3 mg dl The control sample thus has a glucose content (c Kontrolle ) of 92.3 mg/dl, corresponding to a concentration of 5.12 mmol/l. Extinction Patient (P 1 ): 0.66 c P1 mg dl = E Sample E Standard c Standard = mg dl = 275 mg dl The patient s sample thus has a glucose content (c P1 ) of 275 mg/dl, corresponding to a concentration of 15.3 mmol/l. Reference values mg/dl Task 2: Calculation of the deviation of the individual measured value of the control sample (KoPEM) The deviation of the individual measured value of the control sample is used to verify whether errors occurred in the experiment (according to Guidelines of the Federal Medical Council on quality assurance in medical laboratory tests. Please observe statutory provisions in your country - see preliminary remarks). The value of the control samples determined in the experiment is the so-called Actual Value. But we also know the true value of the control sample, called Target value. It is indicated in the package leaflet of the control serum (Trulab P or N). The following formula is used to calculate the deviation of the individual measured value of the control sample (abbr. KoPEM) from the determined actual value and the known target value: 4 PHYWE Systeme GmbH & Co. KG All rights reserved P

5 Quantitative Determination TEAS KoPEM[%] = Target Actual Target 100 Examplary calculation: Actual value of the control sample: 92.3 mg/dl Target value of the control sample: 87.6 mg/dl KoPEM[%] = Target Actual Target 100 = = 3.1% 95.5 Following this, the deviation of the individual measured value of the control sample from the target value is compared with the limits permitted under general rules in the respective country. In the Federal Republic of Germany, this limit for glucose is indicated as +/-11 % in Table B 1a RiliBÄK [Guidelines of the German Federal Medical Council on quality assurance in medical laboratory tests]. In our example, the individual measured value of the control sample fulfils this default. The patient may thus be assessed. Caution: Please observe statutory provisions in the respective country! Please refer to Chapter with preliminary remarks. Questions - Why is a reagent blank value maintained? In order to eliminate the influence of the intrinsic colour of the reagent on the result. - Which system for taking blood specimens permits effective inhibition or prevention of glycolysis? Sodium fluoride (NaF-tubes) - How will the glucose value of a patient turn out, when the blood specimen was centrifuged too late, and why does this occur? The measured sugar value will be too low, because immediately after taking a blood sample, the glycolytic effect of erythrocytic enzymes will initiate a loss of glucose. - Which oxidation product is generated in enzymatic glucose detection with GOD? - H 2 O 2 hydrogen peroxide P PHYWE Systeme GmbH & Co. KG All rights reserved 5

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7 Quantitative Determination TEAS Lab Report Task 1 You can enter your values in the following table: Sample Standard (Std) Extinction Concentration (mg/dl) Control (Ko): Sample 1 Task 2 Record the deviation of the KoPEM and discuss the result. Questions - Why is a reagent blank value maintained? - Which system for taking blood specimens permits effective inhibition or prevention of glycolysis? - How will the glucose value of a patient turn out, when the blood specimen was centrifuged too late, and why does this occur? - Which oxidation product is generated in enzymatic glucose detection with GOD? P PHYWE Systeme GmbH & Co. KG All rights reserved 7

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