Lab_2_-_Blood_Glucose♥

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BZE Laboratory 2 Summer 2023 1 Lab 2 Measuring Glucose Content by the Glucose Oxidase Method AIM When you have completed this lab you will be able to : 1. Define homeostasis and negative feedback 2. Describe blood glucose homeostasis. 3. Describe the oral glucose tolerance test and its use. 4. Describe the Glucose Oxidase-Peroxidase coupled reaction and its specificity in measuring glucose. 5. Construct a standard curve and discuss its importance in quantitative measurements of biomolecules. 6. Use a standard curve to measure the concentration of an analyte in a test sample. INTRODUCTION The main sugar present in the blood is glucose. The body uses an elaborate set of mechanisms (in which hormones play a central part) to maintain a safe level of glucose in the blood between 3.9 and 6.1 mM; it must never be so high as to cause osmotic pressure problems, for example, nor never so low as to deny adequate energy to tissues. Homeostasis works to maintain the organism's internal environment within tolerance limits - the narrow range of conditions where cellular processes are able to function at a level consistent with the continuation of life. It is important to remember that this does not imply that homeostasis is an unchanging state. In fact there are constant adjustments being made to maintain the balance where internal conditions are appropriate. These conditions are always varying but within very narrow limits. The most common way that the internal environment is kept relatively stable, is through negative feedback mechanisms (Figure 1) Immediately after a meal containing carbohydrates, the blood glucose level rises. This stimulates production of insulin by the pancreas, which in turn stimulates muscles and adipose tissue to remove glucose from the blood and to store it away as glycogen or fat. Later on, when blood glucose levels are low, the pancreas releases another hormone, glucagon which stimulates the liver to release glucose into the blood (Figure 1). Figure 1: Blood glucose homeostasis Negative feedback control of blood glucose by the hormones insulin and glucagon A number of disorders can disrupt blood glucose homeostasis with potentially serious consequences, especially for the heart, blood vessels, eyes, and kidneys. The disease diabetes mellitus is caused by a defect in insulin signalling, either an inability to produce enough insulin (Type 1) or a decreased response to insulin in target tissues (Type 2). Blood glucose levels rise, but cells are unable to take up enough glucose Figure I1. Blood Glucose Homeostasis. Maintenance of glucose levels within a normal range by insulin and glucagon. Body cells take up more glucose. Insulin Beta cells of pancreas release insulin into the blood. Liver takes up glucose and stores it as glycogen. Blood glucose level declines. Blood glucose level rises. Homeostasis: Blood glucose level (70 – 110 mg/100mL) STIMULUS: Blood glucose level rises (for instance, after eating a carbohydrate-rich meal). Liver breaks down glycogen and releases glucose into the blood. Alpha cells of pancreas release glucagon into the blood. Glucagon STIMULUS: Blood glucose level falls (for instance, after skipping a meal). 3.9 – 6.1 mM

BZE Laboratory 2 Summer 2023 2 to meet metabolic needs. Instead, fat becomes the main substrate for cellular respiration. In severe cases, acidic metabolites formed during fat breakdown accumulate in the blood, threatening life by lowering blood pH and depleting sodium and potassium ions from the body. Oral Glucose Tolerance Test (OGTT) This medical test assesses an individual’s gl ucose tolerance, the ability to clear glucose in the blood. The test can be used to identify diabetes mellitus or other conditions involving abnormal blood glucose metabolism. The patient receiving the OGTT is asked to fast for 8-14 hours before the test. When the individual arrives for the test, a blood sample is drawn (the 0-hour sample). The patient is then given a standard glucose solution to drink. Blood is drawn 2 hours after the glucose solution was ingested (the 2- hour sample). The glucose concentration in the plasma of both blood samples is determined. Blood plasma is the liquid part of blood and contains salts, carbohydrates, amino acids, proteins, lipids, vitamins, urea, hormones, and other substances. To prepare blood plasma from whole blood, the sample is centrifuged to bring the blood cells down to the bottom of the tube (Figure 2). The yellowish solution on top of the tube is plasma and can be removed from the tube to isolate plasma from the blood cells. The glucose concentration in the 0- hour sample is the patient’s fasting level of glucose in the blood and provides a baseline. The glucose concentration in the 2- hour sample reflects the patient’s glucose tolerance. The criteria for determining an patient’s glucose tolerance condition based on their plasma glucose concentrations in the 2-hr sample listed in Table 1 Table 1. Interpretation of Oral Glucose Tolerance Test (2-hr sample) Normal Glucose Tolerance Impaired Glucose Tolerance Diabetes Mellitus <7.8 mM 7.8 to 11.1 >11.1 In this experiment, you will measure the level of glucose in human blood serum. The level will depend upon how recently the blood donor has eaten and whether carbohydrates were consumed. At its lowest the blood sugar level should be 3.8-5 mM. This is the normal fasting level . After intense exercise or in the case of prolonged starvation the blood sugar level may fall below the normal fasting level, resulting in a state of hypoglycemia. Since the cells of the brain cannot store glucose nor use other substances in the blood for food, the brain is one of the first organs to feel the effects of low blood glucose levels. Hunger produces a feeling of faintness and often a headache. Something sweet, such as chocolate, will relieve these symptoms quickly, as brain cells are able to absorb glucose directly from the blood stream – they are insulin-independent. At its highest, i.e. after a meal, the blood glucose level may rise to about 6.9 mM. This is normally a temporary state of high blood sugar. If the level rises any higher than this the body begins to excrete glucose in the urine, a condition called glucosuria. Such would be the condition of an untreated diabetic, a condition in which the person is unable to produce insulin normally. Glucosuria can also be caused by other diseases and conditions. A biomolecule can be measured directly by using an inherent property of the test substance. For example, the absorption of red light by chlorophyll A can be measured and used to quantitate its concentration in Figure 2 . Centrifugation of a whole blood sample to separate it into plasma and blood cells.

BZE Laboratory 2 Summer 2023 3 solution. A formula based on published measurements for purified pigments can be used to determine concentration from solution absorbance values at 647 nm and 664 nm. The biomolecule can be measured indirectly. Adding a reagent that reacts with the test substance can produce a colorimetric or fluorescent signal that correlates with the amount of the test substance. For example, the reaction of copper(II) ions and peptide bonds in proteins produces a violet color that can be used to quantitate the amount of protein in solution. A spectrophotometer can be used to measure absorbance at 540 nm and a standard curve can be generated to set the relationship between absorbance and protein amount. There are several methods available for measuring glucose. The method used in this lab involves a reagent containing enzymes, phenol and a dye which is colorless (o-toluidine) when reduced and pink when oxidized. This reagent is called Glucose Oxidase Dye reagent, or G.O.D. The enzymes in this reagent catalyze the following reactions: Glucose Oxidase Glucose + O 2 + H 2 O H 2 O 2 + Gluconate Peroxidase with cofactors H 2 O 2 + Reduced dye Oxidized dye (pink) + 2H 2 O 1. In the first reaction, glucose is oxidized with molecular oxygen to gluconate (a carboxylic acid) and hydrogen peroxide: 2. In the second reaction, the hydrogen peroxide produced by reaction 1. oxidizes a reduced dye molecule like o-toluidine (or other) into a colored product which is then measured by the spectrometer: The amount of colour produced depends upon the amount of glucose present. Therefore, by measuring the depth of color (absorbance, optical density, O.D.) with a spectrometer one can determine how much glucose was present in the blood sample. Any method which involves measuring color must involve standardization. In this case, you must find out how much glucose will produce a particular depth of color and whether there is a direct relationship between the amount and the color. To do this you measure the absorbance produced when G.O.D. reagent reacts with various known amounts of glucose. (The standard glucose solution you will use has been very accurately prepared to contain 3mM. ) When plotted, these absorbance values produce what is called a standard curve . You will then determine the absorbance of serum samples (or a serum substitute) reacted with G.O.D. These values can be compared to the standard curve and in this way the concentration of glucose in the serum can be determined.

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BZE Laboratory 2 Summer 2023 4 PROCEDURE: Fill out the table below to make your standard curve For this lab, you will be working as a team with a partner. Make sure that the work is evenly distributed . Your team will make 10 samples & treat them with the GOD-POD reagent to conduct a quantitative specific colorimetric assay for glucose . A. Preparation of Glucose Standards In this step, your team will prepare 6 standard glucose solutions in water. 1. Locate your set of 6 micro test tubes in a rack. Label the cap of the tubes: S5 , S4 , S3 , S2 , S1 , & S0 . Leave the tubes open in a rack in the order described above. 2. Use a P1000 with a clean tip to add 1000 μL (1.0 ml) of deionized water to your S0 tube . Cap this tube & set this sample aside as you will not be adding anything further. This is your blank . 3. Use the appropriate pipettes & clean pipette tips to add the various amounts of deionized water & glucose stock solution (2.8 mM) to tubes: S4 , S3 , S2 , S1 – NOT S5 - as indicated in Table P2 below . Always use a clean tip when taking liquid from a stock solution. This will avoid contamination or dilution of your stock solution. Discard the tips into the “solid” waste container. 4. Use a P1000 with a new clean tip to add 1000 μL of the glucose stock solution (2.8 mM) to tube S5 . 5. Cap the tubes & set them aside in the rack. 6. Complete Table 2. Calculate the quantity of glucose standard to prepare you standard solutions for your standards & enter your values in Table 2. Table 2 Test tube Deionized H 2 O Glucose standard (50 mg/100 ml) (2.8 mmol) Final volume (ml) Final concentration (mmol) 0 1.0 ml (1,000  l) 1.0 0 1 1.0 0.28 2 1.0 0.70 3 1.0 1.4 4 1.0 2.1 5 1.0 ml (1,000  l) 1.0 2.8 7. Before you proceed, verify that each standard tube contains the correct volume to help determine if you made any mistakes. B. Preparation of the OGTT Samples In this step, your team will prepare 3 technical replicates of the 2-hr OGTT sample for either patient A or B. However, before you proceed, you need to decide if you will have to dilute your test sample to prepare your technical replicates & what dilution factor to use. Remember, the concentration of the test sample must be within the concentration range of your standard curve, or you will have to dilute the sample.

BZE Laboratory 2 Summer 2023 5 These samples can contain from 5 to 20 mmol/L of glucose and the standard curve is linear only from 0 to 2.8 mmol thus it will be necessary to dilute the samples. This is a well-known method and a dilution factor of 5 is appropriate Table 3 dilution factor of OGTT sample Dilution factor} 5 dH2O μl Sample μl Total volume μl 500 *Dilution factor = total volume/aliquot of sample Fill out the table below Table 4 preparation of diluted samples Replicate Sample (μl) dH2O (μl) Total volume 1 500 μl 2 500 μl 3 500 μl Label 3 micro test tubes A1, A2, and A3 if your team is testing sample A or Label 3 micro test tubes B1, B2, and B3 if your team is testing sample B Cap the tubes & set them aside in the rack with the other micro test tubes. C. Preparation of the Galactose Specificity Control Sample In this step, you will prepare a sample that contains galactose, a stereoisomer of glucose (Fig. 4). Testing this sample in the analysis method will allow you to determine if the assay is specific for glucose or if other monosaccharides are detected by this assay, giving rise to background. Because you should not have any reaction, galactose can be said to be a negative control for this assay. Figure 4 : Glucose and galactose are steroisomers Procedure 1. Label 1 micro test tube with G for galactose. 2. Use a P1000 with a new tip to add 500  l of the stock solution of galactose (3 mM) to your tube. 3. Cap the tube & set it aside in the rack with the other micro test tubes

BZE Laboratory 2 Summer 2023 6 Transferring an Aliquot of Each Sample In this step, each team will transfer a 200  l aliquot of each of their 10 samples to a glass test tube for processing. 1. Put your 10 samples in the following order in the rack: S0 , S1 , S2 , S3 , S4 , S5 , R1 , R2 , R3 , G . 2. Label each of the 10 glass test tubes with a label corresponding to one of your 10 samples. Put the tubes in the same order to make this step easier & avoid erroneous transfers . 3. Use a P200 micropipette to transfer 200  l of each sample in the micro test tube to the corresponding glass test tube in the other rack. You must change tips between each transfer or else you will be contaminating your samples . Add the sample to the bottom of the tube, not the side! 4. Once the aliquot has been transferred move the glass tube to other side of the rack to avoid mistakes Adding the GOD-POD Reagent In this step, you will add water & the GOD-POD reagent to the tubes with 200  l of each sample you will test. 1. Bring your rack with your 10 tubes each containing 200  l of your samples to the side bench with the dispenser bottle for water & reagent. 2. Add 2 ml of water to each tube using the dispensette attached to the bottle containing deionized water. Bring the mechanism up & down gently to accurately add the correct volume. 3. Add 2 ml of reagent to each tube using the dispensette attached to the bottle containing the GOD-POD reagent. Bring the mechanism up & down gently to accurately add the correct volume. 4. Bring the rack back to your bench, gently swirl the solution to mix, & let the tubes stand at room temperature for 20 minutes.

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BZE Laboratory 2 Summer 2023 7 Reading the Signal Read Before You Proceed: Once you read your samples, DO NOT DISCARD THEM IN THE LIQUID WASTE YET . Return each sample to its test tube. This will allow you to re-read the sample if need be. 1. Make sure your spectrophotometer (spec) is set to 510 nm . 2. Use your S0 sample (blank or zero standard) to blank the spec. Carefully pour the sample into one of the two plastic cuvettes provided. N.B. Keep this blank sample in the cuvette, it will be used to blank the spec again after 5 sample readings. Recalibration of spectrophotometers is often required due to ‘drift’ over time. 3. Start reading the rest of your samples using the other plastic cuvette & fill in the absorbance values in the designated tables in the Data section of this document. Important : wash out the cuvette between sample transfers with the water bottles provided & tap upside-down onto paper towel to dry. N.B. Absorbance values under 0.010 are not reliable & should be recorded as zero . This includes negative absorbance values. Record the absorbance as O.D. values in the table on the data sheet page 8 Using the values obtained for solutions 1 – 5 plot a standard curve of the effect of the amount of glucose on optical density. In the graph on page 9. Plot the absorbance (510 nm) values vs. the glucose concentration (mM) for each of the 5 standards (do not include the tube 0 zero standard). Using a ruler , add a line-of-best-fit that illustrates the linear relationship between absorbance and glucose concentration for this assay show this to your instructor before leaving Label the axes and include a figure description (at the bottom next to Figure 1). N.B. A figure description comprises a descriptive title and any other information you want to include to help a reader understand the graph and make it “stand - alone”. This summary goes at the bottom of the figure next to the figure identifier Which is the independent variable? Which is the dependent variable ? Using either a ruler or excel, add a line-of-best-fit that illustrates the linear relationship between absorbance and glucose concentration for this assay

BZE Laboratory 2 Summer 2023 8 Standard Curve for the Determination of Glucose Content by the Glucose Oxidase Method NAME ____________________________ Date: __________ B. Standards Enter the glucose concentrations used for the standards (see Table P1). Enter the absorbance measurements that you obtained for the standards in Table D1. Table D1. Absorbance values for the glucose standards [Glucose] (mM) Standard tube Absorbance 510 nm 0.00 0 0.000 0.28 1 0.70 2 1.40 3 2.1 4 2.8 5 B. OGTT Samples We are testing patient: _____ Enter the absorbance measurements that you obtained for the 3 technical replicates of the diluted 2-hr OGTT sample in Table D2. Table D2. Absorbance values for diluted OGTT technical replicate samples OGTT Sample Absorbance 510 nm Replicate 1 Replicate 2 Replicate 3 mean C. Galactose Specificity Control Sample Enter the absorbance measurement that you obtained for the galactose specificity control sample in Table D3. Table D3. Absorbance value for the galactose specificity control sample Carbohydrate Concentration (mM) Absorbance 510 nm Galactose 3.00

BZE Laboratory 2 Summer 2023 9 Figure 1: For the lab report on Moodle make an excel file and graph and use the linear regression formula y=mx + b to calculate the concentration in your unknown sample upload the graph (1 per group of two) and data from page 1 of the lab report in the drop file when you complete the Moodle lab report

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BZE Laboratory 2 Summer 2023 10 On the standard curve, mark the positions of the OD values obtained for tubes replicates 1, 2, and 3 of the unknown sample (A or B) read from the x-axis the amount of glucose present in each 0.1 mL serum sample (= mmol glucose) and enter in the table below. Absorbance values for diluted OGTT technical replicate samples OGTT Sample Absorbance 510 nm Replicate 1 Replicate 2 Replicate 3 Table Interpretation of Oral Glucose Tolerance Test (2-hr sample) Normal Glucose Tolerance Impaired Glucose Tolerance Diabetes Mellitus <7.8 mM 7.8 to 11.1 >11.1 Referring to the table above what category would you use to describe the patient ’s condition for glucose metabolism? Referring to the table above what category would you use to describe the patient ’s condition for glucose metabolism? Specificity of the Quantitative Analysis Method Several substances within a test sample can lead to background signal. One source of background is the presence of molecules with similar structures & properties to the analyte if the analysis method is not specific . Any substance that is suspected to be contributing to background can be tested in a control sample. We tested galactose, a stereoisomer of glucose, in this assay to determine whether it would produce background. The background signal can be used to calculate the degree of specificity . The degree of specificity is a measure (expressed as a percentage) of how specific the GOD-POD reagent assay is for detecting glucose when compared to another particular monosaccharide. 1. Complete Table R3. Calculate the glucose concentration measured when galactose is used in the assay & the degree of specificity of the assay. Remember: absorbance values under 0.010 are not reliable & should be recorded as zero . This includes negative absorbance values. Table R3. Specificity of the GOD-POD Assay Monosaccharide concentration (mM) A510 nm [Glucose] Detected (mM) Degree of Specificity (%) * Galactose 3 * Degree of Specificity = (1- ( [Glucose] Detected / [Monosaccharide] Present ) ) x 100%

BZE Laboratory 2 Summer 2023 11 Interpreting the degree of specificity: • A result of 100% indicates that the assay does not recognize the other monosaccharide & suggests that the assay is specific for detecting glucose. • A result of 0% would indicate that the other monosaccharide is detected just as well as glucose & suggests that the assay is not specific for glucose. • A result of 80% would indicate that the assay is mainly specific but the method can also detect the other monosaccharides with 20% efficiency. A degree of specificity under 100% for a particular monosaccharide would indicate that some of the signal detected in your test sample does not correspond to glucose & represents background. Therefore, your calculation of glucose in your test sample might be overestimated if the other monosaccharide is also present. Answer the following questions. 1. What is a standard curve and what is it used for? 2. What is the purpose of using a control in an experiment? 3. Which two (2) samples were used as controls in this experiment? Explain the purpose of EACH of these controls. 4. In an untreated type 1 diabetic person (i.e. lacking insulin) the blood glucose concentration after a meal reaches an abnormally high level. Eventually it slowly falls, however. Knowing that insulin is required for glucose uptake into liver, adipose, and muscle tissues, give two causes why blood glucose levels still fall in an untreated diabetic: 1. 2.

BZE Laboratory 2 Summer 2023 12 5. Briefly describe the homeostatic control of blood glucose, explain the role of insulin and glucagon a) Describe and explain what will happen in the body to regulate the amount of glucose in the blood shortly after a person eats a sugary snack. b) Describe and explain what will happen in the body to regulate the amount of glucose in the blood after a person has not eaten for several hours. Discuss how your answers to parts (a) and (b) relate to the concept of homeostasis.

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BZE Laboratory 2 Summer 2023 13 6. Given the standard curve below what is the concentration of protein in an unknown sample with an absorbance of 0.092 at 595 nm? Show calculations 7. What was the concentration of protein was in the original sample (above) if 250 ml of sample was added to 750ml of buffer? Show calculations [Dilution factor (DF): ratio of final volume/aliquot volume (final volume = aliquot + diluent) 9. Which compound is produced in reaction 1 of the GOD-PAP detection method that will feed into the next reaction? Which compound produced in reaction 1 is just a by-product (not involved in signal production)? Is this a direct or indirect detection method for glucose measurement? Explain. 10. If a test sample contained a high concentration of hydrogen peroxide, could you still use this method to measure the glucose concentration? Explain.

BZE Laboratory 2 Summer 2023 14 11. Give the calibration curve below: If you had an unknown sample that has an absorbance of 0.88 what would you do?

Lab_2_-_Blood_Glucose♥

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