Lab_2_-_Blood_Glucose♥

.pdf

School

Concordia University *

*We aren’t endorsed by this school

Course

364

Subject

Biology

Date

Apr 3, 2024

Type

pdf

Pages

Uploaded by UltraDanger13822

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.

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

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version