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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS LAB 4. CELLULAR RESPIRATION and GLUCOSE MONITORING REMINDER!! Bring a calculator to this lab PRELAB: Answer the questions on diabetes (p 6 Print off lab 4 Journal Article and read before class (see web site under Links); the questions regarding this article are to be answered and handed in at the end of the lab (you may wish to complete these before class!) PART 1. INTRODUCTION TO CELLULAR RESPIRATION Most cellular reactions require a source of chemical energy, ATP. The major source of ATP is the oxidation of glucose. Glucose oxidation can be broken down into two major phases. 1. Glycolysis, an anaerobic process that occurs in the cytoplasm of the cell. Oxygen is not required for glycolysis which occurs in both aerobic and anaerobic organisms. The end product of glycolysis is pyruvic acid. A small amount of ATP is made during glycolysis. 2. Aerobic Cellular Respiration only proceeds when oxygen is available and the cell has enzyme sysytems in place to utilize it. This phase of glucose oxidation, consisting of the Krebs Cycle and electron transport, occurs in the mitochondria of the cell. A large amount of ATP is produced in aerobic respiration -- particularly during electron transport. ANAEROBIC RESPIRATION - Fermentation Some cells carry out glycolysis and then proceed to metabolize pyruvic acid anaerobically by the process of fermentation. When a yeast cell carries out fermentation the end products are ethyl alcohol and carbon dioxide. Other cells will produce lactic acid from pyruvic acid by fermentation. For example, the muscle cells in your body do this when their oxygen supply is temporarily depleted during a period of strenuous exercise. In both cases, the further metabolism of pyruvic acid under anaerobic conditions does not increase the supply of ATP available to the cell. While an anaerobic organism may obtain enough ATP from glycolysis/fermentation to sustain life activities, an aerobic organism requires much more ATP to sustain its activities and can only produce this when pyruvic acid is further metabolized aerobically. Yeasts are facultative anaerobes. This means that while they carry out normal respiration when oxygen is available, they also have the ability to obtain energy via the process of fermentation when oxygen is not available. Under anaerobic conditions yeast will metabolize glucose as follows: C 6H 1 2O 6 2C2H5OH + glucose ethanol + 2CO2 carbon dioxide + ENERGY + 2ATP 4-1 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS PURPOSE 1. To demonstrate the fermentation of sugar by yeast. 2. To determine which sugars yeast can ferment and to what extent. 3. To understand the importance of experimental controls 4. To understand what is meant by a controlled variable, independent variable and dependent variable. PROCEDURE Each group of students will set up one fermentation tube and group results will be compiled. • Practice filling your fermentation tubes with 30 ml of tap water following a demonstration. If not already done, label and colour band them 1 - 12. • Dump out all of the water • Weigh out 0.5g yeast in a small tared weigh boat. • Measure out 30 ml of the stock solution that your group has been assigned into a 50 ml beaker. Add one drop of 0.1 NaOH to each solution and enough phenol red to make the solution pink. NOTE: phenol red is a pH indicator that is yellow in the presence of acid and pink in the presence of base. Orange indicates neutral pH (pH 7) • NOW........ as a class.....take up the 10 weighboats of 0.5g of yeast and on a countdown add the yeast to every beaker except no. 2 ( no yeast ) Mix thoroughly with a stirring rod. • Now, add each mixture to the appropriate labelled, color coded fermentation tube as follows. • Tube 1: Glucose + yeast Tube 7: Galactose + yeast Tube 2: Glucose (no yeast) Tube 8: Fructose + yeast Tube 3: Sucrose + yeast Tube 9: Sorbitol + yeast Tube 4: Maltose + yeast Tube 10: Aspartame + yeast Tube 5: Lactose + yeast Tube 11: Sucralose + yeast Tube 6: Glucose + yeast + NaF Tube 12: Distilled water + yeast 0 Immediately place all tubes on a tray and place it in the incubator at 37 C for about 20 minutes. (While the tubes are incubating, start part 2, Glucose Monitoring on page 6) 4-2 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS • After 20 min. observe the tubes in the incubator and note if gas is appearing anywhere. If any tubes have completely blown; remove the tray and tubes. If not, re-incubate for a further 20 minutes or until the first tube blows! • Remove the tubes for analysis. Measure the heights of the gas columns in each tube and record these data in the table provided. RESULTS Table 1. Fermentation of various sugars and sugar substitutes by yeast Tube No. sugar or substitute Fermentation Tube Results yeast NaF (inhibitor) final colour gas column ht. (mm) 1 2 3 4 5 6 7 8 9 10 11 12 1. Although these data are probably best presented as a table; the results could be also be presented as a graph. What type of figure could you use to present these results? ______________________________ 2. What are the products of aerobic cellular respiration: ________________________________________________ anaerobic fermentation in yeast cells _________________________________________ anaerobic fermentation in muscle cells ________________________________________ 3. What gas was produced and measured in this experiment? ___________________ 4. Phenol red is a pH indicator that turns yellow in the presence of an acid. What acid is produced when carbon dioxide is dissolved in water? ______________________ 4-3 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS 5. What was the purpose of tubes 2 and 11?_ T2________________________________________________________________ T12_______________________________________________________________ 6. Did you get a reaction in either of these tubes? Why or why not? T2________________________________________________________________ T12_______________________________________________________________ 7. Which of the monosacccharide solutions tested reacted the most? ___________________________ 8. List the 3 disaccharides used in the experiment and indicate their component monosaccharides. a)__________________ _______________________ _______________________ b)__________________ _______________________ _______________________ c) __________________ _______________________ _______________________ 9. Which of the disacccharide solutions above reacted the most? _______________________________ 10. What disaccharidase (enzyme that breaks the bond between the 2 monosaccharides in a disaccharide) is lacking in yeast cells? ________________________________ 11. What is sorbitol and did it react? Why or why not? _____________________________________________________________________ 12. What is aspartame and did it react? Why or why not? ______________________________________________________________________ 13. What is sucralose and did it react? Why or why not? ______________________________________________________________________ 4-4 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS 14. NaF (sodium fluoride) is a non-competitive inhibitor of the enzyme enolase. Enolase catalyzes the last step in glycolysis (conversion of phosphoenol pyruvate to pyruvate). Explain your observations in the tube containing NaF. ___________________________________________________________________ ___________________________________________________________________ ___________________________________________________________________ 15. If an experiment were to examine the effect of various concentrations of NaF on CO2 production by yeast a) What would be the independent variable?______________________________ b) What would be the dependent variable? _______________________________ 16. Describe 4 controlled variables that had to be considered in the experiment the class did: ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ 4-5 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS PART 2: GLUCOSE MONITORING INTRODUCTION Most of the glucose in the body is obtained from dietary carbohydrates and is stored as glycogen in the liver and skeletal muscles. Glucose is metabolized by the process of cellular respiration to provide energy in the form of ATP for cells. Insulin and glucagon, two hormones from the pancreas, help control blood glucose levels. Insulin is needed for cellular membrane permeability to glucose and for transport of glucose into cells. Without insulin, glucose cannot enter most cells and remains in the blood. Glucagon stimulates glycogenolysis (conversion of stored glycogen to glucose) in the liver. Decreased blood sugar (hypoglycemia) results from inadequate food intake or too much insulin. When elevated blood sugar (hyperglycemia) occurs, there is not enough insulin or the cells are unresponsive to insulin; this condition is known as diabetes mellitus. A fasting blood sugar of greater than 125 mg/dl or 7 mmol/L usually indicates diabetes. To confirm this when the blood sugar level is borderline or slightly elevated, a fasting (preprandial) blood sugar test and /or a glucose tolerance test (GTT) is performed. Symptoms of hypoglycemia could include: slurred speech, headache, tingling of lips, cool sweating, rapid heartbeat, disorientation, weakness, dizziness, fainting, hunger, nervousness, irritability or tremors. Causes of hypoglycemia include: insulin excess, cancer (stomach, liver, lung) adrenal gland hypofunction, malnutrition, alcoholism, cirrhosis of the liver, strenuous exercise, hemolytic disease. Symptoms of hyperglycemia could include fatigue, dry mouth and skin, increased thirst or hunger, blurry vision, unexpected weight loss, increased urination, rapid deep breathing or a fruity smell to the breath (evidence of ketosis). Causes of hyperglycemia include: diabetes mellitus, diabetic acidosis, adrenal gland hyperfunction (Cushing’s syndrome), acute myocardial infarction, stress, renal failure, hypothermia, acute pancreatitis, cancer of the pancreas, congestive PURPOSE 1. To measure the range of blood sugar levels in the class 2. To understand what diabetes is, and how type I and type II diabetes differ in etiology 3. To understand the clinical significance of the various tests for diabetes Questions: 1. Explain the cause of hyperglycemia in type I diabetes and in type II diabetes: 2. What is the incidence of type I diabetes in Canada? ______________ of type II diabetes in Canada? ______________ 3. List 4 risk factors for development of type II diabetes: 4-6 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS BLOOD GLUCOSE TESTING A simple blood glucose test will be performed on individuals in the class using the Lifescan glucometers provided. This is strictly voluntary; if you do not wish to prick your finger or you have been potentially exposed to any disease transmissible by blood DO NOT DO THE BLOOD GLUCOSE TEST. Only YOU may prick your finger and handle your test strip. SAFETY PRECAUTIONS!! • Disposable items such as lancets, cotton balls, alcohol swabs, etc.) are to be placed in marked containers or autoclave bags as directed. • You must prick your own finger and only you should handle anything contaminated with your blood • Benches must be washed with bleach solution at the end of the lab. Procedure Step #1 preparing the glucometer • • Step # 2 • • • • • • Step #3 follow instructions provided for calibrating the glucometer place a test strip in the glucometer preparing to prick finger GET A BANDAID; OPEN IT UP AND PLACE IT ON A PIECE OF PAPER TOWEL get a sterile lance from the box (a plastic cap will be over the needle; if the cap is not in place or it is loose, the needle is not sterile and should be disposed of in the sharps container!) get an alcohol swab insert the lance (needle) all the way into the auto-lance device twist the cap off the lance clean the end of the auto-lance device with alcohol! getting blood • • • • • • • Step #4 th swab your little finger (5 phalanx) with alcohol press the tip of the finger into the depression at the end of the auto-lance press the button…..you are now bleeding (exercise caution!) hold the glucometer and draw blood into the test strip touching the drop of blood to the end of the test strip (the test strip must remain inserted in the glucometer) by BANDAGE YOUR FINGER NOW! read the result.....in approximately 30 seconds.....in mmol/L record your blood glucose value in the results section lancet removal / clean-up • • • • remove the cap from the lance device, and remove the lancet (needle) clean the end of the lance device again (using the alcohol swab place the used lancet in the proper “sharps” container indicated by your instructor. LEAVING A CONTAMINATED NEEDLE IN THE LANCE DEVICE OR ON THE BENCH IS EXTREMELY IRRESPONSIBLE AND POTENTIALLY DANGEROUS 4-7 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS RESULTS, QUESTIONS, DISCUSSION: 1. Blood Glucose Results: No. (normal post-prandial range is 3.5 – 7.0 mmol/L) time since last eating (hours) blood glucose (mmol/L) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2. What is the range of the blood glucose levels? ___________________ 3. Did anyone fall outside the normal range? ___________________ 4. What is the mean blood glucose level in the class? ___________________ 5. What is the dependent variable? _________________________________ 6. What is the independent variable? ________________________________ 7. Sort the data into 3 groups: ate less than an hour ago, ate 1-3 hours ago, ate more than 3 hours ago. Calculate the mean blood glucose level for each of the 3 groups and the standard deviation. Plot these data as a histogram. group values mean < 1 hr ago ______________________________ ________ > 1 hr but < 3 hr ______________________________ ________ > 3 hr ago ______________________________ ________ 4-8 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS 8. Is any relationship evident? Why or why not? ______________________________________________________________________ ______________________________________________________________________ 9. What hormone is released in the fasted state? ________________________________ What effect does this hormone have on blood glucose levels? _______ ____________ 4-9 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS GLUCOSE TOLERANCE TEST (GTT) The standard test for diabetes is a glucose tolerance test. The individual is asked to fast overnight. In the morning, a fasting (pre-prandial) blood glucose level is recorded. The individual then consumes a very concentrated glucose solution (containing 75 gm glucose), and blood glucose levels are sampled at several intervals for the next 3 hours. Compare the results of a normal and an abnormal GTT as shown below. Table 1. Results of a GTT in 2 patients blood glucose concentration (mmol/L) 0 min 30 min 1 hr 2hr 3hr patient #1 4.7 7.8 8.4 5.6 4.5 patient #2 8.4 12.3 13.7 14.3 14.5 Graph these results below.: 4-10 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS QUESTIONS: 1. How does the fasting blood glucose level differ in these 2 patients? ________________________________________________________ 2. Which of these patients is diabetic? ____________________________ 3. How long does it take for blood glucose levels to peak in a non-diabetic after ingestion of glucose? ________________________________________________________ 4. How quickly does the normal individual’s blood glucose return to baseline levels? ________________________________________________________ 5. How quickly does the diabetic’s blood glucose return to baseline levels? ___________________________________________________________________ 6. When blood glucose levels drop back to baseline levels, where has the glucose gone? ____________________________________________________________________ 7. What does an abnormal GTT usually signify?__________________________________ ______________________________________________________________________ 8. What is the usual reason why blood sugar is elevated?___________________________ ______________________________________________________________________ 9. Would the GTT differentiate between type I and type II diabetics? Explain. 4-11 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS PART 3. CARBOHYDRATE AS FUEL FOR ENDURANCE EXERCISE Depletion of muscle and liver glycogen stores, with resulting hypoglycemia, is thought to be a key factor in fatigue during prolonged exercise. Total carbohydrate stores in the body are limited and have been estimated to last for approximately 70 - 90 minutes when exercising continuously at 65-70% VO2max. It has been shown that consuming carbohydrates during endurance events that exceed 60 minutes duration prolongs time to fatigue and improves performance. Many carbohydrate supplements have been developed (and marketed) for use during endurance exercise. In the development of these supplements, considerations have included: • type of carbohydrate (which monosaccharides, disaccharides, polysaccharides and in what combinations) • how quickly the carbohydrate is absorbed • optimal concentration of carbohydrate • digestibility (tolerance of the user) • palatability (taste, texture, ease of consumption) Purpose: • review a study related to carbohydrate metabolism in the context of exercise • apply knowledge of glucose metabolism (cellular respiration) • apply knowledge of sugar, water and sodium absorption from the small intestine • apply knowledge of normal vs. abnormal glucose levels • interpret experimental results • examine ingredients of carbohydrate supplements marketed for endurance exercise Procedure 1. Examine the exercise fuels / supplements and complete the table on the following page 2. Having read the journal article provided (found under links on Peggy’s web site) and answer the questions that follow. Jentjens R., Venables M.C., and Jeukendrup A.E. Oxidation of exogenous glucose, sucrose, and maltose during prolonged cycling exercise J. Applied Physiology 96: 12851291, 2004 4-12 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS Examine the carbohydrate products displayed on the bench. These products are marketed for consumption during endurance exercise. Complete the following table for 6 of the products. product carbohydrate sources indicate if each of these is a mono, di or polysaccharide any noncarbohydrate energy sources or any caffeine in product? Na+ K+ Questions: 1. Why are Na+ and K+ usually added to products such as these? 2. Based on the results of the Jentjens et al. study, which product(s) would you recommend to a triathlete as fuel during a triathlon? 3. Which product would you choose to use yourself during an endurance event and why? 4-13 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS JOURNAL ARTICLE QUESTIONS: 1. What is CHO? ________________________ 2. What are the products of CHO oxidation in the body? ___________________________ 3. Which of these products was measured to evaluate rate of CHO oxidation in the cyclists? ________ 4. What is exogenous CHO and what is endogenous CHO in the context of this study (be specific; specifically what are the endogenous sources of CHO?) 5. Describe 4 controlled variables in this experiment (see methods): 6. How long did the cyclists exercise for? ___________ At what intensity? ___________ 7. What sugars (or combination of sugars) were tested as energy sources? 8. What solution was the experimental control? ____________________________ 9. What 2 disaccharides were used and what are the monosaccharide components of these disaccharides? 4-14 CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS 10. Over the first 15 minutes of exercise (Fig 3A) plasma glucose rises except in subjects who consumed water. Why? 11. Did plasma glucose levels fall significantly over the duration of the experiment (compared to plasma glucose levels at time 0)? 12. Why do plasma lactate levels rise in the first 15 minutes (Fig 3B)? 13. Which sugar or mixture of sugars provided the most readily available energy to the athletes (as evidenced by exogenous CHO oxidation rates)? 14. What explanation do the authors suggest for the observation above? 15. The authors note that the addition of the electrolyte Na+ aids in gastric emptying and intestinal absorption of glucose. Why? 16. What was the concentration of NaCl in the solutions given to the cyclists? ___________ 17. Did the drinks supply K+ in any form? _______________ 18. Which carbohydrate solution was associated with the fewest gastrointestinal complaints? _________________________ 19. Based on the results of this experiment, can the authors conclude that a mixture of different sugars will enhance performance of cyclists? Explain. 4-15