Blood Glucose Lab #5 SAFETY PRECAUTIONS Blood is a potentially infectious body fluid. Therefore, students are directly responsible for all measurements on their own blood, and must ensure they clean any surface contaminated with their blood. Blood Glucose Lab: Experimental Aim Aim: Observe Effects of Fasted or Fed States on Blood Glucose Homeostasis Blood glucose lab preparation • Three groups of volunteers: • Fasted (+ glucose drink) • Fasted (- glucose drink) • Fed (- glucose drink) • Fast for 8 hours; miss the meal that immediately precedes your lab section NOTE: avoid all stimulant/depressant drinks for 24-48 hours before coming to lab Experimental Procedures Collect baseline blood sample (#1) immediately upon arrival in lab Consume Trutol (dextrose solution), start timer, keep track of time Collect blood samples #2, #3, #4 and #5 about 30 minutes apart Record results on white board General Metabolism & Carbohydrates Energy Storage and Release (i.e. Energy Balance) Figure 58-4, Medical Physiology, 2nd ed., 2012, Boron and Boulpaep Sites of absorption of nutrients. For example, the entire small intestine absorbs carbohydrates, lipids and proteins. This is not the case for other elements of ingestion or metabolism. Fig 45-2 B&B Medical Physiology, 2nd ed., 2012 Dietary Carbohydrates ~45-60% of western diets are carbs Dietary carbohydrates Plant starch Animal starch – monosaccharides – amylose (straight chain glucose polymer) – glycogen is the storage form of carbohydrates – oligosaccharides (short polymers of glucose) -polysaccharides (long polymers of glucose) – amylopectin (a massive, branched chain glucose polymer) Dietary Fiber • Insoluble • Aids bowel movement • Sources = wheat bran, vegetables, whole grains • Soluble • Attracts water and slows digestion • Sources= oat bran, barley, nuts, seeds, beans, lentils, peas • Optimal Intake, 25-50 g/day for general health/well being • Potential Benefits • Improved digestive feelings • Enhanced bowel function • Increased well-being Glucose Metabolism Intestinal Glucose Absorption Diabetes 54(10):3056-62, 2005 Insulin Secretion via Pancreatic Beta Cell A Modern Epidemic: Expert Perspectives on Obesity and Diabetes, 2012 Insulin-Dependent Glucose Metabolism plasma cytoplasm Dutta, 2017 Glucagon Secretion via Pancreatic Alpha Cell Cell Metabolism, 2013 Hormonal Blood Glucose Regulation • High Blood Glucose • Glycolysis • Glycogenesis • Low Blood Glucose • Glycogenolysis • Gluconeogenesis Mayo Clinic Proceedings, 2018 Diabetes Mellitus (DM) • Type 1 Diabetes (insulin-dependent) • Caused by destruction of beta cells which leads to a decrease in insulin production/secretion • Treatment = insulin injection • Type 2 Diabetes (noninsulin-dependent) • Caused by insulin resistance of tissues • Treatment = diet & exercise, oral drug • Symptoms • Polydypsia • Polyuria • Polyphagia • Glucosuria Diabetes & Heart Research Centre, 2007 Diagnosing Diabetes Mellitus • Fasting Plasma Glucose 126 mg/dl or higher • Hemoglobin A1c • >6.5% of hemoglobin glycated • • Oral Glucose Tolerance Test After 2 hours • 200 mg/dl or higher- indicative of diabetes • Urinary Excretion of Glucose (Glucosuria) • Insulin and glucagon are the main regulators of blood glucose • Diabetes Mellitus is caused by Summary destruction of beta cells (Type I) or insulin resistance (Type II) • Once renal threshold is met, excretion of glucose begins to surpass reabsorption, leading to glucosuria. The abstract provides a summary of the experiment you conducted. Readers generally read the abstract to get a feel for the topics that the paper will address. Therefore, the abstract must cover the main points of your experiment. AssignmentAbstract (due next week) The abstract has five parts that must be addressed for every experiment: 1. Introduction: An introductory statement 2. Purpose: The purpose(s) of the study 3. Methods: Methods that were used to obtain data 4. Results: Results from each main experiment 5. Conclusion: Significance of your findings (discussion of mechanism) The abstract must be one single paragraph and no more than 1 page. Blood Glucose In this experiment, we will observe the body’s ability to regulate blood glucose concentration (i.e. to maintain glucose homeostasis) during both fasting and fed states via insulin secretion. Students will be divided into 3 groups: fed, fasted and fasted, ingesting a high glucose solution (glucose tolerance test). Blood samples will be collected at various time points and analyzed for blood glucose concentration. A urine sample will also be analyzed for glucose content at the 60 minute time point.. * Preparation for this experiment: • • • • Fed subjects should come to lab having eaten breakfast, lunch and dinner as they normally would. Fasted subjects should have no food or beverages other than water for at least 4 to 6 hours before the lab begins. Avoid stimulants/depressants (alcohol, coffee, soda, tea, energy drinks, etc.) for at least 24-48 hours prior to lab. Empty your bladder immediately before coming to lab. Background Physiological processing of dietary carbohydrate Dietary carbohydrates including glucose are processed as illustrated in Figures 1-4 below. Energy balance, i.e. the homeostasis between energy/calories ingested and energy/calories expended is illustrated in Figure 6. For today’s exercise we will be focusing on the physiology of glucose and not on either lipid or protein. Figure 1. Most carbohydrate is absorbed in the first 1/3 of the duodenum Figure 2. Differences in Glu transport before/after a meal. Note: On the apical side of the mucosal cell, glucose is cotransported with sodium ion. On the basolateral side of the cell, glucose is transported in the absence of sodium. Figure 3. Roles of insulin/glucagon in Glucose processing in fed (top half) and fasted (bottom half) states. Glycolysis: the first stage of glucose breakdown for energy metabolism. Glycogenesis: conversion of glucose to glycogen in the liver. Glycogenolysis: the breakdown of glycogen into glucose molecules (usually in liver and muscle). Gluconeogenesis: the production of glucose from substrates, such as glycerol (from fat) and protein, usually occurring in the liver. Figure 4. Physiological homeostasis of energy supply and demand, ie, the balance between energy input/output. Role of insulin and symptoms of diabetes including ketoacidosis Insulin is an endocrine hormone secreted by pancreatic beta cells. Its principal function is to assist the transport and metabolism of glucose. When insulin is deficient only small amounts of glucose get processed leading to accumulation of glucose in the blood (hyperglycemia). Excess insulin (hyperinsulinemia) causes hypoglycemia or reduced circulating plasma concentrations of glucose. Hypoglycemia can cause weakness, tremors, hunger, irritability and, in severe cases, coma, hypoglycemic shock (insulin shock) and death. A healthy range of blood glucose concentrations is 80-90 mg percent (mg/100ml of blood), but this can range from 60-140 mg % depending on the individual’s dietary intake of glucose. The disease diabetes mellitus can be caused by a lack of insulin. This fact can be demonstrated either by removing the pancreas of an experimental animal or by destroying the beta cells of the islets of Langerhans. Either of these procedures will produce typical symptoms such as high blood glucose and excretion of glucose in the urine. Urinary excretion of glucose (glucosuria) results when the concentration of blood glucose exceeds the threshold for reabsorption by the kidney. Increased urine osmolality also causes increased excretion of water (polyuria). Excess excretion of water can lead to dehydration and hyperosmolality of body water compartments which in turn stimulates excessive water intake (polydipsia). Polyuria, polydipsia and polyphagia (excessive appetite due to glucosuria) are three major symptoms of diabetes. The disease received its name because the body of the diabetic person was formerly visualized as melting and flowing out in the copious, sweet tasting urine. When insulin is deficient and the cells cannot metabolize glucose for energy, the cells compensate by increasing their metabolism of fats and proteins. Thus, the diabetic is usually thin, owing to the loss of fats and proteins from the body structure. The increased metabolism of fats releases into the blood large quantities of ketone bodies (e.g. acetone), which are intermediate products of fat breakdown. These are excreted in the urine and have the easily recognizable odor of acetone. Also, ketone bodies are acidic and their accumulation will cause a drop in blood pH; the diabetic becomes acidotic (ketoacidosis). Sever ketoacidosis, if uncorrected, can lead to coma and eventually to death. There are two types of diabetes mellitus, Type 1 and Type 2. Type 1 [formerly called “insulin dependent” diabetes (IDDT) or juvenileonset diabetes] is caused by a primary deficiency of insulin due to autoimmune destruction of the beta cells. People suffering from Type 1 diabetes must receive supplemental insulin. Type 2 [formerly known as “non-insulin dependent” diabetes (NIDDM) or adult-onset diabetes] results predominantly when the major target tissues become resistant to the action of insulin. This type of diabetes is more common, and is often linked to obesity. Glucose Tolerance Test In diagnosing diabetes, several tests are used to determine what metabolic error is causing the disease. Such tests include urinary glucose concentrations, concentrations of urinary ketone bodies, fasting blood glucose concentrations, insulin sensitivity, and glucose tolerance tests. The glucose tolerance test evaluates the ability of the pancreas to respond to excess ingestion of glucose. The changes in blood glucose concentrations following glucose ingestion (1g/kg body mass) are markedly different between the healthy and diabetic person. In the healthy person, glucose concentrations rise from about 80-90 mg % to around 140 mg % in one hour and then decline to normal within three hours due to excess insulin release by the pancreas. The diabetic person displays a hyperglycemic response. Glucose concentrations rise from baseline of about 120-160 mg % to as high as 300 mg %. They slowly decline to fasting concentrations after five or six hours. The diabetic’s abnormal response is caused by the inability of the pancreas to secrete additional insulin in response to elevated blood glucose concentrations (Figures 5, 6). Figure 5. One example of insulin-dependent transport of glucose into the cell. Downstream pathways involving glucose metabolism and storage. Figure 6. Data collected in Systems Physiology Laboratory from a previous semester Renal handling of glucose Normally, all glucose filtered at the glomerulus is reabsorbed by the end of the proximal tubule. When plasma glucose increases above about 14-18 mmol/L (~250-320 mg/dL) renal tubular uptake reaches a “transport maximum”, i..e. the kidneys cannot reabsorb further increments in glucose. The excess glucose appears in the urine (glucosuria) causing an osmotic diuresis (excess loss of water). This explains, why diabetics are polyuric ( excess excretion of urine), polydypsic (excess thirst) and polyphagic (excess hunger). Such undiagnosed symptoms can be life threatening. From past experience in this lab, consuming glucose does not elevate circulating concentrations of glucose above about 150-200 mg/dl. Also, students who know they are diabetic have participated in the experiment without any adverse effects. However, if you know that you are diabetic and still want to participate, discuss with your TA which group you should be in. Required instruments and supplies o o o o o o o Alcohol wipes Gauze Lancets Glucometer Contour glucose strips (to test blood glucose) Bayer Diastix Reagent Strips (to test glucose in urine) Trutol glucose tolerance beverage Procedures * This laboratory involves the collection of blood samples by pricking the finger(s) at various time intervals and also testing one urine sample. Subjects should handle only their own blood. Lancets are to be disposed of in red “SHARPS” containers on each lab bench. Dispose of all other blood tainted supplies (alcohol wipes, gauze, contour glucose strips and bench cover) in the red-bagged hazardous waste box at the front of the room. Subjects should clean up their own accidental spills. Reagent strips used to test urine samples must also be disposed of in the red-bagged hazardous waste box. Upon entering the lab, TA’s will demonstrate how to collect and analyze your baseline blood sample. 1. 2. Remove one Contour test strip from the bottle. Hold the test strip with the gray end facing up. Insert the gray end into the test strip port on the Glucometer. The meter will turn on. An image of a test strip with a flashing blood drop will appear, letting you know the meter is ready to test. 3. Use your non-dominant hand for finger pricks. Exercise your hand for a minute or two and ensure that it is warm (e.g. wear gloves for several minutes if you have them) Clean the tip of your finger with an alcohol wipe and allow finger to air dry Position your hand below heart level and use a lancet as instructed by your TA to obtain a blood sample. Remember to place the used lancet in the SHARPS disposal bin. Do not squeeze the finger if sufficient blood flows freely without doing this. Squeeze only as a last resort. 4. 5. 6. Follow instructions below: 7. 8. 9. Immediately after obtaining the baseline reading, subjects in the glucose tolerance test group (fasted, ingesting a high glucose solution) should quickly drink the Trutol solution At the 30, 60 90 and 120 minute time points, blood glucose levels will be analyzed as above. At the 60 minute time point, urine glucose level will also be determined. For this test, take a Bayer Diastix Reagent strip to the restroom with you. Hold the test strip in your urine stream. Carry the stick back to the lab on a paper towel. Compare the color of the square to the colors on the bottle’s glucose chart. Dispose of reagent strip and paper towel in red-bagged hazardous waste box. * ENTER ALL DATA IN THE TABLE ON THE WHITEBOARD. PLEASE MAKE A COPY FOR YOUR OWN RECORDS How to write a laboratory report for Systems Physiology Laboratory (01:146:357) Abstract (≤ 350 words) In short, the abstract provides a summary of the experiment you conducted. Readers generally read the abstract to get a feel for the topics that the paper will address. Therefore, the abstract must cover the main points of your experiment. The abstract has five parts that must be addressed for every experiment: 1. An introductory statement 2. The purpose of the study 3. Methods that were used to obtain data 4. Results from each main experiment 5. Significance of your findings (discussion of mechanism) The introductory statement broadly introduces the main topic to the reader. This section can include an eye-catching statement, encompassing themes, or more specific background information related to the research topic. The purpose of the study is to provide the reader with the rationale for conducting the experiments and must be as specific as possible. In order to write an effective purpose, you must understand WHY you performed the experiment. Ask yourself the following questions: Was the experiment conducted to examine a physiological concept in more detail? Was it to observe the behavior of a tissue/organ in response to a physiological stress? It may also include any primary measures you took to reach your purpose. The abstract must also include a brief methodology. As many of you are aware, various methods can be used to test one hypothesis. Including your methodology in the abstract allows the reader to visualize the way you obtained your data. Be brief and keep in mind that specific information regarding the methods used in the experiment can be found in the methods section. Important information to include would be the subject population (e.g., mice, humans), the medium tested (e.g., blood, urine, muscle), and the manner that it was tested (e.g., glucometers were used to measure blood glucose concentrations). It is also important to mention any information related to equipment setup and if the experiment required repeated testing at specific intervals. The results section of the abstract is to briefly list the main results you obtained. Ask yourself if you saw specific trends or values, which will depend on the experiment being conducted. Be sure to list the results for ALL experiments that you have conducted and will be discussed in the full lab report. The last part of your abstract should include a brief statement about the significance of your findings. What did you learn from the results? Did they support conventional theories? Did they deviate from established findings? When doing this, describe what the conventional theories or established findings are. Describe the mechanism that led to your results. As you continue to read through the guidelines for preparing a laboratory report (i.e., how to write an introduction, methods, results, discussion, and reference sections) refer and use the sections of the journal article(s) posted on Canvas to reinforce your understanding of each section’s content. Introduction (1-2 pages) There are two reasons for writing an introduction: 1. to inform the reader of the basic scientific concepts behind the experiment 2. to state the purpose and hypothesis of the experiment If the reader has a basic proficiency in the scientific concepts studied in the experiment, he/she will be more appreciative of the results and will understand the significance of the findings. To write an effective introduction, imagine explaining the physiological concepts to someone with a basic background in biology. Remember, start gradually and broadly. Describe the anatomy of the system you are addressing in your report. If the experiment requires that the tissue/organ be placed under physiological stress conditions, describe how the tissue/organ would behave under normal conditions. Do not delve into details, as these will only confuse the reader. Importantly, avoid discussion material. Pretend you are writing the introduction to an experiment without already knowing the results. The information provided in the introduction section should eventually lead to a clear and concise purpose statement. Following a purpose statement for each aim, a hypothesis should be included. Again, the hypothesis should be based on information already provided in the introductory material. References should also be used in the introduction to provide support for background information and hypotheses. Methods (1-2 pages) The purpose of the methods section is to summarize all the steps you took as a researcher to conduct the experiment. The methods should not omit important details and should not include superfluous points. The methods should be formatted to include subsection headers such as Participant, Equipment, Procedure, and Data Analysis. DO NOT include the following: ● Unnecessary details, such as, “The frog’s skin was green and the toes had a black mark on them.” This has no relevance to the experiment. ● A numbered/bulleted list. The methods section should be written in paragraph form. ● Pronouns or personal information of participants/subjects. ● Copied sentences from course material, this is considered plagiarism. Organize your methods so that it parallels the experiment. Start with a brief description of the subject(s) and then mention any dissection protocols or equipment preparation. Then proceed to describing each experiment. Include important information relative to each experiment (e.g., doses, time intervals, etc.) and the way you analyzed your data. For the data analysis subsection you should inform the reader of the outcomes you measured and the methods you used to obtain your data. Did you use any specific equations? Did you draw any figures? Be brief but mention these important points. Do not include any results in your methods section. Results (1-3 pages) For the purposes of this lab course, graphs and tables will be used to depict trends in data. The results section is organized in the following manner: ● The results summary describes the data collected in each experiment. This section reports the overall message of the data and addresses more specific details of your data. Are you showing an increase? Decrease? Maintenance of values? You should adequately detail the data you are presenting (i.e. reporting data at different time points and/or between groups). The summary can be limited to a few sentences for each experiment. You can include the summary at the beginning of this section or embed the text between your required figures and tables. ● A pictorial representation of data, this can either be a graph or table. ● The caption describes the figure or table. This information is right beneath the corresponding graph or table. A caption should include a general statement about the data presented. This section also explains aspects of the figure/table such as defining units and reporting sample size. Resources for graphing data: • Calculating mean and standard deviation: https://www.youtube.com/watch?v=39JDN5BNEJ0 • Adding custom standard deviation bars to figures: https://www.ablebits.com/office-addins-blog/add-error-bars-excel/ Discussion (1-3 pages) This section of the report carries the most weight as here you are explaining the reason behind the patterns/trends in your data. Organize your discussion section so it flows to the reader. If your discussion section is not organized, your readers will not follow your explanations even if they are sound. In this section of your report, include a body paragraph for each of the main experiments that you are required to report results on. This section can be organized according to each variable studied, or grouped to explain a theme in the data. The latter can be used when the experiment calls for investigating different aspects of one principle (e.g., muscle activity can be studied by changing the length of the muscle or the frequency). In each body paragraph, remind the reader of your aim(s) and provide an overview of your results. For the purposes of this course, you should address physiological principles when explaining your data. If your data deviate from expected trends, explain the expected trend and why this would occur, then provide an alternative explanation for your data. Following the body paragraphs, you should describe any limitations or sources of error in the experiment within one paragraph. The discussion should end with a conclusion paragraph. Within a few sentences, provide a summary of the study and results to reader. Your conclusion should also acknowledge the overall significance of the experiment and your findings. References (1 page – not included in page count) DO NOT forget to provide citations in your report. These are vital in providing the reader with proof of your claims. Facts discovered by someone other than you have to be credited to the right person. Not providing a citation indicates that this is a fact you are stating based on your own conclusions. Neglecting to give credit where credit is due constitutes plagiarism, which is a serious offense! Bottom line; provide in-text citations throughout your report. Plagiarism – One must do his/her own work when writing reports in System Physiology Laboratory. It is permissible to use short excerpts/quotes from the work of others; however, in any single report, these should be few and short. In all cases where the work of others is cited, credit must be given. When citing, avoid using the same exact words as an author or paraphrasing large segments of writing. Read the information and explain it in your own words. Do not cite Canvas material, lecture notes, manuals, or TAs. Please use the format given to you by your TA when citing textbooks and journal articles in your reports! For instance, if your TA asks you to use APA formatting, be sure to locate a credible source that can guide you with the citation process. Below is a link that provides useful information on APA formatting:https://owl.english.purdue.edu/owl/ resource/560/07/
Purchase answer to see full attachment