BIO340 Laboratory Guide #9
REGULATION OF BLOOD GLUCOSE
IN HUMANS
The concentration of glucose in the blood of mammals is tightly regulated by the endocrine system. Short-term regulation is mediated primarily by insulin and glucagon, peptide hormones secreted by the b and a cells of the Islets of Langerhans in the pancreas, and by epinephrine and norepinephrine, catecholamines released by chromaffin cells of the adrenal medulla. Other hormones, such as the glucocorticoids, thyroid hormone, and growth hormone (via somatomedins) participate in longer-term regulation of blood glucose levels. Insulin is the only one of these hormones that acts to reduce the concentration of glucose in the blood; the other hormones all exert counter-regulatory actions, which tend to drive the blood glucose concentration up.
The secretion of these hormones is highly coordinated and is influenced by a variety of factors, including blood levels of glucose, amino acids, fatty acids, and the hormones themselves. As a result, the concentration of glucose in the blood of a normal individual does not fluctuate widely. Typical levels in a human following an overnight fast are approximately 70-110 mg/dl (milligrams per deciliter) and the maximum level attained after a meal (postprandially) is generally below 180 mg/dl. In some people the consumption of a high-carbohydrate meal leads to a transient elevation of blood glucose concentration, followed by a dip below the normal fasting levels. The latter effect is called "reactive hypoglycemia".
Diabetes mellitus is a disease that disrupts glucose regulation and interferes with other aspects of fuel metabolism. It may be the result of either a deficiency of insulin production by the islet cells or a reduced sensitivity to insulin in hepatic and other somatic cells. Patients with diabetes mellitus have significantly higher fasting blood glucose levels than do normal individuals (hyperglycemia), and they exhibit a much longer-lasting postprandial elevation in glucose concentration. In addition, the hyperglycemia can lead to excretion of glucose in the urine (glycosuria), which may, in turn, stimulate the production of excessive amounts of urine (hyperuria).
Glucose is passively filtered from the blood into Bowman's capsule of each renal nephron, but is subsequently actively reabsorbed into the peritubular capillary blood from the convoluted tubules. This reabsorption involves co-transport with sodium ions and is insulin-dependent. In a non-diabetic person, filtered glucose is generally totally reabsorbed, so that no glucose appears in the urine. In a diabetic person with high blood sugar and low insulin levels (or insulin insensitivity), not all of the filtered glucose is reabsorbed, so glucose appears in the urine ("glycosuria"). This sugar is responsible for the name diabetes mellitus, literally "honeyish urine". Because glucose can't efficiently enter the cells of the body, they oxidize fats for energy. Ketones are a product of fat metabolism, and may also appear in the urine of a diabetic individual ("ketonuria") and may , in extreme cases, be smelled on the breath. In a non-diabetic person, ketones are generally only present in the urine following a very prolonged fast.
In this week's laboratory exercise, you and your classmates will collaborate as a single investigative team to examine the effect of diet on blood and urinary glucose levels. You will also serve as the experimental subjects. The specific details of the experimental protocol will be described later, but in general the experiment will proceed as follows. PLEASE READ THROUGH THIS DESCRIPTION CAFEFULLY, SO THAT YOU CAN MAKE AN INFORMED DECISION ABOUT YOUR OWN PARTICIPATION AS AN EXPERIMENTAL SUBJECT.
You will be instructed to consume a normal breakfast on the morning of your lab section, but to refrain from eating or drinking anything other than water until you come to the lab. At the start of the lab, you will be asked to drink either a standard glucose "tolerance" beverage - 300ml of a cola-flavored drink (caffeine-free Coke®) containing 0.545 grams of glucose per pound of your body weight (~1.2 g/kg), or a control beverage - 300ml of diet cola (caffeine-free Diet Coke®) containing aspartame (NutraSweet™). You will not be told which beverage you have received until after the exercise. You will be asked to refrain from eating or drinking anything else until the end of your laboratory period.
Immediately before drinking the beverage, and at 30 minute intervals for 3 1/2 hours afterwards, you will draw a drop of your own blood and measure the glucose concentration in the blood, using blood glucose test strips and a digital monitor. You will NOT come into contact with blood from any other persons. At approximately the same times you will go to a restroom and collect a sample of your own urine. While in the restroom you will measure and record the volume of the sample, dip a urine glucose/ketones test strip in the sample and then flush the sample down the toilet. You will then compare the colors of the test strip with standard color charts to determine your urine glucose and ketone concentrations. You will handle ONLY your own urine sample, which will never leave the restroom.
To assure the anonymity of your results, the lab assistant will assign each of you a letter and hand out the beverages. She will then give your instructor a list that associates each letter with the type of beverage consumed, but the list will not relate letters with student names. During the experiment, you will record your results on your own data sheet. You should label this sheet with your letter, but not your name. If you do not wish other students or the laboratory assistants to see your results, you may record them in private. At the end of the experiment, you will return your results sheet to the instructor, who will tabulate the findings of the students in each group (glucose and control), omitting both student names and letters, and give everyone in the class a copy of the combined results. The laboratory assistant will not see the completed results sheets.
You may decline to participate in the beverage ingestion, blood sampling, and urine testing portions of this experiment, if you have a legitimate objection to participating. Examples of a legitimate objection would include a family history or person history of diabetes mellitus, current practice of a controlled-carbohydrate diet, hesitance to draw your own blood, a concern about accidental exposure to blood, or a concern about the anonymity of your results, despite the precautions taken. An illegitimate objection would be that you simply prefer to use the class period to do something else. If you decide that you have a legitimate objection to participating in the experiment in the manner described above, you will be free to decline participation without offering any further explanation. If you choose this option, however, you should inform the instructor of your decision no later than Monday of the week of the experiment, so that the instructor will know how many beverages to prepare, and so that you can be assigned other activities to engage in during the laboratory period. Regardless of whether you participate or not, you must read the entire handout and the additional readings for this experiment, complete and hand in the worksheet by the start of the lab period, and complete and hand in the data.
In addition, you must speak to your instructor no later than Monday of the week of the experiment if you Have a family history of, or have been previously diagnosed with either diabetes mellitus (insufficiency of or insensitivity to insulin), or phenylketonuria (sensitivity to phenyalanine - a component amino acid of aspartame). OR IF YOU ARE CURRENTLY PRACTICING A CONTROLLED-CARBOHYDRATE DIET, SUCH AS THE aTKIN'S DIET. in ANY OF THESE caseS you SHOULD NOT, AND WILL not be allowed to participate as a subject, but will BE EXPECTED TO assist in conducting the lab AND COMPILING THE DATA.
Keep in mind that the steps in this lab are not performed sequentially, so read all the way through the lab well in advance to participation.
I. PREPARATION FOR THE LAB SESSION
On the morning of the lab, eat what for you would be a normal mid-week breakfast. If you normally skip breakfast, make an exception and have breakfast on the morning of your lab, as it will be your only meal until late in the day. Finish your breakfast between 8:00 and 9:00 AM, then refrain from eating or drinking anything else, other than water, until you come to the lab.
II. INITIAL PROCEDURES IN THE EXPERIMENT
At the start of the lab period, you should:
1) Make sure that you have read and thoroughly understand the informed consent form. If you intend to participate in the study, then sign the informed consent, have a witness sign it, then turn the signed sheet in to the lab assistant. If you decide not to participate as a subject, please talk to the instructor before proceeding.
2) Draw a letter from the lab assistant. The lab assistant will write down your name and letter, but will not see any of the data. Data which you turn in for inclusion with the class data will be identified by your letter only, not your name.
3) Write your assigned letter at the top of your data sheet.
4) Determine your weight in pounds and calculate the appropriate sugar and Nutrasweet values on Instruction Sheet #2. The sugar dosage will be based on an approximate "industry standard" of 100 grams of ingested glucose for an 85kg (185 lb.) subject.
5) Draw a Subject Letter from the Fishbowl-of-Fate.
6) Transfer your Subject Letter and calculated dosage values to Instruction Sheets #3-5 and give them to the appropriate assistants, as indicated in Instruction Sheet #1).
7) Your instructor will demonstrate the techniques for drawing blood samples, measuring glucose concentrations in blood, and testing for glucose and ketones in the urine. Following the instructions given in sections IV and V below, take your initial blood sample and immediately afterwards your urine sample, or vice versa. MAKE SURE THAT YOU FOLLOW THE PROCEDURES FOR SAMPLING BLOOD EXACTLY, SO THAT THERE IS NO RISK OF EXPOSURE TO THE BLOOD OF OTHER STUDENTS. Record your blood and urine glucose levels, the volume of urine collected, the time at which each of the samples was obtained, and the elapsed number of minutes since you emptied your bladder prior to class. IF YOUR INITIAL BLOOD GLUCOSE LEVEL EXCEEDS 120mg/dl, CONSULT WITH YOUR INSTRUCTOR BEFORE PROCEEDING TO THE NEXT STEP.
8) Receive a beverage from the lab assistant. Double check to make sure that the letter on your beverage cup matches your assigned letter. Drink the beverage, and then return to the lab. We will treat the initial blood and urine samples and the beverage consumption as though they all occurred at the same time, so try to do all of them within a span of 10 minutes. You should take no more than two minutes to finish the beverage.
III. MEASUREMENT OF BLOOD GLUCOSE CONCENTRATION
Blood samples will consist of drops of capillary blood. The easiest and most reliable location to draw these from is the fingers. Quantitative measurements of blood glucose concentration will be obtained with an electronic monitor. You will each have your own area of benchtop covered with plastic-coated paper, and ALL PROCEDURES INVOLVING YOUR BLOOD MUST BE CONFINED TO THAT AREA.
Sampling and Measurement Procedure:
1) Remove a fresh test strip from the vial and immediately reseal the vial. Do not touch sensor area on the tapered end on the test strip. Insert the square end of the strip into the monitor, with the sensor area facing upwards. After a few seconds a code should appear at the top of the display, along with an indication at the bottom of the display that the monitor is ready to receive a drop of blood on the test strip. Make sure that the code matches that on the test strip container. If this fails to happen, contact the instructor. Remove the test strip and return it to its container.
2) Wash your hands/arms with antibacterial soap and warm water, and dry them well. (Warm water helps stimulate good blood flow through the capillaries in your fingers.) Clean your chosen sampling site with an alcohol swab and allow it to air dry.
3) Remove the lancet device from the alcohol bath and shake it dry. Remove the lancet device cap, insert a lancet into the lancet holder, twist off the protective lancet cover, replace the lancet device cap, cock the lancet device, then set the depth dial to an appropriate depth. For fingers chose a moderate to deep depth. For the hand or forearm chose the deepest depth.
4) Remove a fresh test strip from the vial and immediately reseal the vial. Do not touch sensor area on the tapered end on the test strip. Insert the unreacted test strip into the monitor slot. After a few seconds the monitor display should indicate that it is ready for sampling.
5) Use the lancet device to prick the area that you swabbed. (It is generally more comfortable to prick the side of your fingertip, rather than the tip itself.)
6) Say, DO NOT SCREAM, "ouch".
7) Massage the area to produce a moderate size drop of blood. Touch the exposed end of the test strip to the droplet of blood, making sure that the blood wicks over the entire sensor pad surface.
8) The monitor display should switch immediately to a countdown from 10. Within 10 seconds the display should change again to your blood glucose reading in mg/L. Record this value. If you get an error message, the do the following:
a) discard the test strip in the Biohazard container
b) discard the used lancet in a Sharps container
c) mount a fresh lancet in the lancet device
d) go back to step 3 and repeat all steps.
Small blood samples which do not cover the entire sensor area evenly are the most frequent cause of erroneous or failed readings.
9) Wipe the injured area with a fresh alcohol swab and apply an adhesive bandage, if necessary. Discard the test strip in the BioHazard container and discard the used lancet in a Sharps container.
10) Return the lancet device, cap down, to the alcohol bath.
11) Perform any necessary cleanup on your station as soon as possible. All lancets go in the Sharps container. All items potentially contaminated with blood, including alcohol swabs and test strips, go in the BioHazard bag. Any bandages that you decide to remove from your fingers should also be put into a biohazard bag. DO NOT DISCARD BLOOD-CONTAMINATED OR SHARP OBJECTS IN THE GENERAL LAB WASTE BASKETS, AND DO NOT PUT SHARP OBJECTS INTO THE BIOHAZARD BAGS.
IV. EXTENDED MONITORING DURING THE REMAINDER
OF THE LAB PERIOD
1) Collect additional blood and urine samples every 30 minutes for the next 3 and 1/2 hours.
2) Record the glucose concentrations and urine volume for each pair of samples, as well as the time at which the sample was taken. Note any special circumstances or deviations from the protocol at the bottom of the results sheet. You may use your free time between samplings to read or study, but refrain from eating or drinking anything else until the end of the period. If you feel nausea, light-headedness, or other types of discomfort at any time, you may turn in your results sheet and terminate your participation in the experiment.
3) When you finish with all of your blood and urine samples, remove your plastic coated paper and place it in a biohazard bag. Check to make sure that you have filled in all of the spaces on your results sheet, and then give your completed data sheet to your instructor.
4) Eat some pizza and drink some water to "debrief" your digestive system.
V. DATA ANALYSIS
Students who chose not to participate as subjects in this study will be responsible for collecting and tabulating the results.
1) Enter the data from the collected data sheets into the Excel table provided by the instructor.
2) During the class period following the last lab session of the week, your instructor will give you a copy of the tabulated results from all the test subjects in the glucose- and control-beverage groups.
3) Using these results, calculate the urine production rates (in ml/min) for each subject. Then calculate the mean and standard deviation of the blood and urine glucose concentrations and urine production rates at each time point for the two groups. (As an option, you may express each subject's data on a per unit weight basis, prior to computing these group descriptive statistics).
Q1: Was there a significant elevation in blood glucose levels followed by reactive hypoglycemia in those subjects who drank the glucose solution? Are these results what you expected? Explain.
Q2: Assuming that an average person has 5 liters of blood and 10 liters of extracellular fluid, what glucose concentration would you expect to find in the blood of an average individual following ingestion of 100 grams of glucose, if all of the ingested glucose were absorbed from the gut and distributed evenly in the blood and extracellular fluid? How does this calculated value compare to the measured mean peak value for the glucose-beverage subjects? If there is a difference between these two values, what could account for it?
Q3: How large an effect on blood and urine glucose concentration would you expect to see in the "average" subject if he/she ate a single candy bar, or drank a typical can of naturally sweetened soda, given the effects that were produced by the ingestion of a 100 gram glucose beverage?
Q4: What other characteristics of a food besides its total carbohydrate content might influence the magnitude of its effect on blood glucose levels?
Q5: What other controls might be done in an experiment like this, in order to insure greater uniformity of experimental conditions?
VI. PREPARATION OF THE LAB DATA SHEET
Your data sheet should include the ONE item described in the box above. Make sure that the axes of all of the graphs and print-outs are labeled and calibrated. You should certainly discuss your results and the answers to the questions with your partners and others in the lab. However, please work independently when you prepare your data sheet.