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LAB 5 INSULIN AND HUMAN GLUCOSE METABOLISM
I. Introduction:
Insulin is an endocrine hormone secreted by the beta cells of the islets of Langerhans in the pancreas. Its principal function is to assist the transport of glucose across the cellular membrane. When insulin is deficient or lacking, only a small amount of glucose can cross the cell membrane and be used in cellular metabolism. This low rate of transport results in excess accumulation of glucose in the blood, called hyperglycemia. An excess of insulin causes a decrease in the level of blood glucose, or hypoglycemia. The normal concentration for blood glucose is 90 mg/dl (90 mg/100 ml of blood), but it may range from 60 mg/dl to 140 mg/dl, 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 by either removing the pancreas of an experimental animal (difficult to do in rats) or destroying the beta cells of the islets of Langerhans by injecting the chemical alloxan (alloxan is a specific inhibitor of the beta cells). Either of these procedures will produce the typical symptoms of diabetes in the animal: high blood glucose level and excretion of glucose in the urine.
Urinary excretion of glucose (glucosuria) results when the concentration of blood glucose exceeds the threshold level for total reabsorption by the kidney. The increased osmolarity of the urine also causes abnormally large quantities of water to be excreted (polyuria); this increased excretion of water may lead to dehydration, which in turn stimulates excessive water intake (polydipsia). Glucosuria, polyuria, and polydipsia are three major characteristics of diabetes. Diabetes mellitus received its name because the body of the diabetic person was formerly visualized as melting and flowing out via 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. Severe acidosis leads to coma and eventually death. Hyperinsulinism causes weakness, tremors, hunger, irritability, and other symptoms of low blood glucose; insulin shock can occur if blood glucose falls to a very low level.
In diagnosing diabetes, several tests are used to determine as precisely as possible what metabolic error is causing the disease. Such tests are urinary glucose level, urinary ketone bodies, fasting blood glucose level, insulin sensitivity, and glucose tolerance test. The glucose tolerance test assays the ability of the body (especially the pancreas) to respond to an excess ingestion of glucose.
The changes in blood glucose level following glucose ingestion (l g/kg body weight) in the normal and the diabetic person are markedly different. This difference is shown in Figure 12.1. In the normal person, the blood glucose level rises from about 90 mg/dl to around 140 mg/dl in 1 hr and then falls back to normal within 3 hr, or even to below normal due to excess insulin release by the pancreas.
The diabetic person, however, shows a hyperglycemic response in which the blood glucose level rises from about 120 to 160 mg/dl to as high as 300 mg/dl and then slowly falls to the fasting diabetic level after 5 to 6 hr. The diabetic's abnormal response is caused by the inability of the pancreas to secrete additional insulin in response to elevated blood glucose levels.
II. Procedure:
CAUTION! Parts of this lab may involve working with human blood. You should handle only your own blood. Dispose of all supplies (cotton, gauze, lancets, etc.) that come in contact with blood in properly marked containers. ALL BODY FLUIDS AND SUPPLIES MUST BE TREATED AS POTENTIALLY INFECTIOUS.
1. Select one person from each team for this activity, or ask for at least four volunteers from the entire class. These subjects should report to the lab in the fasted state (not having eaten for the last 10-16 hr). For our purpose it will be adequate if they just skip the meal preceding this lab.
2. Determine each subject's normal blood glucose level, using the procedure outlined on the next page for OneTouch-type blood glucose meters or on page 31-32 in the manual for FreeStyle-type blood glucose meters. The subject will also obtain a specimen of his urine and test it for glucose using a Labstix strip.
3. Each subject will then drink a lemon-flavored solution of 25% glucose. The quantity of solution will be based on a quantity of 1 g of glucose per kilogram of body weight. If the glucose solution cannot be made palatable, a good substitute is 30 ml of honey per test. Also, several commercial flavored drinks containing 50 to 100 g of dextrose in 10 oz. are available for this test (e.g., Dextrol, Tritol). Popular soft drinks usually have around 50 g of sugar in a 16-02 serving.
4. After ingesting the glucose or honey, the subject will repeat the test every 30 min. As soon as each blood sample has been taken, the subject will obtain another urine sample and repeat the Labstix test for urinary glucose (see below). Testing will continue in this manner for 2 hr or until the end of the lab period.
5. Record and graph the results of the blood glucose tests in the laboratory report. Also note the time when glucose appears in the urine. How do the results compare with the normal glucose tolerance test curve?
An accurate measurement of blood glucose can be obtained using a blood glucose monitoring system. Many diabetics use these or other glucose meters to monitor the effect of diet, exercise, and so forth on their blood glucose so that adjustments can be made to their insulin injections or regimen of oral medication.
Most are very simple to use but usually depend upon the use of either an electronic coding strip or a test solution with a known glucose content to check the operation of the meter. Below are directions for the meters (OneTouch Mini) that we will most likely use in the lab:
Using the OneTouch-Type Glucose Blood Meters
Start with the meter off. If you have turned the meter on to change settings or review past results, turn it off. Remove a test strip from its vial. With clean, dry hands, you may touch the test strip anywhere on its surface. Do Not bend, cut or modify the test strips in any way. Use each test strip immediately after removing it from the vial. Insert the test strip into the test port. Make sure the three contact bars are facing you. Push the test strip in as far as it will go. Again, Do Not bend the test strip.
After the start-up test screen appears, the meter will display the code from your last test. If a constant and a flashing “––” appear instead of a code number, such as when you are first using the meter, you may need to match the code on the meter with the code on the test strip vial. You can do thin by pressing the ▲ or ▼ to match the code number on the test strip vial.
The new code number will flash on the display for three seconds, and then stay constant for three seconds. The display will advance to the screen with the flashing blood drop icon R. If the codes already match, wait three seconds. The display will advance to the screen with the flashing blood drop icon R. The meter is now ready to perform a blood glucose test.
NOTES:
• If the screen with the flashing blood drop icon R appears before you are sure the codes match, remove the test strip, wait until the meter turns off, then re-start.
• If you press ▲ by mistake so that the control solution test symbol CtL appears on the display, press ▲ again to change it back to the screen with the flashing blood drop icon R.
Once you have a blood sample and your meter shows the screen with the flashing blood drop icon R, you are ready to obtain a blood glucose result. If your meter does not show the screen with the flashing blood drop icon R, remove the unused test strip and re-start the test process.
Keeping your finger extended and steady, move the meter and test strip toward the blood drop. Do not apply blood on the top of the test strip. Also, do not hold the meter and test strip underneath the blood drop. This may cause blood to run into the test port and damage the meter.
Line up the test strip with the blood drop so that the narrow channel on the edge of the test strip is almost touching the edge of the blood drop. Gently touch the channel to the edge of the blood drop. Be careful not to push the test strip against your fingertip or the test strip may not fill completely.
Wait for the confirmation window to fill completely. The blood drop will be drawn into the narrow channel via capillary action and the confirmation window should fill completely. When the confirmation window is full, this means you have applied enough blood. Now you can move the test strip away from the blood drop and wait for the meter to count down from 5 to 1.
You can now read your result on the meter. Your blood glucose level appears on the display, along with the unit of measure, and the date and time of the test. Blood glucose results are automatically stored in the meter’s memory.
Interpitation of Results of the Glucose Tolerance Test
Glucose tolerance tests may lead to one of the following diagnoses:
LabStix Procedure Remove one strip from the bottle and replace the cap tightly immediately. Briefly (no longer than one second) immerse all reagent areas of the test strip into the specimen. Wipe off excess urine on the rim of the container. Then lightly dab off the residual urine with a piece of tissue paper at the rim of the strip.
Hold strip in vertical position. Refer to the bottle label for specific reagent areas on the test strip. Compare the test areas with the color scale on the label. Proper reading times are critical for optimal results. See each reagent time as indicated on bottle label. Coloration appearing only along the edges of the test, or developing after more than two minutes, has no diagnostic value. The regent strips must be kept in the bottle with the cap tightly closed to maintain reagent reactivity. Please refer to bottle label for specific reading time for each reagent.
III. Questions/Data Collection:
1. Record the blood and urine glucose data for the subject from your team and the average values for all team subjects in the class. Generate a graph of your glucose data using Excel or another graphic program.
2. How are the levels of insulin and glucagon regulated in the body?
4. What causes the “insulin shock” seen when an overdose of insulin is given to an organism?
5. Why is there an increase in urine output (diuresis) in diabetes mellitus?
6. Why does a person who has diabetes mellitus have more acidic urine?
7. Briefly list the effect of each of the following hormones on blood glucose and the mechanism producing the effect.
IV. Materials
1. Labstix Reagent Strips for Urinalysis - 100 Each and cups for urine collection
2. Blood glucose test meters with strips
3. Lemon-flavored solution of 25% glucose for students to drink. The quantity of solution will be based on a quantity of 1 g of glucose per kilogram of body weight. If the glucose solution cannot be made palatable, a good substitute is 30 ml of honey per test.
Also, several commercial flavored drinks containing 50 to 100 g of dextrose/ 10 ounce are available for this test (e.g., Dextol, Tritol). Popular soft drinks usually have around 50 g of sugar in a 16 ounce serving
V. References:
1. Most of the lab was adapted from Tharp and Woodman, Experiments in Physiology Ninth Edition 2008. Pearson Benjamin Cummings Pubs.
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