C. Lipids (fats and oil) Large amounts of concentrated lipids leave a translucent spot on absorbent paper after drying

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1 Identification of Biomolecules in Solution and Urinalysis Application Part I: Identification of Biomolecules in Solution Our physical bodies are essentially a collection of both common and some exotic chemicals. Many of these chemicals are simple inorganic combinations such as sodium chloride, hydrochloric acid, molecular oxygen, and carbon dioxide. Most of the different kinds of chemicals comprising our bodies are larger more complex organic molecules. The biochemical reactions that are occurring constantly within our cells synthesize new, larger molecules or decompose larger molecules into smaller pieces. Anabolism is a term used for all the synthesis reactions occurring at any time; Catabolism is a term that refers to all the decomposition reactions occurring at any time. Metabolism is a term that refers to AL of these reactions together. While our bodies can metabolize a wide variety of organic molecules, the vast majority belong to three major groups: carbohydrates, lipids and proteins. Carbohydrates are composed of carbon, hydrogen, and oxygen atoms in a ration of (CH2O)nwhere n can be any number depending on the complexity of the carbohydrate. Simple sugars such as glucose and fructose are called monosaccharides. More complex carbohydrates such as starches are polymers of these monosaccharide units are and called polysaccharides. Simple carbohydrates are broken down or catabolized in a process called glycolysis which provides the cells with most of its energy. Lipids, including fats and steroids are composed of carbon, hydrogen and oxygen atoms. They are important components of cell membranes are used as hormones and for energy storage. Excess food is usually stored as fat in adipose tissue cells. Proteins are constructed from long chains of amino acids and contain carbon, hydrogen, oxygen, nitrogen and sulfur atoms. Proteins provide the major structural components of our cells and therefore our bodies. Other proteins serve as enzymes which are the major catalysts that facilitate complex biochemical reactions in our cells. We can perform simple tests to identify the presence of some of these kinds of molecules by adding indicators to a solution to be tested. A change in color or other physical characteristic indicates the presence or absence of a particular kind of organic molecule. A. Simple carbohydrates (sugars) Benedict s solution causes some sugars to turn green, yellow, orange, or red when heated to boiling. The color of a positive reaction depends on how much sugar is present (green indicates low levels; red high sugar levels) B. Complex carbohydrates (polysaccharides or starches) Lugol s iodine causes a solution containing starch to turn dark blue to black. The more starch there is, the darker the color C. Lipids (fats and oil) Large amounts of concentrated lipids leave a translucent spot on absorbent paper after drying D. Proteins (and Polypeptides) Biuret solution causes a protein solution to turn pink or violet. The first step in learning to detect these chemicals is to perform control tests with substances known to contain or not to contain specific chemicals. You will perform each of the above tests on a positive and a negative solution (the negative is usually water). After completing the tests you will see both the positive and negative results for each of the different kinds of molecule above. Then you can compare your experimental tests to these Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 1 of 9

2 control results to see if any of the different kinds of organic molecules are present in each test (unknown) solution. Control Test Procedures 1. Sugars a. Take two clean test tubes and label one su+ and the other su-. b. Add about 1 cm (use ruler to measure) of glucose solution (10% Karo) to su+ test tube c. Add about 1 cm of DI water to su-. d. Add 5 drops of Benedict s solution to each test tube e. Using test tube clamp, place both test tubes in boiling water bath at your table for about 2 minutes. f. Record the reaction as either + or - in the table on your data sheet. 2. Starches a. Swirl the starch bottle to mix, then add a drop of starch solution (1% starch) to one of the wells in the spot plate and a drop of DO water to another well. b. Add 1-3 drops of Lugol s iodine to each of the wells. c. Record the reaction as either + or - in the table on your data sheet. 3. Lipids a. With a dropper, add a drop of oil (vegetable oil) to a 2 square of a paper towel b. With another clean dropper, add a drop of DI water to the other half of a paper towel c. Place the paper towel in the incubator or on a warming tray for 5 minutes. d. Record the reaction as either + or - in the table on your data sheet. 4. Proteins a. Swirl the protein bottle to mix, then add a drop of protein solution to a clean spot plate b. Then add a drop of Biuret solution to the same well c. Add a drop of Do water to another well on the spot plate. d. Then, add a drop of Biuret solution to the same well. e. Record each of the two reactions as either + or - in the table on your data sheet. Experimental Test Procedures In the second part of this exercise you will be testing each of the unknown solutions that you are given by adding indicators (like you did to the controls) to test for the presence of the above molecules. But, before you actually perform the tests you must first make predictions (hypotheses) by noting which organic molecules you would expect to find in each of the solutions. Indicate which organic molecules you expect to find in each of the test solutions by placing a + sign in the expected results column of your data table. Place a - if you do not expect to find that kind of molecule. AS you make your predictions, be able to explain why you did or did not expect to find a particular kind of molecule in each sample. Now test for the presence or absence of each kind of organic molecule by using the same amounts of indicator solutions that you used in your control tests. Record your results in the experimental results column of your tale on your data sheet. Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 2 of 9

3 Notes: Use the spot plate for the starch and protein tests Use a paper towel for the oil test Use test tubes for the sugar test ***You will need to clean and rinse the test tubes in DI water and reuse them during this lab. At the end of the lab you can WASH the test tubes in the sink using soap and water. Return to test tube rack. Clean up and Disposal at End of lab! Discard all test solutions into the sink with the water running Do NOT empty water from pan on hot plate Make sure the hot plate is unplugged before you leave; leave the boiling water and pan on the hot plate. Keep any unused test tubes in the rack Dispose of paper towels in the trash Clean spot plates with soap and water and return it to the tray on your lab tale. Return all dropper bottles to the tray on your table, make sure caps are screwed on. Wipe down counters. Identification of Biomolecules Lab Data Sheet Control Tests: For each control test below record your results as + or - in the column to the right. Control Tests Results +/- Sugar Test Sugar Solution DI Water Starch Solution Starch Test DI Water Oil Lipid Test DI water Protein Solution Protein Test DI Water Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 3 of 9

4 Questions: Did all the control tests give the expected results, if not explain? Why are these called control tests? What would be the consequences for the rest of this experiment if any of the control tests did not produce the expected results? Describe a specific example. Part II: Urinalysis In this part of the lab, you will be using simulated urine and the indicators to determine ph, color, ammonium, the presence of proteins and glucose. The results from these tests can be used to diagnose various kidney problems. You will compare the results of your tests to the case studies below and determine which urine sample belongs to which case. Case Studies: Unknown Urine Samples Case 1: Mike Smith is 19 years old. He notices that he has increased urine output (polyuria) and increased appetite (polyphagia) and great thirst (polydipsia). He has also experienced unexplained weight loss Case 2: Mr. Magoo is 62 years old. The last couple of weeks, he has been feeling unusually tired. He sometimes feels dizzy and has been finding it increasingly difficult to sleep at night. His face is puffy and his ankles and feet are swollen. He has a burning pain in his lower back, just below the rib cage. He also states that his urine is dark in color. He went to the doctor and found out his blood pressure is high and the area around his kidney is sensitive Case 3: Ms. Ariel is 26 years old and has been experiencing painful and difficult urination (dysuria), increased frequency and urgency of urination. Her urine is a milky color. She also has fever and malaise, which is evidence of infection. Upon seeking medical care, she is given antibiotics. After a couple days on antibiotics, her symptoms disappear. Case 4. Normal Sample (Control) Note: you gathered this data in Part I. Hypothesis: (If then Statement) Case 1: Case 2: Case 3: Case 4: Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 4 of 9

5 Procedure: A. Color: The color of normal urine can range from pale yellow to amber, depending upon the concentration of a pigment called urochrome; which is the end product of the breakdown of hemoglobin. The color of urine is an indicator of pathological conditions. Pale yellow urine may indicate diabetes or may indicate that you have taken in copious amounts of water. After drinking alcohol, alcohol forces the body to increase urine production and your body removes it from other organs along with important electrolytes (this is why it leaves you with a bad headache). A milky color may signify fat globules that indicate a urogenital tract infection. Reddish color urine may indicate the digestion of food pigments (e.g., beets), certain drugs, antibiotics, or that there is blood in the urine. Greenish colors indicate jaundice (bile pigment) or certain bacterial infections. Black-brown urine may indicate phenol or metallic poisonings or hemorrhages due to kidney injury or malaria. 1. Record the color of the urine for each unknown Unknown Urine Sample Results of Color Test Color Description B. ph. ph refers to how acidic or basic something, in this case urine is. The ph of normal urine is approximately 6.0, which is slightly acidic. Urine may be highly acidic in the case of diabetes or dehydration. Highly basic urine is present when there is a urinary tract infection, aspirin overdose, certain types of kidney disease or a diet high in citrus. 1. Place 1 drop of each type of urine on individual strips of ph paper. 2. Record the ph number and indicate whether the urine is acidic or basic. 3. Throw away the ph paper strips in the trash. Results of ph test: Unknown Urine Sample ph number Acidic or Basic? C. Ammonium: Occasionally, people will complain that urine has a smell like ammonia. A person who eats nitrogen-rich food such as proteins and leafy greens the excess nitrogen is released in the urine. Dehydration will cause the smell if the person doesn t take in sufficient water and urinate regularly. The liver is responsible for breaking down ammonia; if it can t then ammonia ends up in the urine. Diabetes, STD s, medications, vitamins and nutritional supplements are other common causes. In woman, menopause, pregnancy, and bacterial infections of the kidneys or bladder may also cause ammonia smell. The presence of ammonia can be detected by the ammonia smell, the urine will appear cloudy, and there may be blood in the urine; indicating heart failure. If ammonium is present in the urine, red litmus paper will turn blue as ammonia vapor leaves the solution. Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 5 of 9

6 1. Place 1 drop of each type of urine on individual strips of red litmus paper 2. Record whether ammonia is present or not (+ or -) 3. Throw away the litmus paper in the trash. Unknown Urine Sample Results of Ammonium Test Urine Cloudy or Clear? Presence of Ammonium (+ or -) D. Glucose: There should be no glucose in the urine. When there is, it often means that the sugar level in blood entering the kidney is so high that it exceeds the kidney s ability to pass it back into the blood. The spillover ends up in the urine. This condition is associated with diabetes, but is also common with pancreatitis and hypothyroidism. During pregnancy, a normal, healthy disease-free woman may present with glucose in their urine. 1. Conduct the appropriate tests. Note: once one sample has turned red/orange all the tests are done. 2. Record your results 3. Dump the liquids into the sink and wash your test tubes with brush in soap and water. Unknown urine sample Results of Glucose Test Test: Test: Type of Saccharide (mono, di, poly) E. Protein: There should be no protein in the urine. Your kidneys are responsible for filtering wasted products from the blood, while retaining components, including proteins, that your body needs. However, some diseases and conditions can allow proteins to pass through the filtering of your kidneys. Proteins in the urine can be temporarily increased due to cold exposure, emotional stress, fever, heat exposure, and strenuous exercise. Persistent elevated levels of protein may suggest kidney disease and be caused by a number of different events including sickle cell anemia, certain drugs, diabetic hypoglycemia, heart disease, heart failure, hypertension, Hodgkin s lymphoma, Leukemia, and kidney infection. 1. Conduct an appropriate test for proteins 2. Record your results. 3. Wash your lab equipment with soap and water. Results of Protein Test Unknown Urine Sample Presence of Protein (+ or -) Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 6 of 9

7 Analysis: 1. The contents of a person s urine can change throughout the day. Explain why you think this might occur. 2. How would a positive result for each of these components in the urine look? a. Glucose b. Proteins c. Ammonium 3.Sometimes people take medicines that may change the appearance of their urine. If a person saw red to red-brown urine, what drug might be responsible? 4. Alcohol causes what change in the color of the urine? Why? 5. What might a healthy, disease-free person have a high amount of glucose present in the urine? Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 7 of 9

8 Results: Note: after you accept or fail to accept your hypothesis: If your experiment did not support your hypothesis, explain specifically why you think you did not get your expected results. Was it a problem with your hypothesis? The data you collected? Be very specific in your answers. I was wrong is NOT an explanation. Unknow n Urine Sample 1. Case Study A, B or C? Did you accept (A) or fail to accept (R) your hypothesis? Why did your experiment fail to support your hypothesis? (use additional paper if needed) Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 8 of 9

9 Part III CHEMICAL ANALYSIS OF YOUR URINE For routine chemical analysis of urine there are several brands of chemical test strips (dip sticks) that are commercially available. These urinalysis test strips have small test patches impregnated with various chemicals in order to detect the presence or absence of certain substances. Qualitative and/or quantitative results can be obtained depending on the particular test. 1. Take a specimen cup, gloves, and dipstick (be careful to not touch testing strip) from the lab to the bathroom; void into the cup. 2. Briefly (one second or less) dip the test strip into the urine. Make sure that all test squares are immersed. 3. Draw the edge of the strip along the rim of the specimen cup to remove excess urine. 4. Dispose of urine in toilet and throw cup away. 5. After the appropriate times (as indicated on the vial of strips) read the tests by comparing to the color chart on the edge of the vial. PLEASE DO NOT TOUCH THE TEST STRIP TO THE COLOR CHART. IF YOU DO SO ACCIDENTALLY, IMMEDIATELY WIPE THE VIAL WITH DISINFECTANT. 6. NOTE: For convenience, all values on the strip may be read between 1 and 2 minutes after immersion. The colors are stable for up to 120 seconds after immersion. Color changes that occur after 2 minutes from immersion are not of diagnostic value. Color changes that occur only along the edge of the test area should be ignored. 7. Results are obtained by direct visual comparison with the color scale printed on the vial label. No calculations are necessary. Record your results. NOTE: You may find it useful to use your cell phone to carefully take a picture of your results to help you describe your results below. 8. NOTE: For such a test to be considered clinically acceptable for a valid diagnosis, careful quality control should be maintained, i. e. expiration dates respected, environmental conditions stabilized, etc. In a teaching lab these conditions are not met. You can learn the procedure and see some variable results among the class members, but do not base any clinical assumptions on the results obtained in this lab. If you have any reason to suspect a clinical problem, go to a licensed medical laboratory for a urinalysis. 9. Please wash your hands when you have completed your test and return to the lab. Describe the color and results of your urine as indicated in urine chart Describe the results of your dipstick test. Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 9 of 9

10 Lab #3 BCC Adapted from Anatomy and Physiology; Lab Manual, Ziser Page 10 of 9