Laboratories 3 and 4Fluid Balance Part 1: The Urine Lab Introduction: The mammalian kidney controls body water balance via a negative feedback loop that involves the hypothalamus and anterior pituitary gland. The blood osmolarity is sensed by osmoreceptors in the hypothalamus. These receptors cause nerve cells in the hypothalamus to increase their rate of firing if the blood osmolarity is high and decrease their rate of firing if it is low. The rate of firing of the hypothalamic cells directly controls the rate of release of antidiuretic hormone (ADH) from the posterior pituitary gland. Diuresis means an increase in urine output. ADH, as its name implies, reduces urine output by increasing the permeability of the collecting duct to water. Since the kidney tissue is hyperosmotic, in the presence of ADH water leaves the collecting duct, and a low volume of concentrated urine is excreted. In the absence of ADH the collecting duct is impermeable to water and a large volume of dilute urine is excreted. The blood volume is also controlled by ADH. When the circulating volume is high, pressure receptors (baroreceptors) in the left atrium of the heart sense this and send messages to the hypothalamus preventing ADH release. This causes an increase in urine volume and a return of blood volume to normal. The purpose of this laboratory exercise is to explore the control of circulating volume and blood osmolarity. You will manipulate your circulating volume by ingesting fluid and manipulate our blood osmolarity by ingesting fluid which is either isotonic or hypotonic to blood. Then you will determine the effects the our manipulations on the volume and specific gravity of the urine you produce. For people with normal kidney function the urine specific gravity is a fairly accurate direct reflection of the urine osmolarity. Changes in urine osmolarity primarily reflect changes in the NaCl content of the urine. The approximate relationship between urine osmolarity and specific gravity is given in the following table: Urine Osmolarity (mOsm/L of water) 200 400 600 800 1000 1200 Urine Specific Gravity 1.006 1.012 1.018 1.024 1.030 1.036 4. Empty bladder again. Measure volume of urine produced and record. Time = 0. You can collect the data from your classmates. Chart the class information on a graph displaying urine output as a function of time. 1. Time = 40 minutes. Average the volumes and specific gravities of urine output for each group during each time period. 1.Materials: Plastic cups Gatorade/H2O Densitometers Methods: The lab section should divide itself into 4 groups of about equal size. Add your results to the lab results on the board. The experimental time line is as follows: 1. Measure volume of urine produced and record. Time = 120 minutes. 5. Describe your results in verbal form comparing the total urine output and specific gravity of urine for the four groups. Measure volume and specific gravity of urine produced and record. Use different symbols for the 4 groups. These groups will consume: Group 1 Group 2 Group 3 Group 4 nothing 330 ml water 330 ml Gatorade 1000 ml water If you have diabetes or a renal problem. 2. Save this urine for Part 2. Draw the negative feedback loop for ADH showing how plasma osmolarity is regulated when pure water is ingested. Empty bladder. Results: Discussion: . Finish the drink within 10 minutes. 2. 3. Empty bladder into cup. please do not participate in this lab. 3. Time = 80 minutes. Begin consuming your treatment drink. Just prior to experiment empty your bladder into a cup. Save the cups for urine collection. 4. 3. Examine your own urine specimen and record the color in the Results section (after Part 3).2. glucose. Materials: Urine sample from Part 1 of this lab. calculate the water volumes in the various body compartments of a 70 kg person. The amount of water in different compartments of the body has been determined for the Bedouin goat. Use this fact to determine what the % decrease in body fluid osmolarity will be when a 70 kg person drinks 1 L of water. Part 2: Urinalysis of your urine In this section of the lab you will perform several routine chemical tests on your urine. Assuming that humans have roughly the same distribution of water as these goats. Give: total body water. so please be careful not to spill or waste any of the supplies. if you are one of the people who drank 1 L of water!). When you drink pure water it is distributed evenly through your body water. 5. Work in groups of four for each of the tests listed below. The values are percentages of body weight. These amounts are as follows: 76% total body water. interstitial and plasma water. why is it dangerous for a person who is lost at sea to drink sea water (sea water is 3% salt and urine is ~2% salt). (pre-experiment) pH indicator strips Hydrometer Clinitest tablets Biuret reagent Methods: Perform the following tests on your urine sample. pH. Given what you have learned in this lab (and in lecture). We have a limited amount of reagents for this lab. Color 1. 27% extracellular water. You will be testing for color.9% blood volume. Table 1. Color Light yellow to amber Clear to light yellow Yellow orange to dark green Red to red brown Smoky red Diet Normal Alcohol Carrots Beets Beets Diseases Uncontrolled diabetes mellitus Bilirubin from obstructive jaundice Hemoglobin in urine Red blood cells from urinary tract . 49% intracellular water. and protein. specific gravity (as in part 1). Use what you know about the effect of plasma osmolarity and volume on ADH secretion to explain the results you obtained in this experiment. and 9. A. These tests can be carried out while you are waiting between the time points in Part A of this lab (it may also help you keep you mind off of your bladder. Table 1 lists some normal and abnormal urine colors and possible causes for the abnormalities. intracellular and extracellular water. Be sure to set the vial down after adding the tablet. 4. severe anemia A large meal or stress Uncontrolled diabetes mellitus High protein diet Severe anemia . 2. Add 2 ml biuret reagent. Determine the specific gravity as described in Part 1. Protein 1. 3. 4.025) Glucose present Protein present Possible causes Diet Disease High protein diet. After about 10 minutes. Caution: do not allow the contents of the vial to come into contact with your skin or eyes! Compare the color to the Clinitest color chart. specific gravity. Shake off the excess urine and closely compare the color of the strip to the colors on the pH chart. place 5 drops of your urine sample into a glass test tube. 5. Table 2.010) High specific gravity (>1. Record the results of the test (positive or negative) in Table 3. 3. Test result Low pH (<4. 2. Record your results (positive or negative) in Table 3. Table 2 lists foods and diseases that can affect pH. Add 1 ml of your urine to a glass test tube. Test the pH of your urine by dipping the pH strip into it three times. 3. 2.) Gently swirl the vial to mix the contents. Glucose 1. Record your pH in Table 3. Rinse the dropper and add 10 drops of water to the vial. Using a dropping pipet. (Note the pale blue color of the biuret reagent. C. pH 1.0) Low specific gravity (<1. presence/absence of glucose. After the reaction has stopped wait 15 seconds and then shake the vial gently to mix the contents. hold the test tube against a white background and observe the color. Specific gravity 1.5) High pH (>8. because it will become hot. A color change from light blue to pale violet indicates the presence of protein. Add your results to Table 3. Drop one Clinitest tablet into the vial. E. The color that most closely matches your strip corresponds to your pH.Dark wine Brown black Green Beets Rhubarb Green food dyes Hemolytic jaundice Melanin pigment from melanoma Bacterial infection B. and presence/absence of protein. cranberry juice Uncontrolled diabetes mellitus Diet rich in vegetables and diary Severe anemia Increased fluid intake Severe renal damage Decreased fluid intake/loss of fluid Uncontrolled diabetes mellitus. D. Urinalysis results. the lab should divide into three groups. When all of the data has been collected. you will make a diagnosis of the diseases that were responsible for the two unknown urine samples. Each group should test all of the following urine samples: Normal High Low Unknown A Unknown B Record your answers on the board and copy all of the class data in Table 4. These specimens will be subjected to the same battery of tests and.Results and Discussion: after Part 3. Part 3: Urinalysis of simulated urines In this section. and Group 3 will test for glucose. Results: Table 3 . Group 2 will test for specific gravity and protein. student urine Urine Test Color pH Specific gravity Glucose Protein Result Table 4 . Materials: Same as for Part B Methods: For these experiments.Analysis of simulated urine samples Test Color pH Specific gravity Normal Low High Unknown A Unknown B . you should be able to determine which disease was responsible for urine in the unknown samples (using the Tables 1 and 2). based on your results. the whole lab will be working together to test several control urine samples as well as two unknown urine samples. Group 1 will test for color and pH. Glucose Protein . What does this comparison tell you about homeostasis? 2. 4.Discussion: 1. Why can there be a spill over of glucose into the urine after a large meal? . You know that glucose is usually recovered from the urine in the kidneys. What diseases might account for the results that you obtained with the two unknown urine samples? 3. Compare the results of each of the urine tests on your urine and the normal simulated urine. Compare the results from the unknown urine samples with Tables 1 and 2. Why did the disease in Specimen A cause the observed change in specific gravity (remember what you have learned about this disease).