Laboratory 4
Fluid 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
Materials:
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•
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Plastic cups
Gatorade/H2O
Densitometers
Urine Specific Gravity
1.006
1.012
1.018
1.024
1.030
1.036
Methods:
The lab section should divide itself into 4 groups of about equal size. These groups will consume:
Group 1
Group 2
Group 3
Group 5
nothing
330 ml water
330 ml Gatorade
1000 ml water
If you have diabetes or a renal problem, please do not participate in this lab. You can collect the
data from your classmates.
The experimental time line is as follows:
1. Just prior to experiment empty your bladder into a cup. Save this urine for Part 2.
2. Time = 0. Begin consuming your treatment drink. Finish the drink within 10 minutes. Save the
cups for urine collection.
3. Time = 40 minutes. Empty bladder into cup. Measure volume of urine produced and record.
4. Time = 80 minutes. Empty bladder. Measure volume and specific gravity of urine produced and
record.
5. Time = 120 minutes. Empty bladder again. Measure volume of urine produced and record.
Results:
1) Average the volumes and specific gravities of urine output for each group during each time
period. Add your results to the lab results on the board. The combined class data will be
available Friday by 5:00.
2) Chart the class information on a graph displaying urine output as a function of time. Use
different symbols for the 4 groups.
3) Describe your results in verbal form comparing the total urine output and specific gravity of
urine for the four groups.
Discussion:
1) Draw the negative feedback loop for ADH showing how plasma osmolarity is regulated when
pure water is ingested.
2) Use what you know about the effect of plasma osmolarity and volume on ADH secretion to
explain the results you obtained in this experiment.
3) The amount of water in different compartments of the body has been determined for the
Bedouin goat. These amounts are as follows: 76% total body water, 49% intracellular water, 27%
extracellular water, and 9.9% blood volume. The values are percentages of body weight.
Assuming that humans have roughly the same distribution of water as these goats, calculate the
water volumes in the various body compartments of a 70 kg person. Give: total body water;
intracellular and extracellular water; interstitial and plasma water.
4) When you drink pure water it is distributed evenly through your body water. Use this fact to
determine what the % decrease in body fluid osmolarity will be when a 70 kg person drinks 1 L of
water.
5) Given what you have learned in this lab (and in lecture), 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).
Part 2: Urinalysis of your urine
In this section of the lab you will perform several routine chemical tests on your urine. You will be
testing for color, pH, specific gravity (as in part 1), glucose, and protein. 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, if you are one of the people who drank 1 L of water!). We
have a limited amount of reagents for this lab, so please be careful not to spill or waste any of the
supplies.
Work in groups of four for each of the tests listed below.
Materials:
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•
•
•
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Urine sample from Part 1 of this lab.
pH indicator strips
Hydrometer
Clinitest tablets
Biuret reagent
Methods:
Perform the following tests on your urine sample.
A. Color
1. Examine your own urine specimen and record the color in the Results section (after Part 3).
Table 1 lists some normal and abnormal urine colors and possible causes for the abnormalities.
Table 1.
Color
Light yellow to amber
Clear to light yellow
Yellow orange to dark green
Red to red brown
Smoky red
Dark wine
Brown black
Green
Diet
Normal
Alcohol
Carrots
Beets
Beets
Beets
Rhubarb
Green food dyes
Diseases
Uncontrolled diabetes mellitus
Bilirubin from obstructive jaundice
Hemoglobin in urine
Red blood cells from urinary tract
Hemolytic jaundice
Melanin pigment from melanoma
Bacterial infection
B. pH
1. Test the pH of your urine by dipping the pH strip into it three times.
2. Shake off the excess urine and closely compare the color of the strip to the colors on the pH
chart. The color that most closely matches your strip corresponds to your pH.
3. Record your pH in Table 3.
C. Specific gravity
1. Determine the specific gravity as described in Part 1. Add your results to Table 3.
D. Glucose
1. Using a dropping pipet, place 5 drops of your urine sample into a glass test tube.
2. Rinse the dropper and add 10 drops of water to the vial.
3. Drop one Clinitest tablet into the vial. Be sure to set the vial down after adding the tablet,
because it will become hot.
4. After the reaction has stopped wait 15 seconds and then shake the vial gently to mix the
contents. 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.
5. Record the results of the test (positive or negative) in Table 3.
Protein
Add 1 ml of your urine to a glass test tube.
Add 2 ml biuret reagent. (Note the pale blue color of the biuret reagent.) Gently swirl the vial to
mix the contents.
After about 10 minutes, hold the test tube against a white background and observe the color. A
color change from light blue to pale violet indicates the presence of protein.
Record your results (positive or negative) in Table 3.
and
Table 2 lists foods and diseases that can affect pH, specific gravity, presence/absence of glucose,
presence/absence of protein.
Table 2.
Test result
Low pH (<4.5)
High pH (>8.0)
Low specific gravity (<1.010)
High specific gravity (>1.025)
Glucose present
Protein present
Possible causes
Diet
Disease
High protein diet, 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,
severe anemia
A large meal or stress
Uncontrolled diabetes mellitus
High protein diet
Severe anemia
Results and Discussion: after Part 3.
Part 3: Urinalysis of simulated urines
In this section, the whole lab will be working together to test several control urine samples as well
as two unknown urine samples. These specimens will be subjected to the same battery of tests and,
based on your results, you will make a diagnosis of the diseases that were responsible for the two
unknown urine samples.
Materials: Same as for Part B
Methods:
For these experiments, the lab should divide into three groups. Group 1 will test for color and pH,
Group 2 will test for specific gravity and protein, and Group 3 will test for glucose. 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. When all of the data
has been collected, you should be able to determine which disease was responsible for urine in the
unknown samples (using the Tables 1 and 2).
Results:
Table 3 - Urinalysis results, student urine
Urine Test
Color
pH
Specific gravity
Glucose
Protein
Result
Table 4 - Analysis of simulated urine samples
Test
Color
pH
Specific gravity
Glucose
Protein
Normal
Low
High
Unknown A
Unknown B
Discussion:
1. Compare the results of each of the urine tests on your urine and the normal simulated urine.
What does this comparison tell you about homeostasis?
2. Compare the results from the unknown urine samples with Tables 1 and 2. What diseases might
account for the results that you obtained with the two unknown urine samples?
3. Why did the disease in Specimen A cause the observed change in specific gravity (remember
what you have learned about this disease).
4. You know that glucose is usually recovered from the urine in the kidneys. Why can there be a
spill over of glucose into the urine after a large meal?