Laboratories 3 and 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

Urine Specific Gravity

1.006
1.012
1.018
1.024
1.030
1.036

Materials:

Plastic cups
Gatorade/H2O
Densitometers

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 4

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.

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.

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.

1.

Draw the negative feedback loop for ADH showing how plasma osmolarity is regulated
when pure water is ingested.

Results:

Discussion:

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.
Workingroupsoffourforeachofthetestslistedbelow.

Materials:

Urine sample from Part 1 of this lab. (pre-experiment)

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

Diet
Normal
Alcohol
Carrots
Beets
Beets

Diseases
Uncontrolled diabetes mellitus
Bilirubin from obstructive jaundice
Hemoglobin in urine
Red blood cells from urinary tract

Dark wine
Brown black
Green

Beets
Rhubarb
Green food dyes

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.
E. Protein
1. Add 1 ml of your urine to a glass test tube.
2. Add 2 ml biuret reagent. (Note the pale blue color of the biuret reagent.) Gently swirl the
vial to mix the contents.
3. 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.
4. Record your results (positive or negative) in Table 3.
Table 2 lists foods and diseases that can affect pH, specific gravity, presence/absence of glucose, and
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

Normal

Low

High

Unknown A

Unknown B

Glucose
Protein

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?