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					         KIDNEY REGULATION OF OSMOLARITY
Urinalysis.doc 30 November 2008


        One of the kidney’s main functions is to regulate the osmolarity of body fluids at around
300mOsm per liter. In these experiments, the kidneys will be presented with excess water and/or
salt load and the response will be monitored by measuring the volume and concentration of the
urine produced.
Overview of Lab:
        1. Empty bladder 2 hrs before lab, and note the time.
        2. When lab period begins, collect bladder contents, label as “control”, and
        3. Drink test solution within 5 minutes.
        4. Analyze “control” urine sample for volume and salt content.
        5. Collect urine at 30 minute intervals to measure volume and salt content.
        6. Determine the percentage of the water and/or salt intake has been excreted during our
            lab.

1. Drink plenty of water on the day of the experiment. Avoid eating or drinking caffeine or
theophylline (all soft drinks, tea, chocolate). EMPTY YOUR BLADDER ONE OR TWO
HOURS BEFORE THE LABORATORY BEGINS and RECORD THE EXACT TIME.
Do not save this urine sample. It is not necessary to measure the volume of this sample.

2. Upon entering the laboratory, each student will take a urine collection bottle to the restroom
and void into the bottle, emptying the bladder completely. Record the exact time. This sample
will be designated as “control” urine. Take the specimen back to the lab and label. You will
analyze this control sample after you have consumed the experimental solution.

3. After collecting and labeling the “control” specimen, each student will return to the laboratory
and immediately drink one of the following solutions as quickly as possible. You and your lab
partners should agree on who will drink which solution.
       800 ml of distilled water
       800 ml of water plus 7 grams of NaCl
       80 ml of water plus 7 grams of NaCl.

These solutions are safe for humans with normal healthy kidneys. Do not drink one of the
solutions containing salt if you have blood pressure problems or if you are on medications that
might be affected by water or salt loading. You may elect not to participate in these
experiments. Once you have consumed the test solution, you may not drink anything until the
last urine sample is collected at about 150 minutes after drinking the test beverage.

4. Every 30 minutes after drinking the solution, each student will empty the entire bladder into a
clean collection bottle. If unable to void, retain the urine in the bladder until the next 30 minute
collection time, and adjust your calculations accordingly.

5. Analyze the urine from each collection, including the “control” for:
      a) Volume: Measure the total volume with a graduated cylinder.
          Express as ml/min excreted. (This is why it is important to know the time interval
          between specimen collections.)
      b) NaCl concentration:
          i) Place 1ml of urine into a test tube.
                                                 1
           ii) Add one drop of 20% potassium chromate and mix thoroughly on Vortexer.
            iii) Titrate with 2.9% silver nitrate (0.171 Normal) solution dropwise from a burette
       while agitating the test tube on a Vortex mixer after each drop until a persistent color
       change is obtained. The end point of titration is a light pinkish brown. Disregard the
       formation of precipitate; you are looking for a light brown solution. If you “overshoot”
       the endpoint of the titration, discard the sample into the labeled waste container and start
       over.
       iv) Each ml of silver nitrate added represents 1 mg/ml of NaC1 in the urine.
       v) Calculate:
                1) the total grams of NaC1 in the urine collected at each 30 minute interval
                2) the NaC1 concentration in mg/ml of urine.

Optional activity: Use the Multistix 9 SG test strips to test the control urine, but no other urine
samples. See instructions on Page 3 of this handout.

6. Record your data on the table on the next page.

Calculations for the control (***) are complicated by the fact that the interval between the last
urination before the lab and the first specimen collection is different for each student. To make
comparisons of NaC1 excretion before and after ingestion of the experimental solution, the NaC1
excreted during the pre-lab interval must be extrapolated to determine how much NaC1 would
have been excreted in a 30 minute interval.
Notation:
tc = time interval between last urination before lab and collection of the control sample (in
minutes)
Vc = volume of control urine sample
V1 = volume of first urine sample after ingestion of the test beverage.
Uc = concentration of NaC1 in control sample of urine
U1 = concentration of NaC1 in first urine sample after ingestion of the test beverage.

A sample calculation:
Time of last urination before lab: 12:48 pm
Time of first urine collection during lab: 2:33 pm
tc = time between 12:48 and 2:33 = 105 minutes (time over which control urine was produced)
For illustration, assume that 62 ml of urine was collected and the concentration was 0.7 mg/ml.
Vc = 62 ml Uc= 0.7 mg/ml

NaC1 excretion for a thirty minute interval during the period before ingestions of the solution =
      (0.7 mg/ml * 62 ml * 30 min)/105 minutes = 12.4 mg
This means that during a thirty minute interval during the control period, 12.4 mg of NaC1
would have been excreted. Note that 12.4 mg is 0.0124 g.

Time                Volume (ml)          Urine flow           NaC1                Urine [NaC1]
                                         (ml/min)             Excreted in         (mg/ml)
                                                              30 min (g)
0 (control          Vc                   Vc/tc                Uc*Vc*30            Uc
sample)                                                           tc
30 min              V1                   V1/30                U1*V1               U1
60 min              V2                   V2/30                U2*V2               U2
                                                 2
Time of last urination before lab period: ________________________
Test liquid (circle one): Pure water, Isotonic saline, and Hypertonic saline.
Time                Volume (ml)         Urine Flow           NaC1                Urine [NaC1]
                                        (ml/min)             Excreted in         (mg/ml)
                                                             30 min (g)
0 (control)                             ***                  ***
30 min
60 min
90 min
120 min
150 min
Total                                   XXXXXXXX                                 XXXXXXXX
Excreted in                             XXXXXXXX                                 XXXXXXXX
2.5 hrs
% of intake                             XXXXXXXX                                 XXXXXXXX
excreted                                XXXXXXXX                                 XXXXXXXX

*** For the values in these cells, you must account for the time interval between your last
urination prior to lab! See sample calculations on the previous page.

Procedure for Analyzing Control Urine Sample with Multistix 9 SG Test Strips.
1. Remove 1 strip from bottle and replace cap. Completely immerse reagent areas of the strip in
Fresh urine and remove immediately to avoid dissolving out the reagents.
2. While removing, run the edge of the strip against the rim of the urine container to remove
excess urine. Hold the strip in a horizontal position to prevent possible mixing of chemicals
from adjacent reagent areas and/or contaminating the hands with urine.
3. Compare reagent areas to the corresponding Color Chart on the bottle label at the times
specified. Hold strip close to color blocks and match carefully. Avoid laying the strip directly
on the color chart, as this will result in the urine soiling the chart.

Proper reading time is critical for optimal results. Read the glucose and bilirubin test at 30
seconds after dipping. Read the ketone test at 40 seconds, the specific gravity at 45 seconds; pH,
protein, urobilogen, blood, and nitrate at 60 seconds; and leukocytes at 2 minutes. The pH and
protein areas may also be read immediately or at any time up to 2 minutes after dipping. Color
changes that occur after 2 minutes are of no diagnostic value.

You could compare your test results with chart in the lab that lists normal values.




                                                 3
Questions
7. Answer these questions:
a) What percentage of the original NaC1 and/or water load was excreted during the 2.5 hour
interval following ingestion of the test liquid? Enter these values in the last row of the data table.
(Exclude data from the “control” urine since this specimen was obtained prior to the
ingestion of the test solution.)

       b) What is the osmolarity of the “strong salt” solution? Which hormone(s) is/are
involved in the response to this solution?

        c) What is the osmolarity of the “dilute salt” solution? Which hormone(s) is/are involved
in the response to this solution?

8. Explain how the results illustrate the kidney’s handling of water and salt loads for each of the
solutions consumed. Your explanation should include the roles of various hormones involved.

9. What are the sources of error in these experiments?




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