• March 4th, 2016

EXPERIMENT TO DETERMINE THE EFFECTS OF VARIOUS SUBSTANCES ON THE RATE OF SODIUM, POTASSIUM & URINE EXCRETION IN THE HUMAN VOLUNTEER

Paper, Order, or Assignment Requirements

– OBJECTIVES
By the end of the practical you should have completed the following learning
objectives:
*Observe and measure the effects of different agents on urine generation and
solute excretion by the kidneys.
*Understand how of these agents to act through different physiological
mechanisms to produce their effects.
*Process clinical data and understand the use of a variety of statistical
methods to analyse the data.
*Prepare a full practical report describing your observations and discussing
these using your knowledge of renal physiology and pharmacology.
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– Should not volunteer to act as a subject in this experiment if:
*they know that they are, or may be, pregnant and/or
*they have a diagnosed kidney disorder and/or
*they are currently receiving medication for the control of blood pressure.
*who are taking any form of prescribed medication (other than oral contraceptives)
*Ideally, subjects (volunteers) should not have consumed alcohol for at least 12 hours
prior to this experiment and should have had only a very light meal together with the equivalent of three cups of fluid prior to the experiment.
*during the course of the experiment the subjects must not take any fluids additional to those indicated, and they must not smoke.
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– EXPERIMENTAL PROCEDURE
(1) Students should divide themselves into pairs with one VOLUNTEER who is
willing to take either placebo, alcohol# or furosemide and one BIOCHEMIST who will
analyse urine samples. Two students can act as DATA COLLECTORS.
#This experiment will be run as double-blind, placebo-controlled study, however,
volunteers who do not wish to take alcohol can be excluded if they see the member of academic staff in charge of the class.
Volunteers only, place complete the declaration attached to this practical schedule and hand in to a member of staff. Then choose a playing card to indicate which treatment you will receive.
(2) Time: 0 min
(a) The Volunteer must empty their bladders and record the total volume of urine produced (to the nearest mL).
To collect a urine sample you will need to visit the appropriate toilet with a litre jug, a measuring cylinder and a clean specimen bottle with cap to return with the sample needed for the photometer reading.
Having recorded the volume of urine produced and retaining the sample, dispose of the excess urine in the usual manner. Remember to wash your jug, measuring cylinder and your hands before returning to the Laboratory with your sample.
Labelled jugs and measuring cylinders may be left in the toilet until the last sample is
taken.
(b) The Biochemist must then measure and record the Na+ and K+ content of the urine sample using the Flame Photometer.
The Flame Photometers will be pre-calibrated. One will be set up to measure Na+ and the other K+.
DO NOT ADJUST THE SETTINGS ON THE FLAME
PHOTOMETERS. Also, the chimneys of the flame photometers are HOT so take care not to place anything on them.
Dilute the urine sample 1:1000 using the Gilson Pipette set at 100 µL and a 100 mL volumetric flask, then aspirate on flame photometers noting the Na+ and the K+ readings.
Clean glassware throughout for every sample.
(c) Give the urine volume and urine biochemistry data to the data collectors and write the results for Cumulative Urine on the table on the board.
(3) Time 30min
All volunteers and biochemists to repeat the above procedure (2).
Volunteers will then receive one of the treatments as below (subjects will not be told which of the treatments they receive).
*1000 mL lime or orange squash & placebo tablet
*1000 mL lime or orange squash containing 45 ml 65% proof vodka (equivalent to two glasses of wine) & placebo tablet
*1000 mL lime or orange squash and 40mg of Furosemide
(4) At half hourly intervals, for up to two hours, volunteers will produce and measure urine production and biochemists will repeat the measurements of urine volume and Na+ and K+ content of each sample.
(5) At the end of the experiment, tabulated Cumulative Urine data will be discussed
with the class to try to work out who received placebo, alcohol and furosemide. The code will then be cracked to reveal what volunteers actually took$…
At the end of the practical class, instructions will be given on how to interpret the data obtained by the class.
Raw data (urine volume, cumulative urine volume, urinary Na+ and K+).
$The effects of furosemide typically last for between 4 and 6 hours. Volunteers who
took furosemide should ensure they take extra fluids to avoid mild dehydration and tiredness.
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– STRUCTURE:
1) Abstract (approximately 250 words)
This is a short summary of the whole study. Briefly outline the aims of the practical, methodology, results (including specific significant data where appropriate) and conclusions. Don’t cite any references in this section.

2) Introduction and Aims (approximately 100 words)
Provide a brief background to the study, e.g. you could highlight here that many substances can modify urine excretion and why diuretics are used clinically. Then list the main aims of the experiment.

3) Materials and Methods (approximately 500 words) in “past tense”
Include a brief description of the materials and methodology used in sufficient detail to allow the study to be replicated.
What kind of study was this? What groups were used? How many subjects were in each
group? Who were excluded from participating? What about subjects who didn’t want to take alcohol? How were the treatments allocated?
This section should also include a brief description of the method used to convert the Na+ and K+ raw data and also the statistical tests you have used. It
should be written in paragraphs as shown in journal articles rather than a step-by-step list of a protocol.

4) Results (500-1,000 words)
(i) Complete the data tables supplied as Excel datasheets which shows the raw data from all four labs groups for the three treatments. These can be added to the end of your report as an Appendix – we can look at them and provide feedback if the data shown on your graphs doesn’t look right.
These tables should also be used to generate the processed data obtained from the Na+ and
K+ concentrations (mM or mmol/L) converted into Output (mmol) based on the urine production every 30 min as discussed in the lab class – you must do this for your Na+ and K+ data to make any sense.

5) Calculate the mean values for each group at each time point. Also calculate standard
deviations (Std Dev) and state the number of subjects in each group (N).
(ii) Perform the statistical analysis of the data using a relevant statistical test such as a one-way ANOVA followed by a post-test such as Bonferroni’s. You can use Excel, SPSS or
Minitab to do this and there is a Powerpoint presentation on studentcentral which provides
advice of statistical tests which may be helpful.
Specifically, you need to check if:
(a) there is a significant change in any parameter measured at 30 min and 150 min (urine
volume, cumulative urine, Na+ output, K+ output) and
(b) there is a significant difference between the values for orange juice, alcohol and
furosemide data obtained at 150 min.
You should then highlight any significant changes between groups (i.e. where P < 0.05) on
the graphs you will draw as described below.
(iii) Draw and provide the 4 graphs as described at the end of the practical:
(a) the average urine output in mL every 30 min
(b) the average cumulative urine output in mL every 30 min
(c) the average Na+ output in mmol every 30 min
(d) the average K+ output in mmol every 30 min
Try to show the standard deviation (SD) for each bar on your graph – these should have
been calculated on your Excel spreadsheet.
Ideally these should indicate any significant changes between groups as discovered
during your statistical analysis which can be shown on your graphs using symbols (e.g. #).
(iv) Provide a commentary of your results. Look at each graph and associated statistical
analysis and describe what they show. Bear in mind that there may not be a significant
change where you would expect one but look for any trends and comment on this. Do not
“discuss” the results here, e.g. the reasons WHY you are seeing effects, leave this for the Discussion section.

6) Discussion (500-1,000 words)
Provide a brief summary here of the main findings of the study. This should include a brief re-iteration of each result together with a discussion of the reasons for the result.
Discuss the results from the experiment in the light of your knowledge of renal physiology and pharmacology. The discussion material can be based on information obtained from academic sources on the Internet (but be sure that your source is scientifically accurate).
You need to show that you understand how furosemide works in a different way to
alcohol, but also note that the 1L orange squash taken by all the volunteers may also have a
diuretic action – why? Could changes in K+ excretion caused by furosemide lead to
unwanted clinical effects? If so, how could these be avoided? Did the alcohol produce a
significant increase compared to the orange juice only? If not then why not? How could
this experiment have been improved? Improvements to the study design here can be
discussed here.

7) Conclusions (maximum 100 words)
A brief conclusions section should be included summarising the most important findings
of the study. Any clinical relevance can be mentioned here. Is furosemide a good diuretic?
References – All sources of information used in the completion of this report should be
cited within the text and appear in the reference list at the end of the report. Please refer to
journal articles for examples of accurate and precise formatting of the references and use a
consistent style in your report and the research article.

8) Referencing and citations.

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