BS2560 Pharmacology and Toxicology : DATA HANDLING EXERCISE.
THE INFLUENCE OF ALKALAEMIA ON THE EXCRETION OF ASPIRIN IN MAN
FOLLOWING ORAL ADMINISTRATION
Learning Objectives.
1. To process data and determine the effects of alkalaemia on aspirin excretion
2. To provide experience of the problems and difficulties of processing experimental data
3. To provide experience in the use of computer aided data processing.
Aspirin is acetylsalicylic acid. After oral administration it is adsorbed, biotransformed and
excreted. In the urine there may be aspirin itself, its hydrolysis product (salicylic acid or its
sodium salt) and conjugates of either aspirin or salicylate (produced in the liver). In order to
estimate these materials easily they are first boiled with ammonia. This hydrolyses all these
excretory products to salicylic acid which can be estimated using the ferric nitrate reagent as
described. The Absorbance is read and from a calibration curve relating the Absorbance
produced by reacting 1 ml of a solution of a known concentration of sodium salicylate with 6 ml
of reagent. Hence the concentration sodium salicylate in the 1 ml of hydrolysed urine solution
is obtained.
This experiment will be done as a data handling exercise. This is to say you will be provided
with the data which would have been collected had the class done the experiment. The data is
provided as an EXCEL spread sheet file.
The practical schedule, which was followed by other students to produce the data, is given
below. The "Introduction", "Procedure" and "Estimation of Aspirin" sections show you how the
data were obtained. READ it and UNDERSTAND what has been done as it has implications
for the calculation and processing of the data.
Requirements.
Calculate the amount of sodium salicylate equivalent excreted in each sample of urine.
Plot:
1. mean (+ SE) urine pH against time for both groups on the same graph.
2. the mean (+ SE) amount of SODIUM SALICYLATE derived from the tablets and excreted in
the urine samples obtained at each time point after taking the tablets.
3. the mean (+ SE) cumulated equivalent amount of ASPIRIN excreted against time for both
groups.
4. Finally, calculate the average percentage of the aspirin dose remaining in the body at each
time period and write it on the above graph.
Explain the form which the graphs take and comment on the results. Is there a statistically
significant difference between the two groups? Think carefully about the data being compared
in each case!
Why is this effect of alkalaemia of clinical significance?
Draw the structure of aspirin (acetylsalicylic acid) and the structures and metabolic relationship
of some of its metabolites excreted in the urine.
Predict the effects which alkalaemia might have on the excretion by the kidney of some other
drugs:
a) quinine;
b) ethanol;
c) penicillin. Explain your predictions.
Note that after the lecture you will not be given any more information by staff or demonstrators
about how to set about the problem, you should already have or be able to find enough
information and knowledge to work out the answers. Part of the assessment is that you have to
think about the possible alternative ways of getting over the problem and make a decision. You
are free to discuss the problems and the answers with your colleagues but remember that you
must not copy each other’s calculations, reports or conclusions!
PRACTICAL SCHEDULE FOLLOWED TO OBTAIN DATA.
Introduction
Subjects will either be rendered alkalotic by ingestion of sodium bicarbonate or will remain in
normal acid-base balance. One half of the group will ingest sodium bicarbonate and the other
half will act as controls. All subjects are given a water load which is maintained throughout the
experiment ensuring that they are able to produce consistent samples of urine at regular
intervals.
*NB. Because of the effects of aspirin on the gastric mucosa, students with any history of gastric
disorder should not act as subjects; nor should students who have not previously taken aspirin,
because a small proportion of people are abnormally sensitive to its effects.
Procedure:

0 min Drink 500 ml water.

0-15 min Construct standard curve of optical density versus concentration, for 100, 200,
300 and 400 g/ml of sodium salicylate using procedure described below.

30 min Collect urine for volume, pH and control salicylate estimation. Drink either an
equal volume of water or water containing sodium bicarbonate (10g).

32 min Take 900 mg aspirin.
Collect urine at 30 min intervals (60, 90, 120, 150, 180, 210 min) drinking an equivalent
volume of water. Record the volume, pH, and sodium salicylate concentration in each 30 min
urine sample.
Estimation of Aspirin
Acetyl salicylic acid and its metabolites are hydrolysed to salicylic acid, which is then estimated
colorimetrically. The colour developed with ferric nitrate is standardized using a solution of
sodium salicylate of known strength.
To a 5 ml sample of urine add a few drops of ammonia (S.G. 0.88). Place the sample in a
boiling water bath for 10 min., cool, neutralize using HCl and make up to 10 ml. Test for
neutrality with indicator paper.
To 1 ml of water, standard or hydrolysed sample of urine add 6 ml of ferric nitrate reagent. (The
ferric nitrate reagent contains mercury and is therefore TOXIC.) Handle with care! Read the
absobance at 525 nm using the spectrophotometers provided. The optical density should be
proportional to the amount of salicylate over the range of concentrations from 0.7 to 3.0 x 10-3M
i.e. 100 to 400 g sodium salicylate per 1 ml sample. If the absorbance indicates a concentration
outside the linear range, dilute the hydrolysed sample and treat the diluted sample with reagent.
Ionization of Drugs
Notes. If a drug is partially ionized in plasma and urine and is able to diffuse through the walls
of the renal tubules only in the non-ionized form, the rate of diffusion will depend on the
concentration gradient of the non-ionized form. Any difference in hydrogen ion concentration
between urine and plasma will affect the gradient, and therefore the rate of excretion of the drug.
Ingestion of acids and alkalis leads to small changes in the hydrogen ion concentration of
plasma, which is well buffered, but to large changes in that or urine, while the renal tubules
correct the acidaemia and alkalaemia.
.
Metabolism of Aspirin
Aspirin is largely unaffected by passage through the stomach and into the small intestine. Once
absorbed, however, it is rapidly hydrolysed by plasma and tissue esterases to give acetic acid
and salicylic acid, some of which is excreted as such and the remainder metabolised in the liver.
ASPIRIN EXCRETION EXPERIMENT DATA.
The data will be given in tabular form and also available on the School website, under
undergraduate materials/course materials/ BS2560/aspirin data.xls
You may use the functions in Excel to calculate statistical data
Individual subjects are numbered 1 to sixteen (one to sixteen) in the alkalotic group and a
different 16 subjects are similarly numbered in the normal group.
The volume is in ml of urine; The salicylate concentration is in ug of SODIUM SALICYLATE
per ml of URINE
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