An investigation to determine how excretion of urea is determined by the consumption of protein

Urea is formed from excess amino acids present in the body, by the process of deamination in the liver. The amino group is first removed from an amino acid by reacting it with oxygen, giving a keto acid (which can be converted in glucose or fat) and ammonia; the ammonia reacts with carbon dioxide to form urea and water.

2 NH2CH(R)COOH + 2 O2 2 CO(R)COOH + 2 NH3

2 NH3 + CO2 C(NH2) 2O + H2O Ref 1

If the consumption of protein is high, when it is broken down into its simplest form of amino acids by enzymes, the liver will convert the excess amino acids into urea, which is filtered out of the blood in the kidney. Urea is then removed from the body in urine.

When urea is in the presence of water and the enzyme urease, a reaction occurs producing ammonium carbonate, a solution with an alkaline pH. This solution can be tested with universal indicator to roughly gauge the concentration of urea present, assuming that it is all catalysed by the urease.

If the pH is very high, it means that there is a high concentration of ammonium carbonate, which has been formed by urea. Accordingly, a lower pH signifies that only a small amount of urea was present to react.

To confirm this, a titration can be done with hydrochloric acid to neutralise the solution, where phenolphthalein acts as a pH indicator. In the pH range 8.2-12.0, the indicator is a pale pink colour, but as it reaches neutral, it turns colourless. Ref 2

Based on my own previous work, I know that by using too much phenolphthalein will cause the solution to turn purple, so for this practical, I shall only use a few drops to prevent anomalous results.

The volume of HCl used to cause the colour change can be plotted against the known concentration of urea in ...

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Based on my own previous work, I know that by using too much phenolphthalein will cause the solution to turn purple, so for this practical, I shall only use a few drops to prevent anomalous results.

The volume of HCl used to cause the colour change can be plotted against the known concentration of urea in the solution for different concentrations, which will allow me to plot a calibration curve against which I can plot future urine samples to determine the concentration of urea.

I predict that as the concentration of urea increases, the volume of HCl needed to neutralise it will proportionately increase; the urea is converted into alkaline ammonium carbonate, so a high concentration of urea will give a high pH level.

Whilst performing the experiment, there are factors that must be kept constant to ensure a fair test; the temperature will be similar to that of urine when it is passed out, and so will be kept at 35°C. The volume of urease added will be in excess to try and ensure that all of the urea is catalysed, and so will always be the same volume.

In effort to gain accurate results, I will use 6 different concentrations of urea solution and repeat each test 4 times so that I may take an average result to plot on the graph to give the calibration curve. Because the urea concentration in urine is often in the range of 1.0 to 2.5g per 100cm3, I will use my concentrations at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0g per 100cm3, to allow for the standard reading and slightly above and below for variation.

I will use a test tube of water as a control, to note the end point of the colour change from pink to colourless, so that all the reactions can end at the same point.

Apparatus

Pipettes Burette Test Tubes

Test Tube Rack Water Bath Bungs

Droppers

Urea solutions Urease Distilled Water

Phenolphthalein Hydrochloric Acid

Method

Place the urea, distilled water and urease in the water bath and allow to heat to 35°C.
Make 6 20cm3 solutions using the table below

To each solution, add 10cm3 of urease and seal the tube using a bung.
Shake well to ensure that the contents are mixed, remove the bung and leave to stand in the water bath for 10 minutes.
Take 4cm3 from test tube A and add 2 drops of phenolphthalein.
Using a burette, add HCl in drops, stirring well after each drop.
Stop when the pink colour fades and so the colour matches the control test tube of water; clear and colourless.
Record the volume of HCl used.
Repeat steps 5-8 for test tubes B to F.
Repeat steps 5-9 4 times.

Take the 5 readings for each concentration and record them in the table below, using the average reading. This can then be used to plot a graph with a calibration curve, as show below.

Now, using an unknown sample of urine, it can be titrated with HCl and the volume plot against the curve on the graph. This value corresponds to a concentration of urea in the urine, and an example of this is included in the graph.

A human subject known to eat a diet high protein is likely to excrete the most urea, given that he is not growing, or has a irregularly high demand for protein, such as a body builder. The subject can have diets progressively increasing in protein levels, each lasting a week long. In week one, the subject has a diet low in protein, and the urea concentration of the urine is measured at the end of the week. Week two, the protein in the diet is increased, and the urea concentration is again measured at the end of the week. This continues for a period of 1 month, and at the end, the urea concentrations can be compared. I predict that the urea concentration in the urine from week four will be the highest, and week one will be the lowest.

This will determine how a change in the consumption of protein in the diet influences the excretion of urea.

Safety precautions should be taken when handling any chemicals. HCl is corrosive, so care should be taken not to make contact with skin, and goggles should be worn. The same principles apply with the urine.

References

Reference 1: Biology 2, page 89

Reference 2: http://en.wikipedia.org/wiki/Phenolphthalein