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# The virtual laboratory: Urea synthesis

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### copyright © 1982 - 2006 David A Bender

### Virtual Laboratory main menu

### Click here to run the program

The program will run in a separate window, and at any time you can minimise the program window to check the theory from this page.

In this program you will investigate the synthesis of urea by isolated liver cells - you will be repeating the experiments that Krebs and Henseleit carried out when they deduced the pathway in the 1930s.

You may enjoy reading an essay by Krebs called "The discovery of the ornithine cycle of urea synthesis",
Krebs HA (1973) Biochemical Education 1: 19-23.

### Formation and metabolism of ammonia
Ammonia toxicity
Treatment of ammonia intoxication
Formation of urea from ammonium
The sources of ammonium ions for urea synthesis
Arginine: the immediate precursor of urea
Addition of ornithine or citrulline
A clinical problem - Argininosuccinic aciduria

# Formation and metabolism of ammonia

Ammonia, as the ammonium ion, is the main immediate product of amino acid metabolism, arising as a result of oxidative deamination of amino acids, catalysed by glutamate dehydrogenase and glycine oxidase, linked to the transamination of a wide variety of other amino acids.

Small aquatic organisms can excrete ammonium directly into their environment; fish excrete some 60% of their nitrogenous waste as ammonium ions and the remainder as trimethylamine oxide.

Earthworms, which have a large surface to volume ratio, excrete a considerable part of their nitrogenous waste as ammonia gas.

Ammonium is highly toxic, and terrestrial organisms need to form a less toxic end-product of nitrogen metabolism.

If water is a problem, as for birds and insects, then the main end-product is uric acid, which is relatively insoluble in water, and can be excreted as a slurry of crystals (by birds) or even as dry crystals (by many insects). This is obviously important in the egg, where there is little water available for storing a soluble nitrogenous end-product, but for adult birds the weight of water required to excrete a soluble metabolite also poses problems. Animals that excrete uric acid are termed uricotelic.

Bats and arachnids excrete guanine as their main nitrogenous end-product; such animals are known as purinotelic.

Mammals, for whom water balance and weight are not generally a problem, excrete most of their nitrogenous waste as urea, which is water-soluble; such animals are termed ureotelic.

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# Ammonia toxicity

Ammonia is highly toxic. Normally blood ammonium concentration is < 50 µmol /L, and an increase to only 100 µmol /L can lead to disturbance of consciousness. A blood ammonium concentration of 200 µmol /L is associated with coma and convulsions.

200 µmol /L is far too low a concentration of ammonium to affect plasma pH or the normal transport of sodium and potassium ions across nerve cell membranes.

The explanation of the toxicity of such (relatively) low concentrations of ammonium lies with the enzyme glutamate dehydrogenase. This enzyme catalyses the oxidative deamination of glutamate to ammonium and ketoglutarate; the reaction is readily reversible, and the direction of reaction (towards deamination of glutamate or glutamate formation) depends on the relative concentrations of the various substrates. As the concentration of ammonium rises, so the reaction proceeds in the direction of formation of glutamate from ketoglutarate.

The effect of forming glutamate from ketoglutarate is to deplete the mitochondrial pool of ketoglutarate, which is a key intermediate in the citric acid cycle. As a result, the rate of citric acid cycle activity falls, so reducing very considerably the rate of formation of ATP.

It is this lack of ATP that affects ion transport across nerve cell membranes, so resulting in disturbance, then loss, of consciousness.

## Formation of glutamine

Ammonium is produced in most cells of the body, as a result of deamination of amino acids and amines. It is exported into the bloodstream as glutamine, formed by the actions of glutamate dehydrogenase, to form glutamate from ketoglutarate and ammonium, then glutamine synthetase, forming glutamine from glutamate and ammonium.

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## Treatment of ammonia intoxication

Ammonia intoxication occurs when blood ammonium rises because the capacity to detoxify it by formation of glutamate and glutamine has been exceeded. Sometimes, in infants whose blood ammonium has risen dangerously high (commonly as a result of a genetic disease affecting amino acid metabolism), the only emergency treatment is a more-or-less complete exchange blood transfusion.

In less critically urgent cases of ammonia intoxication (sometimes in infants, sometimes in adults with liver failure) the treatment of choice is administration of the disaccharide lactulose, either orally or by rectal infusion. Lactulose is a synthetic disaccharide that is not hydrolysed by intestinal enzymes, but (if given orally) passes into the large intestine, where it is a substrate for bacterial fermentation to lactate.

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Ammonia will cross the intestinal wall, since it is lipid soluble, but ammonium ions cannot. At physiological pH the equilibrium between ammonia and ammonium in the bloodstream is well towards ammonium ions, but there is always a small amount of ammonia. This can diffuse across the intestinal wall into the lumen of the gut. Here the result of fermentation of lactulose to lactate is a significant fall in pH, resulting in a shift of the equilibrium further towards ammonium ions, which cannot cross the intestinal wall. As a result, ammonium ions are trapped in the intestinal lumen, and there is a nett flux of ammonia form the bloodstream into the intestinal lumen.

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# Formation of urea from ammonium

If you incubate isolated hepatocytes with increasing concentrations of ammonium, you will see that there is a steady increase in the formation of urea at low concentrations of ammonium, levelling off as the pathway for urea formation becomes saturated. If you perform such studies with isotopically labelled ammonium (15N, a stable isotope) you will find that one of the two N atoms in urea comes from ammonium, but the other does not - it arises from the amino group of the amino acid aspartate.

This is essentially the control for the experiments you will perform in the simulation exercise, when you will look at the effects of adding various compounds, with and without ammonium ions also present, on the formation of urea.

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## The sources of ammonium ions for urea synthesis

It is obvious from the discussion of ammonia toxicity that there is little or no free ammonium in the bloodstream, nor can the intracellular concentration of ammonium be allowed to rise significantly. Therefore there has to be a way of forming ammonium as it is required.

There are two ways in which ammonium is formed in the liver: from glutamine by the action of glutaminase, and from adenosine, by the action of adenosine deaminase. Each provides about half the ammonium that is incorporated into urea directly.

## The utilisation of ammonium ions

Ammonium is formed, more or less as required, by the actions of glutaminase and adenosine deaminase, and is then rapidly used for the formation of carbamyl phosphate, so that there is little or no accumulation of free ammonium in the cell.

There are two isoenzymes of carbamyl phosphate synthetase in liver cells:

carbamyl phosphate synthetase 1 is a mitochondrial enzyme; it uses ammonium as the source of nitrogen, and is induced by feeding a high-protein diet.

carbamyl phosphate synthetase 2 is a cytosolic enzyme; it uses glutamine directly as the source of nitrogen, and is inhibited by pyrimidine nucleotides. (Carbamyl phosphate is the starting substrate for pyrimidine synthesis).

Which isoenzyme is likely to be important for the synthesis of urea?

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# Arginine: the immediate precursor of urea

Having established that the source of half the nitrogen in urea comes from ammonium, by way of carbamyl phosphate, the next step in trying to elucidate the pathway of urea synthesis is to try to discover what might be the final intermediate before the formation of urea. In other words, what compound is likely to yield urea.

One obvious candidate would be the amino acid arginine; as shown below, hydrolysis of the side chain of arginine yields urea.

If you incubate isolated hepatocytes with increasing concentrations of arginine, you will see that there is a steady increase in the formation of urea at low concentrations of arginine, levelling off as the enzyme becomes saturated.

This suggests very strongly that the immediate precursor of urea is indeed arginine, and from the chemistry of the arginase reaction you would expect to form 1 mol of urea for each mol of arginine consumed.

In the simulation you will investigate the effect of adding three different concentrations of arginine to isolated liver cells incubated with a range of concentrations of ammonium. You will be given results for not only the amount of urea formed, but also the amount of ammonium remaining. Think about the results very carefully.

> Is the amount of urea formed in the absence of ammonium what you would expect from the amount of arginine added?

> Is the amount of urea formed in the presence of a high concentration of ammonium what you would expect from the amount of arginine added?

> How does the addition of arginine affect the amount of ammonium remaining at the end of the incubations?

Can you explain your observations?

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# Addition of ornithine or citrulline

The results of experiments in which arginine is added to the incubations are unexpected. Therefore, it would seem to be sensible to look at the effect of adding the product of arginase action, ornithine, to incubations with and without a range of concentrations of ammonium.

Ornithine can react with carbamyl phosphate to form citrulline.

In the simulation you will investigate the effect of adding three different concentrations of ornithine to isolated liver cells incubated with a range of concentrations of ammonium. You will be given results for not only the amount of urea formed, but also the amount of ammonium remaining. Think about the results very carefully.

Would you expect any urea to be formed from ornithine in the absence of added ammonium?
Is any formed?
is the amount of urea formed in the presence of a high concentration of ammonium what you would expect from the amount of ornithine added?
How does the addition of ornithine affect the amount of ammonium remaining at the end of the incubations?

Can you explain your observations?

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The results of experiments in which ornithine is added may also differ from your expectations, so it might be sensible to try the effect of adding the product of the reaction of ornithine with carbamyl phosphate, citrulline.

Citrulline can react with aspartate to form the compound argininosuccinate:

In the simulation you will investigate the effect of adding three different concentrations of citrulline to isolated liver cells incubated with a range of concentrations of ammonium.

You will be given results for not only the amount of urea formed, but also the amount of ammonium remaining. Think about the results very carefully.

Would you expect any urea to be formed from citrulline in the absence of added ammonium?
Is any formed?
is the amount of urea formed in the presence of a high concentration of ammonium what you would expect from the amount of citrulline added?
How does the addition of citrulline affect the amount of ammonium remaining at the end of the incubations?

Can you explain your observations?

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Argininosuccinate can undergo cleavage to remove what had been the carbon skeleton of aspartate as fumarate, leaving arginine.

You should now be able to put together the complete pathway of synthesis of urea from ammonium.

You will notice that while the simulation permits you to work with varying concentrations of ammonium, and various concentrations of arginine, ornithine or citrulline, there is no stage at which you consider the addition of aspartate, yet it is obvious from the diagram of the argininosuccinate synthetase reaction above that aspartate is a substrate, and provides one of the two nitrogen atoms of urea.

What is the likely metabolic fate of the fumarate formed by the reaction of argininosuccinase?
How can the amino groups of various amino acids end up being incorporated into urea?

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# A clinical problem - Argininosuccinic aciduria

A rare genetic defect of the enzyme argininosuccinase leads to the condition of argininosuccinic aciduria; the affected infants excrete large amounts of argininosuccinic acid in their urine.

More importantly, affected infants suffer from hyperammonaemia, and after even a moderate intake of protein can become comatose, and may suffer convulsions, leading to brain damage, or die.

Brusilow & Batshaw (1979) reported the successful treatment of a child with argininosuccinic aciduria using supplements of arginine [Brusilow, S W &. Batshaw, ML (1979). Arginine therapy of argininosuccinase deficiency. Lancet 313 (issue 8108): 124-7].

Can you explain why a child with argininosuccinic aciduria, who lacks argininosuccinase, should become hyperammonaemic after a moderate intake of protein?

Can you explain how supplements of arginine prevent the development of hyperammonaemia and permit the child to consume a more or less normal diet?

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