Introduction
In a healthy individual, intake of nitrogen in food will be balanced by excretion of an equal amount of nitrogen in the urine (as urea), skin, and feces.
Positive Nitrogen Balance: the amount of nitrogen ingested is more than that excreted. Occurs in:
- Growth (in children)
- Pregnancy (due to the growth of the fetus)
- Feeding after starvation
- Convalescence after illness or surgery
Negative Nitrogen Balance: the amount of nitrogen excreted is more than that ingested. Occurs in:
- Old age
- Starvation
- Deficiency of essential amino acids
- Major trauma
- Weight loss
- Diseases involving tissue wasting
- Injuries
Formation Of Ammonia (NH3)
During metabolism, the amino group of amino acids is removed as ammonia. However, small quantities of ammonia may also be formed from the catabolism of purine and pyrimidine bases. Ammonia is highly toxic, especially to the nervous system. Detoxification is by conversion to urea and excretion through urine.
Process Of Urea Formation
Transamination
- Involves the transfer of an amino group from an amino acid to an α-keto acid to form a new amino acid and a new α-keto acid.
- In almost all cases, the amino group is accepted by α-ketoglutaric acid so that glutamic acid is formed.
- Enzymes – aminotransferases (transaminases) catalyze these reactions.
- Pyridoxal phosphate (PLP) is an essential cofactor of all aminotransferases.
Transdeamination
- The amino group of most of the amino acids is released by a coupled reaction, which is transamination followed by oxidative deamination.
- Transamination takes place in the cytoplasm of all cells of the body.
- The amino group is transported to the liver as glutamic acid, which is finally oxidatively deaminated in the mitochondria of hepatocytes.
- So, all amino acids are first transaminated to glutamate and then deaminated in the liver.
Oxidative Deamination
- Only liver mitochondria contain glutamate dehydrogenase (GDH) which deaminates glutamate to α-ketoglutarate & ammonia.
- GDH is an allosteric enzyme.
- It needs NAD+(or NADP+) as a coenzyme.
- It is activated by ADP & ATP and is inhibited by GDP, GTP & NADH.
- The hydrolysis of glutamine also yields NH3 but this occurs mainly in the kidneys where the NH4+ excretion is required for acid-base regulation.
Trapping Of Ammonia
- Being highly toxic, even a very minute quantity of ammonia may produce toxicity in the CNS.
- As such, it must be eliminated or detoxified as and when it is formed.
- But ammonia is always produced by almost all cells, including neurons.
- The intracellular ammonia is immediately trapped by glutamic acid to form glutamine, especially in brain cells.
- The glutamine is then transported to the liver where the reaction is reversed by the enzyme glutaminase.
- The ammonia, thus generated, is immediately detoxified into urea.
Transportation Of Ammonia
- Glutamic acid is the link between amino groups of amino acids and ammonia.
- Glutamine is the transport form of ammonia from the brain and intestine to the liver while alanine is the transport form from the muscles to the liver.
Final Disposal
- In the liver, it is detoxified into urea by liver cells and excreted through the kidney.
- Urea is the end-product of protein metabolism.
Urea Cycle Introduction
- This is the process of formation of urea from ammonia (NH3).
- The urea formed contains two nitrogen atoms, one from ammonia, and the other from aspartate.
Site
- The urea cycle occurs exclusively in the liver.
Equation
The overall stoichiometry of the urea cycle is; CO2 + NH4+ + 3ATP + Aspartate + 2H2O → Urea + 2ADP + 2Pi + AMP + PPi + Fumarate
Pathway
STEP 1: Formation of Carbomoyl Phosphate
- One mole of ammonia condenses with CO2 in the presence of two moles of ATP to form carbomoyl phosphate.
- The reaction is catalyzed by mitochondrial carbomoyl phosphate synthetase-I (CPS-I) which is allosterically regulated.
- This is the rate-limiting step in urea formation.
STEP 2: Formation of Citrulline
- The carbomoyl group is transferred to the NH2 group of ornithine by ornithine transcarbomoylase (OTC) to form citrulline.
- The citrulline diffuses out of the mitochondria into the cytoplasm where further reactions take place.
Step 3: Formation Of Argininosuccinate
- One molecule of aspartic acid adds to citrulline forming a carbon-to-nitrogen bond, which provides the second nitrogen atom of urea.
- Argininosuccinate synthetase catalyzes the reaction.
- This needs hydrolysis of ATP to the AMP level, so two high-energy phosphate bonds are utilized.
Step 4: Formation of Arginine
- Argininosuccinate is cleaved by argininosuccinate lyase (argininosuccinase) to arginine and fumarate.
- The enzyme is inhibited by fumarate, but this is avoided by the cytoplasmic localization of the enzyme.
- The fumarate is funneled into the TCA cycle to be converted to malate and then to oxaloacetate to be transaminated to aspartate. Thus, fumarate links the urea cycle to the citric acid cycle.
Step 5: Formation Of Urea
- The final reaction of the cycle is the hydrolysis of arginine to urea and ornithine by arginase.
- Urea is highly soluble and nontoxic. It enters the blood and is excreted in the urine.
- The ornithine returns to the mitochondria to react with another molecule of carbomoyl phosphate continuing the cycle.
Energetics
- The overall reaction may be summarized as; NH3 + CO2 + Aspartate → Urea + Fumarate.
- 2 ATPs are used in the first reaction.
- Another ATP is converted to AMP and PPi which is equivalent to 2 ATPs.
- The urea cycle consumes 4 high-energy phosphate bonds.
- However, fumarate formed in the 4th step may be converted to malate.
- Malate when oxidized to oxaloacetate produces 1 NADH equivalent to 2.5 ATP.
- So, net energy expenditure is only 1.5 high-energy phosphates.
Regulation
- The enzyme levels change with protein diet – enzyme levels are increased during starvation.
- The rate-limiting step, catalyzed by CPS-I is allosterically regulated by N-acetyl glutamate(+).
- Compartmentalization of the urea cycle enzymes requires that the urea cycle intermediates, ornithine and citrulline, be transported across the mitochondrial membrane.
- Mitochondria contain carbomoyl phosphate synthetase and ornithine transcarbamoylase.
- Cytosol contains argininosuccinate synthetase, argininosuccinate lyase, and arginase.
Clinical Correlates
Genetic defects have been documented for each of the urea cycle enzymes. They are:
- Type I hyperammonaemia – due to a defect in carbomoyl phosphate synthethase-I.
- Type II hyperammonaemia – due to a defect in ornithine transcarbomoylase.
- Citrullinuria – due to a defect in argininosuccinate synthetase.
- Argininosuccinic acidaemia – due to a defect in argininosuccinate lyase.
- Hyperargininaemia – due to a defect in arginase.
