Urea Cycle

Question 1

Disposal of nitrogen - liver

Answer 1

• Ammonia is produced as a result of aa degradation and is potentially toxic – required to be converted into a non-toxic form
• Liver: principle site where most aa’s are degraded leading to the production of: NH3 is TOXIC, Carbon skeleton
• NH3 is detoxified to Urea via the Urea Cycle in liver
• Urea is the major end-product of N metabolism

Question 2

2 possible fates of carbon skeletons

Answer 2

• Used for gluconeogenesis

- Burned in TCA cycle

Question 3

Urea in kidney

Answer 3

• excreted in the urine
• kidneys also excrete ammonia as ammonium ions (important in regulation of the acid base balance)
• amount of ammonium ions produced depends on the amount of protons that need to be excreted
• other non-protein nitrogenous substances are Uric acid (end-product of purine degradation) and creatinine (end-product of creatine degradation)

Question 4

Ammonia in transport of nitrogen

Answer 4

• NH3 is neurotoxic: Creates requirement for a non-toxic transport form of ammonia
• NH3 is transported from peripheral tissues: Glutamine, Alanine

Question 5

Glutamine in nitrogen transport

Answer 5

• Incorporation of ammonia to glutamate
• Glutamate DH: incorporates NH4+ (ammonium) on a- ketoglutarate producing glutamate
• Glutamine synthetase: incorporate 2nd NH4+ ion to produce glutamine

Question 6

Alanine in nitrogen transport

Answer 6

• transport form of ammonia from muscle
• main ammonia transporter produced in muscle due to the high levels of pyruvate generated
• In liver converted back to pyruvate – glucose.
• Ammonia enters urea cycle

Question 7

Urea Cycle

Answer 7

• Converts toxic NH3 to non-toxic urea
• Takes place in liver
• Partly mitochondrial (two reactions) and partly cytosolic (three reactions)
• Requires two nitrogen atoms:
  1st N atom of urea donated by ammonia (GDH & Glutaminase), 2nd N atom is donated by aspartate

Question 8

Formation of Ammonia in Liver

Answer 8

• Transamination: amino group of an amino acid is transferred to α-ketoglutarate to form glutamate
• Oxidative deamination by Glutamate dehydrogenase forms free ammonia
• Glutamate also produced from reverse reaction: removal of free NH3
• Ammonia also formed from glutamine by glutaminase reaction (free ammonia and glutamate are products)

Question 9

How many reactions are there in the Urea Cycle?

Answer 9

• 5 reactions: 2 mitochondrial, 3 cytosolic

Question 10

First reaction of Urea cycle

Answer 10

• catalysed by Carbamoyl phosphate synthetase I
• Catalyses condensation and activation of ammonia (NH3) and bicarbonate (HCO3-)
• Requires hydrolysis of 2ATP
• Rate limiting step of the urea cycle

Question 11

Second reaction of Urea cycle

Answer 11

• Ornithine transcarbamoylase condenses carbamoyl phosphate and ornithine, which forms citrulline
• citrulline is then moved out of the mitochondria into the cytosol of the cell by the transporter SLC25A15

Question 12

Third reaction of Urea cycle

Answer 12

• Argininosuccinate synthetase takes citrulline formed in the mitochondrial stage and condenses it with aspartate to form argininosuccinate
• occurs by the formation of an intermediate, citrulline-AMP

Question 13

Fourth reaction of Urea cycle

Answer 13

• Argininosuccinase breaks Argininosuccinate into arginine and fumarate
• Fumarate is used in TCA cycle, where aspartate is reformed

Question 14

Fifth reaction of Urea cycle

Answer 14

• arginine is then further broken down into urea and ornithine by arginase
• ornithine is then transported into the mitochondria by ornithine translocase
• There, it is used by ornithine transcarbamoylase again to form citrulline, cycle repeats

Question 15

Regulation of Urea cycle

Answer 15

• Need to regulate cycle in response to; changes in protein degradation, high dietary intake
• Increase in arginine indicates substrate availability i.e. NH3
• N-acetyl glutamate (NAG) is an allosteric activator of CPS I (rate limiting enzyme)
• After a protein rich meal & in presence of arginine NAG formation is increased

Question 16

Inherited disorders of Urea cycle

Answer 16

• deficiency exists in all of the 5 enzymes of the Urea Cycle
• results in the accumulation of SUBSTRATE of that enzyme
• All urea cycle disorders are characterized by increased blood ammonia levels (hyperammonaemia) and blood glutamine levels
  there is also decreased urea formation
• Untreated: neurological manifestations, seizures and mental retardation

Question 17

Benzoic acid (management of hyperammonaemia)

Answer 17

• combines with glycine
• Requires activation to CoA derivative
• Product is Hippuric acid
• Excretion of 1 atom of nitrogen

Question 18

Phenylbutyrate (prodrug) (management of hyperammonaemia)

Answer 18

• combines with glutamine
• Prodrug converted to active molecule by oxidation
• Conversion to CoA derivative phenylacetyl-CoA
• Product is Phenylacetylglutamine
• Excretion of 2 atom of nitrogen
• Pharmaceutical invention can be used for all deficiencies

Question 19

Dialysis (management of hyperammonaemia)

Answer 19

• acute emergency situation
• Low protein/high carbohydrate diet to minimize N intake
• Prescribe α-keto acids (expensive) - prevention of stresses that induce a catabolic state
• Long term: Liver transplantation

Question 20

L-arginine supplementation (management of hyperammonaemia due to CPSI deficiency)

Answer 20

• Arginine stimulates the formation of N-acetylglutamate
• High levels of NAG might stimulate deficient CPSI
• L-arginine is also used to treat other deficiencies – those before ariginase
• Promotes excretion of citrulline and argininosuccinate

Question 21

Acquired hyperammonaemia

Answer 21

• Liver disease due to: viral or drug induced hepatitis, alcoholic cirrhosis
• Resultant cirrhosis of the liver leads to porto-systemic shunting of blood
• Portal blood enters the systemic circulation without going to the liver
• Ammonia produced in the intestine directly enters circulation
• Results in neurotoxicity

Question 22

Intestinal formation of ammonia

Answer 22

• Bacterial ureases form ammonia in the colon (also degradation of proteins)
• Ammonia formed enters the portal circulation - delivered back to the liver
• Cirrhotic liver portal blood enters systemic circulation bypassing the liver

Question 23

Ammonia neurotoxicity

Answer 23

• mechanism is unknown - two hypotheses can be considered:
• Energy and Osmotic Effect
• Neurotransmitter Effect

Question 24

Energy and osmotic effect

Answer 24

• α-ketoglutarate converted to glutamate via glutamate DH (reductive amination direction)
• Results in consumption of TCA cycle intermediates i.e. α-KG
• Results in decreased TCA cycle activity = ATP synthesis is reduced
• Subsequent reduced Na+/K+ ATPase activity, a solute pump that pumps sodium out of cells while pumping potassium into cells, both against their concentration gradients
• This results in: Changes in osmotic pressure, Eventual neuronal cell death

Question 25

Neurotransmitters effect

Answer 25

• Glutamate is converted to glutamine via action of Glutamine synthetase
• Results in high circulating glutamine levels: high glutamine levels cause cerebral oedema
• Reduced glutamate, and hence GABA levels in the brain
• Both Glu and GABA are reduced when NH3 is abundant: GABA is major inhibitory neurotransmitter (formed from glutamate), Glu itself is a major excitatory neurotransmitter

Question 26

Treatment of Acquired hyperammonaemia

Answer 26

• Low-protein/high carbohydrate diet - intuitive: less N in, less needed to dispose
• Lactulose: disaccharide (galactose –fructose): resistant to digestion in the small intestine, normal bacterial flora in colon produce lactic acid, acid conditions favour NH4+ formation (can’t be reabsorbed so more N excreted in faeces), promotes more NH3 entering lumen from tissues
• Neomycin: (or other antibiotic treatment): reduction in bacterial urease in the gut