Biochemistry- the urea cycle and its control

Question 1

What is urea

Answer 1

The urea (ornithine) cycle produces urea from ammonia. It takes place primarily in the liver.
 Urea is the main nitrogenous waste of mammals. Most of the nitrogenous waste comes from the breakdown of amino acids
through deamination, producing ammonia (NH3).

Question 2

How does the urea cycle work in different metabolic states eg. fed fasting starvation etc

Answer 2

Fed state: after a high protein meal, excess amino acids are catabolised (as there is no storage of proteins), for energy,
gluconeogenesis and glycogenesis. The activity of the urea cycle therefore increases as more protein is catabolised.
 Fasting: during fasting, as liver glycogen is depleted and insulin levels decrease, proteolysis outstrips protein synthesis.
Excess amino acids are catabolised.
 Starvation: during early starvation, amino acids, particularly from muscle are important gluconeogenic substrates. During
late starvation, protein sparing occurs, switching to ketone bodies.
 Hypercatabolic state: during stress, trauma and certain infections, there is an increase in protein turnover, resulting in
increased proteolysis.
 Postnatal development: during late gestation, and after birth, urea cycle enzyme synthesis begins, with levels increasing
over time.

Question 3

Describe ammonia toxicity

Answer 3

Amino acid catabolism can occur in a number of different metabolic states. Regardless of the metabolic state, nitrogen must
be removed from the carbon skeleton. This results in the formation of ammonia.
 If [NH3
] increases above 50 μM, the individual can suffer from nausea, lethargy, coma and then death. The cause could be
the depletion of α-ketogluterate or the accumulation of Glu/Gln neurotransmitters. Either way, the nervous system is
affected more quickly than other tissues.

Question 4

Explain how waste nitrogen is removed

Answer 4

1. Via deamination
    Removal of nitrogen to produce an ammonium ion
   (NH4
   +), through oxidative deamination. There can be
   toxic at high levels. The liver can take up the free
   ammonium ions and convert them into the less toxic
   urea.
    Example [Right]: glutamate dehydrogenase converts glutamate into 𝛂-ketoglutarate. This enzyme is an exception, in that it
   can use either NAD or NADP+
   .
2. Via transamination (most common)
    The amine group is transferred from an amino
   acid to a keto acid (which can later be
   deaminated, to form ammonium ions). The
   reaction is catalysed by aminotransferase
   enzymes. They typically have glutamate as one substrate/product, and are specific for another particular amino acid.
    The aminotransferase is named after the amino acid (e.g. aspartate aminotransferase).
    Example: [Right]: aminotransferase catalyses the formation of glutamate from 𝛂-ketogluterate. Glutamate is then used by
   the liver to produce urea.

Question 5

Where does urea come from

Answer 5

Urea contains 2 amino groups with nitrogen that comes from:
o Aspartate: formed by aspartate aminotransferase.
o Ammonium ion: formed by glutamate dehydrogenase
and glutaminase which removes ammonium ions from
glutamine.

Question 6

Where is the location of urea production

Answer 6

The liver is the only tissue that expresses all the enzymes of the urea cycle, but not in every hepatocyte.
 Periportal hepatocytes (as opposed to perivenous) are the ones that carry out urea production:
o High oxygenated blood supply via the hepatic artery.
o Blood from the digestive tract contains products of digestion through the portal vein.

Question 7

What are the key points to note in the urea cycle

Answer 7

The first couple of steps occur in the mitochondria and the rest in the cytosol.
 Key points of the cycle:
o Nitrogen enters in the form of ammonium ions. The first reaction uses 2 ATP.
o Ornithine travels around in a cycle, eventually being regenerated.
o Aspartate enters the cycle. 2 ATP is also used (initially only 1 ATP is used, but to convert AMP back another ATP is
also used).
o Fumarate is produced later one.
o Urea is finally produced, containing the two nitrogen atoms, one from ammonium and one from aspartate.

Question 8

What are the inputs and outputs of the urea cycle

Answer 8

Nitrogen enters the cycle in the form of both aspartate and ammonia.
o The aspartate is formed via transamination (from oxaloacetate) with a-KG/Glu shuttling nitrogen in from outside
the mitochondria. Most transaminases have a-KG/Glu as one of their substrates.
o The ammonia predominantly comes from glutamate or glutamine, via glutaminase or glutamate dehydrogenase.
There are also other deaminases.
 Alanine is the predominant form in which muscle transfers waste nitrogen, and therefore is an important gluconeogenic
substrate.

2. Outputs
    The main output is urea, which is disposed of in urine.
    The other output is fumarate. This should be recognised as an intermediate in the citric acid cycle, but since this is in the
   cytosol, it can be used for gluconeogenesis.
    The carbon for aspartate comes from glucogenic amino acids (aspartate) (i.e. the ones that can be converted to pyruvate or
   citric acid cycle intermediates).
    In the fed state, they will come from digestion products. In fasting and starvation, from liver protein and extrahepatic
   sources (i.e. muscle).
    The energy from amino acid oxidation is more than enough to fuel ureagenesis and gluconeogenesis.

Question 9

How are the urea cycle and gluconeogenesis linked?

Answer 9

The urea cycle and gluconeogenesis, both occurring in the liver, are intricately linked.
 During the fed state, the fasting state and early starvation, amino acids are the main source of carbon for gluconeogenesis.

The mitochondrial/cytosolic split is involved in supporting gluconeogenesis.
 The fumarate produced in the cytosol gets converted to oxaloacetate. The cytosolic malate dehydrogenase produces NADH.
The NADH is needed for gluconeogenesis, in the reverse of the glyceraldehyde-3-P dehydrogenase reaction.

Question 10

What are the 2 types of regulation of the urea cycle

Answer 10

Short term: the rate of amino catabolism can fluctuate over short periods of time, most notably in the transition from the
fasted to the fed state.
 Long term: amino acid catabolism also shows fluctuations over long periods of time, such as in the transition from intra to
extra uterine life, or if there is a change from a high to a low protein diet.
 The waste ammonia is toxic, so the urea cycle enzymes must be able to cope with immediate fluctuations, and respond to
long term changes, in order to avoid the dangerous consequences of ammonia toxicity.
 Different mechanisms are used to respond to these types of alterations in amino acid catabolism rates.

Question 11

Describe the enzymes involved in the short term regulation of the urea cycle

Answer 11

Carbonyl-phosphate synthetase requires N-acetyl glutamate in order to be
activated.
 Carbonyl-phosphate synthetase produces carbamoyl phosphate, and
without it, the urea cycle cannot proceed.
 N-acetylglutamate synthetase activity is affected by [Glu] and is also
allosterically activated by arginine.
 Temporary increases in amino acid catabolism results in an increase in [Glu]
and [arginine]. The resultant increase in [NAG} increases the activity of CPS,
allowing the urea cycle flux to increase, before the [NH3
] levels rise.
 NAG entry into the cytosol (where it is broken down) may also be regulated
hormonally. This type of regulation means that ammonia levels are kept constant, rather than fluctuating along with rates
of amino acid catabolism.
 The Kms of other urea cycle enzymes are around the typically physiological concentrations of their substrates, which means
that their capacity to cope with very large changes in flux is limited.

Question 12

Describe in more detail the long term regulation of the urea cycle

Answer 12

In the long term, regulation of the urea cycle enzymes is transcriptional. Transcription is largely controlled by hormones,
although the DNA elements involved have not been completely identified.
 The main hormones involved in stimulating transcription of the urea cycle enzymes are glucagon, adrenaline and the
glucocorticoids (involved in stress). These hormones are either involved in managing fasting/starvation or stress situations.
 Glucocorticoids also increase towards the end of pregnancy, and glucagon is not expressed until after birth. Insulin may be
involved in inhibiting transcription.

Question 13

How do urea cycle disorders occur

Answer 13

Because the human foetus can survive the whole pregnancy without producing urea, genetic defects in the urea cycle are
one of the more common inborn errors of metabolism observed in newborns.
 Affected infants typically die shortly after birth if not immediately diagnosed and treated.
 Some defects do not show up until much later in life, if the enzyme defect is not severe, such as a change in Km, or if waste
nitrogen accumulates in another excretable product.
 Symptoms include hyperammonaemia (high [NH3
] in the blood), vomiting, lethargy, mental retardation, eventually coma
and death.
 Depending on the enzyme defect, other nitrogenous compounds may accumulate in the blood or urine.

Question 14

How does management of the urea cycle occur

Answer 14

Management of urea cycle defects generally involves the following strategies:
o Decrease protein intake and avoiding catabolic states (e.g. fasting). This avoids the need for a urea cycle in the first
place.
o Provide alternative routes for nitrogen
excretion.
o Supplement nitrogen compounds as necessary
due to altered metabolism.

Special diets need to be planned for unavoidable catabolic states (e.g. illness).
 Avoiding hyperammonaemia prevents the most serious complications, but there may be long term effects. Some enzymes
in the urea cycle partake in other cycles in other parts of the body. Defects to these enzymes could therefore have long
term complications.

Question 15

Describe some deficiencies that can lead to urea cycle disorders

Answer 15

2.1. Carbonyl-P-synthetase deficiency
 The most severe of the urea cycle disorders, because it is
the first step in removing NH4
+.
 If carbonyl-P-synthetase is affected, the ammonium levels
in the blood increase.

2.2. Ornithine transcarbomoylase
 The most common hereditary urea cycle disorder, as it is
X-linked. Males are severely affected, and in females, it
depends as there is X-inactivation.
 Excess carbamoyl phosphate feeds into pyrimidine
synthesis, leading to a build-up in orotate, which is useful for diagnosis.
 Nitrogen also accumulates in glycine and glutamine.

2.3. Arginosuccinate synthetase
 Also called citrullinaemia. Not as severe because citrulline can be excreted in the urine (some nitrogen can still be
removed).
 Treatment: arginine supplementation helps to reproduce ornithine, so ammonium can still be excreted to some extent in
the form of citrulline.

2.4. Argininosuccinase
 Argininosuccinate is excretable through the urine.
 Treatment: arginine supplementation.

2.5. Arginase
 Also called hyperargininaemia. Rarely causes hyperammonaemia, as arginine can be excreted, and there is another isoform
in the peripheral tissues.
 Manifests at older age.
 Supplementing arginine would not help anyway, and would not help in removing nitrogen waste.

3. NAGS defects
    NAGS deficiency mimics CPS deficiency: patients present within the first few days of life with severe hyperammonaemia.
    Treatment: N-carbamoyl glutamate. It doesn’t stimulate CPS as efficiently as NAG, but NAG is hydrolysed in vivo, and so
   doesn’t reach the required site of action.
    Less severe defects have been reported:
   o Two siblings with a splice site mutation (truncated, non-functional), and an R to Q mutation (decreased Vmax,
   decreased affinity for substrates, decreased response to arginine). They presented at 4 weeks and 9 years of age.
   o A male with a V to E substitution (very low activity) and a T to I substitution on the other (very low Vmax, very low
   affinity for substrates). He presented at 33 years, after a car accident. Was known to avoid eating meat.