Synthesis of urea and disorders resulting from defects in urea synthesis for medical, biochemistry and biology undergraduates

1. R. C. Gupta
Professor and Head
Department of Biochemistry
National Institute of Medical Sciences
Jaipur, India
Urea cycle
and
its disorders

2. In adult human beings, 1-2% of the body
proteins are broken down every day
Three-fourths of the amino acids released
from breakdown of proteins are reutilized
The remaining amino acids are catabolized
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3. Catabolism of amino acids comprises
catabolism of their:
Amino groups Carbon skeletons
Carbon skeletons of different amino acids
have different fates
But the fate of their amino groups is the
same

4. The amino groups of amino acids are
removed as ammonia
Since ammonia is very toxic, it has to be
converted into a non-toxic metabolite
In ureotelic organisms, e.g. mammals,
ammonia is converted into urea
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5. Conversion of amino nitrogen into urea is
a complex process
This process involves several steps

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• Transfer of amino groups of amino
acids to a-ketoglutarate forming
glutamate
• Oxidative deamination of glutamate to
release ammonia
• Transport of ammonia to liver
• Synthesis of urea in liver
In human beings, disposal of
amino groups involves:

8. a-Ketoglutarate collects the amino groups
of most of the amino acids
The amino groups are transferred to a-
ketoglutarate mostly by transamination
Transfer of amino groups to
a-ketoglutarate

9. Transamination reactions:
Are catalysed by transaminases
(aminotransferases)
Require pyridoxal phosphate
(PLP) as a coenzyme
Are reversible
Do not require energy
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10. There are several transaminases, each
specific for the donor of a-amino group
The acceptor of amino groups is generally
a-ketoglutarate
The enzyme is named after the amino acid
donating the amino group
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12. Oxidative deamination of glutamate
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The amino groups of all amino acids are
concentrated in glutamate
Glutamate is the only amino acid that can
undergo oxidative deamination at a
significant rate
Oxidative deamination of glutamate is
catalysed by glutamate dehydrogenase

13. Glutamate dehydrogenase can use
NAD+ as well as NADP+ as coenzyme
NADP+ is probably used for synthesis
of glutamate from a-ketoglutarate
NAD+ is used for oxidative
deamination of glutamate
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14. Glutamate
a

15. Some NH3 is released by the actions of L-
amino acid oxidase and D-amino acid oxidase
Contribution of L-amino acid oxidase and
D-amino acid oxidase is very small
L-Amino acid oxidase is very low in activity
D-Amino acids do not occur in significant
quantities in human beings
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16. Transport of ammonia
Liver is the major site for catabolism of
amino acids
It is also the only organ having all the
enzymes of urea synthesis
However, some ammonia is produced in
other tissues also
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17. Ammonia produced in extrahepatic tissues
has to be transported to liver
And it must be transported without causing
ammonia intoxication
Normal concentration of ammonia in blood
is 10 - 80 µg/dl
Even a small rise in blood ammonia level
may damage nervous tissue
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18. Brain tissue is extremely sensitive to
ammonia
It possesses a mechanism for immediate
detoxification of ammonia
Ammonia is combined with glutamate to
form glutamine
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20. Thus, ammonia can be transported from
brain in the form of non-toxic glutamine
Some other tissues also use glutamine
as a carrier of ammonia
Glutamine released into circulation is
taken up by liver and kidneys

21. Glutaminase converts glutamine into
glutamate and ammonia
Ammonia formed in kidneys is excreted in
urine as ammonium salts
Ammonia formed in liver is converted into
urea
Glutamine + H2O → Glutamate + NH3

22. Glutamate is another carrier of ammonia
It is formed by transamination of a-keto-
glutarate in many tissues
It transports the amino groups of various
amino acids to liver in a non-toxic form

23. Alanine is also a carrier of ammonia,
especially from muscles
Amino groups of many amino acids are trans-
ferred to pyruvate forming alanine in muscles
Alanine is transported to liver where it is re-
converted to pyruvate
The amino group of alanine is transferred to
a-ketoglutarate

24. NH3 is also formed in the gut by the action of
bacterial enzymes on amino acids and urea
Catabolism of dietary purines and
pyrimidines in the gut also liberates NH3
All this NH3 enters portal blood from the gut
Liver extracts all the NH3 when portal blood
passes through it

25. Ammonia is converted into non-toxic urea
in liver by a cyclic sequence of reactions
The sequence is known as urea cycle or
Krebs-Henseleit cycle
Urea is released into circulation, and is
excreted by the kidneys in urine
Synthesis of urea

26. An adult man excretes about 30 gm of
urea per day
This accounts for nearly 90% of the
nitrogen excretion

28. The C=O group of urea is provided by
CO2, one –NH2 group is provided by NH3
and the other –NH2 group by aspartate
Four high-energy phosphate bonds of
ATP are utilized; two ATP molecules are
converted into ADP and one into AMP

29. After contributing its amino group,
aspartate is converted into fumarate
For continuation of the cycle, fumarate
enters the citric acid cycle
It is converted into oxaloacetate which is,
then, transaminated to aspartate
Thus, urea cycle is linked with citric
acid cycle

30. The first two reactions of urea cycle
occur in mitochondria
The remaining three reactions occur in
cytosol
Conversion of fumarate into oxaloacetate
and conversion of oxaloacetate into
aspartate also occur in mitochondria
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31. Reactions of the urea cycle are:
1. Synthesis of carbamoyl phosphate
2. Transfer of carbamoyl group to
ornithine to form citrulline
3. Conversion of citrulline into
argininosuccinate
4. Cleavage of argininosuccinate into
arginine and fumarate
5. Hydrolysis of arginine into urea and
ornithine

33. Two molecules of ATP are converted into
ADP; one Pi is released and the other is
incorporated in carbamoyl phosphate
N-acetylglutamate is required as an activator
of carbamoyl phosphate synthetase I

34. Carbamoyl phosphate transfers its carbamoyl
group to ornithine to form citrulline
Inorganic phosphate is released
Carbamoyl phosphate is a high-energy
phosphate
Its energy is used to form the covalent bond
between carbamoyl group and ornithine
Transfer of carbamoyl group

36. Ornithine moves from cytosol to mito-
chondria for this reaction
Citrulline leaves the mitochondria for the
next reaction which occurs in cytosol

37. Citrulline combines with aspartate to form
argininosuccinate
A covalent bond is formed between amino
group of aspartate and keto group of citrulline
Hydrolysis of ATP into AMP and PPi provides
energy for the formation of this bond
Conversion of citrulline into
argininosuccinate

39. Argininosuccinate is cleaved into arginine
and fumarate
The reaction is catalysed by arginino-
succinase
The net effect of this and the preceding
reaction is that aspartate loses its amino
group and is converted into fumarate
Cleavage of argininosuccinate

41. Arginase catalyses hydrolytic cleavage of
arginine into urea and ornithine
Ornithine goes back to begin another
cycle by reacting with yet another
molecule of carbamoyl phosphate
Hydrolysis of arginine

43. Glutamate dehydrogenase is an allosteric
enzyme
It is inhibited by ATP and GTP, and is
activated by ADP and GDP
Thus, decreased availability of energy
increases the catabolism of amino acids
and synthesis of urea
Regulation of urea synthesis

44. Mitochondrial carbamoyl phosphate
synthetase is also an allosteric enzyme
It is activated by N-acetylglutamate which
is formed by transfer of the acetyl group
from acetyl CoA to glutamate
N-Acetylglutamate
synthetase
Glutamate N-Acetylglutamate
Acetyl CoA CoA

45. N-Acetylglutamate synthetase is also
subject to regulation
It is activated by arginine

48. Life-threatening neurological
abnormalities can occur in severe liver
diseases e.g. cirrhosis of liver
Disturbed hepatic architecture can lead to
the formation of porto-caval shunt
Portal blood which is rich in ammonia
mixes with systemic blood
Hepatic encephalopathy

49. Decreased number of functioning liver
cells impairs the ability of liver to convert
ammonia into urea
This can result in severe hyper-
ammonaemia

50. Hyperammonaemia causes encephalopathy
manifesting as:
Blurring of vision
Slurring of speech
Flapping tremors
Hepatic coma and death

51. Administration of L-ornithine and L-aspartate
is useful in augmenting urea synthesis
Sterilization of gut by antibiotics is useful in
decreasing ammonia production by bacteria
Low protein diet is also recommended to
decrease ammonia production

52. Lactulose is an indigestible and non-
absorbable disaccharide
It is given orally to convert ammonia into
ammonium ions in the gut
Ammonium ions cannot be absorbed from
the intestines

53. Hepatic encephalopathy
may occur due to:
ATP deficit
Increased level of GABA
Decreased uptake of amino acids

54. Brain possesses a mechanism for
immediate detoxification of ammonia
Free ammonia is combined with
glutamate to form glutamine
Glutamate required for this reaction is
formed from a-ketoglutarate
ATP deficit

55. a-Ketoglutarate is an intermediate of citric
acid cycle
Excessive removal of a-ketoglutarate can
result in impairment of citric acid cycle
This will limit the production of ATP

56. GABA (gamma-aminobutyric acid) is a
neuro-transmitter
Increased synthesis of GABA from
glutamate may be responsible for some
of the neuro-psychiatric abnormalities
Increased level of GABA

57. Synthesis of glutamine is increased in
brain
Increased level of glutamine can inhibit
uptake of some other amino acids by
brain cells
Decreased uptake of amino acids

58. Each of the five enzymes of urea cycle
may be congenitally absent or deficient
This will decrease the synthesis of urea
Decreased urea synthesis causes
ammonia intoxication
Inborn errors of urea cycle

59. Disorder Deficient enzyme
Hyperammo-
naemia, type I
Carbamoyl phosphate
synthetase
Hyperammo-
naemia, type II
Ornithine transcarbamoylase
Citrullinaemia Argininosuccinic acid
synthetase
Argininosuccinic
aciduria
Argininosuccinase
Hyperargininaemia Arginase

60. Disorders of urea cycle are very rare
Clinical abnormalities are common to all
Clinical abnormalities are more severe
when the defect occurs early in the
pathway

61. Clinical features of urea cycle
disorders are:
• Vomiting
• Distaste for protein-rich foods
• Lethargy
• Irritability
• Ataxia
• Mental retardation

62. Small and frequent low-protein meals
are given to prevent sudden and
excessive rise in blood ammonia
Sodium benzoate is used to facilitate
removal of ammonia as hippuric acid