Formation of urea- Urea cycle Urea cycle disorders Variations of urea levels

1. Amino acid catabolism- Part II
(Urea cycle with Clinical
Significance)
Biochemistry For Medics- Lecture
notes
Professor(Dr.) Namrata Chhabra
www.namrata.co

2. Urea cycle
• The continuous degradation and synthesis of
cellular proteins occur in all forms of life.
• Each day, humans turn over 1–2% of their total
body protein, principally muscle protein.
• Of the liberated amino acids, approximately 75%
are reutilized.
• Since excess amino acids are not stored, those
not immediately incorporated into new protein
are rapidly degraded to amphibolic
intermediates.
• The excess nitrogen forms urea.
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4. Urea formation (Urea cycle)
Characteristics of urea cycle
• Urea is the major disposal form of amino groups
• It accounts for 90% of the nitrogen containing
components of urine
• The urea cycle is the sole source of endogenous
production of arginine
• Urea formation takes place in liver,
• Urea excretion occurs through kidney
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5. Urea formation (Urea cycle)
o 6 amino acids participate in urea formation,
which are• Ornithine
• Citrulline
• Aspartic acid
• Argino succinic acid
• Arginine and
• N-Acetyl Glutamate
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6. Urea formation
• Synthesis of 1 mol of urea requires 3 mol of ATP
• 1 mol each of ammonium ion and of the α-amino
nitrogen of aspartate.
• Five enzymes catalyze the reactions of urea cycle
• Of the six participating amino acids, N-acetyl
glutamate functions solely as an enzyme
activator.
• The others serve as carriers of the atoms that
ultimately become urea.
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7. Urea cycle- An overview
• Urea synthesis is a cyclic process.
• The first two reactions of urea synthesis occur in the
matrix of the mitochondrion, the remaining reactions
occur in the cytosol
• Since the Ornithine consumed in 2nd reaction is
regenerated in last reaction, so there is no net loss or
gain of Ornithine, Citrulline, argininosuccinate, or
arginine.
• Ammonium ion, CO2, ATP, and aspartate are, however,
consumed.
• Aspartate can however be resynthesized from the
released fumarate by a series of reactions
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8. Steps of urea formation
Step-1- Formation of Carbamoyl-Phosphate
o Condensation of CO2, ammonia, and ATP to form
Carbamoyl phosphate is catalyzed by mitochondrial
Carbamoyl phosphate synthase I (CPS-1)
o Formation of Carbamoyl phosphate requires 2 mol of
ATP, one of which serves as a phosphoryl donor.
o Carbamoyl phosphate synthase I, the rate-limiting
enzyme of the urea cycle, is active only in the presence
of its allosteric activator N-acetyl glutamate, which
enhances the affinity of the synthase for ATP.
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9. Step-1- Formation of CarbamoylPhosphate
• The reaction proceeds stepwise.
• Reaction of bicarbonate with ATP forms carbonyl
phosphate and ADP.
• Ammonia then displaces ADP, forming carbamate
and orthophosphate.
• Phosphorylation of carbamate by the second ATP
then forms carbamoyl phosphate.
• A cytosolic form of this enzyme, Carbamoyl
phosphate synthase II, uses glutamine rather
than ammonia as the nitrogen donor and
functions in pyrimidine biosynthesis.
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10. Step-1- Formation of CarbamoylPhosphate
• CPS1 is strongly activated by N-acetyl
glutamate, which controls the overall rate of
urea production.
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11. Step-2- Formation of Citrulline
• The Carbamoyl group of Carbamoyl phosphate is
transferred to ornithine, forming Citrulline and Ortho
Phosphate
• The reaction is catalyzed by Ornithine trans
Carbamoylase
• Subsequent metabolism of Citrulline take place in the
cytosol.
• Entry of ornithine into mitochondria and exit of
citrulline from mitochondria involves mitochondrial
inner membrane transport systems
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12. Step-2- Formation of Citrulline
This enzyme has no regulatory significance. The remainder of the
urea cycle steps take place in the cytosol. This requires the
continuous export of citrulline and the uptake of ornithine across
the inner mitochondrial membrane.
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13. Step-2- Formation of Citrulline
Clinical Significance
• Ornithine Transcarbamoylase deficiency causes enhanced
excretion of Uracil.
• Excessive excretion of Uracil or its precursor Orotic acid,
results from an accumulation of Carbamoyl phosphate in the
mitochondria.
• In the absence of Ornithine Transcarbamoylase, Carbamoyl
phosphate accumulates and leaks in to the cytoplasm, where
it can be used to make Carbamoyl Aspartate, the first
intermediate in the pathway of pyrimidine nucleotide
biosynthesis.
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14. OTC Deficiency and Orotic aciduria
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15. Step-3- Formation of Argino succinate
• Argininosuccinate synthase (ASS) links LAspartate and Citrulline via the amino group
of aspartate and provides the second nitrogen
of urea.
• The reaction requires ATP and involves
intermediate formation of citrullyl-AMP.
Subsequent displacement of AMP by
aspartate then forms Argininosuccinate.
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16. Step-3- Formation of Argino succinate
• Production of arginino-succinate is an energetically expensive
process, since the ATP is split to AMP and pyrophosphate.
• The pyrophosphate is then cleaved to inorganic phosphate
using pyrophosphatase, so the overall reaction costs two
equivalents of high energy phosphate per mole.
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17. Step-4- Cleavage of Argino succinate
• Cleavage of argininosuccinate catalyzed by
argininosuccinate lyase (ASL), proceeds with
retention of nitrogen in arginine and release of the
aspartate skeleton as fumarate.
• Addition of water to fumarate forms L-malate, and
subsequent NAD+-dependent oxidation of malate
forms oxaloacetate.
• Transamination of oxaloacetate by glutamate
aminotransferase then re-forms aspartate. carbon
skeleton of aspartate-fumarate thus acts as a carrier
of the nitrogen of glutamate into a precursor of urea
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18. Step-4- Cleavage of Argino succinate
This reaction sequence is very similar to the conversion of
IMP to AMP in the purine biosynthetic pathway. In each
case fumarate is formed as a by-product. Fumarate is not
transported by mitochondria, so this requires the presence
of cytosolic fumarase to form malate.
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19. Relationship of Urea cycle and TCA
cycle
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20. Step-5- Cleavage of Arginine
• Hydrolytic cleavage of the guanidino group of
arginine, catalyzed by liver arginase (ARG1)
releases urea, the other product, Ornithine,
reenters liver mitochondria for additional
rounds of urea synthesis.
• Ornithine and lysine are potent inhibitors of
arginase, competitive with arginine.
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21. Step-5-Cleavage of Arginine
• Arginine also serves as the precursor of the potent muscle
relaxant nitric oxide (NO) in a Ca2+-dependent reaction
catalyzed by NO synthase.
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22. Regulation of Urea formation
• The activity of Carbamoyl phosphate synthase
I is determined by N-acetyl glutamate, whose
steady-state level is dictated by its rate of
synthesis from acetyl-CoA and glutamate and
its rate of hydrolysis to acetate and glutamate.
• These reactions are catalyzed by N-acetyl
glutamate synthase and N-acetyl glutamate
Hydrolase, respectively.
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23. Role of N-Acetyl Glutamate
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24. Synthesis and Degradation of NAG
• N- Acetyl Glutamate Synthase catalyzes the synthesis
of NAG.
• Degradation is catalyzed by NAG- Hydrolase enzyme
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25. Regulation of Urea formation
• Major changes in diet can increase the
concentrations of individual urea cycle
enzymes 10- to 20-fold.
• Starvation, for example, elevates enzyme
levels, presumably to cope with the increased
production of ammonia that accompanies
enhanced protein degradation
• Regulation is also achieved by linkage of
mitochondrial glutamate dehydrogenase with
CPS-1
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27. Fate of Urea
• Urea formed in the liver is transported through
circulation to kidneys for excretion through urine.
• It is also transported to intestine where it is
decomposed by Urease produced by microbial
action.
• Ammonia liberated by this activity is transported by
portal circulation to liver where it is detoxified back
to urea.
• A fraction of ammonia goes to systemic circulation.
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28. Fate of Urea
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29. Urea cycle disorders
Carbamoyl Phosphate synthetase (CPS-1)
deficiency
• Along with OTC deficiency, deficiency of CPS-I is the most
severe of the urea cycle disorders.
• Defects in the enzyme carbamoyl phosphate synthase I are
responsible for the relatively rare (estimated frequency
1:62,000) metabolic disease termed "hyperammonemia type
1."
• Individuals with complete CPS-I deficiency rapidly develop
hyperammonemia in the newborn period.
• Children who are successfully rescued from crisis are
chronically at risk for repeated bouts of hyperammonemia
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30. Ornithine Transcarbamoylase
deficiency (OTC deficiency)
• The disease is characterized as X linked dominant
because most females are also somewhat affected.
• A significant number of carrier females have
hyperammonemia and neurologic compromise.
• The risk for hyperammonemia is particularly high in
pregnancy and the postpartum period.
• The disease is much more severe in males than in
females.
• The enzyme activity can range from 0% to 30% of the
normal.
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31. Citrullinemia (ASS deficiency)
• The hyperammonemia in this disorder is quite
severe.
• Affected individuals are able to incorporate
some waste nitrogen into urea cycle
intermediates,
• which makes treatment slightly easier.
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32. Argininosuccinic aciduria (ASL
deficiency)
• This disorder also presents with rapid-onset
hyperammonemia in the newborn period.
• This enzyme defect is past the point in the metabolic pathway
at which all the waste nitrogen has been incorporated into the
cycle.
• Treatment of affected individuals often requires only
supplementation of arginine.
• Affected individuals can also develop trichorrhexis nodosa, a
node-like appearance of fragile hair, which usually responds to
arginine supplementation.
• ASL deficiency is marked by chronic hepatic enlargement and
elevation of transaminases.
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33. Arginase deficiency
(hyperargininemia; ARG deficiency)
• This disorder is not typically characterized by
rapid-onset hyperammonemia.
• Affected individuals develop progressive
spasticity and can also develop tremor, ataxia,
and choreoathetosis.
• Growth is affected
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34. NAG Synthase deficiency
• Deficiency of this enzyme has been described in a number of
affected individuals.
• Symptoms mimic those of CPSI deficiency; since CPSI is
rendered inactive in the absence of NAG
• N-Acetyl glutamate is essential for Carbamoyl phosphate
synthase I activity
• The NAGS gene encodes N-acetyl glutamate synthase, which
catalyzes the condensation of acetyl-CoA with glutamate.
• Defects in the NAGS gene result in severe hyperammonemia,
which in this specific instance may respond to administered Nacetyl glutamate.
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35. Ornithine Transporter deficiency
• Hyperornithinemia, hyperammonemia, and homocitrullinuria
syndrome (HHH syndrome) results from mutation of the
ORNT1 gene that encodes the mitochondrial membrane
ornithine transporter.
• The failure to import cytosolic ornithine into the
mitochondrial matrix renders the urea cycle inoperable, with
consequent hyperammonemia, and the accompanying
accumulation of cytosolic ornithine results in
Hyperornithinemia.
• In the absence of its normal acceptor ornithine, mitochondrial
carbamoyl phosphate carbamoylates lysine to homocitrulline
with a resulting homocitrullinuria.
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36. Clinical manifestations in urea cycle
disorders
• Infants with a urea cycle disorder often appear normal initially
but rapidly developo cerebral edema
o lethargy
o anorexia
o hyperventilation or hypoventilation,
o hypothermia
o slurring of the speech,
o blurring of vision
o seizures
o neurologic posturing and
o coma.
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37. Clinical manifestations in urea cycle
disorders
• In milder (or partial) urea cycle enzyme deficiencies,
ammonia accumulation may be triggered by illness
or stress at almost any time of life, resulting in
multiple mild elevations of plasma ammonia
concentration; the hyperammonemia is less severe
and the symptoms are more subtle.
• In individuals with partial enzyme deficiencies, the
first recognized clinical episode may be delayed for
months or years.
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38. Laboratory diagnosis of UCD
• The diagnosis of a urea cycle disorder(UCD) is
based on evaluation of clinical, biochemical,
and molecular genetic data.
• A plasma ammonia concentration of 150
mmol/L or higher is a strong indication for the
presence of a UCD.
• Plasma quantitative amino acid analysis can
be used to diagnose a specific urea cycle
disorder
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39. Laboratory diagnosis of UCD
• Plasma concentration of Citrulline helps discriminate
between the proximal and distal urea cycle defects
• as Citrulline is the product of the proximal enzymes (OTC and
CPSI) and a substrate for the distal enzymes (ASS, ASL, ARG).
• Urinary Orotic acid is measured to distinguish CPSI deficiency
and NAGS (N-Acetyl Glutamate Synthase) deficiency from OTC
deficiency.
• The combination of family history, clinical presentation, amino
acid and Orotic acid testing, and, in some cases, molecular
genetic testing is often sufficient for diagnostic confirmation,
eliminating the risks of liver biopsy.
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40. Treatment
•
•
•
•
The mainstays of treatment for urea cycle disorders includeDialysis to reduce plasma ammonia concentration,
Intravenous administration of arginine chloride and nitrogen
scavenger drugs to allow alternative pathway excretion of
excess nitrogenExcess nitrogen is removed by intravenous phenyl acetate and
that conjugate with glutamine and glycine, respectively, to
form phenylacetylglutamine and Hippuric acid, water-soluble
molecules efficiently excreted in urine.
Arginine becomes an essential amino acid (except in arginase
deficiency) and should be provided intravenously to resume
protein synthesis.
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41. Treatment
• If these measures fail to reduce ammonia, hemodialysis
should be initiated promptly.
• Restriction of protein for 24-48 hours to reduce the amount
of nitrogen in the diet, providing calories as carbohydrates
(intravenously as glucose) and fat (intralipid or as protein-free
formula) to reduce catabolism,
• Physiologic stabilization with intravenous fluids
• Chronic therapy consists of a protein-restricted diet, phenyl
butyrate (a more palatable precursor of phenyl acetate),
arginine, or Citrulline supplements, depending on the specific
diagnosis.
• Liver transplantation should be considered in patients with
severe urea cycle defects that are difficult to control
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medically.

42. Treatment of UCD
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43. Genetic counseling
• Deficiencies of CPSI, ASS, ASL, NAGS, and ARG
are inherited in an autosomal recessive
manner.
• OTC deficiency is inherited in an X-linked
manner.
• Prenatal testing using molecular genetic
testing is available for five of the six urea cycle
disorders
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44. Tandem Mass Spectrometry
• The immensely powerful and sensitive
technique of tandem mass spectrometry can
screen for over two dozen metabolic diseases
using only drops of neonate blood.
• The early detection of UCD is of primary
importance.
• Early dietary intervention, however, can in
many instances ameliorate the otherwise
inevitable dire effects.
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45. Differential diagnosis of UCD
• A number of other disorders that perturb the
liver can result in hyperammonemia and
mimic the effects of a urea cycle disorder.
• The most common/significant ones are viral
infection of the liver and vascular bypass of
the liver.
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46. Gene Therapy
• Gene therapy for rectification of defects in the
enzymes of the urea cycle is an area of active
investigation.
• Encouraging preliminary results have been
obtained, for example, in animal models using
an adenoviral vector to treat citrullinemia.
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47. Variations in blood urea levels
• Normal blood urea level ranges between 1540 mg/dl.
• High blood urea level (uraemia)may be
observed ino Pre renal
o Renal and
o Post renal conditions
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48. Variations in blood urea level
• Pre renal conditions
o Salt and water depletion
o Severe vomiting as in pyloric stenosis or intestinal
obstruction
o Severe and prolonged diarrhea
o Addison’s disease
o Ulcerative colitis
o Haemorrhage and shock
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49. Variations in blood urea level
•
o
o
o
o
o
o
o
Renal conditions
Acute glomerulonephritis
Renal failure
Nephrosclerosis
Renal tuberculosis
Mercurial poisoning
Chronic Pyelonephritis
Hydronephrosis
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50. Variations in blood urea level
• Post Renal conditions- There is obstruction to
the outflow of urine. Retention of urine
reduces effective filtration pressure at
glomeruli. The important causes areo Enlarged prostate
o Stones in urinary tract
o Urethral strictures which may be congenital or
surgical
o Bladder tumors
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51. Variations in blood urea level
• Physiological conditions of high blood urea
level
o Advancing age
o Starvation- Proteins are catabolized. The
carbon skeleton of amino acids is used for
glucose, ketone bodies or energy production,
whereas the amino group of amino acids is
removed as ammonia which is later detoxified
as urea.
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52. Variations in blood urea level
• Low blood urea level- is observed in following
conditions
o Liver diseases
o Urea cycle disorders
o Physiologically – in pregnancy and growing
stage
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