1. UREA CYCLE

2. Most mammals convert amino-acid nitrogen to urea for excretion
amino acids
Most mammals convert amino-acid nitrogen to urea for excretion
The carbon chains are broken down to molecules that feed into the TCA cycle.
NH4+
Some animals excrete NH4+ or uric acid.
most terrestrial vertebrates
fish & other aquatic vertebrates
birds & reptiles O HN N H
H2N-C-NH2 urea O
NH4+
ammonium ion
uric acid

3. Ammonia is a toxic substance to plants and animals (especially for brain)
Normal concentration: mol/l ( mg/l)
Ammonia must be removed from the organism
Terrestrial vertebrates synthesize urea (excreted by the kidneys) - ureotelic organisms
Urea formation takes place in the liver
Birds, reptiles synthesize uric acid

4. Major fate of waste nitrogen

5. Why Urea? Non toxic Water soluble
Combines two waste products into one molecule:
CO2
NH3

6. Ammonia is highly toxic
Main reason to form urea is to reduce levels of ammonia
“Ammonia” often refers to (NH3 + NH4+)
NH3 is really ammonia
NH4+ is the ammonium ion

7. Hypotheses toxicity of ammonia A
Hypotheses toxicity of ammonia A. The binding of ammonia in the synthesis of glutamate causes an outflow of α-ketoglutarate from the tricarboxylic acid cycle, with decreased formation of ATP energy and deteriorates the activity of cells. B. Ammonium ions NH4 + caused alkalization of blood plasma. This increases the affinity of hemoglobin for oxygen (Bohr effect), the hemoglobin does not release oxygen to the capillaries, resulting the cells hypoxia occurs. C. The accumulation of free NH4 + ion in the cytosol affects the membrane potential and intracellular enzymes work - it competes with ion pumps, Na + and K +.

8. Hypotheses toxicity of ammonia D
Hypotheses toxicity of ammonia D. The producing ammonia tramsform glutamic acid - glutamine - an osmotically active substance. This leads to water retention in the cells and the swelling that causes swelling of tissues. In the case of nervous tissue it can cause brain swelling, coma and death. E. The use of α-ketoglutarate and glutamate to neutralize the ammonia causes a decrease in the synthesis of γ-aminobutyric acid (GABA) inhibitory neurotransmitter of the nervous system.

9. Ammonia rapidly equilibrates across membranes

10. AMMONIA METABOLISM The ways of ammonia formation
1. Oxidative deamination of amino acids
2. Deamination of physiologically active amines and nitrogenous bases.
3. Absorption of ammonia from intestine (degradation of proteins by intestinal microorganisms results in the ammonia formation).
4. Hydrolytic deamination of AMP in the brain (enzyme – adenosine deaminase)

11. Production of ammonia

12. Summary of sources of ammonia for urea cycle

13. Glutamate is not deaminated in peripheral tissues
Peripheral Tissues Transport Nitrogen to the Liver
Two ways of nitrogen transport from peripheral tissues (muscle) to the liver:
Glutamate is not deaminated in peripheral tissues
1. Alanine cycle. Glutamate is formed by transamination reactions

14. Nitrogen is then transferred to pyruvate to form alanine, which is released into the blood.
The liver takes up the alanine and converts it back into pyruvate by transamination.
The glutamate formed in the liver is deaminated and ammonia is utilized in urea cycle.

15. Closer look at transport of waste N from peripheral tissue to liver via alanine and glutamine
Waste N funnelled to pyruvate
via transaminations
Glucose – Alanine Cycle
Net: N (muscle)  Urea (liver)

16. 2. Nitrogen can be transported as glutamine.
Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH4+ in an ATP-dependent reaction:
Ammonia transport in the form of glutamine.
Excess ammonia in tissues is added to glutamate to form glutamine, a process catalyzed by glutamine synthetase. After transport in the bloodstream, the glutamine enters the liver and NH4 is liberated in mitochondria by the enzyme glutaminase.

17. Synthesis of Glutamine in Peripheral Tissue and Transport to the Liver

19. UREA FORMATION

20. Overview Occurs primarily in liver; excreted by kidney
Principal method for removing ammonia
Hyperammonemia:
Defects in urea cycle enzymes (CPS, OTC, etc.)
Severe neurological defects in neonates
Treatment:
Stop protein intake
Dialysis
Increase ammonia excretion: Na benzoate, Na phenylbutyrate, L-arginine, L-citrulline

21. Overview Key reaction: hydrolysis of arginine
23 April 2017
Overview
Key reaction: hydrolysis of arginine
Arginine + H2O ==> urea + ornithine
arginase
Resynthesis of Arginine

22. Blood Urea Nitrogen Normal range: 7-18 mg/dL
Elevated in amino acid catabolism
Glutamate N-acetylglutamate
CPS-1 activation
Elevated in renal insufficiency
Decreased in hepatic failure

23. 2

24. THE UREA CYCLE
Urea cycle - a cyclic pathway of urea synthesis first postulated by H.Krebs
The sources of nitrogen atoms in urea molecule:
aspartate;
NH4+.
Carbon atom comes from CO2.

25. H2N-C-NH-CH2CH2CH2CH-CO2- O
23 April 2017
The urea cycle
HCO3-
2 ATP ADP + Pi
mitochondria
carbamoyl phosphate
H2N-C-O-P-O- O O-
NH4+
citrulline
NH3+
H2N-CH2CH2CH2CH-CO2-
ornithine Pi
NH3+
H2N-C-NH-CH2CH2CH2CH-CO2- O
cytosol
ATP
-O2C-CH2CH-NH3+
CO2-
Asp O
H2N-C-NH2
AMP + PPi
urea
Historical note: Hans Krebs discovered the urea cycle some years prior to his discovery of the TCA cycle.
-O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-
NH3+
CO2-
NH2+
H2O
argininosuccinate
arginine
NH2+
NH3+
H2N-C-NH-CH2CH2CH2CH-CO2-
-O2C-CH=CH-CO2-
fumarate

26. carbonic-phosphoric acid anhydride
Incorporation of ammonia into urea begins with formation of carbamoyl phosphate
NH HCO3-
2 ATP ADP + Pi
H2N-C-O-P-O- O O-
carbamoyl phosphate
This occurs in the mitochondrial matrix. Carbamoyl-phosphate synthetase-1 catalyzes the reaction in three steps, using two molecules of ATP:
carbamate
HCO3-
ATP ADP Pi
NH4+
carbonic-phosphoric acid anhydride O O-
HO-C-O-P-O-
H2N-C-O-
(1)
(2)
(3)
H2N-C-O-P-O-

27. Carbamoyl phosphate reacts with ornithine to form citrulline
H2N-C-O-P-O- O O-
NH3+
+H3N-CH2CH2CH2CH-CO2-
ornithine
carbamoyl phosphate
NH3+
H2N-C-NH-CH2CH2CH2CH-CO2- O Pi
+ H+
citrulline
This step also occurs in the mitochondrial matrix.

28. H2N-C-NH-CH2CH2CH2CH-CO2- -O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-
Combination of citrulline with aspartate to form argininosuccinate is driven by breakdown of ATP to AMP
aspartate
argininosuccinate
citrulline
NH3+
H2N-C-NH-CH2CH2CH2CH-CO2- O
-O2C-CH2CH-NH3+
CO2-
-O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-
NH2+
ATP
AMP + PPi + H2O
This reaction occurs only in the cytosol, so citrulline first must leave the mitochondria. A transporter exchanges ornithine for citrulline plus a proton across the mitochondrial inner membrane.

29. Argininosuccinate splits into arginine and fumarate
-O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-
NH3+
CO2-
NH2+
argininosuccinate
-O2C-CH=CH-CO2-
fumarate
NH2+
NH3+
H2N-C-NH-CH2CH2CH2CH-CO2-
arginine
This reaction occurs in the cytosol.

30. Hydrolysis of arginine releases urea and regenerates ornithine
NH2+
NH3+
H2N-C-NH-CH2CH2CH2CH-CO2-
arginine
H2O
urea
ornithine H+
NH3+
H2N-CH2-CH2-CH2-CH-CO2- O
H2N-C-NH2
This reaction occurs in the cytosol. To continue the cycle, ornithine must return to a mitochondrion.

31. Formation of urea consumes 4 phosphate anhydride bonds
HCO3-
2 ATP ADP + Pi
carbamoyl phosphate
H2N-C-O-P-O- O O-
NH4+ Pi
citrulline
NH3+
H2N-C-NH-CH2CH2CH2CH-CO2- O
ornithine
ATP
-O2C-CH2CH-NH3+
CO2-
2 Pi O
H2N-C-NH2
PPi + AMP
Asp
H2O
urea
-O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-
NH3+
CO2-
NH2+
argininosuccinate
Formation of urea consumes 4 phosphate anhydride bonds

32. Input-Output

33. -keto acids amino acids aspartate-oxaloacetate aminotransferase
The aspartate consumed in the urea cycle can be regenerated from the fumarate that is produced
2 ATP ADP + Pi
HCO3- + NH4+
carbamoyl phosphate
-keto acids amino acids Pi
aspartate-oxaloacetate aminotransferase
ornithine
citrulline
oxaloacetate
Urea cycle
ATP
urea
aspartate
AMP + PPi
malate dehydrogenase
arginine
argininosuccinate
NADH
malate
NAD+
fumarate
This process also uses both cytosolic and mitochondrial enzymes
H2O

34. Oxidation of malate in mitochondria generates ATP
2 e- to O2 via NADH dehydrogenase generates ~ 2.5 ATP
2 ATP ADP + Pi
HCO3- + NH4+
carbamoyl phosphate
NADH
NAD
oxaloacetate
mitochondrion Pi
aspartate
glutamate
ornithine
malate
citrulline
-ketoglutarate
citrulline
-ketoglutarate
ornithine
glutamate
ATP
aspartate
urea
AMP + PPi
amino acids a-ketoacids
arginine
argininosuccinate
malate
cytosol
fumarate
H2O
NADH, NAD+ and oxaloacetate can’t cross the mitochondrial inner membrane, but there are transporters for malate, aspartate, glutamate and a-ketoglutarate.

35. 23 April 2017
Transport systems in the mitochondrial inner membrane exchange aspartate for glutamate and a-ketoglutarate for malate
mitochondrion
aspartate-
glutamate- + H+
-ketoglutarate
malate
N. Ramoz et al. “Linkage and association of the mitochondrial aspartate/glutamate carrier SLC25A12 gene with autism,” Am. J. Psychiatry 161: (2004)
aspartate-
glutamate- + H+
-ketoglutarate
malate
cytosol
Because the Asp/Glu transporter also moves a proton across the membrane, it can be driven by an electrochemical potential gradient.
Mutations in this transporter have been linked to autism.

36. Alpha--ketoglutarate/malate and aspartate/glutamate transporters also participate in oxidation of cytosolic NADH
cytosol
aspartate
malate
-ketoglutarate
glutamate
oxaloacetate
NAD+
NADH
glycolysis
mitochondrion
2 e- to electron-transport chain

37. Well Fed State
Net: 2 NH CO ATP  urea + 4 ADP + 4 Pi

38. Fasted State
Gluconeogenesis
2 ala + CO2  1 urea + 1 glucose

39. Balancing the levels of ammonia and aspartate for entry into urea cycle

40. The urea cycle is regulated in two ways
1. Allosteric activation of carbamoylphosphate synthetase-1 by N-acetylglutamate
CO2-
+H3N C H
CH2
CO2-
CH3CO-NH C H
CH2
+ acetyl-CoA
N-acetylglutamate
CoA-SH
Glu
In mammals, N-acetylGlu appears to play only a regulatory role. Carbamoylphosphate synthetase-1 is completely inactive in its absence. A genetic deficiency in the enzyme that forms N-acetylGlu can cause a lethal defect in the urea cycle.
carbamoyl- phosphate
NH4+ + HCO3-
2 ATP ADP + Pi O
H2N-C-O-P-O- O-
2. A high-protein diet or starvation leads to increased synthesis of all five enzymes used in the urea cycle, including carbamoylphosphate synthetase-1. Expression of the enzyme that synthesizes N-acetylglutamate also increases.

41. The Linkage between Urea Cycle, Citric Acid Cycle and Transamination of Oxaloacetate
Fumarate formed in urea cycle enters citric acid cycle and is converted to oxaloacetate.
Fates of oxaloacetate:
transamination to aspartate,
conversion into glucose,
condensation with acetyl CoA to form citrate,
conversion into pyruvate.

42. Diagnostic significance of the determination of urea in urine.
25-30 g/day of urea is excreted in normal conditions.
The increase of urea in urine occurs in high fever, malignant anemia, poisoning by phosphorus, intensive decomposition of protein in organism.
The decrease of urea in urine occurs in liver diseases, kidney unsufficiency, acidosis.
23 April 2017
Total slide : 50

43. Urea Cycle Disorders
Deficiency of any of the five enzymes in the urea cycle results in the accumulation of ammonia and leads to encephalopathy.
Episodes of encephalopathy and associated systems are unpredictable and, if untreated, are lethal or produce devastating neurologic sequelae in long-term survivors.
Although these disorders do not produce liver disease, the consequences of hyperammonemia resemble those seen in patients with hepatic failure or in a transient interference with the urea cycle, as seen in some forms of organic acidemias.
Investigate for hyperammonemia in any infant or child with altered mental status

44. The urea cycle Asterisk = N-acetyl glutamate synthetase; 1 = carbamyl phosphate synthetase; 2 = ornithine transcarbamylase; 3 = argininosuccinate synthetase; 4 = argininosuccinate lyase; 5 = arginase

45. UREA CYCLE DISORDERS Disorder Deficient Enzyme Inheritance Pattern
Carbamyl phosphate synthetase deficiency
Carbamyl phosphate synthetase
Autosomal recessive
Ornithine transcarbamylase deficiency
Ornithine transcarbamylase
X-linked
Citrullinemia
Argininosuccinate synthetase
Argininosuccinic aciduria
Argininosuccinate lyase
Argininemia
Arginase

46. Case
The patient is a full-term newborn boy from a normal vaginal delivery. The pregnancy was uncomplicated. At 36 hours the baby became lethargic, irritable, and was hyperventilating. Over the next 24 hours lethargy increased and progressed to coma requiring mechanical ventilation. Hemodialysis was started at 5 days. Patient died
at one week of age.
Laboratory Results
At 36 hours arterial blood pH was 7.50 ( ), carbon dioxide was 25 torr (35-45), and blood urea nitrogen was 2 mg/dl (5-20). Sepsis workup was negative. On day 5 plasma ammonium was 1800 :mol/l (<35). Plasma glutamine was 1500 :mol/l ( ),arginine was below normal, and citrulline undetectable. Orotic acid in the urine was
extremely elevated.
Family History
Two of the mother’s four brothers had died shortly after birth. Cause of death was given as encephalitis.
Biochemical Basis of Disorder , same as..
Diagnosis:
ornithine transcarbamoylase deficiency

47. Biochemical explanations for ornithine transcarbamoylase deficiency
Low BUN
Low blood arginine
Undetectable blood citrulline
Elevated blood ammonia
Elevated blood glutamine
Elevated orotic acid

48. NH4+ + Glu  Gln
Carbamoyl P  orotic acid

49. Carbamoyl P synthetase deficiency

50. NH4+ + Glu  Gln
Carbamoyl P  orotic acid

51. Autism is a neurodevelopmental genetic disorder
23 April 2017
Autism is a neurodevelopmental genetic disorder
Deficits in verbal & nonverbal communication and social interactions
Repetitive or stereotyped behaviors
Incidence ~1 per 1000 people (possibly higher)
Strong evidence for heritability
Polygenic - between 5 & 10 genes may be involved
Single-nucleotide polymorphisms (SNPs) in the gene for a mitochondrial, Ca2+-dependent Asp/Glu exchanger increase the risk by a factor of 3 to 4.
This is the main form of the Asp/Glu exchanger that is expressed in the brain. Mutations in the gene impair the urea cycle.
The SNPs are intronic. Deficiencies in the Asp/Glu exchanger could have other effects on mitochondrial metabolism in addition to impairing the urea cycle.
N. Ramoz et al., Am. J. Psychiatry 161: 662 (2004)
L. Palmieri et al., EMBO J. 20: 5060 (2001)

52. Urea Cycle Disorders (Diagnosis)
Cultured skin fibroblasts may be desirable if prenatal diagnosis is considered in future pregnancies.
Carbamyl phosphate synthetase I and ornithine transcarbamylase (OTC) are not expressed in cultured fibroblasts.
The enzymatic diagnosis of CPSD and OTCD requires liver biopsy.
Biopsy should be done when establishing the diagnosis of the first case in a family.

53. Urea Cycle Disorders (Treatment)
Once hyperammonemia is demonstrated in an infant,
protein-containing feedings should be discontinued immediately,
appropriate supportive care, (mechanical ventilation)
Maximal calories should be provided in the form of intravenous glucose and lipids in an effort to reduce catabolism.
Plans should be immediately made to initiate hemodialysis in infants who are encephalopathic and have plasma ammonia levels over 10 times the upper limit of normal.

54. Urea Cycle Disorders (Treatment)
Maintenance therapy
dietary protein restriction+supplementation with citrulline or arginine+ the use of drugs
The primary drug now used( provides an alternate pathway for waste nitrogen excretion) for maintenance therapy in patients with urea cycle disorders is sodium phenylbutyrate (Buphenyl).
The drug is typically administered four times a day in a dose of 0.4 to 0.6 g/kg/day. It is supplied as a powder, which can be mixed with food or formula, or as a tablet.

55. Urea Cycle Disorders (Treatment)
Liver transplantation for
Severe neonatal OTC and CPS deficiency.
Liver failure and cirrhosis in ASL deficiency.
Failed medical-pharmacologic treatment.
Pretransplant care by
aggressively managing intercurrent hyperammonemia,
vaccinations and prophylaxis are given against infectious
appropriate caloric intake
Gene replacement

56. Genetic deficiencies in some of the urea-cycle enzymes can be treated pharmacologically
benzoate
CO2-
phenylacetate
ATP + CoA-SH
AMP + PPi
ATP + CoA-SH
AMP + PPi O
S-CoA O
S-CoA
benzoyl-CoA
phenylacetyl-CoA
glycine
CoA-SH
glutamine
CoA-SH O N H
CO2- N
NH2 O H
CO2-
phenylacetyl-glutamine
hippurate (benzoylglycine) O
The amide products of these reactions (hippurate and phenylacetylglutamine) are excreted in the urine. Replenishing the Gly or Gln removes ammonia.