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UREA CYCLE. O. H. N. HN. O. N. N. O. H. H. most terrestrial vertebrates. fish & other aquatic vertebrates. birds & reptiles. O. H 2 N-C-NH 2 urea. amino acids. The carbon chains are broken down to molecules that feed into the TCA cycle.

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most mammals convert amino acid nitrogen to urea for excretion

O

H

N

HN

O

N

N

O

H

H

most terrestrial vertebrates

fish & other aquatic vertebrates

birds & reptiles

O

H2N-C-NH2urea

amino acids

The carbon chains are broken down to molecules that feed into the TCA cycle.

Most mammals convert amino-acid nitrogen to urea for excretion

NH4+

Some animals excrete NH4+ or uric acid.

NH4+

ammonium ion

uric acid

slide3

Ammonia is a toxic substance to plants and animals (especially for brain)

Normal concentration:25-40 mol/l (0.4-0.7 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

why urea
Why Urea?
  • Non toxic
  • Water soluble
  • Combines two waste products into one molecule:
    • CO2
    • NH3
ammonia is highly toxic
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
slide7

Hypotheses toxicity of ammoniaA. 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 +.

slide8

Hypotheses toxicity of ammoniaD. 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.

slide10

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)

slide13

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:

1. Alanine cycle. Glutamate is formed by transamination reactions

slide14

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.

closer look at transport of waste n from peripheral tissue to liver via alanine and glutamine
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)

slide16

2. Nitrogen can be transported as glutamine.

Glutamine synthetasecatalyzes the synthesis of glutamine from glutamate and NH4+ in an ATP-dependent reaction:

Ammonia transport in the form of glutamine.

Excessammonia in tissues is added to glutamate to form glutamine, a processcatalyzed by glutamine synthetase. After transport in the bloodstream,the glutamine enters the liver and NH4 is liberated in mitochondriaby the enzyme glutaminase.

overview
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
overview1
Overview
  • Key reaction: hydrolysis of arginine

Arginine + H2O ==> urea + ornithine

arginase

Resynthesis of Arginine

blood urea nitrogen
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
slide24

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.
the urea cycle

O

O

H2N-C-O-P-O-

O-

NH3+

NH3+

O

H2N-C-NH-CH2CH2CH2CH-CO2-

H2N-CH2CH2CH2CH-CO2-

O

H2N-C-NH2

CO2-

NH3+

NH3+

-O2C-CH2CH-NH3+

CO2-

NH2+

-O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-

NH2+

H2N-C-NH-CH2CH2CH2CH-CO2-

The urea cycle

HCO3-

2 ATP 2 ADP + Pi

mitochondria

carbamoyl phosphate

NH4+

citrulline

ornithine

Pi

Asp

cytosol

ATP

AMP + PPi

urea

H2O

argininosuccinate

arginine

fumarate

-O2C-CH=CH-CO2-

incorporation of ammonia into urea begins with formation of carbamoyl phosphate

O

O

O

O

NH4+ + HCO3-

H2N-C-O-P-O-

H2N-C-O-P-O-

carbamoyl phosphate

O-

O-

2 ATP 2 ADP + Pi

O

O

(1)

carbonic-phosphoric acid anhydride

HCO3-

HO-C-O-P-O-

O-

ATP ADP

NH4+

(2)

Pi

ATP ADP

O

carbamate

H2N-C-O-

(3)

Incorporation of ammonia into urea begins with formation of carbamoyl phosphate

This occurs in the mitochondrial matrix. Carbamoyl-phosphate synthetase-1 catalyzes the reaction in three steps, using two molecules of ATP:

carbamoyl phosphate reacts with ornithine to form citrulline

O

O

H2N-C-O-P-O-

O-

NH3+

O

H2N-C-NH-CH2CH2CH2CH-CO2-

+H3N-CH2CH2CH2CH-CO2-

NH3+

Carbamoyl phosphate reacts with ornithine to form citrulline

ornithine

carbamoyl phosphate

Pi

+ H+

citrulline

This step also occurs in the mitochondrial matrix.

slide28

NH3+

O

H2N-C-NH-CH2CH2CH2CH-CO2-

citrulline

aspartate

argininosuccinate

CO2-

NH3+

-O2C-CH2CH-NH3+

CO2-

NH2+

-O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-

Combination of citrulline with aspartate to form argininosuccinate is driven by breakdown of ATP to AMP

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.

argininosuccinate splits into arginine and fumarate

NH3+

NH3+

CO2-

NH2+

-O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-

NH2+

H2N-C-NH-CH2CH2CH2CH-CO2-

Argininosuccinate splits into arginine and fumarate

argininosuccinate

-O2C-CH=CH-CO2-

fumarate

arginine

This reaction occurs in the cytosol.

hydrolysis of arginine releases urea and regenerates ornithine

O

NH3+

NH3+

H2N-C-NH2

H2N-CH2-CH2-CH2-CH-CO2-

H+

ornithine

urea

NH2+

H2N-C-NH-CH2CH2CH2CH-CO2-

Hydrolysis of arginine releases urea and regenerates ornithine

arginine

H2O

This reaction occurs in the cytosol. To continue the cycle, ornithine must return to a mitochondrion.

formation of urea consumes 4 phosphate anhydride bonds

O

O

H2N-C-O-P-O-

O-

NH3+

O

H2N-C-NH-CH2CH2CH2CH-CO2-

O

H2N-C-NH2

CO2-

NH3+

-O2C-CH2CH-NH3+

CO2-

NH2+

-O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2-

Formation of urea consumes 4 phosphate anhydride bonds

HCO3-

2 ATP 2 ADP + Pi

carbamoyl phosphate

NH4+

citrulline

Pi

ornithine

ATP

2 Pi

PPi + AMP

Asp

H2O

urea

argininosuccinate

the aspartate consumed in the urea cycle can be regenerated from the fumarate that is produced
The aspartate consumed in the urea cycle can be regenerated from the fumarate that is produced

2 ATP 2 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

oxidation of malate in mitochondria generates atp

NADH

NAD

Oxidation of malate in mitochondria generates ATP

2 e- to O2 via NADH dehydrogenase generates ~ 2.5 ATP

2 ATP 2 ADP + Pi

HCO3-+ NH4+

carbamoyl phosphate

oxaloacetate

mitochondrion

Pi

glutamate

aspartate

malate

ornithine

citrulline

-ketoglutarate

-ketoglutarate

citrulline

glutamate

ornithine

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.

slide35
Transport systems in the mitochondrial inner membrane exchange aspartate for glutamate and a-ketoglutarate for malate

mitochondrion

malate

aspartate-

-ketoglutarate

glutamate- + H+

malate

aspartate-

glutamate- + H+

-ketoglutarate

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.

slide36

2 e- to electron-transport chain

mitochondrion

NAD+

NADH

oxaloacetate

malate

aspartate

glutamate

-ketoglutarate

malate

aspartate

glutamate

-ketoglutarate

oxaloacetate

NAD+

NADH

cytosol

glycolysis

Alpha--ketoglutarate/malate and aspartate/glutamate transporters also participate in oxidation of cytosolic NADH
well fed state
Well Fed State

Net: 2 NH4+ + CO2 + 4 ATP  urea + 4 ADP + 4 Pi

fasted state
Fasted State

Gluconeogenesis

2 ala + CO2  1 urea + 1 glucose

the urea cycle is regulated in two ways

carbamoyl- phosphate

CO2-

CO2-

2 ATP 2 ADP + Pi

+H3N C H

CH3CO-NH C H

O

O

CH2

CH2

H2N-C-O-P-O-

NH4+ + HCO3-

CH2

CH2

O-

CO2-

CO2-

The urea cycle is regulated in two ways

1. Allosteric activation of carbamoylphosphate synthetase-1 by N-acetylglutamate

+ 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.

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.

slide41

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.
slide42

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.

Total slide : 50

urea cycle disorders
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
slide44

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

slide45

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

Autosomal recessive

Argininosuccinic aciduria

Argininosuccinate lyase

Autosomal recessive

Argininemia

Arginase

Autosomal recessive

slide46

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 (7.35-7.45), 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 (550-650),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

biochemical explanations for ornithine transcarbamoylase deficiency
Biochemical explanations for ornithine transcarbamoylase deficiency
  • Low BUN
  • Low blood arginine
  • Undetectable blood citrulline
  • Elevated blood ammonia
  • Elevated blood glutamine
  • Elevated orotic acid
slide48

NH4+ + Glu  Gln

Carbamoyl P  orotic acid

slide50

NH4+ + Glu  Gln

Carbamoyl P  orotic acid

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.

Autism is a neurodevelopmental genetic disorder

N. Ramoz et al., Am. J. Psychiatry 161: 662 (2004)

L. Palmieri et al., EMBO J. 20: 5060 (2001)

urea cycle disorders diagnosis
Urea Cycle Disorders (Diagnosis)
  • Cultured skin fibroblasts may be desirable if prenatal diagnosis is considered in future pregnancies.
  • Carbamyl phosphate synthetase I and ornithinetranscarbamylase (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.
urea cycle disorders treatment
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.
urea cycle disorders treatment1
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.
urea cycle disorders treatment2
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

genetic deficiencies in some of the urea cycle enzymes can be treated pharmacologically

CO2-

CO2-

ATP + CoA-SH

ATP + CoA-SH

AMP + PPi

AMP + PPi

O

S-CoA

S-CoA

O

glycine

glutamine

CoA-SH

CoA-SH

O

H

CO2-

N

CO2-

N

H

O

NH2

Genetic deficiencies in some of the urea-cycle enzymes can be treated pharmacologically

phenylacetate

benzoate

benzoyl-CoA

phenylacetyl-CoA

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.