Metabolic Disorders of Urea Cycle Metabolic & Molecular Basis of Inherited Disease

Metabolic Disorders of Urea Cycle

Metabolic & Molecular Basis of Inherited Disease



Most mammals convert amino-acid nitrogen to urea for excretion

NH4+

ammonium ion

uric acid

H2N-C-NH2 urea

O

NH4+

O

O

HN

NH

NH

NH

O

most terrestrial vertebrates

birds & reptilesfish & other aquatic vertebrates

Some animals excrete NH4

+ or uric acid.

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



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



4

Major fate of waste nitrogen

O

H2N C NH2urea



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

NH4+

pKa = 9.3

NH3 + H+



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 +.



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.



Ammonia rapidly equilibrates across membranes

NH4+

NH3

pKa = 9.3

NH4+

NH3

Lipid Bilayer



AMMONIA METABOLISMThe 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)



Production of ammonia

NH4+

Amino acids -Ketoglutarate

-Keto acids

-Ketoglutarate

Glutamate GDH

Oxaloacetate

Aspartate Urea cycle

Urea

Other reactions



Summary of sources of ammonia for urea cycle



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

Glutamate is not deaminated in

peripheral tissues



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

Waste N funnelled to pyruvate via transaminations

Net: N (muscle) Urea (liver)

Glucose – Alanine Cycle



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.

2. Nitrogen can be transported as glutamine.

Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH4

+ in an ATP-dependent reaction:



17

Synthesis of Glutamine in Peripheral Tissue and Transport to the Liver





UREA FORMATION



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



Overview

Key reaction: hydrolysis of arginine

Arginine + H2O ==> urea + ornithine arginase

Resynthesis of Arginine



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

COOH

NH

NH2

NH

C

NH2

COOH

CH2

CH2

CH2

CH

H2ONH2

NH2OC

CH2NH2

CH2

COOH

H

COOH

CH2

COOH

CH

CH2

NH2

O

NH2

NH

C

NH2

COOH

CH2

CH2

CH2

CH

CH2NATP

NH

NH

C

NH2

COOH

CH2

CH2

CH2

CH

NH CH

COOH

COOH

CH2

HC

COOH

CH

O

NH2

CH2NH2

COOH

NH2

NH

CH2

C

CH2

NH2

C

COOH

CH2

CH2

H

CH2

CH

O-

O-

O

POC

O

CO2 + NH4

H2N

2 ATP

Mitochondrion

+

ADP + Pi

carbamoylphosphate

synthetase I(CPSI)

1

Carbamoyl phosphate

Ornithineornithine

transcarbamoylase

Pi

Citrulline

2

Ornithine

Citrulline

Aspartate

AMP + PPiArgininosuccinate

Fumarate

Arginine

3

4

5

argininosuccinatesynthetase

argininosuccinase

Urine

Cytosol

urea

2



Urea cycle - a cyclic pathway of urea synthesis first postulated by H.Krebs

THE UREA CYCLE

The sources of nitrogen atoms in urea molecule:- aspartate;- NH4

+.

Carbon atom comes from CO2.



urea

arginine NH2+ NH3

+

H2N-C-NH-CH2CH2CH2CH-CO2-

H2O

argininosuccinate

-O2C-CH=CH-CO2- fumarate

-O2C-CH2CH-NH3+

CO2- Asp

The urea cycle

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

NH3+CO2

- NH2+

OH2N-C-NH2

ATP

AMP + PPi

HCO3-

NH4+

2 ATP 2 ADP + Picarbamoyl phosphate

ornithine

citrulline

NH3+

H2N-CH2CH2CH2CH-CO2-

NH3+


OPi

H2N-C-O-P-O-

O

O-

Omitochondria

cytosol



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

NH4+ + HCO3

-

2 ATP 2 ADP + Pi

H2N-C-O-P-O-

O O

O-

carbamoyl phosphate

carbamate

HCO3-

ATP ADP

Pi

NH4+

ATP ADP

carbonic-phosphoric acid

anhydride

O O

O-

HO-C-O-P-O-

H2N-C-O-

O

(1)

(2)

(3)H2N-C-O-P-O-

O O

O-

Incorporation of ammonia into urea begins with formation of carbamoyl phosphate



carbamoyl phosphate

Pi

ornithine

citrulline

This step also occurs in the mitochondrial matrix.

Carbamoyl phosphate reacts with ornithine to form citrulline

+ H+

H2N-C-O-P-O-

O O

O-

NH3+

+H3N-CH2CH2CH2CH-CO2-

NH3+


O



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.

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

ATP

AMP + PPi + H2O

aspartate

argininosuccinate

citrulline

NH3+


O-O2C-CH2CH-NH3

+

CO2-


NH3+CO2

- NH2+



This reaction occurs in the cytosol.

Argininosuccinate splits into arginine and fumarate

-O2C-CH=CH-CO2-

fumarate

argininosuccinate

arginine


NH3+CO2

- NH2+

NH2+ NH3

+




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

Hydrolysis of arginine releases urea and regenerates ornithine

arginine

H2O

NH2+ NH3

+


urea ornithineH+

NH3+

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

OH2N-C-NH2



urea

argininosuccinate

-O2C-CH2CH-NH3+

CO2-

Asp

Formation of urea consumes 4 phosphate anhydride bonds


NH3+CO2

- NH2+

OH2N-C-NH2

ATP

PPi + AMP

HCO3-

NH4+

carbamoyl phosphate

citrulline

NH3+


O

Pi

H2N-C-O-P-O-

O O

O-

ornithine

2 Pi

H2O

2 ATP 2 ADP + Pi



Input-Output

2 ADP

1. NH4+ + CO2 + 2 ATP

2. carbamoyl phosphate + ornithine

3. citrulline + aspartate + ATP

4. AMP + ATP

5. PPi + ATP

6. argininosuccinate

7. arginine + H2O

SUM: NH4+ + CO2 + 4 ATP + aspartate

2 ADP

carbamoyl phosphate + 2 ADP + Pi

citrulline + Pi

argininosuccinate + AMP + PPi

arginine + fumarate

urea + ornithine

urea + fumarate + 4 ADP + 4 Pi



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

urea

carbamoyl phosphate

arginine

ornithine

citrulline

argininosuccinate

HCO3- +

NH4+

ATP

AMP + PPi

2 ATP 2 ADP + Pi

fumarate

H2O

malate

aspartate

oxaloacetate

NAD+

NADH

-keto acids amino acids

aspartate-oxaloacetate

aminotransferase

Urea cycle

malate dehydrogenase

Pi

This process also uses both cytosolic and mitochondrial enzymes



Oxidation of malate in mitochondria generates ATP

cytosol

urea

fumarate

carbamoyl phosphate

arginine

ornithine

ornithine citrulline

citrulline

argininosuccinate

aspartate

malate

NADH

NAD

malate

-ketoglutarate glutamate

amino acids -ketoacids

aspartate

oxaloacetate

glutamate

-ketoglutarate

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

ATP

AMP + PPi

2 ATP 2 ADP + Pi

NADH, NAD+ and oxaloacetate can’t cross the mitochondrial inner membrane, but there are transporters for malate, aspartate, glutamate and -ketoglutarate.

H2O

Pi

mitochondrionHCO3

-

+ NH4+



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

malate

mitochondrion

cytosol

aspartate- malate-ketoglutarateglutamate- + H+

aspartate- malate-ketoglutarate

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.

glutamate- + H+



Alpha--ketoglutarate/malate and aspartate/glutamate transporters also participate in oxidation of cytosolic NADH

cytosol

aspartate malate-ketoglutarateglutamate

oxaloacetate

NAD+

NADH

glycolysis

oxaloacetate

NADH

aspartate malate-ketoglutarateglutamate

mitochondrion

NAD+

2 e- to electron-transport chain



Well Fed State

NH4

Asp

OAA

Glu KAs

a.a.'s

a.a.'s

CO2 + H2O

KG

+

Mal

ureacycle

urea

fumarate

malate

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



Fasted State

pyr Glu

OAA

Asp

NH4

OAA

ala

ala

Glucose

KG

+ureacycle

urea

fumarate

malate

2 ala + CO2 1 urea + 1 glucose

Gluconeogenesis



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

NADH

NAD

NH3

NAD

NADH

NH3

(a) NH3 in excess

-Ketoglutarate

Glutamatedehydrogenase

GlutamateAspartate

transaminase

Oxaloacetate Aspartate

Carbamoylphosphate

Citrulline

Urea

Urea Cycle

(b) Aspartate in excess

-KetoglutarateGlutamatedehydrogenase

GlutamateAspartate

transaminase

Oxaloacetate Aspartate

Carbamoylphosphate

Citrulline

Urea

Urea Cycle



The urea cycle is regulated in two ways

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

CO2-

CO2-

+H3N C H

CH2

CH2

+ acetyl-CoA

CO2-

CO2-

CH3CO-NH C H

CH2

CH2CoA-SH

N-acetylglutamate

carbamoylphosphate

NH4+ + HCO3

-

2 ATP 2 ADP + Pi

OH2N-C-O-P-O-

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.

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.



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: (1) transamination to aspartate, (2) conversion into glucose,(3) condensation with acetyl CoA to form citrate,(4) conversion into pyruvate.



April 20, 2023 Total slide : 50 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.



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



The urea cycle Asterisk = N-acetyl glutamate synthetase; 1 = carbamyl phosphate synthetase; 2 = ornithine transcarbamylase; 3 = argininosuccinate

synthetase; 4 = argininosuccinate lyase; 5 = arginase



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



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 ResultsAt 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 HistoryTwo 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

Low BUN

Low blood arginine

Undetectable blood citrulline

Elevated blood ammonia

Elevated blood glutamine

Elevated orotic acid



COOH

NH

NH2

NH

C

NH2

COOH

CH2

CH2

CH2

CH

H2ONH2

NH2OC

CH2NH2

CH2

COOH

H

COOH

CH2

COOH

CH

CH2

NH2

O

NH2

NH

C

NH2

COOH

CH2

CH2

CH2

CH

CH2NATP

NH

NH

C

NH2

COOH

CH2

CH2

CH2

CH

NH CH

COOH

COOH

CH2

HC

COOH

CH

O

NH2

CH2NH2

COOH

NH2

NH

CH2

C

CH2

NH2

C

COOH

CH2

CH2

H

CH2

CH

O-

O-

O

POC

O

CO2 + NH4

H2N

2 ATP

Mitochondrion

+

ADP + Pi

carbamoylphosphate

synthetase I(CPSI)

1

Carbamoyl phosphate

Ornithineornithine

transcarbamoylase

Pi

Citrulline

2

Ornithine

Citrulline

Aspartate


Fumarate

Arginine

3

4

5


argininosuccinase

Urine

Cytosol

ureaNH4+ + Glu Gln

Carbamoyl P orotic acid



Carbamoyl P synthetase deficiency



COOH

NH

NH2

NH

C

NH2

COOH

CH2

CH2

CH2

CH

H2ONH2

NH2OC

CH2NH2

CH2

COOH

H

COOH

CH2

COOH

CH

CH2

NH2

O

NH2

NH

C

NH2

COOH

CH2

CH2

CH2

CH

CH2NATP

NH

NH

C

NH2

COOH

CH2

CH2

CH2

CH

NH CH

COOH

COOH

CH2

HC

COOH

CH

O

NH2

CH2NH2

COOH

NH2

NH

CH2

C

CH2

NH2

C

COOH

CH2

CH2

H

CH2

CH

O-

O-

O

POC

O

CO2 + NH4

H2N

2 ATP

Mitochondrion

+

ADP + Pi

carbamoylphosphate

synthetase I(CPSI)

1

Carbamoyl phosphate

Ornithineornithine

transcarbamoylase

Pi

Citrulline

2

Ornithine

Citrulline

Aspartate


Fumarate

Arginine

3

4

5


argininosuccinase

Urine

Cytosol

ureaNH4+ + 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.

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

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



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.



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.




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.



Liver transplantation for Severe neonatal OTC and CPS deficiency. Liver failure and cirrhosis in ASL deficiency. Failed medical-pharmacologic treatment.

Pretransplant care byaggressively 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

benzoate

benzoyl-CoA

hippurate (benzoylglycine)

CO2-

ATP + CoA-SH

AMP + PPi

glycine

CoA-SH

phenylacetate

phenylacetyl-CoAO

S-CoA

phenylacetyl-glutamine

ATP + CoA-SH

AMP + PPi

glutamine

CoA-SH

The amide products of these reactions (hippurate and phenylacetylglutamine) are excreted in the urine. Replenishing the Gly or Gln removes ammonia.

CO2-

O

S-CoA

O

NH

CO2-

O

N

NH2

O

HCO2

-

Documents

Metabolic Disorders of Urea Cycle Metabolic & Molecular Basis of Inherited Disease