Urea Cycle Disorders

Question 1

Citric acid cycle

Answer 1

-Excess amino acids that are not used for the synthesis of nitrogen containing molecules are broken down via amino acid degradation pathways
-Amino acid carbon skeletons are metabolised by citric acid cycle and is useful energy source

Question 2

Urea cycle purpose

Answer 2

Nitrogen component of aa in the form of ammonia has to be eliminated before accumulation to toxic levels and this occurs by the citric acid cycle

Question 3

Urea cycle mechanism

Answer 3

-carried out primarily in liver which converts ammonia to urea which is excreted out as urine
Ammonia is transported in blood from other tissues, not as NH4+ but rather in safe form of glutamine and alanine

Question 4

Glutamine synthesis

Answer 4

Glutamine is synthesised from glutamate by glutamate synthetase and ammonia
it is generated from multiple aa by transaminases

Question 5

Alanine synthesis

Answer 5

Alanine is synthesized from a transaminase reaction via alanine transferase

Question 6

Urea cycle process

Answer 6

Question 7

Normal ammonia levels

Answer 7

15-60micrograms/dL

Question 8

Urea cycle disorders (UCD)

Answer 8

UCD are inborn errors of metabolism resulting from defects in one of the enzymes of the urea cycle or its transporter molecules
UCD lead to accumulation of ammonia

Question 9

Lethality of ammonia build up

Answer 9

Ammonia is extremely toxic, especially to CNS
-newborns with severe mutations in any one of four enzymes of urea cycle can develop severe symptoms as a result of toxic ammonia buildup in blood stream within 36 to 48hr of birth

Question 10

`150-200 micro grams/dL

Answer 10

Hyperammonaemia
-seizures
-loss of consciousness
-Enephalopathy

Question 10

> 100micro grams/dL

Answer 10

Hyperammonaemia
-loss of appetite
-nausea
-insomnia
-Agitation
-Personality change
-Mild enephalopathy

Question 11

200-400 micro grams/dL

Answer 11

Hyperammonaemia
-severe coma
-Respiratory failure
-CNS damage
-Enephalopathy

Question 12

Carbamoyl Phosphate Synthase I deficiency

Answer 12

This is a recessive autosomal disorder
Mutations in CPS1 gene result in defective enzyme

Question 12

citrullinemia and hyperammonemia

Answer 12

-Mutations ins SLC25A13 which codes for citrin also results in citrullinemia and hyperammonemia
-Also the aspartic acid and glutamate transporter citrin transports aspartic acid from mitochondria to cytoplasm where it is needed for urea cycle
-Citrin also transports glutamate from cytoplasm to mitochondria

Question 13

Arginosuccinate synthetase deficiency

Answer 13

Defects in ASS1 in both citrullinemia and hyperammonemia

Question 13

Ornithine Transcarbamylase deficiency

Answer 13

-Most common UCD
-Enzyme is encoded by OTC gene which is on X chromosome so this is an X-linked disease
-Boys are severely affected by deleterious mutations whereas girls have a spectrum of disease ranging from asymptomatic to severe
-Depends on random nature of X inactivation
-Ornithine is transported into mitochondrion by ornithine translocase (ORNT1) protein. Defective ORNT1 also causes hyperammonemia which manifests later in life

Question 14

Argininosuccinate lyase deficiency

Answer 14

Defects in ASL can cause severe UCD in neonates as well as an adult onset form
-Elevated plasma levels of both citrulline and argininosuccinate are characteristic of ASL deficiency

Question 15

Arginase 1 deficiency

Answer 15

Mutations in ARG1 result in both argininemia and hyperammonemia.
-This is a reccessive autosomal disease
-Symptoms are hypotonia, seizures, stunted growth and dehydration

Question 16

Hyperammonemia mechanisms (pathophysiology)

Answer 16

-Acute hyperammonemia (HA) is more toxic to developing CNS then to adult CNS and so is more toxic in young children
-exact mechanism of CNS damage unknown
-Damages multiple neurotransmitters in brain
-Increases extracellular glutamate levels in brain which causes decreased phosphorylation of protein Kinase C that results in activation of Na/K-ATPase which depletes nerve cells of ATP
-It causes cerebral oedama and this occurs due to swelling of astrocytes which are involved in ammonia detoxification in brain

Question 17

Astrocytes

Answer 17

Specialised glial cells in CNS that perform a number of homeostatic functions such as removal of excess neurotransmitters

Question 17

Gene therapy approach

Answer 17

-Involves insertion of a DNA construct that expresses the relevant urea cycle gene into a viral vector. the vector is incapsulated into a viral capsid and these engineered viral particles are injected into the patient
-The particles are designed so that they are taken up by liver cells
-The concept is that the DNA construct introduced in nucleus of hepatocytes will express UC enzyme which cures the disease
-has been seen asn an approach for 30 yrs

Question 18

Treatment of Hyperammonemia

Answer 18

1)Restriction of protein in diet
2) Use of intravenous drugs that sequester nitrogenous waste and facilitate removal by kidneys, independently of urea cycle
3) Haemodialysis
4) Liver transplantation

Question 19

E.Intravenous drugs

Answer 19

2a) Sodium benzoate (forms hippuric acid by conjugating to glycine), reduces nitrogenous waste by reducing glycine ‘nitrogen’
2b) Phenylacetate (forms phenylacetylglutamine by conjugating to glutamine), reduces nitrogen in form of glutamine

Question 20

history of UCD treatment

Answer 20

-First clinical trial in late 1990s using adenoviral vectors
-In 1999, 18 year old patient named Jesse Gelsinger was injected with large amounts of adenovirus-based expression system coding for OTC in clinical trial
-He developed severe inflammation as a result of runaway immune response to vector known as cytokine storm and died due to multiple organ failure
-This caused other viral vector appraoches to be used such as AAV vectors which do not elicit strong immune response

Question 21

Adenoviruses

Answer 21

-Non-enveloped double-stranded DNA viruses and the most common serotype is serotype 5 which has high liver tropism
-adenoviruses trigger strong immune response