3 UREA CYCLE

Reactions of the Urea Cycle

Enzyme Regulation of the Urea
Cycle
Nutritional Regulation of Urea
Synthesis
Urea Cycle Disorders & Treatment

Urea Cycle
1. GDH is the major agency responsible for ammonium
production.
2. Ammonium is toxic (N = 15 - 40M , max 70M)
Urine: organic acids and orotic acid
3. Liver: Principal site but also in small intestine
4. Excretion NH4+ by kidneys important for acid-base balance
but Normally 80-90% N urine as urea.
5. Hyperammonium >500M plasma [NH4+] = TOXIC
related to inborn errors of metabolism (genetic defects) as
well as induced (liver failure)
Usually detected in the newborn period.
Blood: measure ammonium, AA, lactate

Nitrogen-containing components of normal urine

End Product

Excreted %

Urea

86.0

Creatinine

4.5

Ammonium

2.8

Uric acid

1.7

Other compounds

5.0

Urea Cycle
1. The urea cycle was the first metabolic process to be
described as a cycle by Sir Hans Krebs who also
described the TCA cycle.
2. Role of Urea cycle: rid the body of toxic NH 4 + therefore
permitting the use of AA as an energy source.
3. Liver major site of urea synthesis, major source of
arginase, (small amounts in small intestine) and is the
only tissue with the complete set of all 5 enzymes
required

4. Other tissues have enzymes for reactions (iii) and (iv)

only to make ARG or NO (important in blood pressure,
neuro transmission, macrophage antibacterial action)

Urea Cycle

Urea Cycle
I.

Compartmentation:
mitochondria (rxn 1&2) cytosol (rxn 3-5)

II. CP = 20% mitochondrial protein

III. Cyclic inter conversion of ornithine / arginine.
IV. Ornithine is used in the same way as is
oxaloacetate in the TCA cycle. It is the carrier
of a substituent group that undergoes
modification and is subsequently split off.

Mathematics of equation
1. 2 N per urea molecule:
1 NH4+ (start) + 1 transferred from ARG
2. 4 high energy phosphate: 2 ATP ADP + Pi
1 ATP AMP + Ppi
Therefore 2 ATP / amino (N) group
Overall catabolism:
Catabolize 1 Leu 32 ATP (from TCA cycle)
Make urea from N 2 ATP
NET ENERGY 30 ATP produced

Short term Regulation: CPS1

1. NAG(N-acetyl glutamate), a positive allsoteric regulator is absolutely
required.
Alters enzyme conformation
2. NAG is synthesized in liver mitochondria from acetyl CoA and GLU
FA or pyruvate acetyl CoA
Diet or tissue proteins AA GLU and ARG
Acetyl CoA + GLU NAG (enzyme = NAG synthase)
3. NAG synthesis is markedly stimulated by ARG (allosteric) but not
completely dependent ( V max) therefore AA NAG
4. Hyperammonemia that develops with acidemia NAG synthesis
inhibition (propionic acidemia, isovaleric acidemia, nethylmalonic
acidemia) due to competition for CoA (see figure)

Regulation via NAG

Regulation through Mg

2+

(i) Mg2+: CPS1 dependent Mg2+ ( both ATP and free)

Therefore changes in mitochondrial citrate can affect reaction
since citrate chelates Mg2+
(ii) Zn2+ is present in mitochondria
Zn2+ decreases CPSI activity in vitro
However, AA (ornithine) can chelate Zn therefore preventing
inhibition of CPS1.
(iii) CPS1 20% total liver protein (0.4 mM) [substrate] eg
NH4+, HCO3, ATP - Mg2+, NAG
Therefore not operating at maximum capacity and important to
inhibit to keep some NH4+ available to make GLN

Nutritional Regulation
long term regulation
(i) Five Urea Cycle enzymes & NAG synthase
all with low P diets & with high P diets
Therefore regulated nutritionally (over the long term)
(ii) Note also during starvation due to AA catabolism
therefore although muscle and liver protein the
level of these enzymes due to increased urea
synthesis
-increased enzyme synthesis
-decrease enzyme degradation
(iii) Changes take place over 3-7 days.

Urea Cycle Disorders

Prevalence of disorders: 1/30,000 live births but may be
more since some die undiagnosed.
Mode of inheritance = usually autosomal recessive (2 ve genes) OTC (most common) X-linked,
heterozygotes generally asymptomatic
(i) Deficiency enzymes rxn 1-4 hyperammonemia. In
general: concentration of AA metabolites proximal &
distal.
(ii) In all disorders: NH4, GLN, ALA
(iii) Less severe defects: (partial deficiencies) less
side effects, manifested only in later childhood or
adulthood.

Defects of Urea Cycle

orotic acid

V
III

IV

Presentation
Severe Illness: First week
Usually normal first 24h
Symptoms of hyperammonemia within 1-3 days
Include: Feeding intolerance
Vomiting
Lethargy
Irritability
Respiratory Distress (hyperventilation)
Seizures
Coma

Outcome
Mortality
Improvements in treatment have increased 1 year survival rate.
Once past the neonatal period, long term survival rate =
50% OTC (Type II)
75% CPS (Type I)
95% AS and AL (Steps 3+4)
Morbidity
75% mental retardation (mean IQ 50), Seizure disorders, Visual
deficits (proportional to extent of NH4 ), Protein intolerance
Brain: NH4 causes increased permeability and TRP
serotonin behavior abnormalities
quinolininc acid neuronal injury
Also with type V block Arg but ~ NH4+ severely retarded

Treatment: Reduce N Intake

Provide sufficient for growth (need EAA ) but avoid NH4 using a
high calorie low P diet
Provide ARG supplement (except type V) since ARG synthesis
therefore growth, N incorporation into AA therefore NH4
ARG also NAG synthase therefore CPSI (if not type I)
ARG also ornithine (ARG is precursor) especially important in
type III and IV (where citrulline & arginosuccinate are lost in
urine)
ARG also alternate NH4+ excretion (through alternate pathway)
Replacement with EAA (as keto acids to limit N intake) which can
be formed into AA through transamination

Treatment (contd)
1. Compounds to Conjugate AA:( urea load) (see
Diagram)
Benzoate: combines with GLY to generate hippurate
urine
Phenylacetate: +GLN to produce phenylacetyl GLN
urine
2. NAG Permeable Analog:
N carbamoyl glutamate enters mitochondrial.
3. Hemodialysis used to remove both AA & NH4 during
hyperammonemia coma

Treatment:
Stimulate Alternate Pathways

Stimulate Alternate Pathway:

ARG ornithine citrulline arginino succinate
Citrulline & argino succinate can be secreted in urine

Future
(i) Enzyme Replacement Therapy
(Liver Transplant) but expensive and lack donors

(ii) Gene Therapy In mice to date,

In OTC deficient mouse transfection using adeno
successful

virus vector is

(iii) Diagnosis
Molecular Diagnostics (RFLP) can reveal genetic defects by prenatal
diagnosis when indicated.
Direct enzyme determination in amniocytes or chorionic vilus
biopsy to determine presence/absence enzyme
Reactive or anticipatory treatment if defect suspected

Case #3 Discussion
A 6-month-old infant began to vomit occasionally and
ceased to gain weight. At age 8 months he was readmitted to
the hospital. Routine examination and laboratory tests were
normal, but after 1 week he became habitually drowsy, his
temperature rose to 39.4oC, his pulse was elevated, and his
liver was enlarged. The electroencephalogram was grossly
abnormal.
Since the infant could not retain milk given by gavage
feeding, intravenous glucose was administered. He improved
rapidly and came out of the coma in 24 hours. Analysis of his
urine showed abnormally high amounts of glutamine, uracil &
orotic acid but urea, which suggested a high blood ammonium
concentration. This was confirmed by the laboratory.

Discussion:
1. Hereditary hyperammonemia can result from defects in
genes for urea cycle enzymes. Which enzymes might be
affected?
2. Considering the data ( uracil & orotic acid) which enzyme
may be defective in this patient?
3. Why was the urine glutamine concentration elevated?
1. Hyperammonemia is characteristic of all steps
(including NAG synthase) Most frequent OTC
2. N BUN ( blood urea N), ALSO uracil (& orotic
acid) due to carbamoyl phosphate which leaks from
mito cyto increased pyrimidine synthesis.
Unusual: clinical symptoms slow (6 months old)
3 Why? Exceeds kidneys ability GLN GLU + NH4+

Contd
4. Offer a genetic explanation for the observation that this disease
is usually lethal in males but not in affected females.
5. This patient was treated using procedures available at the time.
He was given a daily diet of 1.5 g of protein/kg body weight. After 2 years
on this diet, his height and weight were judged to be normal for his age.
What is the effect of diet on a growing child in terms of nitrogen balance?
6. How would you treat a similar patient today?

4. Disease is x linked, men have only 1 X chromosome,

women have two X chromosomes. Therefore more severe in
men than women (usually).
5. Growing child requires increased N, therefore load on urea
P diet. Balance between P restriction (prevent NH4+) and
enough for growth. Not usually sufficient for patients -ve OTC
6. Hemodialysis / transfusion asap (prevent brain damage)
IV benzoate, phenylacetate to act as NH4 traps