Transcript Nitrogen-containing components of normal urine

# 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 - 40M , max 70M)
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 >500M 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 Mg2+
(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 (cont’d)
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
virus vector is successful
(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 8½ 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+
Cont’d
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