FOR SEMINAR

1. vvydehindraneel
1st YEAR PG
BIOCHEMISTRY
OSMANIA MEDICAL COLLEGE
HYDERABAD
INDIA

3. Hyperammonemia
a metabolic disturbance
characterised by an excess
of ammonia in the blood

6. Ammonia is a normal constituent of all body
fluids.
At physiologic pH, it exists as ammonium ion.
adults 15 - 45g/dL or 11 - 32 mol/L
Children 40 - 80 g/dL or 28 - 57 mol/L
Newborns 90 - 150 g/dL or 64 -107 mol/L

7. Sources of ammonia
1. bacterial hydrolysis of urea and
2. nitrogenous compounds in the
intestine
3. the purine-nucleotide cycle
4. amino acid transamination in
skeletal muscle,
5. metabolic processes in the kidneys
and liver

8. Ammonia is produced from
dietary AA and
by catabolism of
1. amino acids,
2. amines,
3. nucleic acids,
4. glutamine
5. glutamate (nitrogenous wastes)
(skeletalmuscle).

9. coliforms ,anaerobes (colon ,
cecum) convert dietary AA and urea
into ammonia
The ammonia is absorbed into the portal
circulation, taken up by the liver and
converted in the liver, via the urea cycle,
into urea.

10. Urea is then excreted into the
gastrointestinal system (producing a
futile cycle) and into the urine
Of total ammonia ,
80-90% into the urea cycle,
10-20% metabolised by kidney,
heart, and brain.

12. 1. It is a product of the catabolism of
protein.
2. It is converted to the less toxic
substance urea prior to excretion in
urine by the kidneys.
3. The metabolic pathways that synthesize
urea are located first in the
mitochondria and then into the cytosol.
4. The process is known as the urea cycle,.

13. Types
Primary vs. secondary
Primary hyperammonemia
is caused by several inborn errors of
metabolism that are characterised
by reduced activity of any of the
enzymes in the urea cycle.

14. •Secondary hyperammonemia is caused
by inborn errors of intermediary
metabolism characterised by reduced
activity in enzymes that are not part of the
urea cycle e.g.
•.Propionic acidemia,
• Methylmalonic acidemia
•or dysfunction of cells that make major
contributions to metabolism (e.g. hepatic
failure).

15. N-acetylglutamate synthetase deficiency
carbamoyl phosphate synthetase I
ornithine transcarbamylase
Specific types
Type I Hyperammonemia
Type II Hyperammonemia
Hyperammonemia, type III

16. hyperinsulinism
hyperammonemia syndrome
(glutamatedehydrogenase 1)
hyperornithinemia-
hyperammonemia-homocitrullinuria
syndrome (ornithine translocase
hyperlysinuria with hyperammonemia

17. • Methylmalonic acidemia
• Isovalemic acidemia
• Propionic acidemia
• Carnitine palmitoyl transferase II
deficiency
• Transient hyperammonemia of the
newborn, specifically in the
preterm.

19. Causes
Enzyme defects in urea cycle
N -Acetylglutamate Synthetase (Nags)
Deficiency:
↓Nags ↓Nag
Nag is an activator of CPS I
autosomal recessive.
if ↓ acetyl-CoA ↓ NAG

20. Carbamoyl phosphate synthetase I
(CPS I) deficiency:
nag
HCO3 + NH4 ----------------- CARBAMOYL PHOSPHATE
CPS I
2atp mg 2adp + pi

21. • Autosomal recessive
• short arm of chromosome 2
• hepatic mitochondria.
• first day of life.
• infants die in the neonatal period

23. 1. Ornithine transcarbamoylase (OTC)
deficiency: ORNITHINEMIA
mitochondria.
carbamoyl po4 + ornithine ------- ----citrulline
OTC
Citrulline is then transported out of the
mitochondria.

24. absence of enzyme,carbamoyl
phosphate enters cytosol participates
in pyrimidine synthesis in presence of
CPS II.
1. most common urea cycle
defect
2. incidence of 1 case in 14,000
persons.
3. X-linked trait

25. . Neonatal onset is seen in males who have
null mutations , thus no residual enzyme
activity.
Males who and females who are heterozygous
for OTC deficiency
have significant residual enzyme activity
present later with quite variable clinical
pictures.

26. Thus, as many as 60% of OTC deficiency
diagnoses are made in non-neonates. The
oldest reported patient was aged 61 years
Mother of affected child also ehibits
hyperammonemia and aversion to protein
foods
Blood ,urine ,csf :↑glutamine,
↑ ammonia,
↑ ornithine

32. Argininosuccinic acid synthetase
(AS) deficiency: CITRULINEMIA
Citrulline combines with aspartate to
form argininosuccinic acid.
AS deficiency results in citrullinemia.
Onset is usually between hours 24 and
72 of life,
autosomal recessive.
chromosome 9.

33. TWO TYPES OF DEFICIENCY:
ONE TYPE :mutation in regulatory gene
: enzyme is absent in liver .normal for
citulline
Other type : mutation in structural
gene . Amount of enzyme normal in
liver .affects catalytic site and has
abnormally high km value for citrulline

34. Clinically : mental retardation ,
ammonia toxicity
Biochemically : blood and csf : increased
ammonia , ↑citrulline
Urine : large quantities (1—2gm/day ) of
citrulline excreted
Feeding arginine in these patients enhances
citulline excretion

35. Argininosuccinic lyase (AL) deficiency:
1. This enzyme cleaves argininosuccinic acid
to yield fumarate and arginine.
2. The lack of this enzyme leads to
argininosuccinic aciduria.
3. It is the second most common urea cycle
disorder.
4. chromosome 7. autosomal recessive

36. Enzyme deficiency seen in
liver ,kidney brain , RBC.
Early diagnosis can be made by
demonstrating by demonstrating
enzyme defeciency
in RBC from cord blood
and in amniotic fluid by amniocentecis

37. 1. Terminates fatally in early life.
2. Symptoms appear in the neonatal
period or later in life. (2 yrs)
3. Abnormally fragile hair (trichorrhexis
nodosa) observed in these infants of
age 2 weeks.

39. Arginase deficiency:
the final step
arginine---------urea and ornithine.
argininemia,
neurotoxicity
chromosome 6q23.
least frequent

40. •Hyperammonemia is not severe
•uneventful.
•progressive spastic diplegia
• quadriplegia,
• intellectual impairment,
•recurrent vomiting,
•delayed growth,
• seizures.

41. hyperinsulinism hyperammonemia
syndrome (glutamatedehydrogenase 1)
PATIENT: Since the neonatal period, a
white girl had been treated for
hyperammonemia and postprandial
hypoglycemia with intermittent
hyperinsulinism.
ammonia 100 to 300 micromol/L and
was independent of the protein intake.

42. METHODS:
1. Enzymes of the urea cycle
2. glutamine synthetase,
3. glutamate
dehydrogenase (GDH)
were assayed in liver
and/or lymphocytes.

43. RESULTS:
The activity of hepatic GDH ↑
ratio glutamine/blood ammonia low.
Oral -N-carbamylglutamate resulted in
↓ ammonia.

44. CONCLUSION:
glutamate ----------GDH----------------------2-oxoglutarate
↓ glutamate
needed for the synthesis of N-acetylglutamate,
the catalyst of the urea synthesis
↓ glutamate ----------------------↓ glutamine
by glutamine synthetase.
GDH stimulates the release of insulin,.

45. Organic acidemias
ketosis , acidosis , hyperammonemia
accumulation of CoA derivatives of
organic acids, which inhibit the
formation of NAG
the activator of CPSI in liver.

46. Disorders in this group include the
following:
Isovaleric acidemia
Propionic acidemia
Methylmalonic acidemia
Glutaric acidemia type II
Multiple carboxylase deficiency
beta-ketothiolase deficiency

47. Congenital lactic acidosis
↑lactate (10-20 mmol/L),
↑ lactate/pyruvate ratio,
metabolic acidosis, ketosis.
Hyperammonemia and
citrullinemia in some cases.
Pyruvate dehydrogenase deficiency
Pyruvate carboxylase deficiency
Mitochondrial disorders

48. ↓PYRUVATE DEHYDROGENASE
↓ acetyl CoA
↓ NAG

50. Fatty acid oxidation defects
Deficiency of medium- or long-chain acyl
CoA dehydrogenase
hyperammonemia
secondary to hepatic dysfunction.
.

51. Systemic carnitine deficiency:
Carnitine :transport of long-chain fatty
acids into mitochondria.
↑liver transaminases,
hepatomegaly
↑ammonium
liver dysfunction.

52. Dibasic amino acid transport defects
Lysinuric protein intolerance
hyperlysinuria with hyperammonemia
↓ membrane transport
↓ lysine, ↓ ornithine ↓ arginine.
Citrulline( orally)------------ ↓ammonia
it is transported by a different mechanism in
intestine.

53. Hyperammonemia-
hyperornithinemia-
homocitrullinuria (HHH)
1. first few weeks of life
2. seizures,
3. feeding difficulty,
4. altered level of consciousness.

54. A defect in transport of ornithine
cytosol ----│--- → mitochondria
ornithinemia↑
disruption of the urea cycle causes
↑ ammonemia.
In absence of ornithine,
mitochondrial→ carbamoyl po4 +
lysine------→homocitrulline

55. Transient hyperammonemia of the newborn
premature
day 1,day2
before introduction of protein
Hyperammonemia ↑ ↑ ↑
hemodialysis.
30% die
35-45% ↓neurologic development.
slow maturation of the urea cycle function.

56. • seizures
• ↓consciousness,
• fixed pupils
•loss of oculocephalic reflex.
HIE, ICH

57. first 24 hours of life.
↑ ↑ ↑ ammonia
elevated SGOT
Asphyxia

58. Reye syndrome
• acquired
• influenza A or B or
• varicella
• aspirin ingestion.
• cerebral and
•hepatic dysfunction—
•.

59. •vomiting,
• altered level of consciousness,
•seizures,
•cerebral edema, and
•hepatomegaly without jaundice.
•↑ liver transaminases,
• hyperammonemia,
• lactic acidosis

60. Renal
Urinary tract infection with a urease-
producing organism, such as Proteus
mirabilis,
Corynebacterium species, or
Staphylococcus species, can produce a
hyperammonemic state.
high urinary residuals and an
alkaline pH.

61. Other causes
neonatal herpes simplex pneumonitis,
The increase in ammonia level resulted from protein
catabolism caused by prolonged hypoxia.
Parenteral hyperalimentation: Increased nitrogen
load in patients receiving parenteral alimentation can
cause hyperammonemia.
thyroid disease and Hashimoto encephalopathy
Hyperammonemia is a rare but severe complication of
multiple myeloma and is with high mortality.[17]

62. Drugs
Valproate
topiramate
Carbamazepine
Salicylate

63. Pathophysiology
↑ ammonia
↑ECF glutamate in the brain
↑N -methyl D-aspartate (NMDA)
receptor.
↓ATP

64. ↓ phosphorylation
by protein kinase C.
↑ Na+/K+ -ATPase
↓ATP depletion
Activation NMDA receptor →
seizures

66. .
↓
gap–junction channel connexin 43,
water channel aquaporin 4
K channel,
∆ K and water transport
brain edema.

67. ∆ astrocyte morphology

68. p53, a tumor suppressor protein
transcriptional factor,
Activation of p53
astrocyte swelling
glutamate uptake ↓
brain edema

69. ↑lactate,
pyruvate,
glutamine,
glucose,
↓
glycogen,
ketone bodies,
glutamate.

70. ↑inhibitory neurotransmission as
a consequence of 2 factors.
↑ glutamate
↓ glutamate receptors

71. ∆ glutamate-nitric oxide-cGMP
pathway
∆ signal transduction
associated with NMDA
receptors
cognition and learning

73. ↑GABAergic tone from
BZDreceptor overstimulation
Activation of GABA(A) receptors
reduces the function of the
pathway.
↓ intellectual function,
↓consciousness,
coma.

74. ↑ the transport of aromatic AA
(eg, tryptophan) across BBB
↑ serotonin,
anorexia

75. Astrocyte edema
↑ROSand
↑NO species,
RNA oxidation
↑ intracellular zinc.
RNA oxidation
↓postsynaptic proteins involved in
learning and memory consolidation

76. Epidemiology
1 in 25,000 live births

77. Presentation Family history
unexplained neonatal deaths or
undiagnosed chronic illness.
males affected Suggestive of OTC
deficiency, X-linked trait
.
Consanguinity
↑risk of inheriting disorder.

78. Early-onset :
neonatal period.
The baby is well day 1,2
lethargy, irritability, poor feeding, vomiting.
hyperventilation ,grunting respiration,
seizures
ammonia level of 100-150 µmol/L,
2-3 times the reference range.

79. Late-onset hyperammonemia
typically is due to urea cycle disorders,
present later in life.
Adults with partial enzyme deficiency
become symptomatic
postpartum stress,
heart-lung transplant,
short bowel and kidney disease,
parenteral nutrition with high N intake,
gastrointestinal bleeding.

80. Intermittent ataxia:
unstable gait ,dysmetria.
periodic ↑ammonia
Intellectual impairment: Episodic
hyperammonemia may produce subtle
intellectual deficits even in clinically
asymptomatic individuals.
Failure to thrive:
( poor feeding and frequent vomiting )

81. Gait abnormality:
In arginase deficiency,
spastic diplegia, ( toe walking)
Behavior disturbances:
sleep disturbances,
irritability,
hyperactivity,
manic episodes,
psychosis.
Epilepsy
Intractable seizures in a few patients
secondary to urea cycle defect

82. Recurrent Reye syndrome
Episodic headaches
cyclic vomiting
Protein avoidance: Females with
OTC deficiency

83. Physical
• Dehydration ← vomiting
Tachypnea : stimulation of the
medullary center of respiration by
the ammonium ion
• Hypotonia ← acute stress

84. Bulging fontanelle :↑ICP
odor of "sweaty feet" in isovaleric
acidemia
abnormally fragile hair in
argininosuccinic aciduria.
Infants with argininosuccinic lyase ↓:
hepatomegaly

86. Other diagnostic considerations
the neonatal period : nonspecific c/f
c/f indicate distress
1. sepsis,
2. intracranial hemorrhage,
3. cardiac disease,
4. gastrointestinal obstruction
should be ruled out
Plasma ammonium level should be determined
in all such scenarios.

87. NH3 >200 µmol/L),
1. Plasma and urinary amino acids
2. Urinary organic acids
3. Serum glucose
4. Arterial blood gases
5. Bicarbonate
6. Lactate
7. Citrulline , acylcarnitine
8. Urinary ketones
9. Urinary orotate

88. Arterial blood gas analysis:
acid-base status
respiratory alkalosis → a urea cycle defect
stimulation of the central respiratory drive
↓
hyperventilation

89. Serum amino acid tests
↑ Glutamine , alanine ↑
in all urea cycle defects except for
arginase deficiency.
↓Citrulline
mildly in CPS/NAGS and OTC
deficiencies but
↑markedly in AS deficiency and
moderately in AL deficiency.

90. Arginine level
↑arginase deficiency
↓mildly in all the other enzyme
deficiencies of the urea cycle.
Argininosuccinic acid level
↑ in AL deficiency

91. Urinary orotic acid tests:
↑markedly in OTCdeficiency
↑mildly in other enzyme
deficiencies
for CPS/NAGS deficiency, in
which it is ↓ mildly

92. Urinary ketone tests:
ketosis indicates an organic acidemia
Plasma and urinary organic acid tests:
These levels screen for an organic acidemia

93. Enzyme assays:
tissue specimens obtained by
percutaneous liver biopsy
CPS, NAGS, and OTC deficiency
red blood cells
(for arginase deficiency),

94. fibroblast from skin biopsy
(ASS, ASL, and HHH),
intestinal mucosa
(CPS, OTC).
replaced by genetic analysis.
It is still indicated in selected cases
with negative genetic testing

95. DNA mutation analysis is the method of choice
in confirming the diagnosis of UCD as it is
clinically available for all genes of the urea cycle.
Heterozygote identification in OTC-
deficient pedigrees

96. Allopurinol loading test:
establishes the carrier status of
women at risk for OTC deficiency.
After a loading dose of allopurinol,
urinary orotidine excretion is ↑
greatly in carriers.
DNA analysis: determine the presence of
a mutation at the OTC locus.

97. Antenatal diagnosis:
DNA analysis on
chorionic villus or
amniotic fluid cells,
measurements of amniotic fluid
metabolites or enzyme activities in the
amniotic cells, chorionic villi,
fetal liver, fetal RBC

98. Imaging Studies
•Neuroimaging: CT or MRI of the brain
cerebral edema
chronic liver disorders is
hyperintense signal in the globus
pallidum due ↑manganese.

99. Diffuse Brain Edema
Findings: Absence of sulcal markings and poor
differentiation of white and grey matter.

100. •MR spectroscopy:
↑glutamine/glutamate
↓ myoinositol and choline signals.
diffusion tensor imaging
damage to corticospinal tracts :arginase
deficiency.[
Multiple strokelike lesions
MRI finding in a patient with HHH
syndrome

101. Histologic Findings
prominent Alzheimer type II astrogliosis

102. Assay
Measurement of ammonia is
problematic as it is very unstable.
Arterial blood samples are
preferable to venous blood samples
as results are more consistent.
Heparinized plasma samples are
preferable to serum

103. Due to the instability of ammonia
and leakage of ammonia from RBC,
samples need to be assayed for
ammonia as soon as possible after
sample collection and maintained
at 4 C (or on ice) until assay
(stable for a maximum of 3 hours
under these conditions)

104. The sample must be separated from
cells as soon as possible as leakage
of ammonia from erythrocytes
occurs within 30 minutes, resulting
in artifactually high values. This is not
easy to accomplish under most
situations, therefore ammonia assays
are not routinely performed.

105. . Furthermore, any ammonia in the
environment (air, water supply) can
contribute to the ammonia in the
patient sample.
Control samples (from a clinically
healthy animal) should always be run
in conjunction with patient samples, to
ensure that sample collection and
handling are not responsible for
elevations in ammonia.

106. MEASUREMENT OF AMMONIA IN BLOOD
fasted : 6 hours
Plasma ammonia levels
exercise,
smoking,
GI bleeding,
blood transfusions,
high protein intake
medications.
↑

107. .
Heparin is the preferred anticoagulant,
because it has been shown to reduce red blood
cell ammonia production.
The patient’s arm relaxed
,because muscle exertion leads to↑ venous
ammonia levels
Prolonged application of a tourniquet or
fist-clenching while obtaining the blood
sample avoided

108. The blood sample should be drawn into a
chilled,
sodium heparinized vacuum tube that is
immediately placed on ice.
centrifuged and the plasma removed
within 15 minutes of draw.
.

109. It is crucial to keep blood samples
cold after collection,
because the ammonia conc of
standing blood and plasma↑
spontaneously

110. this increase is bcoz of
generation,release of ammonia from rbc
deamination of amino acids,( glutamine.)
capillary blood avoided,
platelet aggregation, clotting →
↑ ammonia levels.

111. Measurements should be taken at the same
time of day ( a diurnal variation )
ammonia levels in whole blood samples
maintained at 4oC are stable for <1 hour.
, plasma ammonia levels are stable at 4oC
for 4 hours.
DO NOT FREEZE
Samples must arrive at laboratory
within 3 hours after collection.

112. Do not use block ice or dry ice, as this will
freeze the specimen.
Hemolyzed specimens and
Specimens received at ambient
temperatures, will not be analyzed, as
falsely increased ammonia concentrations
may result.

113. The decrease in absorbance at 340 nm,
due to the oxidation of NADPH, is
proportional to the ammonia
concentration.
0.2–15 mg/ml.
Spectrophotometer

114. NADH is converted to NAD+ in the
presence of NH3, ketoglutarate and
glutamate dehydrogenase. The
decrease in optical density at 340 nm
or fluorescence intensity
Quantitative Colorimetric/Fluorimetric
Determination of Ammonia

116. bunchman
Flow Diagram to Evaluate
Hyperammonemia
Increased
ammonia
acidosis
No
acidosis
Urine for
organic acids
Plasma amino
acids
Lactate/pyruvate

119. HYPERCITRULLINURIA HYPERORNITHINEMA
HYPERAMMONEMIA

120. ARGINASE DEFICIENCY

121. ↑ removal of nitrogen waste.
convert nitrogen into products other
than urea, which are then excreted;
the load on urea cycle ↓
.

122. Protein intake : stopped.
Calories : hypertonic 10% glucose.
Hemodialysis : all comatose neonates with
plasma ammonium levels greater than 10 times
reference range.
the total dialysis time is shorter with
hemodialysis than with peritoneal dialysis.
Treatment should be started if the
plasma ammonium level is 3 times the
reference level

123. The first sodium benzoate and
arginine.
Later, phenylacetate
now replaced by phenylbutyrate(orally)
. Sodium phenylbutyrate is a prodrug
and is metabolized to phenylacetate

124. arteriovenous or venovenous
hemofiltration as an alternative
method
IV sodiumbenzoate
phenylacetate : ammonium
level falls to 3-4 times the
upper limit
IV arginine

126. Na benzoate with arginine
for
(CPS),
(OTC),
(ASS),
(ASL) deficiencies

128. Antiemetic
control nausea and vomiting associated with
IV administration of sodium benzoate and
phenylacetate
Ondansetron
Granisetron
Palonosetron
Dolasetron

129. •Arginine supplementation: is an
essential AA in patients with urea cycle
defects
. In neonates and in OTC and CPSI
deficiencies, citrulline can be given as a
source of arginine as it gives one less
nitrogen atom;
in late-onset cases, arginine is
acceptable because of increased
nitrogen tolerance.

131. Citrulline levels are ↑in ASS
and ASL deficiencies and
citrulline should not be
administered in patients with
unknown enzyme deficiency.
•Provide enough calories to
meet energy requirements

132. Carglumic acid (Carbaglu)
N -carbamoyl-L-glutamate,
Structural analogue of NAG
enables activation of CPS I (first
enzyme of urea cycle)
ammonia into urea.
More resistant to enzymatic
degradation by hydrolysis
compared with N -acetylglutamate.

133. Corticosteroids are not FOR ↑ ICP
(induce negative nitrogen balance.)

Mannitol is not effective in treating
cerebral edema induced by
hyperammonemia.
Valproic acid should not be used to
treat seizures as it decreases urea cycle
function and
↑ammonia

134. Osmotic demyelination
syndrome
serious complication of standard therapy
for hyperammonemia in patients with
ornithine transcarbamylase
deficiency.[23]

135. Diet
•Low protein intake:
•0.7 g/kg/day of protein
• 0.7 g/kg/day of
essential amino acid mixture.
• first 6 months, an infant may
tolerate 1.5-2 g/kg/day of
protein.

136. •A gastrostomy tube is the
most reliable way to administer
medications and fluids during
illness
•provide adequate nutritional
support to prevent catabolism.

137. Surgical Care
•Liver transplantation correct the
metabolic error.
•, and requirements for medication and dietary
restriction were eliminated.
•Neurologic outcomes correlated closely with status
prior to transplantation.
•Thus, liver transplantation is a good option for
patients with urea cycle defects who have not
suffered major brain injury.

138. •Liver cell
transplantation,
• as multiple intraportal
infusions of cryopreserved
hepatocytes

139. Further Outpatient Care
monitoring growth and development of
the child that would indicate the adequacy
of treatment
.periodic fasting levels of the
1. Plasma ammonium
2. Plasma glutamine (should be
maintained at < 1000 µmol/L)
3. Arginine
4. Total protein

140. Complications
•Cerebral edema
Cortical blindness

141. Prognosis the 5-year survival
22% neonatal-onset
41% late-onset group.
neonatal-onset 90% severe
neurologicdeficits,
late-onset 28%