Roanna George

Cardiff and Vale University Health Board

ACB Training Course June 2014
Produced primarily from amino acid catabolism
De-amination of amino acids in liver, muscle and kidney

Amino Acid -ketoglutarate NADH + NH4+

-keto acid Glutamate NAD+ + H2O

TRANSAMINASES GLUTAMATE
DEHYDROGENASE
 Mitochondria Cytoplasm
HCO3 + NH4 + 2 ATP
CPS1 N-acetylglutamate
Aspartate
Carbamyl Phosphate
Citrulline Citrulline
OTC
Orotic Acid
ASS
Uracil
Ornithine

Argininosuccinic acid

Ornithine ASL

Arginine
Fumarate
UREA ARG
Glutamine synthase converts ammonia to glutamine:

Glutamate + ATP + NH3 Glutamine + ADP + Pi

High ammonia concentration drives the reaction

Glutamine is used to store, buffer and transport

ammonia
Ammonia concentration is higher in
neonates:
Sick neonates <180 umol/L
Prem neonates <150 umol/L
Healthy neonates <100 umol/L
Infant/Child <40 umol/L

Urgent action is required if ammonia is:

>200 umol/L in a sick neonate
>150 umol/L in children
Sample requirements:
Must be uncuffed (free flowing)
Transport to the lab preferably on ice
Analyse within 15 minutes of sample collection
Advisable to measure the ammonia even if the above
criteria is not met

If ammonia is raised:
CONFIRM BY TAKING A SECOND SAMPLE
Take the repeat within four hours of the initial test
The trend can give an indication of the cause
 Cause Ammonia (umol/L)
Transient >1500
Urea cycle defect >600
HHH syndrome >600
Lysinuric Protein Intolerance >600
Valproate therapy >600
Organic acid disorder 200-600
FAO Disorder 200-600
HIHA syndrome 200-600
Severe liver failure <200
Raised muscle activity <200
Spurious <180
Blood gases
Resp alkalosis
Mixed resp alkalosis and met acidosis
Urea low
LFTs
Severely deranged in some causes of hyperammonaemia
Mildly deranged in some UCDs/OADs
Glucose
Low in FAODs, hyperinsulinism, liver failure
Lactate
Raised in metabolic disorders and liver failure
Calcium
Hypocalcaemia is a feature of some organic acid disorders
Urine ketones
High in OADs and low or absent in FAODs & liver failure
Urine and plasma amino acids
Urine organic acids
Urine orotic acid
Plasma or bloodspot acylcarnitines

Enzyme studies
Mutation analysis
 Mitochondria Cytoplasm
HCO3 + NH4 + 2 ATP
CPS1 N-acetylglutamate
Aspartate
Carbamyl Phosphate
Citrulline Citrulline
OTC
Orotic Acid
ASS
Uracil
Ornithine

Argininosuccinic acid

Ornithine ASL

Arginine
Fumarate
UREA ARG
Rapidly progressive symptoms after short
symptom free period:
Lethargy
Poor feeding
Hyperventilation
Seizures
Progressive encephalopathy
Temperature instability
Loss of reflexes
Intracranial haemorrhages
 ! " "

Presenting features can include:

Failure to thrive
Feeding problems
Vomiting
Chronic neurological symptoms
Episodic encephalopathy
Ataxia
Seizures
 "

UCDs can present at any age with:

Chronic neurological symptoms
Behavioural problems
Episodes of disorientation
Lethargy
Psychosis
Recurrent encephalopathy
Routine Ix:
Ammonia
Blood gases (respiratory alkalosis) beware as can
get a concurrent lactic acidosis due to poor perfusion
for UCD patients with circulatory failure

If ammonia is high request:

Repeat to confirm
Plasma amino acids
Organic acids
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Argininosuccinic acid
(ASA)
Internal Standard

Argininosuccinic acid
(ASA)
 $
History: Male
Term baby
Uneventful pregnancy

Day 2 lethargy
poor feeding
grunting
?sepsis
convulsions
&
What is the diagnosis?
 '
Mitochondria Cytoplasm
HCO3 + NH4 + 2 ATP
CPS1 N-acetylglutamate
Aspartate
Carbamyl Phosphate
Citrulline Citrulline
OTC
Orotic Acid
ASS
Uracil
Ornithine

Argininosuccinic acid

Ornithine ASL

Arginine
Fumarate
UREA ARG
 3500

haemodialysis
3000

cool body tx
2500
A m m onia uM ol/L

2000

1500

1000

500

60 umol/l 0
12/12/2000 00:00 12/12/2000 12:00 13/12/2000 00:00 13/12/2000 12:00 14/12/2000 00:00 14/12/2000 12:00 15/12/2000 00:00 15/12/2000 12:00 16/12/2000 00:00
( (

Block urea synthesis by:

Acetyl-CoA deficiency
NAGS inhibition

Ammonia concentration cannot always distinguish

between UCD and other disorders

Usually associated with acidosis (possible mixed

acid base disturbance):
Organic acids for organic acid disorders
Lactate (possibly ketones) for fatty acid oxidation disorders
 )
* +
Gain of function mutation of glutamate
dehydrogenase
Leucine activates glutamate dehydrogenase in the
pancreatic -cell which stimulates insulin secretion

Features:
Children have normal birth weight
Recurrent hypoglycaemia later in infancy with
asymptomatic hyperammonaemia

Condition is responsive to diazoxide

Inhibits insulin release at sulphonylurea receptor/K+
channel complex of -cell
 , ,

Causes of severe liver failure in the neonatal

period:
Galactosaemia
Hereditary fructose intolerance
Tyrosinaemia type 1
Neonatal haemochromatosis
Respiratory chain defects
NOTE: elevated transaminases or reduced
PT can also be seen in UCDs

Blood ammonia should be measured in

EVERY case of unclear encephalopathy
whatever the age of presentation of the
patient
 Case
Examples
 '

Background:
Female
10 days old
Born term normal delivery
No known family history
3 other siblings, all well

History:
Presented to A/E
Drowsy and unresponsive
Poor feeding
Reduced urine output
 "

On admission
Bradycardic at 90/min
Responsive only to pain
Poorly perfused and required fluid resuscitation

On examination:
Fixed and dilated pupils, but her pupils normalised
subsequently
Marked hypotonia
Tendon reflexes were difficult to elicit
Not tolerating feeds
- # "
Calcium 1.17 mmol/L * 2.10-2.70
Albumin 24 g/L * 30-45
Adj Calcium 1.41 mmol/L * 2.10-2.70

Alk.Phos. 187 IU/L 100-300

ALT 37 IU/L <50
Bilirubin 32 umol/L <200
Total Protein 49 g/L * 50-70
Calc Globulin 25 g/L 17-37
Phosphate 3.53 mmol/L * 1.30-2.75

Sodium 147 mmol/L * 133-146

Potassium 7.5 mmol/L * 3.4-5.5
Urea 23.3 mmol/L * 1.0-5.5
Creatinine 146 umol/L * 30-70

CRP 2 mg/L <6

( ,
Blood pH 6.80 * 7.35-7.45
pCO2 5.5 kPa 4.7-6.0
Blood pO2 19.9 kPa * 11.1-14.4
Base Excess -25 mmol/L

Ionised Calcium 0.82 mmol/L

Plasma Glucose 15.4 mmol/L
Serum Potassium 4.9 mmol/L 3.4-5.5
Serum Sodium 147 mmol/L * 133-146
Serum Chloride 119 mmol/L * 95-108

Blood Lactate 0.4 mmol/L * 0.5-1.6

 " # "

Ammonia measured on two consecutive days:

Ammonia 268 umol/L * 20-100
Ammonia 620 umol/L * 20-100

CK 616 IU/L
Urate 1.34 mmol/L * 0.10-0.36

3-OHB 2.05 mmol/L

NEFA 0.53 mmol/l
Glucose 1.0 mmol/L
Evidence of lipolytic and ketotic response to hypoglycaemia
 ,

An EEG was performed which showed a

burst suppression pattern in keeping with
metabolic encephalopathy

Biochemistry:
Profound metabolic acidosis
Severe hyperammonaemia
Hypocalcaemia
Hypoglycaemia
Acute renal failure
. / ,

Amino Acids:
Sample degradation
No evidence of an amino acid disorder

Organic Acids:
Marked increased excretion of 3-hydroxypropionic acid,
propionyl glycine, tiglylglycine and methyl citrate
Profile consistent with a diagnosis of Propionic acidaemia

Blood Acylcarnitines:
Profile shows increased propionylcarnitine (C3)
Consistent with a diagnosis of propionic acidaemia
.
Commenced on sodium benzoate and sodium
phenylbutyrate

Carnitine was added

Despite this ammonia continued to rise

When ammonia reached 900 mol/L she was started

on haemofiltration

Ammonia remained severely elevated for several days

Haemofiltration discontinued as ammonia decreased

 0
Background:
Female
2 weeks old
Born term normal delivery
No known family history
3 other siblings, all well

History:
Poor feeding
Floppy
Becoming drowsy

On Examination:
In casualty she was unconscious
 ,
Blood Gas:
pH 7.5 7.35-7.45
pCO2 2.1kPa 4.7-6.0
pO2 18kPa 11.1-14.4

Ammonia 1850mol/L <40

Sodium 145 mmol/L 133-146

Potassium 3.3 mmol/L 3.4-5.5
Urea 0.6 mmol/L 1.0-5.5
Creatinine 55 mol/L 30-70

ALP 436 IU/L 100-300

ALT 25 IU/L <50
Bilirubin 18 mol/L <200
Albumin 38 g/L 30-45
. / ,

Amino Acids:
Increased glutamine
Low citrulline and arginine

Organic Acids:
Significantly increased orotic acid

Acylcarnitines
Normal profile
 (
Mitochondria Cytoplasm
HCO3 + NH4 + 2 ATP
CPS1 N-acetylglutamate
Aspartate
Carbamyl Phosphate
Citrulline Citrulline
OTC
Orotic Acid
ASS
Uracil
Ornithine

Argininosuccinic acid

Ornithine ASL

Arginine
Fumarate
UREA ARG
 1
Background:
Male
11 days old
Born 38+2 by planned c-section
Birth weight 3.46 kg
Initially low BMs
Some jaundice but not severe or prolonged
Breast fed well - home at 5 days old

Family History:
Non-consanginous white Welsh family
Maternal grandmother 2 babies shortly after birth - ? Cause
Two healthy brothers
Nil else of note
On admission:
Faltering growth
Lost 16.4% of birth weight (weight now 2.89kg)
Initially gained a small amount of weight but losing weight since
Baby not as alert as other children
Breast feeding 2.5-3 hourly but needs to be woken

On examination:
Looks well
Small/scrawny
Not dysmorphic
Abdo soft, no hepatomegaly
Normal palmar creases
?? Slightly hypotonic
Cataracts
( ,
Lactate 1.9 mmol/L * 0.5-1.6
Chloride 114 mmol/L * 95-108

Blood pH 7.26 * 7.35-7.45

pCO2 6.3 kPa * 4.7-6.0
Blood pO2 4.6 kPa * 11.1-14.4
SxO2 76.1 %
CxO2 19.6 ml/dL
CarboxyHb 0.6 % <1.5
Bld Met-Hb 0.8 % <1.5

Standard Bicarb 19 mmol/L 18-24

Base Excess -5 mmol/L
# " &
Adj Calcium 3.04 mmol/L * 2.10-2.70
Phosphate: Haemolysed
TP: Haemolysed
Albumin 34 g/L 30-45
Alk.Phos. 1362 IU/L * 100-300

Glucose 4.2 mmol/L

ALT: Haemolysed
Bilirubin 65 umol/L * 1-22

Sodium 140 mmol/L 133-146

Potassium: Haemolysed
Urea 11.5 mmol/L * 1.0-5.5
Creatinine 75 umol/L * 20-35

Ammonia 130 umol/L (<40)

Raised urea, creatinine and ALP noted
Metabolic acidosis with hypercholoraemia
Anion gap = Na (140) (Cl 114 + bicarb 18) = 8 so lower end of
normal

Impression:
Faltering growth not 2ry to intake

Differential diagnosis:
IEM: UCD or organic acidaemia
RTA
Dehydration/sepsis

Hypercalcaemia not typically associated with organic acidaemia

. / ,

Acylcarnitines: Normal

Organic acids:
Mild excretion of p-hydroxyphenyl-lactate
reflecting liver immaturity/dysfunction. No succinyl
acetone/succinylacetoacetate making
Tyrosinaemia type I unlikely.

Amino Acids: Normal

Phenylalanine, methionine and tyrosine normal
Lysosomal Enzymes :
Several enzyme activities outside normal
reference ranges
Secondary to delay in sample reaching external
laboratory
Advise sending a repeat specimen for analysis if
clinically indicated
Analysed at R. Manchester Children's Hospital.
 &
CT Head:
White matter changes

MRI ordered secondary to white matter changes

MRI Head:
Hyperintensity in white matter
There is no evidence of involvement of the deep grey
nuclei

MR Spectroscopy shows a large peak at

approximately 3.7
The appearance is in keeping with galactosaemia
 " . / &

Red Cell GAL-1-PUT:

Galactosaemia screen shows reduced galactose-1-
phosphate-uridyl transferase activity.

Biotinidase 12.6 U/L * 2.5-10.5 Normal biotinidase

activity detected.

Organic Acids:
Mild ketonuria with appropriate dicarboxylic aciduria.
Nothing diagnostic. Persistent/recurrent ketosis must be
investigated further
 ,
Red Cell GAL-1-PUT:
Repeat galactosaemia screen confirms reduced gal-1-PUT activity. Send
a lithium heparin sample for diagnostic quantitation of enzyme activity

Gal-1-PUT activity :
<0.5 umol galactose-1-phosphate converted/hr/g Hb
Reference ranges:
Normal: 18-28
Heterozygote: 8-14
Galactosaemic: <0.5

Genetics:
Heterozygous for GALT exon 3 mutation c.292G>A (Asp98Asn)
and common exon 6 missense mutation c.563A>G (Gln188Arg)
 , $ 2

September 2006:
Outline the biochemical pathways responsible for the
disposal of waste nitrogen. Discuss the clinical causes of
hyperammonaemia

March 2001:
Outline the clinical disorders and the disturbances in
biochemical mechanisms which result in elevation of
plasma ammonia
Suspect hyperammonaemia with unexplained respiratory
alkalosis or encephalopathy:
X-Linked disorders can present in females
UCDs can present at any age

Ammonia > 200 umol/L strongly suggests a metabolic

disorder but it can be lower than this

Specialist investigations required:

Amino acids (urine and plasma can be helpful)
Urine organic acids
Dried bloodspot or plasma acylcarnitines
Any Questions?