Investigation of Amino

Acid Metabolism
Disorders
 Lecture Outline
The amino acids
Glucogenic & ketogenic amino acids
Amino acid metabolism
Urea cycle
When things go wrong
Symptoms of amino acid metabolism disorders
Suspecting an inborn disorder of metabolism
Aminoaciduria
Laboratory evaluation of aminoaciduria
Amino acid laboratory tests
Amino acid metabolism disorders
 The Amino Acid

H Central carbon
Amino group
-
H3 N +
C COO
Acidic carboxyl group

Hydrocarbon-
R
containing group
 The Amino Acid

Hydrocarbon-containing group
- Hydrocarbon chain H
- COOH
- NH2
-
- OH H3 N +
C COO
- Sulphur groups
- 6-carbon aromatic ring
- Indole ring
- Imino ring
R
 Amino Acids

Serve several purposes in the body

Amino acids are:
 the building constituents of proteins

 the precursors to other biochemicals →

nucleotides and neurotransmitters


the carbon skeletons used as fuel by the
body
 Amino Acids
Proper diet contains adequate amounts of protein
Severe metabolic diseases when there are:

Deficiencies in dietary protein
 Upsets / blockages in metabolism of amino acids

Twenty standard amino acids ⇒ protein synthesis

Humans can make only 10 of the 20
The other 10 must be obtained through the diet

Essential AA: the 10 amino acids that must be

obtained through the diet
Nonessential AA: the other 10 amino acids
Essential vs. Nonessential Amino Acids
Nonessential Essential
Alanine Arginine

Asparagine Histidine

Aspartate Isoleucine

Cysteine Leucine

Glutamate Lysine

Glutamine Methionine

Glycine Phenylalanine

Proline Threonine

Serine Tyrptophan

Tyrosine Valine
Essential vs. Nonessential Amino Acids
Essential
*Arginine, methionine and phenylalanine:
Arginine* considered essential for reasons not
Histidine directly related to lack of synthesis
Isoleucine
Arginine is synthesized by mammalian cells
Leucine but at a rate that is insufficient to meet the
Lysine growth needs of the body; the majority that
is synthesized is cleaved to form urea
Methionine*
Methionine is required in large amounts to
Phenylalanine* produce cysteine if cysteine is not
Threonine adequately supplied in the diet
Tyrptophan Phenyalanine is needed in large amounts to
form tyrosine if tyrosine is not adequately
Valine supplied in the diet
 Amino Acid Metabolism
Where?

All tissues have some capability for:

 synthesis of the non-essential amino acids

 amino acid remodeling


conversion of non-amino acid carbon
skeletons into amino acids and other
derivatives that contain nitrogen
Liver ⇒ major site of nitrogen metabolism
Amino Acid Metabolism
 Amino Acid Metabolism

Formation of urea occurs predominantly in

liver
Urea then excreted mainly in urine, sweat

Breakdown of ingested protein ⇒

gastrointestinal tract ~ digestive enzymes
→→→→ essential amino acids
 Amino Acid Metabolism
When there is dietary surplus → potentially toxic nitrogen
of amino acids is eliminated:
 Transaminations
 Deaminations
 Urea formation
Carbon skeletons of amino acids ⇒
 Conserved as carbohydrates ~ gluconeogenesis

 Or: conserved as fatty acids ~ fatty acid synthesis

pathways
∴ Amino acids fall into three categories:
1. Glucogenic
2. Ketogenic
3. Glucogenic and ketogenic
 Amino Acids’ Who’s Who

Glucogenic amino acids can convert to

glucose
All amino acids except lysine and leucine are at
least partly glucogenic
Ketogenic amino acids convert to acetyl CoA
or acetoacetate that cannot convert to glucose
Lysine and leucine are the only amino acids
that are solely ketogenic
 Amino Acids’ Who’s Who

A small group of amino acids comprised of

isoleucine, phenylalanine, threonine,
tryptophan, and tyrosine give rise to both
glucose and fatty acid precursors and are
thus characterized as being glucogenic
and ketogenic
Amino Acid Metabolism
 Amino Acids

During times of starvation the reduced carbon

skeleton is used for energy production
↓
oxidized to CO2 and H2O
 The urea cycle

In mitochondrion

All enzymes are in red,

CPS-I is carbamoyl
phosphate synthetase-I,
OTC is ornithine
transcarbamoylase.
 The urea cycle

Body does not keep stores of nitrogen

Function ⇒ eliminate excess nitrogen
Excess amino acids → degraded →
ammonia
Ammonia → urea NH2CONH2 → excreted
 The urea cycle

When dietary proteins increase significantly, enzyme

concentrations rise
On return to a balanced diet, enzyme levels decline
Under conditions of starvation, enzyme levels rise as
proteins are degraded and amino acid carbon skeletons
are used to provide energy ⇒ increasing the quantity of
nitrogen that must be excreted
 Amino Acid Metabolism

Metabolism of amino acids:

 Amino acid synthesis

 Amino acid breakdown

Block in metabolism ← defect or absence of activity in

an enzyme or cofactor involved in the synthetic or
degradative pathway

Metabolic pathway blockage ⇒ metabolite levels ↑

∴ interfere with other metabolic processes
⇒ disease
 Amino Acid Metabolism

enzyme
SUBSTRATE (S) PRODUCT (P)
 When things go
wrong….
SUBSTRATE Normal metabolic
enzyme product does not
deficiency form

Secondary
metabolites
accumulate Normal: these metabolites present
in very low levels
 Disorders of Amino Acid Metabolism
Hyperammonaemia
Phenylketonuria (deficiency of phenylalanine hydroxylase)
Alkaptonuria (defect in homogentisate oxidase)
Tyrosinaemia (accumulation of tyrosine and its metabolites)
Albinism (tyrosinase deficiency)
Parkinson’s disease (dopamine deficiency)
Cystinuria (cysteine excess)
Hartnup’s disease (defect in transport of neutral amino acids)
Excess xanthunerate [ in urine ]
Maple Syrup Urine Disease (elevated leucine, isoleucine, valine)
Hypervalinaemia (defect in valine transaminase)
Isovaleric acidemia (leucine metabolism defect)
Methylcrotonic aciduria (defect in enzyme in course of leucine
breakdown)
Homocysteinuria (defect in cystathionine synthase)
Hypermethioninurua (decrease in methionine adenosyl transferase)
Homocysteinuria variant (homocysteine → methionine)
Cystathioninuria (deficiency of cystathioninase)
Histidinaemia (lack of histidase)
When do you suspect an inborn
error of metabolism?

Bizarre, inexplicable clinical features

Abnormal laboratory findings in infancy &
early childhood – especially if more than
one affected infant in family
Symptoms of disorders of amino acid metabolism
Newborn
Failure to thrive, poor feeding
Neurologic disorder; seizures,
irratability, lethargy
Vomiting, diarrhea, dehydration
Hepatomegaly
Metabolic acidosis, increased
anion gap
Hyperglycemia, hypoglycemia
Neutropenia, thrombocytopenia
Infants and older children
Mental retardation
Seizures
Developmental delay (mental,
physical, motor)
Haematologic disorders, anaemias
CNS defects, speech, behaviour
problems
Hepatomegaly, splenomegaly
Renal calculi, defects
Blindness
Recurrent infections, fever of
unknown origin
Disorders of Amino Acid Metabolism

Aminoaciduria: a condition in which

one or more amino acids are
excreted in excessive amounts
 Aminoaciduria
Primary
 Rare, genetically determined and generally
inherited in an autosomal recessive manner
 An enzyme deficiency or defect in a specific
amino acid metabolic pathway or a defect in
renal amino acid reabsorption mechanism

More than 50 primary aminoacidurias

Secondary
 Arise secondary to another condition
 Aminoaciduria

Under normal conditions:


Amino acids filtered by glomeruli in kidneys
 Concentration of amino acids at renal
proximal tubules are equal to those in plasma

Almost completely reabsorbed
 Aminoaciduria
Overflow
 Plasma level of one or more amino acids exceeds the renal
threshold, and excess amino acids spill into the urine
No-threshold

Owing to an inborn error of metabolism, excessive amounts of
one or more amino acid are present in the urine, but plasma
levels remain essentially normal because all the amino acid is
excreted
Renal

Plasma amino acid levels are essentially normal but the renal
tubular reabsorption system has congenital or acquired defects
resulting in excessive amino acid excretion in the urine
 Aminoaciduria

Must be diagnosed and treated as early as

possible to prevent permanent damage such as
mental retardation or early death

Diagnosis chiefly dependent on laboratory

testing since many disorders produce non-
specific symptoms in affected newborns, infants
and older children
Laboratory evaluation of aminoaciduria
Laboratory evaluation of aminoaciduria

A component of routine neonatal screening in most

countries
Diagnosis of a symptomatic infant or child
Routine follow up of treatment for aminoaciduria
What do you evaluate?

Detect amino acids and metabolites in the urine,
blood, and body fluids
Laboratory evaluation of aminoacidurias

Level of testing includes screening,

quantitation, and identification methods
Measure urinary organic acid excretion +
enzyme analysis
Prenatal diagnosis may be requested if
mother is a carrier or if there is a positive
family history
Laboratory evaluation of aminoaciduria
Why the test is performed:

To assess amino acid levels in the urine

Common for some of each amino acid to be in the
urine, but elevated levels of individual amino acids
indicates possible inborn enzyme deficiency
Increased levels of amino acids in the urine ⇒ ⇒
24-hour quantitative urine chromatography is
necessary to accurately measure the elevated levels
of the specific amino acids
Increased levels also show up in the blood plasma
 Laboratory evaluation of
Normal Values:
Alanine aminoaciduria
Children: 65 to 190
Adults: 160 to 690
Alpha-aminoadipic acid Beta-amino-isobutyric acid Glycine
Children: 25 to 78 Children: 25 to 96 Children: 195 to 855
Adults: 0 to 165 Adults: 10 to 235 Adults: 750 to 2,400
Alpha-amino-N-butyric acid Carnosine Histidine
Children: 7 to 25 Children: 34 to 220 Children: 46 to 725
Adults: 0 to 28 Adults: 16 to 125 Adults: 500 to 1,500
Arginine Citrulline Hydroxyproline
Children: 10 to 25 Children: 0 to 13 Children: not measured
Adults: 13 to 64 Adults: 0 to 11 Adults: not measured
Asparagine Cystine Isoleucine
Children: 15 to 40 Children: 11 to 53 Children: 3 to 15
Adults: 34 to 100 Adults: 28 to 115 Adults: 4 to 23
Aspartic acid Glutamic acid Leucine
Children: 10 to 26 Children: 13 to 22 Children: 9 to 23
Adults: 14 to 89 Adults: 27 to 105 Adults: 20 to 77
Beta-alanine Glutamine Lysine
Children: 0 to 42 Children: 150 to 400 Children: 19 to 140
Adults: 0 to 93 Adults: 300 to 1,040 Adults: 32 to 290
 Laboratory evaluation of
Methionine
Children: 7 to 20
aminoaciduria
Adults: 5 to 30
1-methylhistidine Proline
Children: 41 to 300 Children: not measured
Adults: 68 to 855 Adults: not measured
3-methylhistidine Serine
Children: 42 to 135 Children: 93 to 210
Adults: 64 to 320 Adults: 200 to 695
Ornithine Taurine
Children: 3 to 16 Children: 62 to 970
Adults: 5 to 70 Adults: 267 to 1,290
Phenylalanine Threonine
Children: 20 to 61 Children: 25 to 100
Adults: 36 to 90 Adults: 80 to 320
Phosphoserine Tyrosine
Children: 16 to 34 Children: 30 to 83
Adults: 28 to 95 Adults: 38 to 145
Phosphoethanolamine Valine All measurements in
Children: 24 to 66 Children: 17 to 37 micro mole per deciliter
Adults: 17 to 95 Adults: 19 to 74 (µmol/dl)
Laboratory evaluation of aminoaciduria

Why the test is performed:

If the test is performed early enough on infants

and the defect is treated, the severe mental
retardation that can result may be prevented
 Aminoaciduria
What abnormal results mean
Increased total urine amino acids may indicate any of the following:

• Alkaptonuria • Methylmalonic acidemia

• Canavan disease
• Cystinosis
• Cystathioninuria
• Fructose intolerance
• Galactosemia
• Hartnup disease
• Homocystinuria
• Hyperammonemia
• Hyperparathyroidism
• Maple syrup urine disease
• Multiple myeloma
• Ornithine transcarbamylase
deficiency
• Osteomalacia
• Propionic acidemia
• Rickets
• Tyrosinemia type 1
• Tyrosinemia type 2
• Viral hepatitis
• Wilson's disease
 Aminoaciduria

Comparison is necessary with the

plasma amino acids to develop a
complete picture
 Amino acid screening tests

2D TLC
Guthrie microbiological tests
Colorimetric spot tests
Amino acid quantitative tests
Chromatography
HPLC-ion exchange
GLC
 Amino acid identification
Gas chromatography-mass spectroscopy
(GC-MS)
The Disorders

Phenylketonuria
Tyrosinaemia
Alkaptonuria
Albinism
Histidinaemia
Cystinosis
Homocystinuria
Maple Syrup Urine Disease (MSUD)
Cystinuria
Hartnup’s disease
Familial iminoglycinuria
Hyperammonaemia