What
is the significance of the "ketonuria" part of the name?
=>
One of the clinical signs of the disease is the significant buildup of phenylketones
in the bloodstream and their appearence in the urine.
Is
PKU an inherited disease?
=>
Yes, PKU is an inherited disease.
What
does PAH stand for?
=>
Phenylalanine hydroxylase.
What
is PKU caused by?
=>
The most common cause of PKU is a mutation in the gene coding for the enzyme phenylalanine
hydroxylase (PAH). The mutation causes a deficiency in the activity of this enzyme
and leads to aberrant amino acid metabolism.
What
are typical general symptoms in children with severe PKU?
=>
Hypotonia, mental retardation, skin rashes, musty odor, fairer complexion than
parent or siblings.
Is
mental retardation necessarily severe in all cases of PKU?
=>
There is a spectrum of severity in mental retardation, but most cases (if untreated)
are associated with an IQ below 30. Microcephaly is present in 68% of untreated
cases; seizures present in 25% of untreated cases.
Clinical
Tests
What
tests may be used to determine blood levels of Phe?
=>
The Guthrie test is most commonly used. One may also
use HPLC. There is also a fluorometric method. An older, simple but inaccurate
method depends on the formation of a blue-green complex of urinary phenylpyruvate
with ferric ion (from a ferric chloride solutions.) This method is inaccurate
because other compounds in the urine may also form colored complexes and mask
the color of the ferric-pyruvate complex.
In
a suspected case of PKU, what information could be gained from a liver biopsy?
=>
PAH is expressed only in the liver; a biopsy could be used to determine its activity.
However, this is a stressful and invasive procedure, and it would not normally
be done with children.
What
are typical serum levels of phenylalanine (Phe) in cases of PKU?
=>
Benign: blood Phe at 120 - 480 micromol/L
Atypical: blood Phe at 480 - 1200 micromol/L
Severe: blood Phe at 1200 micromol/L or higher
What
other aberrant serum or urine levels may be noted?
=>
Elevated levels of phenylpyruvate, phenylacetate, phenyllactate, phenylethylamine.
Does
hyperphenylalaninemia always indicate a deficiency in the enzyme phenylalanine
hydroxylase?
=>
No, it does not. There may be other defects present, such as those involved
in regeneration of the cofactor tetrahydrobiopterin (BH4),
that would cause elevated levels of phenylalanine. In cases where the defect
is not in the enzyme PAH, but involves production or regeneration of BH4
, there may be aberrant levels of neopterin and biopterin. The elevation or depression
of the urinary pterins can in fact be used to deduce the exact defect in the metabolism
of BH4. Neurotransmitter levels might also be affected,
since the synthesis of certain neurotransmitters depends on the oxidation of tyrosine
and tryptophan in reactions that use the cofactor BH4.
What
is the Guthrie test for PKU, and how does it work?
=>
The Guthrie test is a bacterial assay for the presence of high levels of phenylalanine.
For details of this
and other tests for PKU, click
here.
How
might a carrier for PKU be diagnosed?
=>
One could use a genetic, rather than a biochemical, test. An allele-specific
oligonucleotide hybridization test, if positive, would be conclusive. One could
also use full length human cDNA that has the gene sequence for PAH, as a probe
for restriction fragment length polymorphism (RFLP).
Genetics
Is
PKU recessive or dominant?
=>
It is recessive.
In
order for a child to have PKU, must both parents have a defective PAH gene?
=>
In the "classical" form of the disease involving a defective PAH gene (not the
forms involving aberrant tetrahydrobiopterin metabolism), since the mutation in
PAH is recessive, then the child must have two (not just one) defective copy of
the PAH gene in order to suffer from PKU. This situation can arise most
commonly when both parents have a defective PAH gene and pass it along to the
child. Much more rarely there could be a spontaneous mutation in an otherwise
fully-functional PAH gene that would cause loss of enzyme activity. If this
occurred in combination with inheritance of a defective PAH gene from one parent
or the other, then the child would suffer from PKU.
A
mother is described as a "carrier" for PKU. What does this mean?
=>
She has one fully functional copy of the gene for PAH, and one defective copy.
She may be normal with respect to phenylalanine metabolism.
What
is the approximate overall frequency of defective PAH genes in the general
population?
=>
Approximately 2%.
What
is the approximate frequency of occurrence of PKU in live births?
=>
It occurs approximately once in 10,000 live births.
Approximately
how many different mutations (haplotypes) are known for the PAH gene?
=>
European populations show at least 46 different haplotypes, 4 of which account
for over 80% of the cases of PKU. Among Chinese and Japanese populations, one
haplotype predominates.
What
is the genetic location of the human PKU locus?
=>
On chromosome 12, at 12q22-q24.1
How
might an ASO (allele-specific oligonucleotide) hybridization test be used
to diagnose a specific genetic mutation in the PAH gene?
=>
DNA from the patient is extracted and denatured, then a portion combined with
the ASO in a stringent hybridization test. The ASO has a sequence matching
one specific mutation in the PAH gene, so that if the ASO hybridizes with the
patient's DNA under stringent conditions, it must be fully complementary to the
patient's PAH gene sequence. If the ASO does not hybridize, then the patient
does not carry that particular mutation. This process can be repeated with different
ASOs to identify the particular mutation carried by the patient.
Biochemical
Defects and Consequences
Is
phenylalanine an essential amino acid? Can humans synthesize phenylalanine,
or must it all come from dietary intake? Can they synthesize tyrosine,
or must it all come from dietary intake?
=>
Phenylalanine is essential for humans; humans do not have all the enzymes needed
for de novo synthesis of this amino acid. Thus, for humans all phenylalanine
must be obtained through the diet. Humans can make tyrosine from phenylalanine,
so tyrosine is not considered an essential amino acid.
What
are the physical characteristics (subunit stoichiometry and composition, and
molecular weights) of the PAH enzyme?
=>
Human PAH is a dimer of identical 50-kDa subunits.
What
reaction does this enzyme catalyze?
=>
It catalyzes the hydroxylation of phenylalanine on the phenyl ring at the 4 position,
to make tyrosine.
What
cofactor is required for this reaction?
=>
Tetrahydrobiopterin.
Why
is the enzyme dihydropteridine reductase needed for the conversion of phenylalanine
to tyrosine?
=>
During the conversion reaction, the cofactor is co-oxidized to the quinonoid form.
Dihydropteridine reductase is needed to reduce this and regenerate the tetrahydrobiopterin,
so that the conversion reaction can continue.
What
is the importance of this reaction in general amino acid metabolism in humans?
=>
In humans, this is the major biosynthetic route to tyrosine.
In
what organ is most of the PAH activity located?
=>
The liver.
What
is an alpha-keto acid? Give some examples from biochemistry?
=>
An alpha-keto acid has a carbonyl groups located immediately adjacent to
the carboxylic acid group:
R-CO-COOH
Examples from primary metabolism include pyruvate, oxaloacetate, and alpha-ketoglutarate.
How
can phenylalanine be converted into phenylpyruvate by the body?
=>
Enzymes known as transaminases can catalyze the removal of the alpha-amino
group (and a hydrogen from the alpha-carbon) from phenylalanine, and convert
that carbon to a carbonyl, making phenylpyruvate.
In
PKU, why are there high levels of phenylpyruvate in the plasma and urine?
=>
The lack of PAH activity leads to an accumulation of phenylalanine. As the
concentration of this amino acid rises, side reactions with the phenylalanine
generate byproducts. These are normally are present only in vanishingly
small concentrations, but under these conditions, their concentrations become
appreciable. One of the side reactions is the transamination reaction of
phenylalanine with alpha-ketoglutarate, which generates glutamate and (more
importantly) phenylpyruvate. The reaction is the analog of the transamination
of alanine to pyruvate. This leads to higher-than-normal concentrations
of phenylpyruvate in the bloodstream, and then in the urine.
How
might excessive levels of phenylpyruvate interfere with pyruvate metabolism?
=>
Pyruvate is transported into the mitochondria for oxidation; this transport involves
a specific carrier protein. Phenylpyruvate inhibits the transport of pyruvate
(but not acetate) into human red blood cells, and into rat liver and brain mitochondria.
The results with rat tissue, supported by the results with human red blood cells,
suggest the existence of a similar carrier in humans. They further suggest
that this carrier would be specifically inhibited by phenylpyruvate in humans.
Such inhibition would block much of the formation of acetyl CoA in the mitochondria,
though some could be derived from fatty acid metabolism. The acetyl CoA
is needed for TCA cycle turnover and is a key part of energy generation in the
mitochondria. Thus a block of the entry of pyruvate into mitochondria would
reduce the cell's ability to generate energy.
Maple
syrup urine disease (MSUD) leads to high serum levels of alpha-ketoisocaproate.
How might high levels of this metabolite interfere with pyruvate metabolism?
=>
Inhibition of pyruvate transport into mitochondria could be produced by
alpha-ketoisocaproate using the same sort of mechanism as for phenylpyruvate.
The consequences would be the same.
High
concentrations of phenylalanine can inhibit the enzyme that decarboxylates
5-hydroxytryptophan to form serotonin. How might this lead to mental
retardation?
=>
Serotonin is a neurotransmitter. Blockage of its synthesis could easily
upset CNS function and, if prolonged, could lead to mental retardation.
Apart
from its effects on neurotransmitter levels, explain how a deficiency in PAH
activity can lead to mental retardation.
Since phenylpyruvate may block entry of pyruvate into mitochondria, and so upset
TCA cycle function, it would tend to weaken the cell's capacity for generating
energy, e.g. ATP. Brain tissue consumes much ATP, and any interference with
ATP production in this tissue could be expected to lead to CNS effects.
If prolonged, the interference could then cause mental retardation, and psychological
and emotional disturbances, etc.
What
metabolites of phenylalanine might you expect to see in the blood from a child
with PKU?
=>
Common metabolites of phenylalanine would be phenylpyruvate, phenylacetate, phenyllactate,
and phenylethylamine.
What
causes the musty odor associated with PKU?
=>
The odor is probably due to phenylacetate, one of the products of side-reactions
of phenylalanine.
How
would a deficiency in tetrahydrobiopterin production or regeneration lead
to symptoms similar to those caused by a deficiency in PAH activity?
=>
If there is insufficient tetrahydrobiopterin, then the phenylalanine hydroxylase
cannot turn over for renewed catalytic cycles; in effect, the enzyme is inhibited
by lack of the properly-reduced cofactor. The consequences are much the
same as if the enzyme were simply deficient in activity, that is, elevated levels
of phenylalanine and byproducts of its metabolism.
What
other upsets in amino acid metabolism might arise if there were a deficiency
in tetrahydrobiopterin production or regeneration?
=>
This cofactor is used in the hydroxylation of tryptophan, so there could be a
deficiency of neurotransmitters made from either tryptophan or of course from
tyrosine.
Treatments
What
is the basic treatment for an infant with PKU?
=>
The intake of phenylalanine must be greatly restricted, to supply just the amount
needed for replacement of phenylalanine lost during protein turnover. One
should use foods that are low in Phe and that provide adequate tyrosine; high
protein foods (meat, milk, eggs, cheese, and legumes) should be avoided.
What
are the recommended blood levels of Phe for patients with PKU?
=>
In general, blood levels in the range 120 - 600 ?mol/L are usually considered
appropriate, though some authorities recommend the lower and more restricted ranges
of 120 - 360 micromol/L or 120 - 240 micromol/L.
How
long must dietary restrictions continue?
=>
It was originally thought that in later childhood or adolescence, the dietary
restrictions could be relaxed. Recent studies indicate that the restricted
diet must continue indefinitely (though perhaps with some modification in adulthood),
to avoid learning disabilities and psychological and emotional difficulties later
in life.
Why
does tyrosine become an "essential" amino acid for children with PKU?
=>
Since they lack the enzyme activity to make tyrosine from phenylalanine, the tyrosine
must be supplied through their diet, making it effectively "essential".
What
treatment is recommended for patients with hyperphenylalaninemia due to a
defect in BH4 metabolism?
=>
They should receive oral supplements of the active form of the cofactor, along
with supplementation for the neurotransmitter precursors 5-hydroxytryptophan
and L-DOPA.