Screening for
phenylketonuria (PKU) –
laboratory methods
Péter Monostori
Phe & BH4 metabolism pathway
During the
hydroxylation of Phe
by Phe hydroxylase
(PAH) to form
tyrosine (Tyr),
tetrahydrobiopterin
(BH4) is oxidized to
a 4a-hydroxy-BH4
intermediate (when
molecular oxygen
and iron (Fe2+) are
present).
q-Dihydrobiopterin
Phe & BH4 metabolism pathway
This intermediate is
subsequently
regenerated back to
BH4 via quinonoid
(q)-dihydrobiopterin
by the enzymes
pterin-carbinolamie4a-dehydratase
(PCD) and by the
NADH-dependent
dihydropteridine
reductase (DHPR). Dihydropteridine
reductase (DHPR)
q-Dihydrobiopterin
Pterin-4α-carbinolamine
dehydratase (PCD)
Phe & BH4 metabolism pathway
GTP cyclohydrolase (GTPCH)
BH4 is synthesized
from guanosine
triphosphate (GTP)
by three additional
enzymes: GTP
cyclohydrolase I
(GTPCH), 6-pyruvoyltetra-hydropterin
synthase (PTPS),
and sepiapterin
reductase (SR).
Mutations in genes Dihydropteridine
coding enzymes for reductase (DHPR)
30%
GTPCH, PCD, SR,
q-Dihydrobiopterin
DHPR and PTPS
result in BH4
Pterin-4α-carbinolamine
deficiency.
dehydratase (PCD)
5%
5%
6-Pyruvoyl-tetrahydrobiopterin
synthase (PTPS)
60%
Sepiapterin reductase (SR)
Which markers can be useful in the
diagnosis of PKU and BH4 deficiency?
 PKU:
Phe
(from DBS)
Tyr (DBS)
Phe/Tyr ratio (DBS)
 BH4 deficiency:
Neopterin,
biopterin and pterin levels (urine, DBS)
DHPR activity (DBS)
Homovanillic acid (HVA) (liquor)
5-Hydroxy-indoleacetic acid (5-HIAA) (liquor)
Definitions of positive/negative predictive
value, sensitivity and specificity
Sensitivity

The proportion of affected subjects that have a positive
test result
Specificity

The proportion of unaffected subjects that have a negative
test result
Positive predictive value


The chance that a positive test result actually indicates an
affected individual
The proportion of „real” positive samples within all
positive results
Negative predictive value


The chance that a negative test result actually excludes
the disorder
The proportion of „real” negative samples within all
negative results
The beginnings…
 1920: A child with developmental delay was
born to American parents living in China. No
one could help in finding the disease.
 The mother wrote a book, describing the
symptoms.
 The child was later diagnosed as having
classical PKU.
Overview of the methods for PKU screening –
The Folling-test
 1. In the 1930s: Asbjorn Folling: a mother
noticed a strange smell of her mentally
retarded child’s urine → Folling analyzed the
urine with various tests including the ferric
chloride test (for aromatic hydroxyl groups,
such as those in ketones):
When
ketones are present, urine develops a redbrown colour.
This time the urine turned into dark-green.
Folling isolated a substance from the urine
which was confirmed to be phenylpyruvate.
The Folling-test
The ferric chloride test (for urine):
 not sensitive: usually positive at
plasma Phe concentrations above
900 μM (dark green coloration)
 not specific: a slightly altered color
reaction may be indicative of other
metabolic disorders/medication:
maple syrup disease (MSUD)
 tyrosinemia
 salicylates, L-DOPA metabolites…
traditionally, the reagent was
dropped on the diaper of the baby


The Guthrie-method
 2. From the 1960s: Robert Guthrie and Ada Susi
developed a bacterial inhibition assay, suitable
for the screening of PKU for the first time.
This
assay monitors the growth of a mutant strain of
Bacillus subtilis with a requirement for exogenous Phe
for growth.
DBS samples are placed onto agar plates containing
mutant bacteria and an inhibitor.
The sizes of the colonies are assessed after
incubation.
The Guthrie-method – principals
 The growth of Bacillus subtilis is inhibited by an
appropriate amount of β-2-thienylalanine added to the
agar.
 This inhibition is reversed when a dried blood spot
(DBS) containing the blood of a patient with PKU is
placed on the agar → Phe in the blood permits the
growth of bacteria around the DBS.
 The test is positive if the diameter of the growth zone is
between the 2 mg% (120 μM) and the 4 mg% (240 μM)
standard points (marked).
 The amount of growth is proportional to the level of Phe
in the DBS.
The Guthrie-method
Standards: 2
4
8
16
32 (mg%)
The Guthrie-method
 Control agar plate without β-2-thienylalanine inhibitor
 Rationale: antibiotic therapy can prevent the growth of
Bacillus subtilis, resulting in false-negative results
 A new blood sample is obtained if a zone with signs of
inhibited bacterial growth is found (marked)
The Guthrie-method – characteristics
 inexpensive
 specific
 semiquantitative
 not very sensitive:

limit of detection ≈180-240 μM (=3-4 mg%)
Fluorimetric assays
 3. From the 1960s: Fluorimetric assays
McCaman
and Robins (1962):
 for the determinaton of Phe only
 principals: the reaction of Phe, ninhydrin and copper yields
a weakly fluorescent product
 the fluorescence is increased by the addition of a dipeptide,
L-leucyl-L-alanine
Wong,
O’Flynn and Inouye (1964):
 modified the above method to measure Phe, and added
another method to determine Tyr
Ambrose,
Ingerson, Garrettson and Cliung (1967):
 optimized the Phe-assay by changing several parameters
Fluorimetric assays – characteristics
 quantitative
 automatization is possible
 sensitivity is good:
of detection may be as low as 6 μM
(0.1 mg%)
limit
 not specific (other substances may also
yield some degree of fluorescence)
Enzymatic colorimetric assays
 4. From the 1980s: Enzymatic colorimetric assays
Wendel, Hummel and Langenbeck (1989):
 for the measurement of Phe using
dehydrogenase, NAD and a chlorophore
Campbell
L-phenylalanine
et al. (1992):
 modified the method to reach greater specificity (lower
cross-reactivity with Tyr)
Enzymatic colorimetric assays –
characteristics
 quantitative
 automatization is possible
 sensitivity is acceptable:
limit of detection is about 43 μM (0.7 mg%)
(higher than that of the fluorescence assay)
the
 specific
Liquid chromatography-tandem mass
spectrometry (LC-MS/MS)
 5. From the 1990s: liquid chromatography-
tandem mass spectrometry (LC-MS/MS)
allows the simultaneous measurement of a number
of disorders of amino acid, organic acid and fatty
acid metabolism, including PKU
deuterated internal standards are used
derivatization
with butanol-acetyl chloride is
employed
selected ratios of the amino acids (or acylcarnitines)
are used to help the evaluation

LC-MS/MS assays – characteristics
 quantitative
 automated
 rapid
 very sensitive: lower than 1 μM (0.07 mg%)
 very specific
 the
false-positive rate is the lowest,
highlighting the advantage of using the Phe/Tyr
ratio (Tyr levels are simultaneously measured):
example:
parenteral amino acid supplementation or
too much blood on the filter paper: Phe ↑, but Phe/Tyr
is normal → PKU can be excluded
PKU screening – Blood sampling
 primary sample: blood spots dried on filter
paper (DBS)
 stability of DBS:
≈10
days at room temperature for amino acids
(≈7 days for acylcarnitines)
 problems associated with blood sampling:
inappropriate
timing
inappropriate technique
delayed delivery
insufficient data on the patient/parent
Problems with blood sampling (DBS)
 a)
Inappropriate timing of
blood sampling (rule: 48-72 h
of age; earlier: < 5 days)
the
catabolic state associated with
birth is the main trigger of most
amino acid (incl. Phe) and
acylcarnitine elevations in the first
few days of life (and not feeding)
(this is not true for galactosaemia
and some other disorders)
delayed
blood sampling may
cause false-negative results
Problems with blood sampling (DBS)
 b) Inappropriate technique of blood sampling
is mainly responsible for the SD of the MS/MS method
insufficient blood
excess blood
DBS has not dried

Safe
NOT safe
Safe
Problems with blood sampling (DBS)
 c) Delayed delivery of samples
 d) Insufficient data on the child/parent
about
drugs, parenteral feeding/glucose/middlechain triglycerides given to the newborn
contact address and telephone number of the
parent
The diagnostic value of Phe assays
 a positive screening result in a Phe assay is
generally sufficient to conclude that some
form
of
hyperphenylalaninemia
(PKU,
transient hyperphenylalaninemia or BH4
deficiency) is present
 confirmation by means of genetic testing or
gas
chromatography-mass
spectrometry
(GC/MS) is not essential
Analysis of PKU with (GC/MS): urine samples
Plus: 4-hydroxy-phenyllactate, 4-hydroxy-phenylpyruvate, mandelic acid
The diagnostic value of Phe assays
(continued)
 PKU
and BH4 deficiency cannot be
distinguished from each other by Phe levels
plus Phe/Tyr ratios
 for the differential diagnosis of BH4 deficiency:
BH4
loading test,
pterin profile analysis (from urine or DBS),
dihydropteridine
reductase (DHPR) activity
measurement (from DBS) should be performed
Phe & BH4 metabolism pathway
GTP cyclohydrolase (GTPCH)
Mutations in
genes coding
enzymes for
BH4 synthesis
(GTPCH, PCD,
SR), and BH4
recycling
(PTPS, DHPR)
result in BH4
deficiency.
5%
6-Pyruvoyl-tetrahydrobiopterin
synthase (PTPS)
60%
Sepiapterin reductase (SR)
Dihydropteridine
reductase (DHPR)
30%
q-Dihydrobiopterin
Pterin-4α-carbinolamine
dehydratase (PCD)
5%
Diagnosis of BH4 deficiency
 1. BH4 loading test
useful
in all forms of BH4 deficiency
single Phe dose plus a single BH4 dose 3 h later
blood sampling: -3; 0; 4; 8; 12; 16; 24 h
(if basal Phe level is low (e.g. < 360 μM), a 24 h Phe
loading test may be performed prior to the BH4 test)
Diagnosis of BH4 deficiency
 2.
Analysis of pterins with HPLC plus
fluorescent or MS/MS detection
Levels
of neopterin, biopterin and pterin are
measured from urine or DBS.
Chromatographic separation is needed.
Identifies variants: 65-70% of cases.
 3. DHPR activity measurement
Primary
sample: DBS
Identifies a single variant: 30-35% of cases.
Pterin levels and DHPR activity in
variants of BH4 deficiency
Phe
(plasma)
Biopterin
(urine)
Neopterin
(urine)
DHPR
activity
(blood)
Homovanillic
acid (HVA,
liquor)
5-hydroxyindoleacetic acid
(5-HIAA, liquor)
GTPCH1
(recessive)



N


GTPCH1
(dominant)
N
N ( in
liquor)
N ( in
liquor)
N

N/
PTPS



N


PCD


N/ 
primapterin
N
N
N
DHPR


N



SR
N
N ( in
liquor)
N ( liquor
sepiapterin)
N


Summary
 The
ideal method for PKU screening is
sensitive, specific, rapid and reliable.
 Of
the numerous techniques for the
measurement of Phe, the Guthrie method,
fluorimetric and enzymatic colorimetric assays,
and LC-MS/MS (the most recent technique) are
used widely for screening purposes.
 For
the
differential
diagnosis
of
hyperphenylalaninemias, BH4 loading test,
pterin profile analysis, or measurement of
DHPR activity can be performed.
Thank you for your attention!