The measurement of phenylalanine in biological fluids for the diagnosis of phenylketonuria (PKU)
The measurement of phenylalanine in biological fluids for the diagnosis of phenylketonuria (PKU) 2ge5A
The measurement of phenylalanine in biological fluids for the diagnosis of phenylketonuria (PKU) in newborns and the monitoring/therapeutic drug monitoring of individuals with PKU are especially important. Owing to the importance of PKU monitoring in clinical medicine, a new fluorometric method was developed for the determination of L-phenylalanine
in serum samples. This method is based on the relationship between phenylalanine
ammonia-lyase (PAL) and o-phthalaldehyde (OPA). PAL catalyzes the conversion of phenylalanine
to ammonia and trans-cinnamic acid. The formed ammonia reacts with OPA in
the presence of sodium sulfite, giving a fluorescent product. The presence of sulfide in an
alkaline environment prevents OPA from reacting with other amino acids while allowing
it to react only with ammonia. Method characterization and optimization studies, such as
the effects of pH, temperature, and interferents, were carried out. The amount of L-phenylalanine
in a human serum sample was successfully determined by using the fluorescence
emission intensity of the fluorescent product formed as a result of the reaction between
OPA and ammonia. The linear range of the method is between 10 μM and 10 mM. The
obtained result showed good agreement with the results of the biochemistry analysis laboratory.
Recoveries of 95. 41% and 73. 39% were obtained for phenylalanine and ammonia,
respectively. This PAL–OPA–based fluorometric method for phenylalanine is practical,
easy to operate, low cost, highly sensitive, and selective and can also be used for the simultaneous
determination of ammonia in human serum samples.
The measurement of
phenylalanine
in biological fluids for the diagnosis of
phenylketonuria
(
PKU
) in newborns and the monitoring/therapeutic drug monitoring of individuals with
PKU
are
especially
important
. Owing to the importance of
PKU
monitoring in clinical medicine, a new
fluorometric
method
was developed
for the determination of
L-phenylalanine
in serum samples. This
method
is based
on the relationship between
phenylalanine
ammonia-lyase
(PAL) and
o-phthalaldehyde
(OPA). PAL catalyzes the conversion of
phenylalanine
to ammonia and
trans-cinnamic
acid. The formed ammonia reacts with OPA in
the presence of sodium
sulfite
, giving a fluorescent product. The presence of sulfide in an
alkaline environment
prevents
OPA from reacting with other amino acids while allowing
it to react
only
with ammonia.
Method
characterization and optimization studies, such as
the effects of pH, temperature, and
interferents
,
were carried
out. The amount of
L-phenylalanine
in a human serum sample was
successfully
determined by using the fluorescence
emission intensity of the fluorescent product formed
as a result
of the reaction between
OPA and ammonia. The linear range of the
method
is between 10
μM
and 10
mM
.
The
obtained result
showed
good
agreement with the results of the biochemistry analysis laboratory.
Recoveries of 95. 41% and 73. 39%
were obtained
for
phenylalanine
and ammonia,
respectively
. This PAL–OPA–based
fluorometric
method
for
phenylalanine
is practical,
easy to operate, low cost,
highly
sensitive, and selective and can
also
be
used
for the simultaneous
determination of ammonia in human serum samples.
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