CLINICAL TESTS FOR PKU
What is the Guthrie test?
The Guthrie test is a microbiological assay for the presence of phenylalanine,
phenylpyruvate, and phenyllactate in blood or urine.
What is it used for?
It is used to screen newborn infants for phenylketonuria (PKU).
How does the test work?
The test uses the growth of a strain of bacteria on a specially-prepared agar plate as a sign for the presence of high levels of phenylalanine, phenylpyruvate, and/or phenyllactate. The compound B-2-thienylalanine will inhibit the growth of the bacterium Bacillus subtilis (ATCC 6051) on minimal culture media. If phenylalanine, phenylpyruvate, and/or phenyllactate is added to the medium, then growth is restored. Such compounds will be present in excess in the blood or urine of patients with PKU. If a suitably-prepared sample of blood or urine is applied to the seeded agar plate, the growth of the bacteria in the test will be a positive indicator for PKU in the patient.
To prepare the sample for application, a small amount of blood (from a heel
puncture, for example) or urine (from a diaper, for example) is applied to a
piece of filter paper. Then a small disc is punched from the center of
the spot of blood or urine, and the disc applied to the surface of a seeded,
minimal-medium agar plate that contains added beta-2-thienylalanine.
If the sample contains phenylalanine, phenylpyruvate, and/or phenyllactate then
these compounds will diffuse into the agar medium. If their concentrations
are high enough (as with the excess levels seen with PKU), bacteria will grow
under the disc, but not elsewhere. Generally an overnight incubation is
enough to determine whether phenylalanine, phenylpyruvate, and/or phenyllactate
are present in unusual concentrations in blood or urine.
Guthrie, R., & Susi, A. (1963) Pediatrics 32, 338-343 HPLC ANALYSIS OF AMINO ACIDS
FERRIC CHLORIDE TEST
What is the ferric chloride test?
This tests for the presence of high levels of phenylpyruvate in urine. Since high phenylpyruvate levels are usually the result of PKU, this is a qualitative, indirect test for PKU.
How does it work?
Ferric ion forms a colored complex with phenylpyruvate. The complex has a characteristic blue-green color.
A small amount of a solution of ferric chloride is added to a urine sample. Development of the characteristic blue-green color indicates the presence of metabolites of phenylalanine, which would presumably be present only in patients with PKU.
Why is it no longer used?
The ferric ion may be complexed with other compounds, producing interfering colors. Other compounds in blood or urine may mask the blue-green color of the ferric complex with phenylpyruvate. Also, relatively high concentrations of phenylpyruvate must be present in order for there to be appreciable development of the characteristic color change. Thus the assay is less sensitive, and more prone to misinterpretation, than the Guthrie test.
GENETIC TESTS FOR PHENYLKETONURIA
What types of tests are used?
see Daiger et al Am J Hum Genet 1989: 45, 310
see DiLella et al 1987 Nature 327, 333
What advantages do these tests offer?
The ASO hybridization test will allow identification of specific mutant PKU
allele. Tests can be done on minute samples of blood or tissue, since
PCR can very substantially amplify critical DNA sequences to permit easier detection
of mutant alleles. Also, the tests can be done using as sources of DNA
the blood samples already taken as standard procedure. Also, the tests
can be done using amniotic fluid, thus permitting prenatal diagnosis of PKU.
Also, the tests can be used for genetic counseling for prospective parents who
are concerned about their status as possible carriers of one or another allele
Why are they not used now in place of the Guthrie test?
1. Not all mutations in the PAH gene that cause PKU are known.
2. The PCR amplification and the hybridization steps still require special technical expertise not yet regularly found in typical clinical laboratories.
3. The tests are expensive compared to the Guthrie test.
Most PKU patients are genetic compounds; that is, they have two different PKU alleles, one from each parent. Suppose that you had available an ASO for each of the two most common PKU alleles. Using an ASO dot blot test, how would you verify the genetic compound status of a patient with PKU? What would be the dot blot results for the patient's parents?
This is precisely the situation described in the paper by DiLilla et al., and
which is summarized in the reivew by Woo. See Figure 2 in the review article
al. (1989) Am. J. Hum. Genet. 45, 310
DiLella et al. (1987) Nature 327, 333
Woo, S.L.C. (1989) Biochemistry 28, 1-7