| Analysis
of pterins
Liquid urinary samples or, even better, a random
urine specimen dried on filter paper can be used1. Reduced
urinary pterins are extremely unstable and are affected by
daylight, temperature, and storage time. The recovery of
urinary pterins in dried filter paper and stored at room
temperature for one and two weeks showed good correlation
with standard methods. A significant advantage of this method
is that the samples can be mailed unfrozen in an envelope,
speeding delivery and minimizing costs. Typical urinary pterin profiles are as follows: in GTPCH
deficiency neopterin and biopterin are very low; in PTPS
deficiency neopterin and monapterin (isomer of neopterin)
are very high and there are only traces of biopterin; in
DHPR deficiency neopterin is normal or slightly increased
and biopterin is very high; and in PCD deficiency neopterin
is initially high, biopterin is in the subnormal range and
primapterin (7-substituted biopterin) is present. Patients
with the classical PKU excrete generally more neopterin and
biopterin in urine than normal controls2.
By two-dimensional plotting of total biopterin versus percentage
of biopterin, of the sum of neopterin plus biopterin, variants
of HPA can be separated (Figure).
Collection of liquid urine specimens:
Pipette 5 ml of fresh
random urine into a centrifugation vial and adjust pH to
1.0-1.5 with 6M HCl. Add 100 mg of manganese dioxide (MnO2,
No. 5957, Merck, Darmstadt, Germany) and shake for 5 min
at room temperature. Centrifuge for 10 min at 4000 rpm.
Immediately transfer 1 ml of the clear supernatant into a
vial for shipment.
Wrap the vial in aluminum foil to protect from light.
Collection of dried urine specimens
Fresh random urine is processed as described for liquid specimen. Filter paper strips (3x5 cm, filter paper backing 165-0921, Bio-Rad, Richmond, USA) are dipped into the clear supernatant of the oxidized urine up to 1 cm below the upper edge. Excess urine is wiped off and the filter paper is left to completely dry at room temperature in dim light. The filter strip is then sent to the laboratory in an envelope by express mail.
Preconditions
The analysis of urinary pterins must be performed at elevated plasma phenylalanine levels (>400 umol/l) and not during treatment with a low-phenylalanine diet. Healthy newborns excrete relatively higher levels of pterins than children or adults3. Handling
Native urine specimens should
be stored in the dark at -20°C. Store oxidized samples
in the dark at RT.
Pitfalls
Viral infections increase neopterin
excretion in urine dramatically.
REFERENCES
- Blau N, Kierat L, Heizmann CW, Endres W, Giudici
T, Wang M. Screening for tetrahydrobiopterin deficiency in
newborns
using dried urine on filter paper. J. Inherit. Metab.
Dis. 1992;15:402-404.
- Blau N, Thöny B, Spada M, Ponzone
A. Tetrahydrobiopterin and inherited hyperphenylalaninemias.
Turk J Pediatr 1996;38:19-35.
- Niederwieser A, Curtius HC, Gitzelmann R, Otten
A, Baerlocher K, Blehova B, Berlow S, Grobe H, Rey F,
Schaub J, Scheibenreiter
S, Schmidt H, Viscontini M. Excretion of pterins in
phenylketonuria and phenylketonuria variants. Helv.
Paediatr. Acta. 1980;35:335-342.
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