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Technical Briefs |
1
The Children’s Hospital, Medical Center of Bonn University, D-53113 Bonn, Germany
a address
correspondence to this author at: Universitätskinderklinik Bonn, Adenauerallee 119, D-53113 Bonn, Germany; fax 49-228-287-3444, e-mail
a.boekenkamp{at}uni-bonn.de
With an incidence of 1 in 6600 newborns, phenylketonuria (PKU)
is among the most common inborn errors of metabolism. PKU is caused by
a deficiency of hepatic phenylalanine hydroxylase (1). The
increase in the blood Phe concentration leads to permanent structural
damage of the central nervous system as a result of disturbed
myelination and neurotransmitter deficiency (2). If plasma
Phe concentrations are normalized by a low-protein diet with
supplementation of essential amino acids before 3 weeks of age,
irreversible mental retardation is prevented (2). Still,
strict metabolic control is mandatory throughout childhood
(2) and perhaps into adult life (3). This is
achieved by regular measurement of blood Phe and Tyr concentrations.
Tyr is monitored because Phe hydroxylase deficiency renders it an
essential amino acid in PKU. Recommendations for the duration and
intensity of dietary control are not uniform (2); likewise,
there is some variation in the local practices of PKU
monitoring. The current guidelines of the German
"Arbeitsgemeinschaft für Pädiatrische
Stoffwechselerkrankungen" (The German Working Group for Metabolic
Diseases) set a target range for Phe concentrations of
40–250 µmol/L, at least until age 10 (
900 µmol/L is
acceptable during adolescence) (4). These
recommendations were based on data from samples that were analyzed
immediately after sampling (Udo Wendel, University Children’s
Hospital, Düsseldorf, Germany, personal communication). Pregnant
women with even mild hyperphenylalaninemia also require strict dietary
control of Phe concentrations to prevent PKU-induced fetopathy
(1).
To ensure optimal metabolic control, patients are monitored in specialized clinics where dietary Phe intake recommendations are adjusted according to weekly to monthly Phe and Tyr measurements. To reduce the inconvenience of regular outpatient visits, most German metabolic centers (like centers in other countries) have established methods that allow patients to have capillary samples collected at home and mailed to the laboratory as whole blood (5). Postal transfer of whole-blood samples takes 24–48 h. Storage of whole blood for 6 h at 20 °C has been shown not to significantly affect the recovery of Phe and Tyr (6). To our knowledge, a delay of 48 h until sample preparation, which is introduced by the mailing process, has not been formally evaluated. We therefore studied the effect of delayed sample preparation on Phe and Tyr serum concentrations from whole blood.
Forty-nine blood samples from 35 PKU patients (11.9 ± 10.1 years of age) were obtained by venipuncture during routine monitoring at the metabolic unit of Bonn University Children’s Hospital. Blood was collected into tubes that contained bead-activating coagulation (Serum Monovette; Sarstedt). After informed consent, Phe and Tyr were measured immediately in one aliquot (Pheearly, Tyrearly), whereas the second aliquot was stored as whole blood at room temperature for 48 h until amino acid analysis (Phelate, Tyrlate), thus simulating the mailing process.
Serum sample preparation included centrifugation for 10 min at 500g and deproteinization with 50 g/L sulfosalicylic acid (1:1 by volume). Amino acid analysis was performed by ion-exchange chromatography using a Pharmacia 4151 alpha plus automated analyzer (LKB) as described previously (7). Chromatograms were read using a Shimadzu C-R6A Chromatopac Integrator (Shimadzu). Day-to-day CVs were 6.0% and 2.1% for Phe concentrations of 50 and 330 µmol/L, respectively. For Tyr, the CV was 2.7% at a mean concentration of 31 µmol/L.
Data are presented as means ± SE, and statistical analysis was performed using the Wilcoxon test. To check for a potential concentration effect on deviations between immediate (early) and delayed (late) measurements, Bland-Altman analysis was performed (8).
Pheearly (350 ± 40 µmol/L) and
Tyrearly (66 ± 6.5 µmol/L) were
significantly lower than Phelate (372 ± 40
µmol/L) and Tyrlate (75.6 ± 6.5
µmol/L), respectively (P <0.0001). Bland-Altman
analysis (Fig. 1
) showed that the difference between
Pheearly and Phelate was
inversely related to Phe serum concentrations (r =
0.327; P = 0.022). Residuals for Tyr did not correlate
with Tyr serum concentrations.
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In the present study, storage of whole-blood samples at room temperature for 48 h substantially increased the serum concentrations of Phe and Tyr. For Phe, this effect was most prominent at low concentrations. Similar changes have been observed for Phe and Tyr in EDTA plasma (Sabine Scholl, Hannover Medical School Children’s Hospital, Hannover, Germany, personal communication). The increase in amino acid concentration probably reflects proteolysis of proteins and peptides in whole blood and a transfer from blood cells into serum. Studying amino acid transfer between erythrocytes and plasma in vitro, Schaefer et al. (9) showed a transfer of Phe and Tyr along the concentration gradient. Under physiological conditions, Phe and Tyr concentrations were 30–40% higher in red blood cells than in plasma. The authors noted that there was an inverse correlation between the changes and the original intracellular amino acid concentrations after incubation of erythrocytes with a solution that contained amino acids at concentrations fourfold higher than physiological concentrations. They postulated that there are intracellular saturation concentrations that cannot be exceeded. This might explain why the increase in Phelate diminished with increasing Phe concentrations in our experiment. This effect was negligible for Tyr because Tyr concentrations are not increased in PKU and therefore the concentration range was much narrower.
Do the observed increases in Phe and Tyr concentrations induced by delayed sample preparation challenge the current practice in PKU monitoring? The quality of metabolic control during childhood has been shown to correlate directly with intellectual outcome in PKU patients (10)(11). With age, higher Phe concentrations are tolerated without adverse effects on intelligence; however, sustained attention, as well as novel problem-solving capabilities, are inversely correlated with high concurrent Phe concentrations (12)(13). In the absence of reliable home monitoring of Phe concentrations, dietary management and patient dietary education has to rely on frequent Phe and Tyr measurements in specialized laboratories (5). In this setting, the advantage of frequent short-term measurements in mailed whole-blood samples has to be weighed against the inaccuracy introduced by delayed sample preparation. The mailing process leads to an overestimation of Phe and Tyr concentrations, which may cause mild overtreatment, i.e., a reduction in dietary Phe allowances in excess of metabolic tolerance. This is not critical with Phe concentrations at or above the upper limit of normal. Still, neurological outcome has also been demonstrated to be adversely affected by Phe deficiency (14). In view of an overestimation by up to 60 µmol/L in the low concentration range and a target range of 40–250 µmol/L in fresh samples, it would appear prudent to aim at Phe concentrations >100 µmol/L for samples measured after delayed preparation from whole blood.
In conclusion, the clinical practice of delayed measurement of Phe and Tyr in whole-blood samples mailed to the specialized laboratory leads to slightly increased readings. This is clinically safe, as long as Phe concentrations are kept above 100 µmol/L to prevent Phe deficiency.
Acknowledgments
We thank Ulla Aberfeld and Elisabeth Salvay for logistic help and Claudia Ullmann and Sonja Gross for performing the amino acid measurements.
References
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