Aspartame loading test in PKU heterozygous individuals bearing severe and moderate mutations

To the Editor: Phenylketonuria (PKU) is a disease of amino acid metabolism caused by mutations in the liver enzyme phenylalanine hydroxylase (PAH). The interindividual variation in disease severity observed among PKU patients is the result of extensive allelic heterogeneity at the PAH locus. To date, more than 400 mutations in the PAH gene have been reported worldwide (1, 2). Mental retardation is the main symptom observed in non-treated PKU patients (3). Several methods have been developed for the detection of PKU heterozygous individuals, based on measurements of plasma phenylalanine (Phe) and tyrosine (Tyr) levels or analysis of the Phe/Tyr and Phe/Tyr ratios (4, 5). However, results obtained in most of these studies showed an important overlap among heterozygous and normal individuals. Several studies have been performed in order to analyze possible effects of aspartame sweetener and related components on Phe and Tyr levels in heterozygous individuals for PAH deficiency (6– 8). The correlation among results of Phe loading tests and mutations in the gene have not been extensively studied (9–11). This study investigated the extent to which mutant PAH alleles influence Phe and Tyr metabolism in PKU heterozygotes after oral aspartame loading. Mutations carried by 12 PKU heterozygous individuals were identified by restriction endonuclease or sequence analyses, following DNA extraction from peripheral blood by standard methods. After an overnight fast (12 h), carriers and six controls were submitted to an oral aspartame loading test (100 mg/kg of aspartame dissolved in 300 ml of orange juice). Blood samples were collected prior to and 30 min after aspartame loading, and Phe and Tyr plasma concentrations were determined fluorimetrically (12, 13). Individuals were divided into three different groups: heterozygous individuals bearing a severe mutation, which leads to an enzyme activity below 1% (P281L, R408W or IVS12nt1g\a); heterozygous individuals bearing a moderate mutation, which leads to significant (15–50%) residual enzyme activity (V388M, R261Q or I65T); and controls. Table 1 shows Phe and Tyr plasma concentrations and Phe/Tyr and Phe/Tyr ratios before and after the loading test for each individual tested. Results were statistically compared by the Student’s t-test for paired and independent samples. Results obtained show a significant increase of Phe levels and Phe/Tyr and Phe/Tyr ratios in individuals with severe mutations after the loading test. In individuals with moderate mutations only, the Phe/Tyr ratio was significantly higher (pB 0.05) after the loading test. Comparison of measurements across groups of heterozygotes revealed the only statistically significant difference to be a higher mean level of Phe before aspartame loading (pB0.05) in heterozygotes with moderate mutations. No significant differences were seen between both groups of heterozygotes after aspartame loading. Phe levels, Phe/Tyr and Phe/Tyr ratios of both groups of heterozygotes were significantly different from the control group (pB0.05) before and after the loading test, and no overlapping values were detected. In addition, significantly higher values (pB0.05) of Tyr were seen in controls than in either or both groups of heterozygotes after aspartame loading. A recent study showed that Tyr monitoring following oral Phe loading appears to be very effective in the detection of heterozygotes, but such a scheme is not useful for evaluation of mutation severity in PKU heterozygotes (10). According to our results, Tyr level after aspartame loading is similarly not useful for predicting severity of mutations. Results presented here show that the baseline Phe plasma concentration determined in heterozygotes bearing moderate mutations was significantly higher than in heterozygotes bearing severe mutations, although overlap of values was observed. These results are in agreement with a previous study, which reported that plasma Phe and Tyr