Although the phenylalanine/tyrosine ratio in blood has been the gold standard for diagnosis of phenylketonuria (PKU), the disadvantages of invasive sample collection and false positive error limited the application of this discriminator in the diagnosis of PKU to some extent. The aim of this study was to develop a new standard with high sensitivity and specificity in a less invasive manner for diagnosing PKU. In this study, an improved oximation-silylation method together with GC/MS was utilized to obtain the urinary metabolomic information in 47 PKU patients compared with 47 non-PKU controls. Compared with conventional oximation-silylation methods, the present approach possesses the advantages of shorter reaction time and higher reaction efficiency at a considerably lower temperature, which is beneficial to the derivatization of some thermally unstable compounds, such as phenylpyruvic acid. Ninety-seven peaks in the chromatograms were identified as endogenous metabolites by the National Institute of Standards and Technology (NIST) mass spectra library, including amino acids, organic acids, carbohydrates, amides, and fatty acids. After normalization of data using creatinine as internal standard, 19 differentially expressed compounds with p values of <0.05 were selected by independent-sample t test for the separation of the PKU group and the control group. A principal component analysis (PCA) model constructed by these differentially expressed compounds showed that the PKU group can be discriminated from the control group. Receiver-operating characteristic (ROC) analysis with area under the curve (AUC), specificity, and sensitivity of each PKU marker obtained from these differentially expressed compounds was used to evaluate the possibility of using these markers for diagnosing PKU. The largest value of AUC (0.987) with high specificity (0.936) and sensitivity (1.000) was obtained by the ROC curve of phenylacetic acid at its cutoff value (17.244 mmol/mol creatinine), which showed that phenylacetic acid may be used as a reliable discriminator for the diagnosis of PKU. The low false positive rate (1-specificity, 0.064) can be eliminated or at least greatly reduced by simultaneously referring to other markers, especially phenylpyruvic acid, a unique marker in PKU. Additionally, this standard was obtained with high sensitivity and specificity in a less invasive manner for diagnosing PKU compared with the Phe/Tyr ratio. Therefore, we conclude that urinary metabolomic information based on the improved oximation-silylation method together with GC/MS may be reliable for the diagnosis and differential diagnosis of PKU.
Keywords: Diagnosis; Gas chromatography/mass spectrometry; Phenylacetic acid; Phenylketonuria; Phenylpyruvic acid; Urinary metabolites.