GP15 Phenylalanine measurements in human blood using NIR spectroscopy and DBS, a preliminary study

Introduction Phenylketonuria (PKU MIM 261600) is a human metabolic disease caused by mutations in phenylalanine hydroxylase gene (PAH) and is inherited in an autosomal recessive Mendelian fashion. Phenylalanine hydroxylase (PAH also known as phenylalanine 4-monooxygenase EC 1,14,16,11) catalyzes the rate-limiting step in L-Phenylalanine (L-Phe) catabolism in liver, using tetrahydrobiopterin (BH4) and dioxygen as additional cosubstrates. Untreated PKU is associated with an abnormal phenotype which includes growth failure, poor skin pigmentation, microcephaly, seizures, global developmental delay and severe intellectual impairment. However, since the introduction of newborn screening programs and with early dietary intervention, children born with PKU can now expect to lead relatively normal lives.Dried Blood Spot, (DBS) is the golden method used to acquire specimen for neonatal screening of PKU. It is fast, not expensive and give the possibility to collect samples far from the reference centre and to quickly delivery to the analytical laboratory, where the Phe content is evaluated by Tandem Mass Chromatography. However, because PKU needs a continous followup, would be really worth to set a new, fast, reproducible and chipper biochemical approach to monitor patients and to optimize the dietary intervention. Here we describe a novel fast and non-destructive method to assay phenylalanine in human blood using Near Infra-Red (NIR) spectroscopy. Methods By using a mobile infrared micro sensor (SCIO sensor) calibrated in the Near Infrared spectrum (700nm to 1000nm), we acquired by scanning and elaborated spectra from DBS card alone, DBS card with a reference concentration scale for Phe alone, and Phe plus Tyr. Based on these data we selected the most sensitive wavelentgh window within the NIR and exploited this parameter to acquire spectra from DBS containingμ blood spiked with 0 to 1200 μM Phe, 0 to 1200 Tyr and with different Phe/Tyr.ratios. An additional experiment using 100 calibrators with 5 datapoints for phenylalaninemia was performed. For each sample, at least 20 scans in duplicate were acquired. Principal component analysis (PCA) was conducted. Results The analysis of the ‘scrubbed’ data both by a specific image elaboration software and by data point algorithms, clear show that it is possible to correlate the nature of the analyte, and its relative concentration with significative differences in the acquired spectra, to obtained the so called specific Phe NIR fingerprint of the sample These observation open the exciting possibility to design a chemiometric model to assay the amount of Phe in DBS without elution and the expensive chromatographic procedures.