Pterin Profiling in Serum, Dried Blood Spot, and Urine Samples Using LC-MS/MS in Patients with Inherited Hyperphenylalaninemia

Background: Hyperphenylalaninemia (HPA) is defined as blood phenylalanine (Phe) levels exceeding the normal values (>120 mu mol/L or >2 mg/dL) and is caused by a deficiency in the enzyme phenylalanine hydroxylase (PAH). The widespread screening of Phe levels in newborn screening programs has led to a very high number of patients with HPA.Methods: The samples were collected at various ages, not at the point of diagnosis. Nine pterin derivatives, including isoxanthopterin, sepiapterin, xanthopterin, primapterin, biopterin, neopterin, 7,8-dihydrobiopterin, 7,8-dihydroneopterin, and tetrahydrobiopterin (BH4), were analyzed in different HPA classes in serum, dried blood spots (DBS), and urine samples. A total of 18 patients, including six classical phenylketonuria (PKU), eight BH4-responsive PKU, and four mild HPA patients, were included in the study.Results: Among the nine pterin derivatives measured, a significant increase was observed in the levels of isoxanthopterin, biopterin, and 7,8-dihydrobiopterin in serum, dried blood spots (DBS), and urine samples of patients with HPA compared to the control group. However, elevations in isoxanthopterin, biopterin, and 7,8-dihydrobiopterin were observed in all HPA groups, although the extent of elevation varied among the different disease groups. There were also significant differences between HPA subgroups among these high values.Conclusion: In this study, it might be suggested that pterin profiling shows promising potential for its effective utilization in the differential diagnosis of HPA. Pterin profiling demonstrated its efficacy in accurately categorizing patients into distinct subtypes. This approach offers several notable advantages, including the ability to simultaneously screen multiple HPA subtypes through a single test, establish disease decision limits for pterins, shorten the time required for HPA differential diagnosis, reduce the risk of misdiagnosis, and increase overall diagnostic accuracy. This study is the most comprehensive study examining the association between HPA pterin in the literature. In our study, samples obtained from BH4-responsive PKU patients were on treatment. This may have affected the results. Preliminary findings on pterin profiles may need to be replicated in a prospective cohort of samples collected at the time of diagnosis to confirm the results.