CONICET | Buscador de Institutos y Recursos Humanos

Background: Considering the unavailability of phenylalanine (Phe) free human blood, we have recently described a procedure for obtaining low Phe serum to be used in the preparation of reference materials and calibrators for newborn screening (NBS), using L-Phenylalanine Ammonia Lyase, (PAL, EC 4.3.1.25). (JIEMS. doi:10.1177/2326409815613261. 2015).Objective: To present the recent modifications introduced to the original protocol for obtaining low Phe serum and their corresponding results.Methods: PAL from Rhodosporidium toruloides (NBRC 0559) catalyzes the non-oxidative deamination of L-Phe producing trans-cinnamic acid (t-CA). The original protocol consists in treating a normal serum pool with PAL immobilized on calcium alginate beads (483 mU/mL of reaction mixture) at 37 °C for 5 h, and the following recovery of the serum without addition of exogenous substances. In the modified procedure, PAL activity was increased twice (966 mU/mL), the incubation time was reduced at 90 min, and most of the t-CA produced was eliminated by centrifugation after keeping the serum overnight at 2?8 °C. Phe was measured by Tandem Mass Spectrometry. The resulting serum was freeze dried and kept at room temperature.Results: The modified procedure allowed obtaining an almost free-Phe serum, with a negligible final concentration lower than 0.4 µmol/L. The serum lyophilization allowed to have a time-stable material easy to be transported and suitable to be used in the preparation of Phe reference materials and calibrators for NBS.Conclusions: The obtained results indicate that the freeze dried Phe-free serum is an appropriate material for the intended purpose. The modified procedure demonstrates to be reproducible, inexpensive and easy to perform, offering a new alternative to improve the assignment of Phe values to reference materials and calibrators provided for commercial reagent kits.

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