We report herein a chiral microchip electrophoresis-mass spectrometric (MCE-MS) platform for monitoring cellular uptake and release. Previous work has established that D-serine (D-Ser) plays important roles in various neurological processes. However, study on its uptake /storage and release by neuronal cells has been inadequate. In this work, PC-12 cells were incubated with a racemic serine solution, and then both intra- and extracellular levels of D-Ser and L-Ser were quantified simultaneously by the proposed chiral MCE-MS. A significant advantage of using the proposed MCE-MS analytical platform for serine quantification is that the extracellular levels of serine stereoisomers could be monitored against incubation time, thus exploring the kinetics of the uptake and release. Figure 1A illustrates the MCE-MS analytical platform. All incubation tests were carried out in the microfluidic chip. The reservoir containing the cell suspension was equipped with a piece of 0.22 mm membrane, preventing cells from getting into the microfluididc channels. The incubation solution could be injected at time intervals into the system for chiral quantification as shown in Fig. 1B. Peak identification was verified by MS (Fig. 1C). It was found that the cells preferably took up D-Ser over L-Ser. After 80 min incubation, the ratio of L-Ser /D-Ser in the culture media changed to 9 from 1 while the ratio inside the cells decreased to 0.7 from 9. Small neutral amino acids, including alanine and threonine impaired D-Ser uptake to various degrees, indicating that amino acid hetero-exchange is involved in D-Ser uptake by the cells. To study of D-Ser release, PC-12 cells loaded with D-Ser were treated with PBS containing various simulants. KCl, Glu, and Gly evoked D-Ser release. While KCl evoked a release of D-Ser / L-Ser (1:1) mixture, exposure to Glu caused release of a mixture with D-Ser being the predominant component at the first several min. of release. Interestingly, L-Ser became prodominant in the release mixture after 6 min exposure to Glu.

Chiral microchip electrophoresis-mass spectrometric platform for studying cellular uptake and release of D-/L-serine

249th National Meeting (2015)

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