Analysis of D-amino acids secreted from murine islets of Langerhans using Marfey’s reagent and reversed phase LC-MS/MS

Abstract

D-amino acids (D-AAs) are important signaling molecules due to their ability to bind ionotropic N-methyl-D-aspartate receptors. D-serine (D-Ser), D-alanine (D-Ala), and D-aspartate (D-Asp) have been found individually in the endocrine portion of the pancreas, the islets of Langerhans, and/or their secretions. However, there has been no report of a comprehensive assessment of D-AAs in islet secretions. To evaluate the release of these compounds, the effectiveness of both 1-(9-fluorenyl)-ethyl chloroformate (FLEC reagent) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey’s reagent, MR) in separation of D/L-AA enantiomeric pairs in islet-specific buffers were evaluated. MR-derivatized D/L AAs showed greater than baseline resolution (Rs ≥ 1.5) of 13 enantiomeric pairs when using a non-linear gradient and an acidic mobile phase system, while FLEC-derivatized AAs exhibited limited resolution on both biphenyl and C18 columns. The optimized MR method yielded highly reproducible separations with retention times less than 1% RSD. Excellent linearity between the analyte concentrations and response (R2 > 0.98) were obtained, with less than 15% RSD for all analyte responses. Most analytes had an LOD at or below 100 nM, except for L-Ala (200 nM). The optimized MR method was used to quantify D-AAs in secretions of 150 murine islets after incubation in 3- and 20-mM glucose. In response to both solutions, D-Ser and D-glutamine were tentatively identified via comparison of retention time and quantifier-to-qualifer ion ratios with standards, and from spiking experiments. Both were secreted in low quantities which did not differ significantly in either low (D-Ser: 44 ± 2 fmol islet-1h−1; D-Gln: 300 ± 100 fmol islet-1h−1) or high (D-Ser: 23 ± 1 fmol islet-1h−1; D-Gln: 120 ± 50 fmol islet-1h−1) glucose across 3 biological replicates. The method developed is robust and can be applied to further examine the release of D-AAs and their potential roles in islet physiology.