Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL 32306, United States.
Program in Molecular Biophysics, Florida State University, 95 Chieftain Way, Tallahassee, FL 32306, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Dec 1;1231:123928. doi: 10.1016/j.jchromb.2023.123928. Epub 2023 Nov 10.
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 (R > 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 isleth; D-Gln: 300 ± 100 fmol isleth) or high (D-Ser: 23 ± 1 fmol isleth; D-Gln: 120 ± 50 fmol isleth) 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.
D- 氨基酸（D-AAs）是重要的信号分子，因为它们能够结合离子型 N- 甲基-D-天冬氨酸受体。D-丝氨酸（D-Ser）、D-丙氨酸（D-Ala）和 D-天冬氨酸（D-Asp）已分别在胰腺内分泌部分、胰岛和/或它们的分泌物中被发现。然而，目前还没有关于胰岛分泌物中 D-AAs 的全面评估报告。为了评估这些化合物的释放，评估了 1-（9-芴基）-乙基氯甲酸酯（FLEC 试剂）和 1-氟-2,4-二硝基苯-5-L-丙氨酸酰胺（Marfey's 试剂，MR）在胰岛特异性缓冲液中分离 D/L-AA 对映体的有效性。当使用非线性梯度和酸性流动相系统时，MR 衍生的 D/L AA 对 13 对映体的基线分辨率（Rs≥1.5）大于基线分辨率，而 FLEC 衍生的 AA 在联苯和 C18 柱上的分辨率有限。优化的 MR 方法得到了高度可重复的分离，保留时间的相对标准偏差小于 1%。分析物浓度与响应之间具有极好的线性关系（R>0.98），所有分析物响应的相对标准偏差小于 15%。除 L-Ala（200 nM）外，大多数分析物的检出限（LOD）均为 100 nM 或更低。优化的 MR 方法用于在 3-和 20-mM 葡萄糖孵育后定量分析 150 个胰岛分泌的 D-AAs。对于这两种溶液，通过与标准品的保留时间和定量器到定量器离子比进行比较，并通过加标实验，初步鉴定出 D-Ser 和 D-谷氨酸。两者都以低量分泌，在低（D-Ser：44±2 fmol isleth；D-Gln：300±100 fmol isleth）或高（D-Ser：23±1 fmol isleth；D-Gln：120±50 fmol isleth）葡萄糖浓度下，在 3 个生物学重复中没有显著差异。所开发的方法稳健，可进一步用于研究 D-AAs 的释放及其在胰岛生理学中的潜在作用。
