The work in this dissertation presents a chiral separation method for the quantitative measurement of primary amines secreted from murine islets of Langerhans along with whole brain tissue and astrocytes. Primary amines, including amino acids, can have a chiral center which leads to nomenclature of the resulting enantiomers, L- or D-. Most alpha amino acids exist in either form, however, prior to the 1980’s D-amino acids (DAAs) were thought to not be utilized by cells.1 Of the alpha amino acids, D-serine (D-Ser)2, D-alanine (D-Ala)3,4, and D-aspartate (D-Asp)5,6, to name a few, have been identified throughout the body in considerably lower concentrations to the corresponding enantiomer. Even at low concentrations, DAAs are important to the overall homeostasis of the mammalian body. For example, D-Ser can activate the Gly binding site on N-methyl-D-aspartate receptors (NMDARs). D-Ser appears throughout the brain in varying concentrations depending on the region.7,8 Unfortunately, detection of the D-enantiomers is difficult when the sheer abundance of the L-enantiomer is considered. Several methods have monitored primary amines from numerous biological systems, including murine islets of Langerhans, whether directly or indirectly.9-14 In recent years, direct methods for monitoring cellular content have gained some ground in effort to have quantitative methods to measure chiral amines. Direct measurements of chiral amines would elucidate the roles of DAAs better compared to the indirect methods that require assumptions about pathways or signaling mechanisms. In this work, the optimized separation conditions utilized four internal standards to quantify 17 primary amines, of which were 5 D-amino acids, with limits of detection (LOD) ranging from 0.3 nM to 8 nM. The normalized migration times had relative standard deviations (RSD) less than 0.6% and the majority of the normalized peak areas were less than 10% RSD. The effects of glucose were tested on small batches of islets and a small shoulder corresponding to the same migration time as D-Ser standard was observed under high glucose. While the islet samples did not yield any D-amino acid peak, other tissues are known to contain numerous D-amino acids such as whole brain tissue. Utilizing the optimized chiral separation method, changes in D-Ser secretion and content from three different brain regions under two conditions were investigated. D-Ser is proposed to be in lower concentrations, secreted or content, in murine brains after identified with the status condition compared to the non-status brains. Samples were treated in the same manner as the islet samples with one caveat, β-Ala was no longer considered an internal standard due to its presence in the samples. The chiral separation method was capable of observing D-Ser in most of the samples, which was identified through the use of D-Ser standard spikes and D-amino acid oxidase (DAAO) to eliminate of the peak in question. The role of pure populations of astrocytes has yet to be fully examined in relation to the effects of drug abuse, specifically the direct effect of D-Ser concentration in cellular content. Due to differences in astrocytic populations based on brain region, a different set of three brain regions were selected for the next experiment. Content from whole brain regions from the hippocampus, cortex, and olfactory bulb were examined utilizing the optimized chiral separation method for the presence of D-Ser. To elucidate if astrocytes contained D-Ser in the absence of neurons, cultures of only astrocytes and co-cultures of astrocytes with neurons from the previously mentioned regions were stimulated with high potassium to induce release of cellular content. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / September 10, 2019. / capillary electrophoresis, Chiral, D-amino acids, separation / Includes bibliographical references. / Michael G. Roper, Professor Directing Dissertation; Paul Trombley, University Representative; Yan-Yan Hu, Committee Member; Geoffrey Strouse, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_752423 |
| Contributors | Evans, Kimberly Celeste (author), Roper, Michael Gabriel (professor directing dissertation), Trombley, Paul Q. (university representative), Hu, Yan-yan (committee member), Strouse, Geoffrey F. (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Chemistry and Biochemistry (degree granting departmentdgg) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text, doctoral thesis |
| Format | 1 online resource (96 pages), computer, application/pdf |
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