Analysis of endogenous D-amino acids in the central nervous system and endocrine system via capillary electrophoresis with biochemical sample treatment

Ota, Nobutoshi

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https://hdl.handle.net/2142/45614 Copy

Description

Title

Analysis of endogenous D-amino acids in the central nervous system and endocrine system via capillary electrophoresis with biochemical sample treatment

Author(s)

Ota, Nobutoshi

Issue Date

2013-08-22T16:55:28Z

Director of Research (if dissertation) or Advisor (if thesis)

Sweedler, Jonathan V.

Doctoral Committee Chair(s)

Sweedler, Jonathan V.

Committee Member(s)

• Scheeline, Alexander
• Bailey, Ryan C.
• Gillette, Martha U.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• D-amino acid
• chiral separation
• capillary electrophoresis
• nervous system

Abstract

Chirality has significant functional effects in biological system including the central nervous system. Due to their unique chirality, endogenous D-amino acids are considered to play different roles from their corresponding L-isomers. In the central nervous system, a few D-amino acids have been found and they have increasingly gained research interest because their localization suggests physiological function such as involvement in learning and memory. A well-studied D-amino acid is D-serine that works as a signaling molecule in the nervous system and its abnormal concentration can cause some pathological states. However, the other endogenous amino acids still require further study to establish their metabolic pathways, molecular/cellular functions, and physiological role(s) including pathological effect. These endogenous D-amino acids include D-aspartate, D-alanine, and D-glutamate that are found in a diverse range of animals. A major challenge in endogenous D-amino acid research is to distinguish D-amino acids from abundant L-amino acids. Furthermore, neuronal sample volume is usually limited due to heterogeneity of neuronal cells; biochemical compositions of neighboring neuronal cells can be different. To overcome these difficulties, capillary electrophoresis (CE) has been used because CE requires small sample volume and possesses chiral separation capability. Chiral CE separation can be achieved by choosing an appropriate chiral selector and/or chemical additives working as a pseudo stationary phase. Another advantage of CE is its compatibility with various detection methods. In my thesis research, I have frequently employed laser-induced fluorescence (LIF) detection to couple with CE to achieve low limit of detection and reliable quantitative analysis. For identification of D-amino acid, biochemical methods are excellent options. There are two enzymes that take D-amino acid as substrate: D-aspartate oxidase and D-amino acid oxidase. Combining with CE analysis, D-amino acids can be identified from migration time and enzyme digestion. Besides enzymatic treatment, antibody-based D-amino acid identification has been used. These biochemical sample treatments provide complementary information to help D-amino acid analysis via CE separation and detection. Using these approaches with emerging new knowledge, formation and function of these enigmatic molecules have been uncovered. Overall, various chiral CE separation modes have been developed in this study to detect D-amino acids in a complex chemical matrix. Sensitive and selective D-amino acid measurements have been achieved by employing amino acid derivatization, LIF detection and biochemical sample treatment methods besides chiral CE separation. Combining these techniques together, identification and quantitation of endogenous D-amino acids have been performed to address the presence, localization, and putative roles of D-amino acids in nervous and endocrine systems.

Graduation Semester

2013-08

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http://hdl.handle.net/2142/45614

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Copyright 2013 Nobutoshi Ota

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