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Title page for ETD etd-10092009-085052


Type of Document Dissertation
Author Greene, Jason
URN etd-10092009-085052
Title Rapid Online Measurement of Amino Acid Flux of Continuously Perifused Cells
Degree PhD
Department Biological and Applied Chemistry
Advisory Committee
Advisor Name Title
John P Wikswo Committee Chair
Darryl J Bornhop Committee Member
David E Cliffel Committee Member
David L Hachey Committee Member
Franz J Baudenbacher Committee Member
Owen P McGuinness Committee Member
Keywords
  • metabolic dynamics
  • amino acid metabolism
  • microbioreactor
  • capillary electrophoresis
  • amino acid quantification
  • high pressure liquid chromatography
Date of Defense 2009-09-18
Availability unrestricted
Abstract
Amino acid metabolism is intricately linked to multiple intracellular metabolic pathways. By measuring dynamic changes in cellular amino acid flux, the pattern of changes correlates to changes in cellular metabolism. To that end, an instrument which continuously measures amino acid concentration with less than one-minute time resolution was fabricated and effluent from a custom-designed microbioreactor (MBR) was measured.

A transparent ~4 µL MBR was designed and constructed which evenly perifuses ~2×106 attached AML12 hepatocytes. The polydimethylsiloxane cell chamber was clamped to a glass top. Live and dead cells in the bioreactor were counted and the effluent collected for offline quantitation of amino acid flux rates by a new application of high-pressure liquid chromatography. Flux rates for nine amino acids correlated well to literature values of primary hepatocytes in day-long experiments.

An online amino acid flux analyzer (AAFA) was designed and constructed which separates analytes by micellar electrokinetic chromatography and quantitates the amino acid concentration by laser-induced fluorescence detection. Samples from control cell media were analyzed every 30-45 s. The detector response was found to be linear with amino acid concentration with relative standard deviations of less than 10% for 13 of 15 amino acids over a 2.5 hr continual run.

A proof-of-concept experiment was performed in which AML12 cells in the MBR were exposed to aminooxyacetate, a transaminase inhibitor, or arsenite, an inhibitor of the citric acid cycle. Dialyzed effluent was analyzed by the amino acid flux analyzer and significant differences were found between basal metabolism and aminooxyacetate exposure with glutamine and alanine flux, and alanine for arsenite exposure.

The AAFA can reproducibly quantitate changes in amino acid concentrations in less than one minute. The MBR, dialysis, and AAFA systems had a collective response time to a step change in the input perifusate composition of ~10 minutes. The cumulative transit time was ~37 minutes. Designing a larger MBR with faster flow rates, using stop-flow perifusion, or shortening tubing length should reduce the time lag. This approach offers a powerful tool for near-real-time analysis of amino acid metabolism.

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