High-Speed Microdialysis-Capillary Electrophoresis Assays for Measuring Branched Chain Amino Acid Uptake in 3T3-L1 cells

Rachel K. Harstad, Michael T. Bowser

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

We have developed a high-throughput microdialysis-capillary electrophoresis (MD-CE) assay for monitoring branched chain amino acid (BCAA) uptake/release dynamics in 3T3-L1 cells. BCAAs (i.e., isoleucine, leucine, and valine) and their downstream metabolites (i.e., alanine, glutamine, and glutamate) are important indicators of adipocyte lipogenesis. To perform an analysis, amino acids were sampled using microdialysis, fluorescently labeled in an online reaction, separated using CE, and detected using laser-induced fluorescence (LIF) in a sheath flow cuvette. Separation conditions were optimized for the resolution of the BCAAs isoleucine, leucine, and valine, as well as 13 other amino acids, including ornithine, alanine, glutamine, and glutamate. CE separations were performed in <30 s, and the temporal resolution of the online MD-CE assay was <60 s. Limits of detection (LOD) were 400, 200, and 100 nM for isoleucine, leucine, and valine, respectively. MD-CE dramatically improved throughput in comparison to traditional offline CE methods, allowing 8 replicates of 15 samples (i.e., 120 analyses) to be assayed in <120 min. The MD-CE assay was used to assess the metabolism dynamics of 3T3-L1 cells over time, confirming the utility of the assay.

Original languageEnglish (US)
Pages (from-to)8115-8122
Number of pages8
JournalAnalytical Chemistry
Volume88
Issue number16
DOIs
StatePublished - Aug 16 2016

Bibliographical note

Funding Information:
The authors gratefully acknowledge funding from the National Institutes of Health (Grant GM063533) and the National Science Foundation (CHE-1152022).

Publisher Copyright:
© 2016 American Chemical Society.

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

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