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Thread-based assay for quantitative small molecule analysis of mice tear fluid by capillary electrophoresis

Abstract

Tear fluid plays blood-like roles in the cornea, and changes in its chemical composition may be reflective of ocular surface disease pathogenesis. Studies of mice tears are limited by the small volume available for collection and difficulty in obtaining representative samples. Here, we establish a non-invasive assay for small volume analysis of small molecules in mice tears that requires no pre-treatment of mice. To the best of our knowledge, this is the first small molecule analysis of mice tears. Nanoliters of mice tears (70 ± 25 nL) was collected via a single insertion of phenol red thread in the corner of the eye without anesthesia to prevent any tear production alteration. The processing and elution of tear samples were optimized for minimal sample handling and dilution while maintaining high separation resolution. A capillary electrophoresis separation with light-emitting diode-induced fluorescence detection was developed for the analysis of primary amine-containing small molecules. The levels of arginine, alanine, aspartate, and glutamate after elution were in the micromolar range as seen in human tears. However, taurine and histamine levels were decreased and increased, respectively, compared to human tears, which may be indicative of restraint-induced emotional stress. No significant differences were seen for any of the small molecules between 20-week-old ND4 Swiss Webster females and 12-week-old CD-1 males (N = 3). The developed assay represents a means to assess the chemical composition of tear fluid in mouse models of human disease, which could significantly improve our understanding of ocular surface diseases.

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Acknowledgments

The authors thank C.R. Adams from the UIC Biologic Resources Laboratory for providing the mice used in this study and assisting with tear collection. A.B. acknowledges support from the UIC President’s Award Program STEM Initiative and Herbert E. Paaren Research Award. S.A.S. acknowledges project funding support from UIC Chemistry and College of Liberal Arts and Sciences.

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Correspondence to Scott A. Shippy.

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Research involving human and/or animal subjects

Phenol red threads that were previously used for the collection of human tears for educational purposes were procured for this study. Institutional review determined this project did not meet the definition of human subject research. Informed consent was collected from human subjects. All experiments involving animals were conducted under a protocol reviewed and approved by the UIC Institutional Animal Care and Use Committee. The animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

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The authors declare that they have no conflict of interest.

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Barmada, A., Shippy, S.A. Thread-based assay for quantitative small molecule analysis of mice tear fluid by capillary electrophoresis. Anal Bioanal Chem 411, 329–338 (2019). https://doi.org/10.1007/s00216-018-1488-6

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  • DOI: https://doi.org/10.1007/s00216-018-1488-6

Keywords

  • Tear
  • Ocular surface
  • Amino acids/peptides
  • Capillary electrophoresis/electrophoresis
  • Bioanalytical methods
  • Sampling