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Threads for tear film collection and support in quantitative amino acid analysis

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Abstract

The collection of tears for chemical composition analysis is complicated by both the difficulty in sampling the tear film and the relatively low microliter volumes available for analysis. The experiments in this study are focused on the demonstration of a method for determining amino acids from tear samples. Phenol red thread was used to absorptively collect tear fluid for qualitative and quantitative analyses of amino acids in basal, reflex, and emotional tears. The thread is also used as a support for sample preparation followed by elution with a buffer. The phenol red indicator on the thread turns from yellow to red with 15-s tear absorption and allows accurate volume measurement from 100 nL to over 1 μL. Derivatization of amino acids was performed directly on the thread with primary amine reactive fluorescamine for fluorescence detection. Analyte elution was performed via centrifugation with the thread in a pipet tip suspended in a centrifuge tube. Collected tear eluate was analyzed via capillary electrophoresis with LED-induced fluorescence. Glycine, glutamate, and aspartate were baseline resolved and used for method characterization. Recoveries were at 50 % for a single derivation and elution step but average recoveries near 90 % were found with two-step processing. Glutamate and aspartate are shown to be stable stored on thread for 3 days. Basal, reflex, and emotional tears were analyzed from three subjects showing distinct amino acid profiles for each tear type. The demonstration of this method may facilitate the development of routine tear compositional analysis to assess ocular health.

Schematic drawing of thread-based tear collection and quantitative analysis

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References

  1. Zhou L, Beuerman RW. Tear analysis in ocular surface diseases. Prog Retin Eye Res. 2012;31(6):527–50.

    Article  CAS  Google Scholar 

  2. Nakatsukasa M, Sotozono C, Shimbo K, Ono N, Miyano H, Okano A, et al. Amino acid profiles in human tear fluids analyzed by high-performance liquid chromatography and electrospray ionization tandem mass spectrometry. Am J Ophthalmol. 2011;151(5):799–808.

    Article  CAS  Google Scholar 

  3. Karns K, Herr AE. Human tear protein analysis enabled by an alkaline microfluidic homogeneous immunoassay. Anal Chem. 2011;83(21):8115–22.

    Article  CAS  Google Scholar 

  4. Dumortier G, Chaumeil JC. Lachrymal determinations: methods and updates on biopharmaceutical and clinical applications. Ophthalmic Res. 2004;36(4):183–94.

    Article  CAS  Google Scholar 

  5. Small D, Hevy J, Tang-Liu D. Comparison of tear sampling techniques for pharmacokinetics analysis: ofloxacin concentrations in rabbit tears after sampling with Schirmer tear strips, capillary tubes, or surgical sponges. J Ocul Pharmacol Ther. 2000;16(5):439–46.

    Article  CAS  Google Scholar 

  6. Posa A, Bräuer L, Schicht M, Garreis F, Beileke S, Paulsen F. Schirmer strip vs. capillary tube method: non-invasive methods of obtaining proteins from tear fluid. Ann Anat. 2013;195(2):137–42.

    Article  CAS  Google Scholar 

  7. Gelstein S, Yeshurun Y, Rozenkrantz L, Shushan S, Frumin I, Roth Y, et al. Human tears contain a chemosignal. Science. 2011;331(6014):226–30.

    Article  CAS  Google Scholar 

  8. Vingerhoets A. Why only humans weep : unravelling the mysteries of tears. 2013

  9. Georgakopoulos CD, Lamari FN, Karathanasopoulou IN, Gartaganis VS, Pharmakakis NM, Karamanos NK. Tear analysis of ascorbic acid, uric acid and malondialdehyde with capillary electrophoresis. Biomed Chromatogr. 2010;24(8):852–7.

    CAS  Google Scholar 

  10. Choy CK, Cho P, Chung WY, Benzie IF. Water-soluble antioxidants in human tears: effect of the collection method. Invest Ophthalmol Vis Sci. 2001;42(13):3130–4.

    CAS  Google Scholar 

  11. Saijyothi AV, Angayarkanni N, Syama C, Utpal T, Shweta A, Bhaskar S, et al. Two dimensional electrophoretic analysis of human tears: collection method in dry eye syndrome. Electrophoresis. 2010;31(20):3420–7.

    Article  CAS  Google Scholar 

  12. Stuchell RN, Feldman JJ, Farris RL, Mandel ID. The effect of collection technique on tear composition. Investig Ophthalmol Vis Sci. 1984;25(3):374–7.

    CAS  Google Scholar 

  13. Markoulli M, Papas E, Petznick A, Holden B. Validation of the flush method as an alternative to basal or reflex tear collection. Curr Eye Res. 2011;36(3):198–207.

    Article  CAS  Google Scholar 

  14. Esmaeelpour M, Cai J, Watts P, Boulton M, Murphy PJ. Tear sample collection using cellulose acetate absorbent filters. Ophthalmic Physiol Opt. 2008;28(6):577–83.

    Article  Google Scholar 

  15. Kaye SB, Sims G, Willoughby C, Field E, Longman L, Brown MC. Modification of the tear function index and its use in the diagnosis of Sjögren’s syndrome. Br J Ophthalmol. 2001;85(2):193–9.

    Article  CAS  Google Scholar 

  16. Tomlinson A, Blades KJ, Pearce EI. What does the phenol red thread test actually measure? Optom Vis Sci. 2001;78(3):142–6.

    Article  CAS  Google Scholar 

  17. Saleh T a, McDermott B, Bates a K, Ewings P. Phenol red thread test vs Schirmer’s test: a comparative study. Eye (Lond). 2006;20(8):913–5.

    Article  CAS  Google Scholar 

  18. Storey ES, Carboni DA, Kearney MT, Tully TN. Use of phenol red thread tests to evaluate tear production in clinically normal Amazon parrots and comparison with Schirmer tear test findings. J Am Vet Med Assoc. 2009;235(10):1181–7.

    Article  Google Scholar 

  19. Saito A, Kotani T. Tear production in dogs with epiphora and corneal epitheliopathy. Vet Ophthalmol. 1999;2(3):173–8.

    Article  Google Scholar 

  20. Rajaei SM, Sadjadi R, Sabzevari A, Ghaffari MS. Results of phenol red thread test in clinically normal Syrian hamsters (Mesocricetus auratus). Vet Ophthalmol. 2013;16(6):436–9.

    Article  Google Scholar 

  21. Borra S, McCullagh EA, Featherstone DE, Baker PM, Ragozzino ME, Shippy SA. Determining striatal extracellular glutamate levels in xCT mutant mice using LFPS CE-LIF. Anal Methods. 2014;6(9):2916–22.

    Article  CAS  Google Scholar 

  22. López-Cisternas J, Castillo-Díaz J, Traipe-Castro L, López-Solís RO. Use of polyurethane minisponges to collect human tear fluid. Cornea. 2006;25(3):312–8.

    Article  Google Scholar 

  23. Fischer FH, Wiederholt M. Human precorneal tear film pH measured by microelectrodes. Graefes Arch Clin Exp Ophthalmol. 1982;6:168–70.

    Article  Google Scholar 

  24. Rocha E, Alves M, Rios D, Dartt DA. The aging lacrimal gland: changes in structure and function. Ocul Surf. 2012;29(6):997–1003.

    Google Scholar 

  25. Smith D, Chen F. The fluorescence of fluorescamine-amino acids. Biochem Biophys. 1978;189(2):241–50.

    Article  Google Scholar 

  26. Company P, Nakamura H, Pisano JJ. Derivatization of compounds plates with fluorescamine. 1976;121

  27. Shippy SA, Jankowski JA, Sweedler JV. Analysis of trace level peptides using capillary electrophoresis with UV laser-induced fluorescence. Anal Chim Acta. 1995;307(2-3):163–71.

    Article  CAS  Google Scholar 

  28. Robert N, Stuchell R, Linsy Farris IDM. Basal and reflex human tear analysis. Ophthalmology. 1981;88(8):858–62.

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Chicago, 845 W Taylor St., SES 4500, M/C 111, Chicago, IL, 60607, USA

    Vitaly Avilov, Qi Zeng & Scott A. Shippy

  2. Laboratory of Integrative Neuroscience, University of Illinois at Chicago, 840 W Taylor ST., SEL 4311, M/C 067, Chicago, IL, 60607, USA

    Scott A. Shippy

Authors
  1. Vitaly Avilov
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  2. Qi Zeng
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  3. Scott A. Shippy
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Corresponding author

Correspondence to Scott A. Shippy.

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Research involving human and/or animal subjects

The phenol red threads previously used for the collection of tears for educational purposes were procured for this work. Institutional review determined this project did not meet the definition of human subject research.

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The authors declare that they have no conflicts of interest.

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Avilov, V., Zeng, Q. & Shippy, S.A. Threads for tear film collection and support in quantitative amino acid analysis. Anal Bioanal Chem 408, 5309–5317 (2016). https://doi.org/10.1007/s00216-016-9624-7

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  • DOI: https://doi.org/10.1007/s00216-016-9624-7

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