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.
Similar content being viewed by others
References
Scherz W, Doane MG, Dohlman CH. Tear volume in normal eyes and keratoconjunctivitis sicca. Graefes Arch Clin Exp Ophthalmol. 1974;192:141–50.
Versura P, Nanni P, Bavelloni A, Blalock WL, Piazzi M, Roda A, et al. Tear proteomics in evaporative dry eye disease. Eye. 2010;24:1396–402.
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;151:799–808.
Jäger K, Garreis F, Posa A, Dunse M, Paulsen FP. Functional relationship between cationic amino acid transporters and beta-defensins: implications for dry skin diseases and the dry eye. Ann Anat. 2010;192:65–9.
Zhou L, Beuerman RW. Tear analysis in ocular surface diseases. Prog Retin Eye Res. 2012;31:527–50.
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;195:137–42.
Calderone L, Grimes P, Shalev M. Acute reversible cataract induced by xylazine and by ketamine-xylazine anesthesia in rats and mice. Exp Eye Res. 1986;42:331–7.
VanDerMeid KR, Su SP, Krenzer KL, Ward KW, Zhang JZ. A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex. Mol Vis. 2011;17:1056–63.
Avilov V, Zeng Q, Shippy SA. Threads for tear film collection and support in quantitative amino acid analysis. Anal Bioanal Chem. 2016;408:5309–17.
Nicholson JK, Lindon JC, Holmes E. “Metabonomics”: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica. 1999;29:1181–9.
Hasin Y, Seldin M, Lusis A. Multi-omics approaches to disease. Genome Biol. 2017;18:83.
Johnson CH, Ivanisevic J, Siuzdak G. Metabolomics: beyond biomarkers and towards mechanisms. Nat Rev Mol Cell Biol. 2016;17:451–9.
Wishart DS. Emerging applications of metabolomics in drug discovery and precision medicine. Nat Rev Drug Discov. 2016;15:473–84.
Wishart DS. Quantitative metabolomics using NMR. Trends Anal Chem. 2008;27:228–37.
Dunn WB, Bailey NJC, Johnson HE. Measuring the metabolome: current analytical technologies. Analyst. 2005;130:606–25.
Zhang A, Sun H, Wang P, Han Y, Wang X. Modern analytical techniques in metabolomics analysis. Analyst. 2012;137:293–300.
Barbas C, Moraes EP, Villaseñor A. Capillary electrophoresis as a metabolomics tool for non-targeted fingerprinting of biological samples. J Pharm Biomed Anal. 2011;55:823–31.
Cabay MR, Harris JC, Shippy SA. Impact of sampling and cellular separation on amino acid determinations in Drosophila hemolymph. Anal Chem. 2018;90:4495–500.
Cabay MR, McRay A, Featherstone DE, Shippy SA. Development of μ-low-flow-push-pull perfusion probes for ex vivo sampling from mouse hippocampal tissue slices. ACS Chem Neurosci. 2018;9:252–9.
Bergquist J, Gilman SD, Ewing AG, Ekman R. Analysis of human cerebrospinal fluid by capillary electrophoresis with laser-induced fluorescence detection. Anal Chem. 1994;66:3512–8.
Thongkhao-On K, Kottegoda S, Pulido JS, Shippy SA. Determination of amino acids in rat vitreous perfusates by capillary electrophoresis. Electrophoresis. 2004;25:2978–84.
Graves SW, Woods TA, Kim H, Nolan JP. Direct fluorescent staining and analysis of proteins on microspheres using CBQCA. Cytometry A. 2005;65:50–8.
Udenfriend S, Stein S, Böhlen P, Dairman W, Leimgruber W, Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972;178:871–2.
Van Haeringen NJ. Clinical biochemistry of tears. Surv Ophthalmol. 1981;26:84–96.
Lam SM, Tong L, Duan X, Petznick A, Wenk MR, Shui G. Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles. J Lipid Res. 2014;55:289–98.
Stuchell RN, Feldman JJ, Farris RL, Mandel ID. The effect of collection technique on tear composition. Invest Ophthalmol Vis Sci. 1984;25:374–7.
Saleh TA, McDermott B, Bates AK, Ewings P. Phenol red thread test vs Schirmer’s test: a comparative study. Eye. 2006;20:913–5.
Rentka A, Koroskenyi K, Harsfalvi J, Szekanecz Z, Szucs G, Szodoray P, et al. Evaluation of commonly used tear sampling methods and their relevance in subsequent biochemical analysis. Ann Clin Biochem. 2017;54:521–9.
Sullivan DA, Krenzer KL, Sullivan BD, Tolls DB, Toda I, Dana MR. Does androgen insufficiency cause lacrimal gland inflammation and aqueous tear deficiency? Invest Ophthalmol Vis Sci. 1999;40:1261–5.
Nakamachi T, Ohtaki H, Seki T, Yofu S, Kagami N, Hashimoto H, et al. PACAP suppresses dry eye signs by stimulating tear secretion. Nat Commun. 2016;7:12034.
Inaba T, Tanaka Y, Tamaki S, Ito T, Ntambi JM, Tsubota K. Compensatory increases in tear volume and mucin levels associated with meibomian gland dysfunction caused by stearoyl-CoA desaturase-1 deficiency. Sci Rep. 2018;8:3358.
Rusciano D, Roszkowska AM, Gagliano C, Pezzino S. Free amino acids: an innovative treatment for ocular surface disease. Eur J Pharmacol. 2016;787:9–19.
Venza I, Visalli M, Ceci G, Teti D. Quantitative determination of histamine in tears during conjunctivitis by a novel HPLC method. Ophthalmic Res. 2004;36:62–9.
Mazurkiewicz-Kwilecki IM. Single and repeated air blast stress and brain histamine. Pharmacol Biochem Behav. 1980;12:35–9.
Eutamene H, Theodorou V, Fioramonti J, Bueno L. Acute stress modulates the histamine content of mast cells in the gastrointestinal tract through interleukin-1 and corticotropin-releasing factor release in rats. J Physiol. 2003;553:959–66.
Nakamura Y, Ishimaru K, Shibata S, Nakao A. Regulation of plasma histamine levels by the mast cell clock and its modulation by stress. Sci Rep. 2017;7:39934.
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
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.
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
ESM 1
(PDF 304 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-018-1488-6