Combined Targeted Analysis of Metabolites and Proteins in Tear Fluid With Regard to Clinical Applications

Abstract

PurposeTo establish a robust workflow for combined mass spectrometry–based analysis of metabolites and proteins in tear fluid with regard to clinical applicability.MethodsTear fluid was taken from 12 healthy volunteers at different time points using specially designed Schirmer strips. Following the liquid extraction of metabolites from standardized punches, the remaining material was processed for bottom-up proteomics. Targeted metabolite profiling was performed adapting a metabolomics kit, which targets 188 metabolites from four different analyte classes. Proteomics was performed of the identical samples targeting 15 tear proteins relevant to ocular health.ResultsSixty metabolites could be consistently determined in all tear samples (98 metabolites were detectable in average) covering acylcarnitines, amino acids, biogenic amines, and glycerophospholipids. Following normalization, the majority of metabolites exhibited intraindividual variances of less than 20%, both regarding different times of sampling, and the individual eye. The targeted analysis of tear proteins revealed a mean intraindividual variation of 23% for the three most abundant proteins. Even extreme differences in tear secretion rates resulted in interindividual variability below 30% for 65 metabolites and two proteins.ConclusionsThe newly established workflow can be used for combined targeted detection of metabolites and proteins in one punch of a Schirmer strip in a clinical setting.Translational RelevanceOur data about intra- and interindividual as well as intereye variation provide a valuable basis for the design of clinical studies, and for the applicability of multiplexed “omics” to well accessible tear fluid with regard to future routine use.

Novel Polyvinyl Pyrrolidone–Loaded Olopatadine HCl–Laden Doughnut Contact Lens to Treat Allergic Conjunctivitis

“…The efficiency of the selected PVP-loaded DNT-OL-500 lens in stabilizing the tear film was investigated using Schirmer tear test (New Zealand rabbit model). [56][57][58] The study include 4 groups (n ¼ 6): The test was started before 3 days of contact lens insertion and continued for next 7 days (total 10 days study). The Schirmer strip (Tianjin Jingming New Technological Development Co., Ltd. Tianjin, China) of 5 Â 35 mm was placed inside the margin of the inferolateral (third) of the lower eyelid for 2 min (every 24 h) and the strip wetting was measured in mm and plotted versus time (days).…”

Short-Term Reproducibility of MUC5AC Measurement in Human Tear Fluid

“…In these experiments we identified a high variability in the levels of MUC5AC among different individuals, with coefficients of variation greater than 50%. Previous studies have shown that the inter-individual variability appears to be dependent of the specific component being analyzed in the tear film and its secretion rate [ 12 , 18 ]. In this regard, MUC5AC is produced by goblet cells in the conjunctiva but the mechanisms regulating its secretion remain largely unknown.…”

Quantifying Sample Collection and Processing Impacts on Fiber-Based Tear Fluid Chemical Analysis

“…6 Another study has used a specially designed Schirmer strip with standardized punches of 4 mm in diameter that were cut out for protein and metabolite analysis, discarding the remaining wetted part of the Schirmer strip. 7 Although these approaches standardize collection volume, discarded tear-absorbed portions may include significant concentrations of some chemical components, and these discarded portions should be always assessed to evaluate the validity of the research protocol.…”

“…4,5 The most commonly used methods for noninvasive tear analysis in both clinical and research settings from humans and animal models involve collection and processing via absorbent material, such as thread and filter paper. [6][7][8][9][10][11][12][13][14][15][16][17] Yet the impact of these analytical methods on the chemical composition of tear fluid is largely unexplored. It is crucial to define the impacts of collection and processing methods on tear analysis to standardize results drawn from different studies.…”