Human Tears Research Articles

Characterization of Human Tear Fluid by Means of Surface-Enhanced Raman Spectroscopy.

Tears are exceptionally rich sources of information on the health status of the eyes, as well as of whole body functionality, due to the presence of a large variety of salts and organic components whose concentration can be altered by pathologies, eye diseases and/or inflammatory processes. Surface enhanced Raman spectroscopy (SERS) provides a unique method for analyzing low concentrations of organic fluids such as tears. In this work, a home-made colloid of gold nanoparticles has been used for preparing glass substrates able to efficiently induce an SERS effect in fluid samples excited by a He–Ne laser ( = 633 nm). The method has been preliminary tested on Rhodamine 6G aqueous solutions at different concentrations, proving the possibility to sense substance concentrations as low as few M, i.e., of the order of the main tear organic components. A clear SERS response has been obtained for human tear samples, allowing an interesting insight into tear composition. In particular, aspartic acid and glutamic acid have been shown to be possible markers for two important human tear components, i.e., lactoferrin and lysozyme.

Ellipsometry of human tears

PurposeThe outer surface layer of tears is presumably composed of lipid. The thickness of this layer is considered critical to retard evaporation. Prior thickness measurements differ widely. Advances in ellipsometry have availed more precise and accurate measurements for thin films. The range in thickness of the surface layer of tears was studied by ellipsometry to uncover the source of prior discrepancies. MethodsTear surface layers of normal and dry eye subjects were measured by in-vitro ellipsometry. Lateral and Z resolutions of ∼1 μm and 0.1 nm, were achieved respectively. Thicknesses were derived from matrices and a Levenberg–Marquardt multivariate regression algorithm to Fresnel equations for multi-layered films. ResultsEllipsometric measurements of pooled and individual human tears in-vitro revealed a larger overall range (0–500 nm) of surface film thicknesses than previously reported by any one study. Each sample showed thin areas (0–2.6 nm) with interspersed thicker regions (∼200–500 nm). Repeat measurements of a single donor collected at weekly intervals showed a broad range of surface thicknesses within and between samples. Thickness measurements from a dry eye subject overlapped that of normal subjects. ConclusionThe data show that published disparity in surface film thickness may be attributable to limitations of prior methodologies. The range and overlap of surface film thicknesses challenge less rigorous methodologies that claim to segregate normal and dry eye.

Thermal Effusivity of Human Fluids

In the present study, the thermal effusivities of human fluids, such as human blood serum, urine, saliva and tears, were obtained by the photopyroelectric (PPE) technique in inverse configuration (IPPE). Human blood serum and urine were obtained from studies carried out in a government hospital for public servants in Mexico City, whereas saliva and tears were artificial. Making use of both, the amplitude and phase of the PPE signal, the thermal effusivities of each one of the mentioned human fluids were obtained. In order to obtain them, the experimental PPE data were normalized to the PPE signal with air as sample and the theoretical PPE equation was fitted to the normalized experimental data.

Immunoresolvent Resolvin D1 Maintains the Health of the Ocular Surface.

The present review focuses on the role of one of the D-series resolvins (Rv) RvD1 in the regulation of conjunctival goblet cell secretion and its role in ocular surface health. RvD1 is the most thoroughly studied of the specialized proresolution mediators in the goblet cells. The anterior surface of the eye consists of the cornea (the transparent central area) and the conjunctiva (opaque tissue that surrounds the cornea and lines the eyelids). The secretory mucin MUC5AC produced by the conjunctival goblet cells is protective of the ocular surface and especially helps to maintain clear vision through the cornea. In health, a complex neural reflex stimulates goblet cell secretion to maintain an optimum amount of mucin in the tear film. The specialized pro-resolution mediator, D-series resolvin (RvD1) is present in human tears and induces goblet cell mucin secretion. RvD1 interacts with its receptors ALX/FPR2 and GPR32, activates phospholipases C, D, and A2, as well as the EGFR. This stimulation increases the intracellular [Ca2+] and activates extracellular regulated kinase (ERK) 1/2 to cause mucin secretion into the tear film. This mucin secretion protects the ocular surface from the challenges in the external milieu thus maintaining a healthy interface between the eye and the environment. RvD1 forms a second important mechanism along with activation of a neural reflex pathway to regulate goblet cell mucin secretion and protect the ocular surface in health.

Analysis of amino acids in human tears by hydrophilic interaction liquid chromatography and quadrupole orbitrap mass spectrometry.

Amino acids in human tears play certain physiological roles and their determination is challenging due to complicated chemical properties. This study described a fast and sensitive method for the simultaneous determination of 15 amino acids (AAs) in tears by hydrophilic interaction liquid chromatography and quadrupole orbitrap mass spectrometry (HILIC-Q-Orbitrap-MS). Amino acids in tears were extracted by methanol, and then cleaned up with a solid phase extraction (SPE) cartridge. Chromatographic separation was performed on a 1.7 μm BEH Amide column within 8 min. Tear samples spiked with free AAs were tested in terms of linearity, sensitivity, repeatability, and recovery. Two stable isotope-labeled amino acids were used as internal standards to improve the method performance. Recoveries for all analytes ranged from 89 to 107%. Intra-day and inter-day precision, expressed as relative standard deviations, were all below 10%, and the method detection limits ranged from 0.02 μmol L−1 to 0.11 μmol L−1. The developed method with high throughput and high analyte specificity shows good promise for consistent analysis of free amino acids in tears.

Differential involvement of glycans in the binding of Staphylococcus epidermidis and Corynebacterium spp. to human sweat

Sweat is a secretory fluid that can be a source of unpleasant body odour due to interaction of resident bacteria with sweat components. Identification of glycoproteins in sweat suggests that protein-conjugated glycans may act as binding epitopes for bacteria, as found in other secretory fluids such as human milk, tears and saliva which help to protect epithelial surfaces from infection.We conducted proteomic and glycomic analysis of sweat to reveal an abundance of glycoproteins, predominantly carrying bi-antennary sialylated N-glycans with or without fucose. A fluorescent plate assay was used to determine whether glycans on sweat proteins provide binding epitopes for odour-producing skin commensals Staphylococcus epidermidis and Corynebacterium. Sialic acid and fucose were found to be important binding epitopes for S. epidermidis 3-22-BD-6, a strain recently isolated from human sweat, whereas fucose (but not sialic acid) contributed to the binding of Type strain S. epidermidis ATCC 12228. In contrast, our results indicate that sweat N-glycans do not provide binding epitopes for Corynebacterium.Synthetic sugar mimics of Lewis blood group antigens were investigated as potential inhibitors of the binding of S. epidermidis 3-22-BD-6 to sweat. Pre-incubation of the bacterium with LeB, LeX, LeY and sLeX (pentaose) resulted in a significant reduction in sweat protein adhesion indicating that terminal fucose is a key binding epitope, particularly when linked to a Type 2 chain (Galβ1-4GlcNAc) configuration (LeY).Our results form an impetus for future studies seeking to elucidate the role of glycans in sweat associated malodour, with possible implications for cosmetic and medical fields.

Manganese oxide nanoparticles electrodeposited on graphenized pencil lead electrode as a sensitive miniaturized pH sensor

pH monitoring in micro volume samples is required for environmental and clinical analysis. Low cost, miniaturized and stable metal oxide based pH sensors could be a suitable alternative to glass electrodes. In this study, a sensitive potentiometric solid state pH sensor based on manganese oxide nanoparticles electrodeposited on graphenized pencil lead electrode (MnO2/GPLE) was reported. The prepared MnO2/GPLE was carefully characterized by SEM, XRD and electrochemical techniques. To miniaturize the prepared pH sensor, a stainless steel 304 needle was used as a reference electrode. Selectivity, response time, stability and reproducibility of the miniaturized pH sensor were studied and compared with conventional glass pH electrode. According to experimental results, a near-Nernstian slope of − 57.051 mV/pH and linearity over the pH range of 1.5–12.5 were obtained for the developed MnO2/GPLE pH sensor. The prepared sensor represented high ion selectivity to mono-valence and multi-valence ions with $$- \log K_{{{\text{A}},{\text{B}}}}^{{{\text{Pot}}}}$$ values around 6.49. A fast response time of 20 s in acidic medium and 60 s in alkaline medium, long-term stability and reproducibility in 2 months, the simplicity of fabrication, low cost and accuracy makes this sensor as a suitable choice for rapid pH recording in micro volume samples. The MnO2/GPLE pH sensor was successfully used for the pH monitoring of human tear, human blood, saliva, apple juice, lemon juice, milk, and vinegar samples with satisfactory results.

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

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 ± 25nL) 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. Graphical abstract ᅟ.

Comparative limitations and benefits of liquid chromatography – mass spectrometry techniques for analysis of sex steroids in tears

Sex steroids impact regulation of the ocular surface tissues and thus influence dry eye. Most tissues in the human body synthesise and metabolise active sex steroids at levels required by the tissue. This is likely to also be the case for humans in ocular surface tissues. This study investigated the presence and quantities of selected sex steroids, in addition to sex steroid precursors and metabolites, in human tears. Detection of sex steroids in tears is challenging due to trace level analyte concentrations and low volumes of available tears. Immunoassays have previously been employed to assess sex steroids in tears, however, this approach only allows a single analyte to be measured and can overestimate concentrations. This study evaluated ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) and tandem mass spectrometry (MS/MS) methods for the concurrent detection and estimation of fourteen sex steroid and metabolite compounds in human tears.Basal tears were collected and pooled from 5 healthy pre-menopausal women (total 100 μL). Following protein precipitation and centrifuging, extract volumes equivalent to 14 μL of pooled tears were analysed.A Thermo Scientific Q Exactive™ Plus MS was used to compare novel high-resolution MS atmospheric pressure chemical ionization (APCI) and electrospray ionisation (ESI) methods for detection of fourteen target sex steroids, including dehydroepiandrosterone–sulphate (DHEAS), testosterone, oestradiol and their metabolites, using standards and pooled tears. The MS was programmed to switch between positive and negative polarity ionization modes at designated times through the UPLC run, in order to detect each analyte at optimal sensitivity. Analytes were analysed using APCI in standard mixtures at concentrations of 0.1, 0.3, 1, 3, 10, 30, 100 and 1000 pg/mL, per component, to determine the limits of detection (LOD) and quantification. Both parallel reaction monitoring (PRM) MS/MS and selected ion monitoring (SIM) MS, were evaluated by plotting narrow range mass-to-charge ratio (m/z) chromatograms for each analyte.An APCI UPLC-MS method to simultaneously measure 14 sex steroids using standards was successfully developed following comparative evaluation of all available LC-MS techniques. Preliminary experiments found APCI to be more sensitive than ESI using standards. Narrow m/z range SIM MS resulted in better sensitivity, in tear samples, than PRM. One sex steroid and two androgen metabolites were detected, with the developed APCI UPLC-MS method, and their concentrations estimated in human tears that had been extracted with a protein crash. Progesterone, androsterone–glucuronide (ADT-G) and 3αDiol-G were successfully detected in the tear extract and their concentrations in the pooled tear sample were estimated (with 95% confidence intervals) to be 0.10 ± 0.03 pg/μL, 30.9 ± 18.3 pg/μL and 9.8 ± 4.3 pg/μL respectively. The concentrations of the remaining 11 sex steroids in the tear sample were below the LODs of the method.This work shows that high mass resolution UPLC-MS can detect certain sex steroids and metabolites in tears, but that sensitivity of the technique and the low available tear volumes limit its application to a broader range of sex steroids. The investigation of sex steroids in tears and ocular surface tissue will aid understanding of the influence of sex steroids on ocular surface tissues facilitating better targeted treatment for dry eye.

Extracts of different pollen species and their effect on human tear fluid and an epithelial cell line

Purpose Hazelnut and birch pollen are known to destroy tear film components and attack ocular surface cells. We investigated further pollen species from different plant families, whether they show similar effects on human tear fluid and an epithelial cell line in vitro, to provide a broad basis for further research on pollen reactions affecting the tear film and ocular surface. Materials and methods Regional pollen species from different plant families (Adoxaceae, Betulaceae, Fagaceae, Juglandaceae, Malvaceae, Oleaceae, Pinaceae, Plantaginaceae, Poaceae, Salicaceae, Sapindaceae) were collected. Their proteolytic activity was evaluated by Zymography. Human tear fluid and cells of an epithelial cell line were incubated with pollen extracts. Tear fluid was analyzed by Polyacrylamide gel electrophoresis (PAGE). Cytomorphology was assessed microscopically and cell viability by proliferation (MTS), water-soluble tetrazolium (WST-1) assay and the impedance-based xCELLigence real-time analysis (RTCA). Results Zymography revealed significant protease activity and PAGE showed the degradation of tear proteins by different pollen species. Cells incubated with pollen extracts presented dose- and time-dependent cytomorphological changes. MTS, WST-1, and RTCA revealed cytostatic as well as cytotoxic effects of pollen extracts. Conclusions Pollen species from different plant families exert proteolytic activity and degrade human tear fluid as well as epithelial cells, which may play a crucial role in the pathogenesis of allergic and non-allergic reactions affecting the ocular surface.

The Effect of Aging on Nerve Morphology and Substance P Expression in Mouse and Human Corneas.

Aging impairs corneal nerve density and sensitivity. Substance P (SP), a neuropeptide secreted by sensory nerves, regulates nerve morphology and nociception. Here, we investigate the relationship between aging, nerve morphology, and SP expression in mouse and human corneas. SP levels in mouse corneas (wild type and substance P-knockout) and human corneas and tears were quantified with an ELISA assay. Corneal total nerve length (TNL) was measured with whole-mount β3-tubulin immunofluorescence in mouse and in vivo laser corneal confocal microscopy in humans. SP and β3-tubulin stained cross-sections were used to assess the colocalization of SP and nerves in human and mouse corneas. Ocular surface nociception was assessed with a wiping test in mice. SP colocalizes with sub-basal neurons in mice and humans. In WT mice, SP levels decrease with age (P = 0.0045, 8 vs. 52 weeks; P = 0.004, 26 vs. 52 weeks) as well as TNL (P = 0.018, 8 vs. 26 weeks; P = 0.0001, 8 vs. 52 weeks). Knockout mice show a greater TNL reduction (8 vs. 26 weeks, P = 0.0016) than WT mice. In the oldest WT and age-matched KO mice, nociception is impaired (P = 0.007 and P < 0.0001, respectively), and KO mice sensitivity is restored by topical SP treatment. In humans, SP levels are reduced in old subject corneas and correlate, in tears, with age (P = 0.0368); TNL also decreases in older patients (P = 0.0002). Age-associated corneal nerve loss is paralleled by reduction of SP expression in mice and humans. SP promotes the maintenance of normal nerve morphology in the long term and modulates nociception in the cornea.

Investigation of the global protein content from healthy human tears

Considering absence of invasiveness and side effects, tears emerge as a particularly attractive fluid for biomarker discovery and therefore for daily clinical use. However, to date this fluid remains poorly studied in healthy condition. Here, we present an updated in-depth characterisation of the human healthy tear protein composition using proteomics approach. Both eyes of 8 healthy controls (4 men and 4 women, average age: 38 ± 18) were collected using the Schirmer's strip method. After liquid digestion and off-gel electrophoresis fractionation, three independent proteomics analyses were performed on a LTQ-Orbitrap Velos Pro. Globally, 1351 proteins were identified with 2 unique peptides and 1% FDR. Gene ontology analyses showed up that 39% of the tear proteins were enzymes, with high numbers of dehydrogenases, phosphatases, kinases and ligases. Immunoglobulins, serpins and 14-3-3 domains proteins also emerged as the main tear protein families. The glycolysis and the coagulation and complement cascades, which were already shown in tears as involved in ocular and systemic diseases, were highlighted performing pathway analyses. Our study therefore complements the existing data on healthy tears proteome. Nevertheless, extensive studies for deeply and definitively characterise this promising fluid are required in the near future in order to be able to routinely use this fluid in clinics. A better understanding of its protein content will probably open new avenues in the biomarker discovery and clinical practice in the near future.

The feasibility of finger prick autologous blood (FAB) as a novel treatment for severe dry eye disease (DED): protocol for a randomised controlled trial

IntroductionPatients with severe dry eye disease (DED) often have limited treatment options with standard non-surgical management focused on the use of artificial tears for lubrication and anti-inflammatory drugs. However, artificial...

Label-Free Detection of Tear Biomarkers Using Hydrogel-Coated Gold Nanoshells in a Localized Surface Plasmon Resonance-Based Biosensor.

The dependence of the localized surface plasmon resonance (LSPR) of noble-metal nanomaterials on refractive index makes LSPR a useful, label-free signal transduction strategy for biosensing. In particular, by decorating gold nanomaterials with molecular recognition agents, analytes of interest can be trapped near the surface, resulting in an increased refractive index surrounding the nanomaterial, and, consequently, a red shift in the LSPR wavelength. Ionic poly( N-isopropylacrylamide- co-methacrylic acid) (PNM) hydrogels were used as protein receptors because PNM nanogels exhibit a large increase in refractive index upon protein binding. Specifically, PNM hydrogels were synthesized on the surface of silica gold nanoshells (AuNSs). This composite material (AuNS@PNM) was used to detect changes in the concentration of two protein biomarkers of chronic dry eye: lysozyme and lactoferrin. Both of these proteins have high isoelectric points, resulting in electrostatic attraction between the negatively charged PNM hydrogels and positively charged proteins. Upon binding lysozyme or lactoferrin, AuNS@PNM exhibits large, concentration-dependent red shifts in LSPR wavelength, which enabled the detection of clinically relevant concentration changes of both biomarkers in human tears. The LSPR-based biosensor described herein has potential utility as an affordable screening tool for chronic dry eye and associated conditions.

Detecting Glucose Levels in Blood Plasma and Artificial Tear by Au(I) Complex on the Carbopol Polymer: A Microfluidic Paper-Based Method.

We report on a selective paper-based method and a microfluidic paper-based analytical device (μPAD) for the detection of human plasma glucose and tear glucose using carbopol polymer-encapsulated Au(I) complex (AuC2C6H4OMe)2(Ph2P(C6H4)3PPh2), (B5). To the best of our knowledge, this demonstrates for the first time the glucose sensing based on dual emission, i.e., fluorescence and phosphorescence, of a single type molecule on the carbopol polymer. Upon addition of human blood treated with anticoagulants to μPADs, plasma is separated from the blood and flows into the response region of the μPADs to react with carbopol polymer-encapsulated B5, in which the ratiometric luminescence is analyzed. The plasma glucose concentration can be quantitively detected at 1.0–50.0 mM on paper, and tear glucose can be detected at 0.1–4.0 mM on μPADs. Owing to the structural design, this device has superior ratiometric changes of dual emission over other Au(I) complexes for signal transduction. The encapsulation of carbopol polymer also offers long-term storage stability. In tear measurement, carbopol polymer is not only used to encapsulate enzyme to remain the enzyme’s activity, but also played as a glue (or media) to connect microfluidic channel and response region. This further improves the sensitivity and limit of detection for glucose. Moreover, this sensor provides a faster response time, a wider range for glucose sensing than reported previously, and no statistical difference of the data from a commercial glucometer, allowing for practical diagnosis of diabetes and healthy individuals.

Femoral entheseal shape and attachment angle as potential risk factors for anterior cruciate ligament injury

Although non-contact human ACL tears are a common knee injury, little is known about why they usually fail near the femoral enthesis. Recent histological studies have identified a range of characteristic femoral enthesis tidemark profiles and ligament attachment angles. We tested the effect of the tidemark profile and attachment angle on the distribution of strain across the enthesis, under a ligament stretch of 1.1. We employed a 2D analytical model followed by 3D finite element models using three constitutive forms and solved with ABAQUS/Standard. The results show that the maximum equivalent strain was located in the most distal region of the ACL femoral enthesis. It is noteworthy that this strain was markedly increased by a concave (with respect to bone) entheseal profile in that region as well as by a smaller attachment angle, both of which are features more commonly found in females. Although the magnitude of the maximum equivalent strain predicted was not consistent among the constitutive models used, it did not affect the relationship observed between entheseal shape and maximum equivalent strain. We conclude that a concave tidemark profile and acute attachment angle at the femoral ACL enthesis increase the risk for ACL failure, and that failure is most likely to begin in the most distal region of that enthesis.

Intra-articular treatment with corticosteroids increases apoptosis in human rotator cuff tears

ABSTRACTPurpose: The aim of this study is to evaluate in vivo the level of apoptosis in human rotator cuff tears and the relationship it might have with tendon degeneration.Methods: Rotator cuff biopsies from 19 male and female patients, ages between 38 and 68 years, with and without previous corticosteroid infiltrations were collected via arthroscopy. Biopsies from seven patients with healthy rotator cuffs were used as a control group. An in situ terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed to detect the level of apoptosis, which was expressed as a percentage of apoptotic cells (PAC).Results: PAC in patients with corticosteroid infiltrations was 76.97 ± 16.99 in all tendon rupture zones, in non-infiltrated patients was 35.89 ± 22.96, whereas in control patients was 14.48 ± 8.15. Likewise, the tendency of PAC reveals that apoptosis in control and non-infiltrated groups was different and dispersed in all tear zones; while in corticosteroid treated patients, the tendency was similar in all rupture sites.Conclusions: This investigation leads us to conclude that the administration of corticosteroid is associated with a higher amount of apoptosis at the insertion site of the rotator cuff (rupture edge).

Comparison of Collection Methods for the Measure of Human Meibum and Tear Film-Derived Lipids Using Mass Spectrometry

ABSTRACTPurpose/aim: To assess the effectiveness of polytetrafluoroethylene (PTFE) tubes in the collection of human tears and meibum.Materials and methods: This was a prospective study that enrolled 10 healthy human subjects. Both the tear film and meibum were sampled using PTFE tubes in the right eye of all subjects. In the left eyes, either 5-µL or 1-µL glass microcapillary tubes were used to collect tears, and 0.5-µL glass microcapillary tubes were used to collect meibum. The lipids from the samples were extracted and analyzed using mass spectrometry (SCIEX TripleTOF 5600, Framingham, MA, USA). The absolute peak intensities of the omega-acyl hydroxy fatty acids (OAHFA), cholesterol esters (CE), and wax esters (WE) obtained for both methods were summed and compared between collection methods.Results: A total of 10 subjects completed the study (five female, mean age: 35.7 ± 7.9 years). Using the mass spectrometer output, the median (first quartile, third quartile) summed intensity units of OAHFA, CE, and WE collected associated with tears using PTFE were 516 (125, 1315), 7946 (2571, 19,915), and 38,892 (139,630, 174,082), all of which were significantly higher (all p ≤ 0.04) than those collected from glass microcapillaries (91 (41, 408), 2463 (1389, 6042), and 11,109 (7465, 37,371), respectively). The median summed intensity units of OAHFA, CE, and WE associated with meibum (1958 (1417, 3502), 11,726 (8434, 87,691), and 84,771 (52,657, 206,050), respectively) using PTFE were not significantly different (all p ≥ 0.13) than those associated with glass microcapillaries (1502 (699, 4407), 10,781 (3287, 38,205), and 77,381 (26,590, 178,213), respectively).Conclusions: PTFE tubes, which are thought to be lipophilic, were associated with more measurable lipids from the tear film than glass microcapillaries. There was no difference between collection methods in lipid profiles when used with meibum.

Clusterin from human clinical tear samples: Positive correlation between tear concentration and Schirmer strip test results

PurposeTo investigate the relationship between tear concentration of the homeostatic protein clusterin (CLU) and dry eye signs and symptoms, and to characterize tear CLU protein. MethodsTwo independent studies were conducted, one in Tucson (44 subjects), the other in Los Angeles (52 subjects). A cohort study design was employed to enroll patients without regard to dry eye diagnosis. Dry eye signs and symptoms were assessed using clinical tests. Tear samples were collected by Schirmer strip, and also by micropipette at slit lamp when possible. CLU from both sample types was quantified by immunoassay. The relationship between CLU concentration and clinical test scores was determined by Pearson's correlation coefficient (for individual eyes) and multiple linear regression analysis (including both eyes). CLU was also evaluated biochemically by western blotting. ResultsIn the Tucson cohort, a positive correlation was observed between tear CLU concentration and results of the Schirmer strip test, a measure of tear flow (p = 0.021 includes both eyes). This result was corroborated in the Los Angeles cohort (p = 0.013). The mean tear CLU concentration was 31 ± 14 μg/mL (n = 18 subjects, 33 eyes; range = 7–48 μg/mL). CLU from clinical tear samples appeared biochemically similar to CLU from a non-clinical tear sample and from blood plasma. ConclusionsResults support the hypothesis that an optimal concentration of tear CLU is important for ocular surface health, and that this drops below the effective threshold in dry eye. Tear CLU measurement might identify patients that could benefit from supplementation. Information about concentration will aid development of therapeutic dosage parameters.

Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips.

PurposeReliable measurement of MUC5AC in human tears is essential for elucidation of the pathophysiological role of MUC5AC in dry eye disease. The purpose of this study was to develop a sensitive and reliable method for measurement of MUC5AC in human tear samples extracted from Schirmer strips by modifying a commercially available ELISA.MethodsMUC5AC was extracted from Schirmer strips containing human tears by PBS with various concentrations of polysorbate 20. The extracts were treated with neuraminidase A to cleave the sialic acids in MUC5AC. An ELISA plate was blocked to prevent nonspecific binding. The rate of extraction of MUC5AC from Schirmer strips, linearity of dilution, limit of quantification, calibration range, and intra-assay and inter-assay reproducibility were examined.ResultsMUC5AC was extracted using polysorbate 20 in a concentration-dependent manner. Extraction was more efficient at 37°C than at 25°C. The signal-to-noise ratio of the assay was dramatically increased by treatment with neuraminidase A. Treatment with a blocking reagent before incubation produced good linearity of dilution. The inter-assay and intra-assay coefficients of variation were ≤16.6%. The relative error was within 13%.ConclusionWe developed an efficient method for extraction of MUC5AC from Schirmer strips and a highly sensitive, reliable assay for MUC5AC in human tear samples using a commercially available ELISA kit. This method will aid in our understanding of the pathophysiology of dry eye, assessment of the effects of treatment in daily practice, and selection of appropriate therapeutic agents for patients.