Tear Fluid Proteins Research Articles

Chitosan/alginate based multilayers to control drug release from ophthalmic lens

In this study we investigated the possibility of using layer-by-layer deposition, based in natural polymers (chitosan and alginate), to control the release of different ophthalmic drugs from three types of lens materials: a silicone-based hydrogel recently proposed by our group as drug releasing soft contact lens (SCL) material and two commercially available materials: CI26Y for intraocular lens (IOLs) and Definitive 50 for SCLs. The optimised coating, consisting in one double layer of (alginate – CaCl2)/(chitosan+glyoxal) topped with a final alginate-CaCl2 layer to avoid chitosan degradation by tear fluid proteins, proved to have excellent features to control the release of the anti-inflammatory, diclofenac, while keeping or improving the physical properties of the lenses. The coating leads to a controlled release of diclofenac from SCL and IOL materials for, at least, one week. Due to its high hydrophilicity (water contact angle≈0) and biocompatibility, it should avoid the use of further surface treatments to enhance the useŕs comfort. However, the barrier effect of this coating is specific for diclofenac, giving evidence to the need of optimizing the chemical composition of the layers in view of the desired drug.

Lacrimal and salivatory glycoproteins in ophthalmic herpes

to compare tear, saliva, and plasma levels of acute phase proteins (APPs) of inflammation in patients with herpes keratitis and to use the RESULTS in treatment evaluation. APPs were measured in tears, oral fluid, and blood plasma from 22 adults and 34 children with ophthalmic herpes as well as 68 healthy controls using immunoturbidimetric and spectrophotometric methods of detection. High levels of C-reactive protein and orosomucoid, low levels of ceruloplasmin, α1-antitrypsin, and transferrin in tears from patients with herpes keratitis as well as abnormal tear, saliva, and plasma APPs levels at discharge are poor prognostic signs. They all indicate that corneal inflammation is still intense and that the treatment should not be ceased yet. Severity of APPs concentration changes in tear from patients with herpes keratitis correlates with the depth of corneal lesions, recurrence rate, and disease dynamics. Quantitative determination of acute phase proteins in tear and oral fluid is an early and sensitive inflammation test and may be also used for non-invasive monitoring and antiviral treatment evaluation. Oral fluid allows to extend the capabilities of non-invasive diagnostics of ophthalmic herpes.

Modern approach to early diagnosis and pathogenetic treatment of diabetic retinopathy

To improve the approach to pathogenetic treatment of diabetic retinopathy (DR) through early diagnosis and a new method for predicting disease progression. The study enrolled 330 type 2 diabetes patients with DR (660 eyes), of whom women constituted 64.6%, men - 35.4%. The mean patient age was 62.3±2.3 years. Three groups were formed: the controls - 30 healthy volunteers (60 eyes) and 30 type 2 diabetes patients without ocular involvement (DR 0, 60 eyes); group 1 - 30 type 2 diabetes patients with DR I but no diabetic macular edema (DR I without DME, 60 eyes) that were treated with calcium dobesilate; group 2 - 240 type 2 diabetes patients, who had diabetic retinopathy of different stages (DR I, II, or III with DME, 480 eyes) and received laser retinal photocoagulation (LRP). The groups were all alike in terms of sex and age distribution. All patients underwent ophthalmic examination, including best corrected visual acuity (BCVA) and critical flicker fusion frequency (CFFF) testing, tonometry, biomicroscopy, MAIA fundus microperimetry, optical coherence tomography (OCT), and fluorescein angiography (FAG) of the retina. Traditionally we also determined blood sugar and glycated hemoglobin levels as well as vascular endothelial growth factor (VEGF-A) and monocyte chemoattractant protein (MCP-1) in tear fluid by ELISA. In group 1, which was under conservative therapy with calcium dobesilate, there was an increase in BCVA by the average of 0.95±0.02 and CFFF by 42.5±0.2 Hz (p<0,05). The mean central retinal thickness decreased reliably down to 265.1±12.1 µm (p<0.05). Light sensitivity of the macula improved and scored 24.13±12.3 dB (p<0.05). In group 2, the mean central retinal thickness appeared to be 383.1±221 µm, which was reliably higher than that in healthy individuals (p<0.05) and in type 2 diabetes patients without diabetic retinopathy (DR 0) (p<0.05). Tear assessment 12 months after the treatment revealed a significant decrease in VEGF-A and MCP-1 concentrations - down to 655.1±86.1 pg/ml and 1133 pg/ml, respectively (p<0.05). Conservative treatment with calcium dobesilate has proved effective in patients with DR I without DME as it ensures improvement and stabilization of the state of the retina (clinical and morphological) in one month already (judging from FAG and OCT findings). Laser treatment is rational in DR I, DR II, and DR III patients, whose condition is complicated with DME. Improvement and stabilization take, however, longer to be achieved - up to 1 year (according to FAG and OCT). Tear fluid assessment for particular participants in disease pathogenesis, such as VEGF-A and MCP-1, is a unique method for disease control and patient follow-up with account to different treatments. A new method for predicting the progression of diabetic retinopathy and diabetic macular edema has been suggested (RF patent for invention №2520826).

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Aims Dry eye disease (DED) is a distressing condition that is commonly associated with ageing, contact lens wear and autoimmune disease. DED is one of the fastest-growing eye disorders in older populations (over-50s) and is significantly underdiagnosed. Multiplex studies of tear fluids from DED patient have suggested a role for inflammatory cytokines in its pathology. In this study, we investigated tear fluids from DED and normal subjects for a panel of 7 cytokines, using the multiplex bead assay.

Analysis of Graves’ ophthalmopathy patients’ tear protein spectrum

Background Graves’ ophthalmopathy/orbitopathy (GO) patients often suffer ocular surface damages and tear fluid proteins play a significant role in maintaining healthy ocular surfaces, while changes in tear protein components reflect the changes ocular surface abnormalities. In this study proteomics techniques were used to investigate tear protein compositions in GO patients. Methods We carried out a case-control study by comparing tear fluid contents of GO patients with that of healthy subjects. In the first step the tears were subjected to SDS-PAGE electrophoresis and then single protein bands were analyzed by to in-gel trypsin digestion and nano-flow liquid mass spectrometry (LC-MS/MS) using a MS software. Results In tear samples of GO subjects, the protein fractions of inflammation-related protein immunoglobulin kappa chain C region (IgKC) and serum albumin were essentially reduced, whereas a novel isoform of complement component 3 (C3), which we detected in control subjects, was completely absent in the GO patients’ tears. Conclusions Reduced protein concentrations of particularly IgKC and complement C3 as well as albumin in the tears of GO patients may contribute to changes in their ocular surfaces via diminished reactive oxygen species (ROS) depletion and adaptive immune responses. The completely absent of C3 in the GO patients’ tears, may imply that an important inflammatory signaling pathway is affected, which needs further investigation.

Lysozyme transport in p-HEMA hydrogel contact lenses

Protein binding in hydrogels adversely affects their performance and can interfere with their usage in several biomedical applications including contact lenses. In this study we focus on understanding and modeling the mechanisms of protein transport in hydrogels, specifically focusing on the effect of protein concentration and gel crosslinking on transport. Specifically, we focus on lysozyme, the most abundant protein in tear fluid, and hydrogels of poly-hydroxyethyl methacrylate (p-HEMA), a common contact lens material. Protein uptake experiments with gels of different thicknesses showed a time scale increase as the square of the thickness suggesting diffusion controlled transport. Partition coefficient was found to be dependent on the equilibrium concentration of lysozyme, and also on the degree of crosslinking. Since transport is related to mesh size, gel modulus was obtained for various crosslinkings and utilized to estimate the mesh size. The transport data were fitted to a diffusion model and the fitted diffusivity was compared to diffusivity predicted from a model based on hydrogel mesh size. Both protein absorption and desorption data fitted the diffusion model with the same value of diffusivity, but the experimentally measured diffusivities were significantly smaller than those estimated on the basis of the gel mesh size. Models were modified to take into account protein binding to the polymer but the modified predictions were still larger than the measured values. The results of this study could assist in the development of contact lens materials that exhibit minimal protein binding, in designing cleaning regimens for protein removal from contact lenses, and in applications related to protein binding in several other biomaterials.

Wax-tear and meibum protein, wax–β-carotene interactions in vitro using infrared spectroscopy

Protein–meibum and terpenoids–meibum lipid interactions could be important in the etiology of meibomian gland dysfunction (MGD) and dry eye symptoms. In the current model studies, attenuated total reflectance (ATR) infrared (IR) spectroscopy was used to determine if the terpenoid β-carotene and the major proteins in tears and meibum affect the hydrocarbon chain conformation and carbonyl environment of wax, an abundant component of meibum. The main finding of these studies is that mucin binding to wax disordered slightly the conformation of the hydrocarbon chains of wax and caused the wax carbonyls to become hydrogen bonded or experience a more hydrophilic environment. Lysozyme and lactoglobulin, two proteins shown to bind to monolayers of meibum, did not have such an effect. Keratin and β-carotene did not affect the fluidity (viscosity) or environment of the carbonyl moieties of wax. Based on these results, tetraterpenoids are not likely to influence the structure of meibum in the meibomian glands. In addition, these findings suggest that it is unlikely that keratin blocks meibomian glands by causing the meibum to become more viscous. Among the tear fluid proteins studied, mucin is the most likely to influence the conformation and carbonyl environment of meibum at the tear film surface.

Detection of ICP0 protein in tear fluid of individuals with active herpetic epithelial keratitis

Infected cell protein 0 (ICP0) has been detected in the tear fluid of rabbits with herpetic keratitis. Here, we investigated whether ICP0 of herpes simplex virus 1 is detectable in the tear fluid of patients with herpetic epithelial keratitis. Seven patients with herpetic epithelial keratitis (age range, 51-76 years) and 11 healthy volunteers (age range, 48-85 years) were enrolled in the study. Tear fluid was collected with the use of Schirmer test strips and subjected to immunoblot analysis with antibodies to ICP0. ICP0 was not detected in the tear fluid of the healthy controls, whereas it was detected in 4 of the 7 patients with herpetic keratitis. The tear fluid of the 4 ICP0-positive patients was collected within 3 days of the onset of keratitis, whereas that of the ICP0-negative patients was obtained at least 7 days after disease onset. ICP0 was detected in the tear fluid of patients in the early phase of herpetic epithelial keratitis, but not in that of patients in the later stages of the disease nor in that of the healthy controls. ICP0 expression may thus be dependent on the disease phase.

Interaction of phospholipid transfer protein with human tear fluid mucins

In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.

The importance of fine dust for the development of an environmentally induced dry eye

Abstract Purpose Environmental conditions promote dry eye by destruction of tear fluid and induction of cell damage. Fine dust is not only present in an urban polluted environment, but also in a cigarette fume or candle light dust loaded home environment. We wanted to determine, if fine dust affects the tear fluid components and conjunctival cells. Methods Human tear fluid from volunteers was obtained and incubated with fine dust suspensions. Possible tear protein changes were analysed by polyacrylamidegel‐electrophoresis (PAGE). Cultivated human conjunctival cells (CHANG‐cells) were incubated with fine dust suspensions as well as exposed to ultraviolet light (UV‐A). Cytomorphological changes and cell vitality were analysed via CASY1 Cell Counter and MTS‐assay. Results Fine dust suspensions destroy tear fluid proteins, particularly in combination with UV‐A‐light irradiation. Moreover, the incubation of human conjunctival cells with fine dust suspensions leads to a statistically significant decrease of cell vitality, depending on the fine dust concentration and incubation period. Additional UV‐A‐irradiation induces a further reduction of cell vitality. Conclusion Due to the alterations of human tear proteins and conjunctival cells caused by fine dust and exaggerated by UV‐A‐irradiation, fine dust can also be responsible for the development of an environmentally induced dry eye.

A lubricious formulation exhibiting reduced thrombogenicity, cell proliferation, and protein adsorption

The adhesion of human platelets, erythrocytes, and leukocytes, the adsorption of protein, and the proliferation of human umbilical vein endothelial cells (HUVEC) on the surface of electropolished stainless steel and the lumen of polyurethane tubing coated with Hydromer's lubricious Duality T8B formulation was evaluated. Following exposure to a platelet-enriched suspension from citrated human whole blood, stainless steel coated with this formulation exhibited significantly reduced adhesion of platelets, erythrocytes, and granulocytes. This reduction in adhesion was confirmed using an immunohistochemical method utilizing antibodies to CD41, CD235, and CD15, respectively. The proliferation of HUVEC cells were significantly reduced when cultured on coated stainless steel. This formulation was also able to significantly reduce the adsorption of plasma proteins and the major protein in tear fluid (lysozyme) to the surface of stainless steel. The nonthrombogenic properties of Duality T8B after application to the lumen of polyurethane tubing were also examined. Following a short-term (3 h) static exposure to citrated human whole blood, microscopic examination revealed that the adhesion of platelets and erythrocytes was reduced significantly, a finding confirmed using anti-CD41 and anti-CD235 antibodies in the immunohistochemical method. A long-term (12 day) study yielded essentially identical results indicating a significant reduction in the adhesion of blood components on the luminal surface of coated polyurethane tubing. In summary, these data indicate that the application of Duality T8B onto surfaces of medical devices, such as catheters, extracorporeal circuitry, and coronary stents, could aid in reducing or preventing not only thrombus formation but also the process of restenosis.

Serum and Tear Levels of Nerve Growth Factor in Diabetic Retinopathy Patients

To measure serum and tear nerve growth factor (NGF) concentrations in diabetic retinopathy (DR) patients to determine whether the NGF correlated with parameters associated with DR. Cross-sectional comparative study. The study enrolled 254 DR patients and 71 nondiabetic controls. Patient demographic characteristics and diabetic parameters, including blood sugar levels, HbA1c, liver and renal function, were evaluated in two separate university hospitals. Serum and tear NGF concentrations and the ratio of NGF to total protein (TP) in serum and tear fluid were determined. Serum and tear NGF levels were found to be higher in proliferative diabetic retinopathy (PDR) patients (98.7+/-12.1 ng/ml in serum, 45.6+/-6.3 ng/ml in tear) than in nondiabetic controls (18.5+/-6.1 ng/ml in serum and 8.3+/-4.7 ng/ml in tear) and nonproliferative diabetic retinopathy (NPDR) patients (26.6+/-5.1 ng/ml in serum and 8.6+/-4.2 ng/ml in tear; p<.001 for both serum and tear differences). Similarly, NGF levels were higher in PDR patients than in controls and NPDR patients after adjusting for possible confounding factors such as age, gender, serum blood urea nitrogen, creatinine, and diabetic parameters. In addition, the NGF-to-TP ratio for both serum and tear fluid was higher in the PDR group compared with the control and NPDR groups. NGF levels correlated well with diabetes duration, HbA1c, and blood sugar levels and diabetic nephropathy. NGF concentration may be a good parameter for evaluating DR status. In addition, serum and tear NGF concentrations correlated strongly, indicating that tear fluid assays may offer an effective, accurate, and noninvasive option for NGF measurement.

Single eye analysis and contralateral eye comparison of tear proteins in normal and dry eye model rabbits by MALDI-ToF mass spectrometry using wax-coated target plates.

A study of rabbit tear protein expression in a dry eye rabbit model was performed to determine if a pattern in expressed proteins could be identified. The uniqueness of the model allows the comparison of normal (control) eye tear protein expression with surgically induced dry eye tear protein expression in individual animals. The sensitivity of the method allows for single eye analysis. One-dimensional mini-gel electrophoresis of the tear proteins did not show substantial differences between band patterns of the normal versus the dry eye, but was used to assess the molecular weight ranges of the major proteins. Specific assignments of some of the predominant proteins were obtained by tandem mass spectrometry (MS) which showed that the lower molecular weight lipid-binding proteins (approximately 10 kDa to 36 kDa) constitute a considerable amount of the observed protein, followed in lesser quantities by the transferrins which have higher molecular weights ranging from 70 kDa to 85 kDa. Enhancement of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS linear mode analysis of intact proteins in tear fluid was demonstrated through the use of wax-coated MALDI plates and spot washing. MALDI-ToF MS analysis of the expressed tear proteins illustrates that differences between normal eye tear and dry eye tear protein content are manifested in changes in the lower molecular weight lipid-binding proteins such as lipophilin which exhibits an increase in concentration in the dry eye, and beta-2 microglobulin which undergoes a decrease.

Characterization of the in vivo forms of lacrimal‐specific proline‐rich proteins in human tear fluid

The tear film is complex and is rich in both peptides and proteins. Physiological factors have been shown to alter the balance of the protein components in the tear film, however, little is known of the precise stimuli that initiate these changes, or their nature and extent. Attention has been directed at the role of tear proteins in the protection of the external ocular surface, and their potential role in the pathogenesis of inflammatory and autoimmune diseases, but few lacrimal-specific proteins have been identified and demonstrated to offer a protective function at the ocular surface. The biological importance of proline-rich proteins is uncertain, although there is some evidence to indicate a potential antimicrobial function for these proteins in saliva. Despite the detection of mRNA for proline-rich proteins in lacrimal gland, the translated protein product has not been detected in tear fluid. In this study we investigate the presence of proline-rich proteins in the tear film. Human reflex tear fluid was examined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry directly, and following size exclusion high performance liquid chromatography. This revealed significant levels of a truncated form of lacrimal proline-rich protein, and a series of peptides derived the C-terminus of this protein. None of these had previously been identified in tear. Our study highlights the dangers inherent in proteomic strategies that assign an identity to a protein based on limited coverage of tryptic peptides.

Capillary electrophoresis off‐line matrix‐assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic‐coated capillaries

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.

The effect of sample treatment on separation profiles of tear fluid proteins: qualitative and semi-quantitative protein determination by an automated analysis system.

Qualitative and quantitative determination of tear fluid components is of increasing interest in ophthalmology. Until now, for diagnosis and course control of some diseases of the anterior parts of the eye, different methods for tear fluid protein analysis are available. Results can be obtained by polyacrylamide gel electrophoresis (PAGE), immunochemistry, and high-performance liquid chromatography (HPLC). A new method for protein separation, identification and semi-quantitative determination on a chip-based micro-fluidic technique is used for the first time to investigate tear fluids. Normal human reflex tears were obtained by stimulation with China mint oil and collected using glass capillary tubes. A lab-on-a-chip technology (developed by Agilent Technologies, Waldbronn, Germany, in co-operation with Caliper Technologies, Mountain View, California, USA) was used for separation and semi-quantitative determination of tear proteins. Tear fluid was separated on the Agilent 2100 Bioanalyzer in combination with the Protein 200 LabChip kit and the dedicated protein assay software. Time and temperature of the incubation with sample buffer were varied, and the influence of these parameters on protein separation profiles was studied. Tear proteins were also analysed by PAGE, and the results obtained by both methods were compared. The different proteins of tear fluid can be separated by the Agilent 2100 Bioanalyzer method in very short time. By this method, the molecular weight as well as the concentration of proteins can be determined. Data are automatically stored in digital format and can be retrieved and shared. Results of this technology were comparable with the protein pattern obtained by PAGE. It was confirmed by both methods that, depending on incubation time of tear fluid with sample buffer and on temperature, different protein pattern can be obtained from the tears of one specimen. Tear proteins - in contrast to serum or aqueous humour proteins - are very sensitive to changes in sample buffer temperature as well as incubation time with buffer. To obtain comparable results for tear fluid proteins, the sample buffer applied and the incubation time and temperature must be observed carefully. This can be demonstrated by both the new Agilent 2100 Bioanalyzer method and PAGE. These results are of importance when comparing tear fluid protein pattern for the diagnosis and course control of dry-eye syndrome and of other diseases of the anterior part of the eye.

P2Y2 receptor stimulation increases tear fluid secretion in rabbits

Purpose. The purinergic P2Y 2 receptor agonists stimulate active Cl - transport across the excised rabbit conjunctival tissue in vitro. We determined whether UTP or ATP could increase the tear volume and change tear fluid composition in normal rabbits in vivo. Methods. Fifty µL was applied to rabbit eyes of UTP, ATP at concentrations of 0.1, 0.3, 1, 3, 8.5% (1.8–154 mM) or saline. A modified Schirmer test with topical anesthesia was performed 5, 15, 30 and 60 min after the instillation. In studies lasting 30 days, 50 µL of 0.5% UTP was applied 6 times a day for 4 weeks. Tear samples were collected from the conjunctival sac with a glass microcapillary. The protein profile of the tear fluid was analyzed by SDS-polyacrylamide gel electrophoresis and total protein was measured with the Bradford assay. The Easy-Titer rabbit IgG assay kit was used for the determination of immunoglobulin G (IgG). Results. UTP had dose-dependent stimulatory effects on tear secretion. It maximally increased tear secretion about 4-fold 15 min after its application. Similar effects were obtained with ATP. Repeated treatment with UTP reproducibly increased tear volume. Furthermore, UTP did not decrease total protein and IgG concentration in tear fluid and it had no effect on the protein profile. Conclusion. These data indicate that activation of P2Y 2 receptor increases tear fluid secretion accompanied with some proteins in normal rabbits. The purinergic agonists, UTP and ATP, have potential therapeutic value in the treatment of dry eye.

A Novel Method of Tear Collection

To develop a rapid, user-friendly method of tear collection to facilitate tear-protein analysis. Tears were collected from a total of 19 normal volunteers without evidence of ocular-surface disease with either porous polyester rods or glass-capillary micropipettes. Tear-collection rate and recovery of two tear proteins, epidermal growth factor (EGF, low abundance) and lactoferrin (LFR, high abundance) were compared between polyester rods and glass-capillary micropipettes. The recovery of LFR and EGF and the stability of these proteins after storage at -70 degrees C were quantitated by specific monoclonal enzyme-linked immunosorbent assay (ELISA). Polyester rods collected tears an average of 3.9-fold faster than glass-capillary micropipettes (p < 0.001). Both methods were comparable in efficacy of protein recovery. The polyester rods demonstrated a trend toward enhanced recovery, but this difference was not statistically significant (p = 0.12, LFR; p = 0.055, EGF). Analysis of the reliability and reproducibility of the tear-collection assay system revealed that ELISA analysis is highly reproducible, but there is significant day-to-day variation in tear-protein levels of both LFR and EGF for a given volunteer. Both LFR and EGF displayed a trend toward enhanced detection by ELISA shortly after freezing at -70 degrees C and slow decay after storage at -70 degrees C for up to 72 and 105 days, respectively. After stimulation of reflex tearing via the nasolacrimal reflex, LFR levels remained relatively constant, whereas EGF levels for most patients declined and then plateaued. Polyester rods provide a more rapid, user-friendly alternative to glass-capillary micropipettes for the collection and analysis of tear fluid and tear proteins. Polyester rods may have greater clinical utility, facilitating routine analysis of the preocular tear film.

Establishment of the human reflex tear two‐dimensional polyacrylamide gel electrophoresis reference map: New proteins of potential diagnostic value

To understand the changes in protein expression associated with various physiological states as well as the development of pathological eye disease, we have begun to map the protein components of normal human reflex tears. An analytical reference map of normal human reflex tears was created using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with pH 3.5-10 immobilized pH gradients (IPGs). Micropreparatively loaded gels were transferred to polyvinylidene difluoride (PVDF) and analysed by a combination of N-terminal sequence tagging and amino acid compositional analysis. Thirty spots were sequence tagged, resulting in identification of six different proteins (lipocalin, lysozyme, lactotransferrin, zinc-alpha-2 glycoprotein, cystatin S, cystatin SN) that matched to entries in the SWISS-PROT database. A group of N-terminally blocked proteins was clearly identified from SWISS-PROT by amino acid analysis, isoelectric point (pI) and molecular weight (Mr). A number of highly expressed protein components remain unidentified despite being subjected to amino acid analysis and Edman sequencing. A majority of the abundant proteins showed varying degrees of charge heterogeneity attributed to post-translational processing such as glycosylation and N-terminal truncation. We have identified a previously undescribed protein that we have named lacryglobin. This protein displays strong homology with mammaglobin, a protein overexpressed in breast cancer. The discovery of this homologue in tears offers the potential for disease diagnosis by screening tear fluid proteins.