ORIGINAL ARTICLE

Protein Deposition and Clinical Symptomsin Daily Wear of Etafilcon Lenses

Lakshman N. Subbaraman*, Mary-Ann Glasier†, Jalaiah Varikooty‡, Sruthi Srinivasan§, and Lyndon Jones�

ABSTRACTPurpose. To determine the relationship between clinical signs and symptoms and protein deposition over 8 h of wear ofetafilcon A lenses in symptomatic and asymptomatic contact lens wearers.Methods. Thirty adapted soft contact lens wearers (16 symptomatic and 14 asymptomatic) were fitted with etafilcon Alenses. In vivo wettability, non-invasive tear break-up time, and subjective symptoms (vision, comfort, and dryness) wereassessed at baseline and after 2, 4, 6, and 8 h. After 2, 4, 6, and 8 h time points, lenses were collected, and total protein,total lysozyme, and active lysozyme deposition were assessed.Results. There was a significant reduction (p � 0.032) in the non-invasive tear break-up time at 8 h in both groups. In thesymptomatic group, there was a significant reduction in subjective comfort and dryness ratings at 6 and 8 h measurementwith respect to baseline (p � 0.05). There was a significant increase in total lysozyme and total protein deposition (p �0.027) across all time points in both groups; most of the lysozyme remained active (�94% at 8 h). Pearson’s correlationsbetween subjective symptoms and protein deposition showed poor correlations for total protein/lysozyme and anysubjective factor (r � 0.3; p � 0.05), and only weak correlations between dryness and % active lysozyme (r � 0.3 to 0.5for all time points). However, stronger correlations were found between active lysozyme and subjective comfort (r � 0.6to 0.7; p � 0.001).Conclusions. In addition to investigating total protein deposited on contact lenses, it is of significant clinical relevance todetermine the conformational state of the deposited protein.(Optom Vis Sci 2012;89:1450–1459)

Key Words: comfort, contact lens, deposition, lysozyme, protein, protein activity, tears

Dryness and discomfort are considered to be one of theprimary reasons for contact lens intolerance and discon-tinuation of lens wear.1–3 Contact lens-related dry eye has

often been associated with changes in functional visual acuity,4,5

reduced lens wearing time,6 increased risk of bacterial adhesion,ocular surface desiccation, and infection.7,8 Several studies haveattempted to determine the potential mechanisms for contact lens-related dry eye, and it has been observed that increased evaporationof the tear film,9 rapid pre-lens tear film thinning,10 limbal injec-tion,10 inflammation,11–13 reduced lacrimation with concurrentincreased osmolality,14,15 and an increase in tear film osmolality10

are all significant factors that may be associated with contact lens-related dry eye. In addition, higher water content contact

lenses,10,16 reduced wettability of the lens surface,17–19 or any ofthe aforementioned factors could also be associated with contactlens-related dry eye, confirming that this condition is multifacto-rial. Several studies have demonstrated that dryness and discomfortratings become worse independently of the amount of dehydrationor water content of the lens material.2,20,21

The tear film is by far the most dynamic unit in the lacrimalfunctional unit, which consists of a variety of components, includ-ing proteins, lipids, mucins, peptides, electrolytes, and salts. Usinga proteomic technique, 97 proteins have been identified in the tearfilm,22 and many of these proteins are known to sorb onto contactlens materials.23,24 Protein deposition on contact lens materials ishighly material dependent, with water content and surface chargehaving significant impacts on the amount of protein depos-ited.25–36 One of the major tear proteins that is recovered from groupIV contact lens materials is lysozyme.28,34,36–40 Lysozyme is abacteriolytic enzyme with a relatively small molecular weight (14kDa) and a positive charge at neutral pH. Once lysozyme firmlyadsorbs onto contact lens materials, it tends to undergo conforma-

*PhD, BSOptom, FAAO†MSc‡MBBS, MSc§PhD, BSOptom, FAAO�PhD, FCOptom, FAAOCentre for Contact Lens Research, School of Optometry, University of Water-

loo, Waterloo, Ontario, Canada.

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Optometry and Vision Science, Vol. 89, No. 10, October 2012

tional changes,28,40–42 which might potentially result in a varietyof immunological responses, including contact lens-associatedpapillary conjunctivitis.43–46

Several studies have determined changes that occur in tear filmprotein or lipid levels in contact lens wearers, with some of thesestudies classifying contact lens wearers as being either “tolerant” or“intolerant.”11,13,47–61 However, these studies did not quantify theprotein deposited on the lens materials per se; therefore, it was notpossible to determine the relationship between various clinical pa-rameters and the amount of protein deposited on the lenses. An-other study38 that quantified the protein deposited on the lensmaterial determined the effect of overnight eye closure on the rateand composition of protein deposition on the probable change inthe rate of reflex-type tear secretion associated with eye closure.Some studies have determined the link between protein depositionon contact lenses and subjective symptoms reported by lens wear-ers.62–66 However, all these studies determined the deposition onlenses by using relatively insensitive techniques, such as visibledeposition or video image analysis.64–66

Although studies have speculated that the conformational stateof the deposited protein could have an influence on various sub-jective symptoms in contact lens wearers,62,67 to date, no study hasdetermined the relationship between subjective symptoms and theconformational state of the deposited protein, or indeed the differ-ences in these factors in symptomatic and asymptomatic contactlens wearers. Thus, the main purpose of this study was to investi-gate the impact of lens wearing time on clinical signs, subjectivesymptoms, and the quantity of total protein and lysozyme deposi-tion and the conformational state of the lysozyme deposited overan 8 h wear period of a high water content ionic lens material(etafilcon A; Acuvue; Johnson & Johnson, Jacksonville, FL) in agroup of symptomatic and asymptomatic contact lens wearers andto determine whether there is any association between the clinicalsigns and symptoms and the protein deposition measured on thelenses.

MATERIALS AND METHODS

Ethics clearance was obtained from the Office of Research Eth-ics at the University of Waterloo before commencement of thestudy. The study was carried out in accordance with the tenets ofthe Declaration of Helsinki. Informed consent was obtained fromall participants before enrollment. The study was conducted at theCentre for Contact Lens Research, School of Optometry, Univer-sity of Waterloo. This was a non-dispensing randomized studyinvolving previously adapted soft contact lens wearers. Participantswere classified as being “symptomatic” or “asymptomatic” basedon their responses to a prescreening questionnaire, and the exam-iners were masked to the presence of symptoms. Participants whowere classified as being symptomatic with their soft lenses werethose subjects who reported reduced comfortable lens wear withtheir existing soft lens materials after a minimum of 6 h of lens wearand who needed to resort to ocular lubricants to sustain their lenswear. Participants were eligible to participate in the study if theywere at least 16 years or older. Participants who were �18 years(and �16 years of age) were eligible to participate with the parent’s

or guardian’s permission and after reading the Information andConsent Letter for adolescents. Self-consent was applicable forthose participants who were �18 years of age. All participants werefitted with etafilcon A contact lenses (group IV lens material; Acuvue;Johnson & Johnson), and the participants were not permitted to useany rewetting drops during the course of the study. The partic-ipants were advised not to wear any contact lenses or use any form oflubricants for 5 days before the initial lens-dispensing visit.

Each participant attended on two consecutive days, with a base-line and two study visits on each day. On each day, the first visitwas the baseline visit, and this was followed by second and thirdvisits anytime between 2 and 8 h, and all the study visits wererandomly determined based on a randomization table. During thebaseline visit on day 1, contact lenses were inserted into both eyes,and, after the lenses had settled, objective and subjective measure-ments were determined. During the first study visit (which wasrandomly determined) on day 1, objective and subjective measure-ments were determined, and at the end of this visit, one lens wasrandomly removed from one of the participant’s eyes for proteinanalysis. A new lens was reinserted into that eye to ensure thesubject was binocularly corrected. During the second study visitlater that day (which was again randomly determined), objectiveand subjective measurements were taken, and at the end of thisvisit, the lens from the other eye was collected for protein analysis.On the following day, the same procedures were repeated, for theremaining two time points, which were determined using a ran-domization table. Thus, each participant had lenses collected foranalysis after four time periods (two per day), with the time periodsbeing after 2, 4, 6, or 8 h of lens wear.

Clinical Measurements

Objective Measurements

The objective measurements were performed on the partici-pants at baseline and after 2, 4, 6, and 8 h of lens wear. Tear filmstability was assessed by determining the non-invasive tearbreak-up time (NITBUT) using the ALCON Eyemap modelEH-290 topography system (ALCON Inc., Forth Worth, TX).Participants were asked to blink three times before each mea-surement was taken. NITBUT was determined by measuringthe time taken for distortions or discontinuities to appear in thereflected image of the concentric ring pattern. The time (inseconds) for the tear film to rupture (and thus distort the rings)was measured to the nearest 0.1 of a second. Three measure-ments were taken on each eye, and the average of these was usedfor analysis purposes.

Overall wettability of the contact lenses was assessed in vivousing the grid viewed on the ALCON Eyemap (ALCON Inc.).The image of the Placido disc was viewed on the monitor of theEyemap, and the in vivo wettability of the contact lenses was graded ona five-point scale (0 to 4), where “0” related to a lens exhibiting “se-verely reduced” wettability and “4” a lens with “perfect” wettability.68

In vivo wettability of the contact lenses was assessed according to thefollowing schema: Grade 0: One or more non-wetting areas�0.5 mm in size; Grade 1: Several non-wetting areas, 0.1 to 0.5mm in size; Grade 2: Single non-wetting area 0.1 to 0.5 mm insize; Grade 3: Small (�0.1 mm), discrete non-wetting areas;Grade 4: 100% of anterior lens surface wettable.

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Assessment of Subjective Symptoms

Participants completed visual analog scales at baseline, 2, 4, 6,and 8 h study visits. Participants rated the subjective symptoms ofvision, comfort, and dryness on a scale of 0 to 100 (0 � worstrating, 100 � best rating).

After 2, 4, 6, and 8 h of lens wear, lenses were collected by agloved examiner, and the lenses were briefly rinsed with salineto remove any residual loosely adhered tear film and placed inindividual sealed glass vials containing a 50:50 mix of 0.2%trifluoroacetic acid and acetonitrile (ACN/TFA), as describedpreviously.37,42 The vials were incubated in the dark at roomtemperature for 24 h, after which the aliquots of lens extractswere transferred to sterile Axygen microcentrifuge tubes andevaporated to dryness in a Savant Speed Vac (Halbrook, NY).Dried protein pellets were stored at �80°C for up to 2 weeksbefore reconstitution.

Analytical Measurements

Reagents and Materials

Immuno-Blot polyvinylidene difluoride membranes were pur-chased from Bio-Rad Laboratories (Mississauga, ON, Canada).Polyclonal rabbit anti-human lysozyme was purchased from Ce-darlane Laboratories (Hornby, ON, Canada), and goat anti-rabbitIgG-HRP was purchased from Sigma (St. Louis, MO). Humanlysozyme (neutrophil) and lyophilized Micrococcus lysodeikticuscells were also purchased from Sigma. Bovine serum albumin stan-dard was obtained from Pierce Biotechnology Inc (Rockford, IL).All other reagents purchased were of analytical grade.

Measurement of Total Lysozyme Deposition—Electrophoresis and Immunoblotting

Before electrophoresis/Western blotting and lysozyme activityanalysis, lyophilized protein pellets were reconstituted in modifiedreconstitution buffer—MRB (10 mM Tris-HCl; 1 mM EDTA,with 0.9% saline), pH 12.0, and BioStab Biomolecule StorageSolution (Sigma-Aldrich).69 Human lysozyme standard curveswere run on each Western blot so that four points falling within thelinear range of detection were produced, to facilitate regressionanalysis of sample extracts. Lysozyme standards were preparedfresh on the day of analysis from a 1.0 �g/�l frozen stock ofpurified human neutrophil lysozyme with modified reconstitutionbuffer, pH 8.0, and subjected to sodium dodecyl sulfate polyacryl-amide gel electrophoresis followed by Western blotting to polyvi-nylidene difluoride membranes. Lysozyme was identified using arabbit anti-human lysozyme polyclonal antibody (Calbiochem),followed by a peroxidase-conjugated goat anti-rabbit secondaryantibody (Sigma-Aldrich). Individual standard curves of puri-fied human neutrophil lysozyme (Sigma-Aldrich) were run oneach gel to facilitate regression analysis. The entire procedure isdescribed in detail elsewhere.42,69,70

Negative Control—Extraction and Western BlotAnalysis of Unworn Lenses

Three new unworn etafilcon A lenses were extracted in ACN/TFA solution and were subjected to sodium dodecyl sulfate poly-

acrylamide gel electrophoresis and Western blotting, as describedearlier.

Measurement of Lysozyme Activity

The contact lens extracts were assayed for lysozyme activity us-ing a fresh suspension of M lysodeikticus for each sample, as de-scribed previously.40,42,70 Micrococcal cells were suspended in 50mM sodium phosphate buffer (pH 6.3) to an initial optical densityof 1.0 at 450 nm (Multiskan Spectrum ELISA Plate Reader, fittedwith a microcuvette, Thermo Labsystems). Human neutrophil ly-sozyme standard (2.5, 5, 12.5, 50, 150, 250 ng) was run concur-rently with the samples. The mass of active lysozyme in contactlens extracts was extrapolated from the native lysozyme standardcurve, as described previously.40,42,70 The final calculation was thepercent of active lysozyme: % active lysozyme � (active lysozyme/total lysozyme) � 100.

Measurement of Total Protein Deposition

The total protein extracted from the lenses was determinedusing the Micro-BCA assay. Manufacturer’s instructions werefollowed for the Micro-BCA Protein Assay Reagent Kit (PierceBiotechnology). Phosphate buffered saline was used as the buf-fer. Each data point was the average of three determinations.

Statistical Analysis

Statistical analysis was conducted using Statistica 7 software(StatSoft Inc., OK). All data are reported as mean � standarddeviation and range, unless otherwise indicated. A two-way re-peated measures analysis of variance was performed, with timecourse and groups as the factors, and post hoc multiple comparisontesting was undertaken using the Tukey-HSD test. Pearson’s cor-relations were performed to determine the relationship betweenvarious clinical signs and symptoms vs. the analytical measures. Inall cases, a p value of �0.05 was considered significant.

RESULTS

Based on the participants’ responses to the prescreening ques-tionnaire, 16 participants (mean age, 24.73 � 5.31 years, 9 femaleand 7 male) were classified as symptomatic and 14 participants(mean age, 25.31 � 4.78 years, 13 female and 1 male) were clas-sified as asymptomatic.

Objective Measurements

Fig. 1 shows the NITBUT values over time for the symptomaticand asymptomatic groups. There was no significant difference inthe NITBUT values between the two groups at any time point(p � 0.05), but the 8 h time point was significantly lower than thebaseline measurement in both the symptomatic and asymptomaticgroups (p � 0.032). Fig. 2 shows the in vivo wettability over timefor the symptomatic and asymptomatic participants. Although invivo wettability is seen to reduce over the course of the day for bothgroups, this reduction was not statistically significant for eithergroup (p � 0.05). There was also no significant difference betweenthe two groups at any time point (p � 0.05).

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Subjective Symptom Ratings

Fig. 3 shows that there was no significant difference forsubjective vision ratings for both groups over time (p � 0.05)and also between the two groups at any time (p � 0.05), al-though the symptomatic group showed lower ratings at all timepoints.

Fig. 4 shows that there was no significant decrease incomfort over time in the asymptomatic group (p � 0.05); how-ever, in the symptomatic group, the 6 and 8 h ratings weresignificantly lower than the baseline measurement (p � 0.013).The symptomatic group had significantly lower comfort ratings

than the asymptomatic group at the 6 and 8 h time points (p �0.035). However, there was no significant difference betweenthe two groups at other time points (all p � 0.05), althoughthe symptomatic group had lower comfort ratings at thesetimes.

Fig. 5 shows that there was no significant reduction in dry-ness ratings over time in the asymptomatic group (p � 0.05);however, in the symptomatic group, the 6 and 8 h ratings weresignificantly lower than the baseline measurement (p � 0.012).The symptomatic group reported significantly more drynessthan the asymptomatic group at the 6 and 8 h time points

FIGURE 1.Non-invasive tear break-up (in seconds) over time in symptomatic and asymptomatic contact lens wearers. Error bars represent mean � SD. * representsp � 0.05 within the same group from 0 h (baseline visit).

FIGURE 2.In vivo wettability (on a scale of 0 to 4) over time in symptomatic and asymptomatic contact lens wearers. Error bars represent mean � SD.

Protein Deposition and Symptoms with Daily Wear Etafilcon Lenses—Subbaraman et al. 1453

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(p � 0.024). However, there was no significant differencebetween the two groups at other time points (all p � 0.05),although the symptomatic group had lower dryness ratings atthese times.

Analytical Measurements

Table 1 shows the total protein, total lysozyme deposition, andpercentage active lysozyme recovered from the lenses at varioustime points in the asymptomatic and symptomatic participants.There was a gradual increase in total protein deposition and totallysozyme deposition on the lenses across the four time points (p �0.05) both in the asymptomatic and symptomatic group of partic-ipants. However, there was no significant difference between the

two groups at any time point (p � 0.05). There was a gradualreduction in the activity of lysozyme deposited across the four timepoints, albeit it was not statistically significant (p � 0.05). Thepercentage active lysozyme recovered from the symptomatic lenswearers was lower at all time points, although this was not statisti-cally significant (all p � 0.05).

Correlations

Pearson’s correlations between clinical signs and any of the pro-tein deposition measures showed poor (r � 0.2) insignificant cor-relations (p � 0.05). Pearson’s correlations between subjectivesymptoms and protein deposition showed poor correlations fortotal protein/total lysozyme and any subjective factor (r � 0.3; p �

FIGURE 3.Subjective vision ratings (on a scale of 0 to 100) over time in symptomatic and asymptomatic contact lens wearers. Error bars represent mean � SD.

FIGURE 4.Subjective comfort ratings (on a scale of 0 to 100) over time in symptomatic and asymptomatic contact lens wearers. Error bars represent mean � SD.* represents p � 0.05 within the same group from 0 h; � represents p � 0.05 between groups.

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0.05), and only weak correlations between dryness and active ly-sozyme (r � 0.3 to 0.5 for all time points) as shown in Table 2.However, significant linear correlations were found between theactive lysozyme and subjective comfort (r � 0.6 to 0.7; p � 0.001)as shown in Table 2.

DISCUSSION

To date, this is the only study that reports on the relationshipbetween clinical signs, subjective symptoms, and the conformationalstate of lysozyme deposited on contact lenses. These results clearlysuggest that there is a linear correlation between lysozyme activityrecovered from contact lenses and subjective comfort, even over shortperiods of lens wear. Previous studies have shown that tolerant contactlens wearers have fewer symptoms of discomfort and a more stable tearfilm (as measured by a higher maximum forced interval betweenblinks, tear meniscus height and volume, and NITBUT).71 In thepast, studies have also shown that the tear film of tolerant lens wearersshowed lower levels and activity of secretory phospholipase A2, lowerconcentration of lipocalin, and lower levels of peroxidized lipids.47 Ithas also been shown that in the absence of lens wear, there were nodifferences between tolerant and intolerant lens wearers in conjuncti-val or limbal redness, lipid layer appearance, tear flow rate, tear filmosmolality, and total protein, lactoferrin, lysozyme, or secretory im-munoglobulin A concentrations in the tear film.71

Non-invasive Tear Break-Up Time

When a contact lens is inserted into the eye, the tear film isdisturbed and tear film break-up time reduces significantly.72,73

FIGURE 5.Subjective dryness ratings (on a scale of 0 to 100) over time in symptomatic and asymptomatic contact lens wearers. Error bars represent mean � SD.* represents p � 0.05 within the same group from 0 h; � represents p � 0.05 between groups.

TABLE 1.Total protein, total lysozyme, and percentage active lysozyme (mean � standard deviation) recovered from etafilconcontact lenses worn by asymptomatic and symptomatic subjects at four time points

Time

Total protein (�g/lens) Total lysozyme (�g/lens) Percentage active lysozyme

Asymptomatic Symptomatic Asymptomatic Symptomatic Asymptomatic Symptomatic

2 h 147.87 � 43.41 183.89 � 55.04 123.23 � 36.18 141.45 � 42.34 99.15 � 1.06 97.95 � 1.964 h 307.45 � 83.56 347.26 � 78.24 227.74 � 61.90 253.94 � 64.66 98.79 � 2.12 96.87 � 2.426 h 371.78 � 79.57 417.66 � 86.93 288.21 � 61.68 321.28 � 66.87 98.19 � 3.06 95.98 � 2.628 h 465.48 � 79.32 478.48 � 90.53 349.99 � 59.64 382.78 � 72.43 97.69 � 4.20 94.16 � 4.32

TABLE 2.Correlation between active lysozyme recovered from eta-filcon contact lenses and various subjective symptoms atdifferent time points

Comfort DrynessSubjective

vision

Active lysozyme(2 h)

R � 0.64(p < 0.001)

R � 0.37(p < 0.001)

R � 0.08(p � 0.162)

Active lysozyme(4 h)

R � 0.70(p < 0.001)

R � 0.36(p < 0.001)

R � 0.05(p � 0.097)

Active lysozyme(6 h)

R � 0.69(p < 0.001)

R � 0.47(p < 0.001)

R � 0.13(p � 0.214)

Active lysozyme(8 h)

R � 0.71(p < 0.001)

R � 0.31(p � 0.067)

R � 0.35(p � 0.141)

Bold font indicates p values that are statistically significant.

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Previous studies have shown the NITBUT of soft contact lenswearers to be in the range of 3 to 10 s,59,74,75 which is similar tothat found in the current study. A study by Guillon et al. showedthat there was no difference in the tear film stability betweenasymptomatic and symptomatic contact lens wearers, althoughthey found a significant difference between asymptomatic andsymptomatic non-contact lens wearers.73 However, another studyby Glasson et al. showed that contact lens wear affected the stabilityof the tear film in tolerant contact lens wearers more than in intol-erant contact lens wearers.76 In their study, NITBUT decreasedmore dramatically in the tolerant contact lens wear group, and itwas also shown that the NITBUT of intolerant subjects was sig-nificantly lower initially before lens wear and remained low over6 h of lens wear. Another study by Fonn et al. also demonstrated astatistically significant decrease in pre-lens NITBUT in symptom-atic lens wearers during a 5 h period, regardless of soft lens type,compared with no significant change in asymptomatic subjects.20

In Vivo Wettability

Although high levels of total protein and total lysozyme depos-ited on etafilcon lens materials within a few hours of lens wear(Table 1), it is clear that these deposits do not modify the measuredin vivo wettability of these lenses to any appreciable extent. In thepast, studies that determined the wettability of etafilcon lens ma-terials over short periods of lens wear using indirect methods suchas pre-lens NITBUT also showed similar findings.77 Previous invitro and ex vivo studies that determined the influence of tearproteins on wettability of etafilcon lens materials also found thatthese deposits do not reduce the wettability of etafilcon lens mate-rials over short periods of lens wear or over short periods of in vitroincubation,78–80 and this can be attributed to the fact that ly-sozyme penetrates into the bulk of the etafilcon lens material ratherthan remaining on the surface of these lens materials.81

Subjective Symptoms

As expected, symptomatic contact lens wearers in this studyshowed a significant reduction in subjective comfort and drynessratings over 8 h of lens wear, whereas the ratings of asymptomaticlens wearers remained relatively constant (Figs. 4 and 5). Theseresults are consistent with those from previous studies where symp-tomatic lens wearers showed a decrease in comfort and drynessratings using visual analog scales over time.2,20,71

Protein Deposition on Lenses

It is clear from Table 1 that etafilcon lenses attracted substantialquantities of total protein and total lysozyme even after 8 h of lenswear in both the symptomatic and asymptomatic participants.This finding is in accordance with other previous in vitro and exvivo studies that evaluated protein and lysozyme deposition onetafilcon contact lenses.30,31,36,39–42,82–87 The increased affinityof lysozyme to the etafilcon material occurs because methacrylicacid imparts a negative charge to the material and thus thermody-namically favors the deposition of lysozyme, which is a positivelycharged species at physiological pH.

After 8 h of lens wear, the percentage active lysozyme recoveredfrom etafilcon lenses worn by symptomatic and asymptomatic lens

wearers was �94% (Table 1), which is significantly higher thanthose seen from silicone hydrogel lens materials.40–42,70,88,89 Pre-vious ex vivo41,42 and in vitro40,89 studies have also demonstratedthat the percentage active lysozyme recovered from conventionalhydrogel group IV etafilcon lens materials is significantly higherthan those seen in novel silicone hydrogel and other groups ofconventional hydrogel lens materials. Denaturation of a protein onany polymeric surface is dependent on several factors, includingcontact time of the protein with the substrate, chemical composi-tion of the substrate, surrounding pH, type of protein, temperatureof the surrounding medium, and also the location of the protein ina polymer.28,67,82,90–93 Using confocal microscopy, it has recentlybeen shown that lysozyme is primarily located within the bulk ofetafilcon lens materials, with relatively little surface-located ly-sozyme,81 resulting in significantly increased levels of lysozymeremaining active.

Relationship between Protein Deposition andSubjective Symptoms

A previous study by Lever et al. that investigated the relationshipbetween total protein deposition and patient-rated lens comfortfound that there was no statistical correlation between these twofactors.62 This was the only study that attempted to determine therelationship between protein deposition on contact lenses and sub-jective comfort by quantifying the total protein deposited on lensesusing biochemical techniques62; other studies estimated the rela-tionship by evaluating the visible deposition/video image analysisof deposits on the lenses.4,63–66 Most studies that used visibledeposition/video image analysis showed that there was an associa-tion between visible deposition and comfort4,64–66; however, onestudy that surveyed 50 comfortable and uncomfortable contactlens wearers did not show a difference in the amount of visibledeposition on the lenses between the two groups.63 A recent studythat looked at correlating clinical responses during contact lenswear with the amount of protein or cholesterol extracted fromlenses after wear suggested that the quantity of protein that depos-its onto contact lenses during wear may have more effect on lensperformance on eye94; however, this study did not look at theconformational state of the protein deposited on these contactlenses. Furthermore, protein deposition has a significant potentialto cause problems, as these deposits do play a significant role inmodulating microbial adherence to lens materials.95,96 Therefore,it is important that practitioners advise their patients regarding theimportance of lens disinfection and cleaning and appropriate lensreplacement schedules.

This study is the first to demonstrate that a significant correla-tion exists between subjective comfort and active lysozyme recov-ered from etafilcon lens materials, even over short periods of lenswear. However, these results should be interpreted with caution, asit would be erroneous to conclude that denatured lysozyme oncontact lenses is solely responsible for the symptoms experiencedby symptomatic contact lens wearers. Rather, they should beinterpreted as lysozyme deposited on the contact lenses of symp-tomatic lens wearers tends to denature more than that seen inasymptomatic lens wearers. This is likely to happen because of thebiochemical changes that occur in the tear film of symptomaticlens wearers,71,76 resulting in altered properties of the lens material,

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potentially leading to a change in the conformational state of thedeposited lysozyme. Therefore, in addition to the other factorsmentioned earlier, the conformational state of the deposited pro-tein, inflammatory and subinflammatory mediators, and the secre-tomotor response of the lacrimal system could also be significantfactors in contact lens-induced dry eye, reiterating that this condi-tion is multifactorial.

In conclusion, the results from this study suggest that even overa short period of contact lens wear, a significant correlation existsbetween subjective symptoms of comfort and dryness and the ac-tivity of lysozyme recovered from etafilcon contact lenses, withlittle correlation being shown with total amounts of either totalprotein or total lysozyme. Therefore, in addition to investigatingthe total quantity of the deposited protein, it is of significant clin-ical relevance to study the conformational state of the depositedprotein. These results have tremendous implications, in that thenovel contact lens materials that are being developed should pos-sess properties that can retain the activity of the deposited protein,in addition to being deposit resistant. Care regimens and multi-purpose solutions should be capable of removing denatured pro-teins that are deposited on the lens materials or be manufactured tomaintain protein activity at the material surface.

Further work is required to determine whether this importantclinical finding is transferable to those patients who use siliconehydrogel lens materials. It would also be of interest to determinewhether there is a difference in the activity of lysozyme recoveredfrom the tears of symptomatic and asymptomatic contact lenswearers. Although it is interesting to note that a significant corre-lation exists between the conformational state of the depositedprotein and clinical symptoms, further studies with a better samplesize and validated instruments that clearly identify symptomaticand asymptomatic groups are warranted. In addition to determin-ing the activity of lysozyme, it would also be of interest to deter-mine the activity of lactoferrin and other tear proteins with highcationicity, such as the defensin peptides.

ACKNOWLEDGMENTS

This work was funded by a Collaborative Research and Development Grantfrom Natural Sciences and Engineering Research Council of Canada(NSERC) and Alcon Research Limited, U.S.

Received January 3, 2012; accepted June 18, 2012.

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Lakshman N. SubbaramanCentre for Contact Lens Research, School of Optometry

University of Waterloo200 University Avenue West

Waterloo, OntarioCanada

e-mail: lnagapat@uwaterloo.ca

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