Despite the 140 million contact lens wearers worldwide, lens wear discomfort stands as a major impediment in the growth of the contact lens industry.1–3 This has stimulated much interest, and the recent workshop on contact lens discomfort by the Tear Film & Ocular Surface Society was one such initiative to understand and address this issue.4 According to the report by “the contact lens interactions with tear film subcommittee,” a person’s tear film plays an important role in lens wear discomfort.5,6 The three-layered structure of the tear film includes a glycocalyx layer covering the corneal epithelium, an intermediate aqueous layer, and an outer lipid layer.7 The structure of the lipid layer is described by McCulley and Shine8 as a bilayer consisting of an upper nonpolar layer and a lower polar layer. However, King-Smith et al.9 proposed a “multilamellar sandwich model” of the tear lipid layer with major components including long, saturated chains of wax esters (WE) and cholesterol esters (CE) and other nonpolar components such as triacylglycerols (TAG), diesters, free sterols, free fatty acids, and a polar lipid interface between nonpolar lipids and the aqueous layer. The barrier function of lipid layer in preventing evaporation of tears from the aqueous phase makes it crucial in maintaining tear film stability.10
The clinical, functional, and biochemical aspects of the tear film lipid layer during soft contact lens wear have been reviewed previously.6,11,12 A thicker lipid layer is associated with increased stability of the tear film and increased interblink period and also a decreased tear evaporation rate.10 Reduced concentrations of phospholipids and cholesterol have been observed in tears of soft contact lens wearers.13,14 The secretory phospholipase A2 enzyme (sPLA2), which hydrolyzes the sn-2 acyl chain of phospholipids, has been found to be associated with reduced tear film stability among chronic blepharitis patients.15 Deposition of sPLA2 on contact lens surface catalyzes lipid degradation that results in free fatty acid formation such as arachidonic acid.14 In many inflammatory diseases,16–19 including ocular surface inflammation20 and contact lens intolerance,21 increased levels of sPLA2 activity have been reported. Fatty acid catabolism can lead to the formation of phospholipid aldehydes, which are the by-products of lipid peroxidation.22 Lipid aldehydes can alter cellular function in many human diseases.23 Higher levels of lipid aldehydes were found in the tears of symptomatic contact lens wearers compared with asymptomatic wearers.21
The most common intervention used by subjects with dry eye in attempts to alleviate their symptoms is exogenous tear supplementation.24 Some of these products contain lipids to address potential deficiencies in tear film lipids. Supplements containing castor oil in an emulsion in water resulted in increased lipid layer thickness and reduced tear evaporation rate and persisted in tears for up to 4 hours after the initial instillation.25–29 A phospholipid eye spray (Tears Again, BioRevive) improved tear breakup time and decreased inflammation of eyelid margins in a dry eye population when compared with a saline spray.30 Additionally, the phospholipid eye spray demonstrated the same clinical improvements for contact lens wearers when compared with a hyaluronate-based tear supplement.31 Craig et al.32 observed an increased tear breakup time in healthy eyes and improved subjective comfort from baseline after the use of a phospholipid spray containing phosphatidylcholine when compared with application in the contralateral eye of a saline spray. A preliminary evidence on the beneficial effects of the spray during silicone hydrogel contact lens wear was also observed.33
The current study aimed to evaluate the effect of short-term contact lens wear on the clinical and biochemical aspects of tear film lipids among symptomatic contact lens wearers. A secondary objective was to determine the effect of a liposomal spray on tear film lipids among symptomatic contact lens wearers. The study hypothesis is that an exogenous lipid supplement will improve contact lens wear comfort by modulating the clinical and biochemical aspects of tear film lipid layer.
Participants with a history of soft contact lens wear were recruited through e-mail and poster advertisements. Twenty participants (18 women and 2 men) with a mean (±SD) age of 25 (±4) years were enrolled. The sample size was based on an outcome variable of tear breakup time32 with an expected SD of 9 seconds, to detect a clinically significant difference of 10 seconds, assuming an estimate of type 1 error α = 0.05 and a power of 80%. The study protocol was approved by the Human Research Ethics Advisory panel at the University of New South Wales and was conducted in accordance with the Declaration of Helsinki. Signed informed consent was obtained from all study participants at the start of the study. A screening of the participants was conducted before enrolment. The screening involved a routine slit lamp examination, which included an evaluation of meibomian gland expressibility. Participants who expressed a clear meibum with mild to moderate pressure were then enrolled for the study. Using the Contact Lens Dry Eye Questionnaire (CLDEQ), each participant was categorized as either a symptomatic or an asymptomatic contact lens wearer. The questionnaire34 consists of nine habitual symptom subscales and a self-diagnosis question. The analysis of CLDEQ involves an algorithm that produces a dichotomous outcome for the diagnosis of contact lens–related dry eye.34 If a participant scored greater than −0.13 on the CLDEQ and ticked “yes” to the self-diagnosis question for dry eye, he or she was categorized as symptomatic. On the other hand, if a participant ticked “no” or “unsure” to the self-diagnosis question for dry eye, he or she was categorized as symptomatic if the scores were greater than 1.27 and greater than 1.44, respectively.
The study had two stages. In stage 1, participants were asked to wear soft hydrogel lenses (Focus Dailies; Ciba Vision, Duluth, GA) bilaterally for 6 to 8 hours. At the end of the lens wear, with lenses in situ, clinical parameters of lipid layer thickness and stability were assessed and basal tears were collected using microcapillary tubes (see below). Tear samples were assayed for the concentration and activity of sPLA2 and also the concentration of malondialdehyde (MDA, a product of lipid peroxidation). Mass spectrometric analysis was conducted to characterize the tear lipidome. In stage 2, symptomatic lens wearers (n = 10) were subjected to a single-masked, randomized crossover trial during which a liposomal spray (Tears Again, BioRevive) and a control saline spray were sprayed over the upper eyelids of each subject during their down gaze during lens wear. The washout period was 48 hours between experiments. Lipid layer appearance and stability, as well as basal tear collection along with ocular comfort scores using a numeric rating scale were obtained soon after spraying and again at 2 and 6 hours after spraying. Each visit occurred at the same time of the day for each stage for each subject.
Lipid Layer Grade
Tear lipid patterns were assessed noninvasively using the Tearscope Plus (Keeler Ltd, Berkshire, UK) in conjunction with a slit lamp biomicroscope and then graded into one of five categories based on lipid layer thickness.35 The absence of a lipid layer was graded as 0, open or tight meshwork lipid interference patterns were graded as 1 or 2, a wavy appearance was graded as 3, and amorphous or color fringes were graded as 4 or 5, respectively.
Noninvasive Surface Drying Time
Noninvasive surface drying time (NISDT) was also measured using the Tearscope Plus (Keeler Ltd). Participants were asked to blink normally three times and then to refrain from blinking until the examiner noticed the development of an initial dry spot on their contact lens surface. A minimum of three readings was recorded for each eye and these were averaged at the conclusion of the experiment. Lipid layer grading and surface drying time were taken for both right and left eyes in a random order.
Tear Collection and Storage
Basal (nonstimulated) tear samples were collected using disposable microcapillary tubes (Blaubrand intraMARK, Wertheim, Germany). Up to 5 μl of tears was collected from each eye, by asking the participants to tilt their heads to one side and resting the capillary tube gently on the lower lid in the temporal region of a randomly selected eye. Care was taken in collecting only basal tears and not reflex tears. Precautions included providing regular breaks in between tear collection and avoiding contact of microcapillary tube with the conjunctiva. If reflex tearing occurred, tear collection was stopped immediately and then continued with a fresh capillary tube after a few minutes break. The time allotted for tear collection was 30 minutes for all participants. On average, 8 ± 2 μl of basal tears was collected from each participant. However, tear collection rate was slower in symptomatic contact lens wearers (0.26 μl/min) than in asymptomatic wearers (0.5 μl/min). After collection, tears from both eyes were pooled, centrifuged at 5000g for 10 min to remove any cellular debris, and placed into smaller aliquots and stored at −80 °C until analysis.
Secretory Phospholipase A2 Enzyme
The concentration of phospholipase A2 in tear samples was quantified using a double antibody sandwich enzyme-linked immunosorbent assay. Samples and standards were prepared according to the manufacturer’s instructions (Cayman Chemical Company, Ann Arbor, MI). The concentration of sPLA2 standards ranged from 0 to 1000 pg/ml and the minimum detectable level was 15.6 pg/ml. Tear samples were diluted in enzyme immunoassay buffer (1:100,000). After incubation, Ellman’s reagent was added to each well including blank, samples, and standards resulting in a yellow color reaction. Absorbance was read at 405 nm after 2 hours. The intensity of this color, determined spectrophotometrically, was proportional to the concentration of sPLA2. A linear equation was generated by plotting absorbance versus concentration of standards and was used to calculate the concentration of each tear sample.
The catalytic activity of sPLA2 was measured using the 1,2-dithio analog of diheptanoyl phosphatidylcholine, which serves as a substrate for most acidic PLA2 enzymes/isoforms. Free thiols produced by sPLA2 activity are detected using 5,5′-dithio-bis-(2-nitrobenzoic acid). Sample and standard preparations were prepared according to the manufacturer’s instructions (Cayman Chemical Company). Tear samples were diluted (1:10) using the assay buffer (25 mM Tris-HCl, containing 10 mM CaCl2, 100 mM KCl, and 0.3 mM Triton X-100). Ten microliters of 10 mM 5,5′-dithio-bis-(2-nitrobenzoic acid) in 0.4 M Tris-HCl and 15 μl of assay buffer constituted the blank well. The detection range of this assay was 0.02 to 0.2 μmol min−1 ml−1 of sPLA2 activity. The substrate was added to each well and carefully shaken and the absorbance was read at every minute at 414 nm for at least 5 minutes. By plotting the absorbance values as a function of time, the catalytic activity of sPLA2 was calculated.
Derived from polyunsaturated fatty acids, lipid peroxides decompose to form compounds such as MDA. Measurement of these reactive carbonyl compounds is widely used as an indicator of lipid peroxidation.36 The assay is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA at 45 °C, yielding a stable carbocyanine dye. The standards and reagents were prepared as per manufacturer’s instructions (Biokits.com Bioxytech MDA-586, Dublin, Ireland). Samples were diluted (1:10) using 20 mM Tris-HCl buffer and, with the recommended reagents, were incubated at 45 °C for an hour and then centrifuged at 10,000g for 10 min to obtain a clear supernatant. Absorbance of the supernatant was read at 586 nm and a calibration curve was drawn by plotting absorbance versus concentration of standards and was used to calculate the concentration of MDA in each tear sample.
Tear lipid extraction was performed as described previously using a methyl-tert-butyl-ether/methanol/ammonium acetate two-phase extraction.37 Tear extracts were characterized by targeted chip-based nanoelectrospray tandem mass spectrometry.37–39 About 199 individual lipid species were quantified by comparison to class-specific internal standards. Lipid species were quantified from the classes of CE, WE, TAG, phosphatidylcholine, sphingomyelin, and (O-acyl)-ω-hydroxy fatty acids (OAHFA). To normalize for individual variation in total lipid concentration, the mole percentage of each lipid species in the total lipidome was calculated.
A paired t test and the Wilcoxon signed-rank test were conducted between the right and left eye for NISDT and lipid layer grades, respectively. There was no significant difference between the eyes and hence right eye results are presented. In stage 1, comparisons between symptomatic and asymptomatic groups were performed using the Student t test or the Mann-Whitney U test. Continuous variables such as NISDT and mole percentage of each lipid class were compared using the Student t test, whereas a categorical variable such as lipid layer grade was compared using the Mann-Whitney U test. Associations between variables were examined using either the Pearson or the Spearman correlation coefficient test where appropriate. In stage 2, a repeated-measures analysis of variance was conducted to compare the variables at three time points. A Bonferroni correction was applied to control the overall type 1 error rate. Statistical analyses were performed using SPSS (IBM SPSS Statistics 19, New York), and a p value of less than 0.05 was considered statistically significant.
The appearance of the lipid layer did not differ between symptomatic and asymptomatic contact lens wearers, whereas the NISDT was lower in symptomatic contact lens wearers than in asymptomatic contact lens wearers (4.5 ± 0.6 vs. 9.9 ± 3.1 seconds, p = 0.01). The proportion of each lipid class in the total tear lipidome in symptomatic and asymptomatic contact lens wearers is shown in Table 1. Cholesterol esters and WE dominated the tear lipidome, whereas phospholipids (phosphatidylcholine and sphingomyelin) were at low concentrations in both groups. Lipid ratios were compared between symptomatic and asymptomatic wearers (Table 2). The ratio of WE to TAG in symptomatic contact lens wearers (30.4:1) was significantly lower (p = 0.03) than that in asymptomatic contact lens wearers (49.3:1). As the NISDT increased, so did the percentage of WE in the total lipidome (R2 = 0.70, p = 0.01), whereas the percentage of CE decreased (R2 = 0.59, p = 0.04). Phospholipase (sPLA2) activity was associated with increased MDA (R2 = 0.65, p = 0.01) and shorter NISDT (R2 = 0.84, p = 0.001).
The mean NISDT of symptomatic lens wearers treated with liposomal spray or control saline spray is shown in Fig. 1. Noninvasive surface drying time measured immediately after spraying and 2 and 6 hours after spraying were 9.5 ± 2.2, 7.9 ± 2.5, and 5.8 ± 2.3 seconds for the liposomal spray group and 8.8 ± 1.8, 6.5 ± 3.1, and 5.9 ± 2.1 seconds for the saline spray group, respectively. The repeated-measures analysis of variance showed that the NISDT of those treated with liposomal spray was not affected by time (p = 0.06), whereas the NISDT of those treated with saline spray was affected by time (p = 0.01). The Bonferroni multiple pairwise comparisons showed that the NISDT of those treated with liposomal spray differed (p = 0.03) between 0 and 6 hours but not between 0 and 2 hours (p = 0.34), whereas the NISDT of the lens wearers treated with control spray differed between 0 and 2 hours (p = 0.05) and also between 0 and 6 hours (p ≤ 0.01). As the NISDT increased, there were improved ocular comfort scores for those subjects treated with liposomal spray (R2 = 0.25, p = 0.005) but not with saline spray. Use of the lipid spray did not change the activity of sPLA2 or the concentration of MDA compared with the saline spray, although a marginal but not statistically significant reduction in the concentration of sPLA2 was observed at 6 hours after use of the liposomal spray (Table 3).
According to the Tear Film & Ocular Surface Society report from “the contact lens interactions with tear film subcommittee,”6 a person’s ocular environment is one of the factors that contribute to lens wear discomfort. The various elements in a person’s ocular environment include the biophysical and biochemical properties of the tear film.21,40–46 In the current study, a potential role of the tear film lipid layer in contact lens wear comfort among a small group of symptomatic wearers is reported. Considering the wide use of exogenous lipid supplements among subjects with dry eye, a biochemical basis of the effectiveness of these supplements in contact lens wearers was explored.
The relationship between clinical variables, such as tear volume and stability, and lens tolerance has been discussed in the literature.47 Contact lens wearers with lower tear volume and reduced tear breakup time are more susceptible to contact lens intolerance.47 Noninvasive surface drying time measured in the current study is associated to stability of tear film over the lens surface and was lower among symptomatic contact lens wearers. Interestingly, when symptomatic wearers were treated with a liposomal spray, their NISDT did not change until 2 hours after the initial instillation when compared with a saline spray. As the NISDT of symptomatic wearers did not change until 2 hours after instillation of the liposomal spray, we speculate that the spray does not affect the lipid layer of tears immediately; the spray may not be sufficiently migrating into the tear film from the eyelid and hence needs some time to interact significantly with this layer. Improved clinical symptoms and ocular comfort using the same liposomal spray have been shown previously among healthy subjects, in a dry eye population, and among contact lens wearers.30,31,33
Patients with meibomian gland dysfunction were found to have increased levels of CE and decreased levels of WE in their meibum.48 It was proposed that the differences observed in ester composition could increase the melting point of meibomian gland secretions, which might lead to blocked glands and a destabilized tear film. Similarly, alterations in wax and sterol ester fractions of patients with chronic blepharitis were observed and the differences in ester fractions were regulated either by the meibomian gland or by bacterial lipases.49 About 50% of patients with chronic blepharitis and 33% of patients with meibomian gland dysfunction had an associated dry eye disease.48–51 The current study demonstrates that at the end of 6 to 8 hours of lens wear, a higher proportion of WE and a lower proportion of CE are associated with improved tear film stability.
The effect of lipid ratios in regulating the stability and distribution of lipid films has been observed in vitro using Langmuir films, X-ray diffraction, and coarse-grained simulations.52–54 Neutral lipid classes (CE, TAG, and fatty acids) increase tear film stability53; however, a decreased phospholipid-to-neutral lipid ratio can reduce the stability of the lipid film in vitro.54 In the current study, lipid ratios were compared between symptomatic and asymptomatic wearers in vivo, and the asymptomatic group had a higher WE-to-TAG ratio (49.3:1) in their tear lipidome than symptomatic wearers (30.4:1). This further emphasizes the significance of lipid ratios in the stability of the tear lipid layer, thereby regulating ocular comfort.
The secretory phospholipase A2 enzyme catalyzes the hydrolysis of the sn-2 fatty acyl moiety from phospholipid, producing lysophospholipid and free fatty acid. In the case of arachidonic acid containing phospholipids, the free arachidonic acid can then be metabolized to produce reactive oxygen species, which can react with cellular lipids generating lipid peroxides.22,55 One of the major by-products of lipid peroxidation is MDA, which in the current study was found to have an association with increased sPLA2 and reduced tear film stability. The role of sPLA2 in regulating ocular surface inflammation was observed in a mouse model where it was concluded that sPLA2 could enhance ocular inflammation.20 These findings further support the observations of increased enzyme activity and degraded lipids among symptomatic lens wearers.21
Increased tear film stability was associated with improved ocular comfort after the application of liposomal spray. However, there was no association between the improved ocular comfort and phospholipase activity or formation of lipid aldehyde. This could be attributed to the small sample size. Despite this limitation, the clinical and biochemical associations observed in stage 1 indicate a potential role of phospholipids in maintaining ocular comfort. In a different group of subjects, a substantial association was observed between reduced concentrations of tear phospholipids and increased tear evaporation rate during contact lens wear.56
Similar to phospholipids, OAHFA are potential lipid classes that need to be explored for their role in maintaining tear film stability and ocular comfort. The presence of OAHFA in meibum was first reported by Nicolaides et al.57; however, a structural description was initially provided by Butovich et al.58 and later confirmed by several investigators.37,59,60 An in vitro investigation of OAHFA using synthesized (O-oleyl) ω-hydroxy palmitic acid as the model confirmed the surfactant and bridging properties of OAHFA to spread and stabilize lipid films.61,62 In addition, an association of reduced concentration of OAHFA with increased severity of dry eye disease59 signifies the importance of this amphiphilic lipid class.
This pilot study confirms the clinical and biochemical changes in the tear film lipid layer during contact lens wear. Moreover, it provides a preliminary observation on the effectiveness of an exogenous lipid supplement on the tear film stability and the concentration of phospholipase enzyme used during contact lens wear. A study using a larger sample size and appropriate power is recommended to draw further conclusions about the effect of lipid supplementation on tear lipidome.
School of Optometry and Vision Science
University of New South Wales
Level 3, North Wing, RMB
Gate 14 Barker St
Sydney, New South Wales 2052
This work was partly supported by the University of New South Wales (UNSW), the Brien Holden Vision Institute, and the Vision Cooperative Research Centre (CRC). There are no proprietary interests for any of the authors. Following Australian Government policy related to CRC Grants, the Vision CRC and partners including the Brien Holden Vision Institute receive royalties on the sales of certain silicone hydrogel contact lenses. AR’s candidature was supported by the Tuition Fee Remission Scholarship from the UNSW and an additional top-up postgraduate grant from the Vision CRC, Australia.
Received December 16, 2013; accepted September 12, 2014.
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