Lens care systems were assigned for each study phase according to a randomization table. Participants began with one system and then switched to the other system in the second phase. Subjects were instructed to wear the study lenses on a daily wear basis for at least 12 h/d and at least 6 d/week for the duration of the 4-week study. Participants were instructed to clean and disinfect their lenses, using the appropriate study lens care system, after each day of lens wear. As far as possible, participants were masked from which care regimen they were using. All identifying labels were removed, but it was not possible to mask the embossed “Alcon” from the care system preserved with Polyquad and Aldox. Participants using lubricating drops with their habitual contact lenses were allowed to continue to use their habitual brand of drops. The use of drops was recorded along with the subjective symptom ratings throughout the study (not reported here). Study visits were scheduled after at least 4 h of wear and at approximately the same time for a given study participant in each study phase. At the 2-week visit for each phase, the participants were required to return all remaining study products. The study lenses were collected at the end of each phase (Fig. 1), at which time lenses were stored at −80°C until lipid deposits on the lenses were analyzed.
Reagents and Materials
The lipids used for standards were cholesterol, cholesterol oleate (CO), oleic acid (OA), OA methyl ester (OAME), and triolein. Lipid standards, chloroform, and methanol for extraction solutions were purchased from Sigma-Aldrich (Oakville, ON, Canada) and HPLC solvents were purchased from Fisher Scientific (Ottawa, ON, Canada).
Lipid Deposit Extraction From Lenses
Lens Collection and Storage
Study lenses were carefully removed by the investigator, with clean powder-free nitrile gloves (SemperCare Nitrile PF) at the end of each 2-week phase and stored dry at −80°C in amber glass vials. Lens collection was randomized, with either the left or right lens being collected for lipid analysis. The same randomization was used for both phases to allow for pair-wise phase comparisons for each participant.
Lipid Extraction From Participant-Worn Lenses
Lipids were extracted from each lens by gentle shaking in 1.5 ml of a 2:1 chloroform:methanol solution at 37°C for 3 h. The extracts were evaporated to dryness under a stream of nitrogen, then sealed and stored at −80°C before resolubilization.
Preparation of Unworn Lenses Used for Extract Controls
To control for detergent carry over and possible polymer extraction, unworn lenses were prepared as follows and extracted in parallel with participant-worn lenses. Unworn senofilcon A lenses were prepared by soaking the lens for 8 h intervals in one of the two study solutions, followed by a 16 h soak in phosphate-buffered saline. The solutions were replaced 14 times to approximate the number of cleaning cycles of the participant-worn lenses. At the end of the cleaning cycle, each lens was soaked in 5 ml of phosphate-buffered saline for 5 min to remove any loosely bound care solution components. The lens was blotted to remove excess moisture and stored at −80°C.
Extract Resuspension Solvent
Lipid samples and extracts from both patient-worn lenses and unworn lens controls were suspended in mobile phase (100 hexane: 0.5 isopropanol: 0.01 acetic acid), sonicated and filtered through a Nalgene 4-mm nylon syringe filter (0.2 μm pore).
Lipid Standard Curves
The range of standard lipid concentration was from 0.01 to 5 μg. Lipid standards and lens extract controls were run for every 12 participant-worn lenses (Fig. 2).
Lipid samples of 10 μl were separated isocratically in a mobile phase of 100 hexane:0.5 isopropanol:0.01 acetic acid on a Hiasil normal phase silica gel column (4.6 × 150 mm) from Higgins Analytical (Mountain View, CA) using a guard column of the same material. The column temperature was held constant at 25°C, the flow rate set to 1.5 ml/min. Chromatograms were collected at 210 nm (Fig. 3).
The peaks of the chromatograms were analyzed with Chromquest 4.2 software. The area under the peak was compared with lipid standards and corrected for the entire extract. Unworn lens extracts had a small peak that co-eluted with the peak for CO. The area of the appropriate control lens was subtracted from the CO peak and then quantified. The co-eluted peak was <10% of the average CO peak from the care system preserved with Polyquad and Aldox-exposed lenses and <5% from the peroxide-based care system lenses.
Data analysis was conducted using SigmaStat 3.1 (Systat Software, San Jose, CA). Data were presented as means ± SD (unless otherwise stated) and a significance level of p = 0.05 was used for all analyses. All pair-wise comparisons were made with paired Student t-tests. Data were analyzed using a one way analysis of variance, and Tukey HSD post hoc comparisons were performed, where applicable. Thirty participants were enrolled in both phases of the study, totaling a maximum of 60 possible lenses for analysis. Two lenses disinfected with the peroxide-based care system were not analyzed because they were damaged during wear and had to be redispensed, resulting in less wearing time than was acceptable for the study. Two lenses cleaned using the care system preserved with Polyquad and Aldox were accidentally put into the same vial by the investigator and had to be excluded from analysis. One participant discontinued early from the study. Thus, 27 peroxide-based care system and 28 samples disinfected using the care system preserved with Polyquad and Aldox were analyzed. Paired t-test analysis comparing the two care systems for each participant was performed. Only complete pairs could be used for this analysis, hence the sample size was 26 per system.
Lipid Deposited on the Lens
The mean and standard deviation of the lipids deposited on the lenses are summarized in Table 4. The values are given as microgram/lens, along with the ranges (in parentheses) for each lipid. The p values for the paired Student t-tests are also listed. In cases where the amount of lipid detected was less than the lower limit of quantification, only the range of values are presented. In these cases, most of the lenses did not have detectable lipid. The total lipid values are calculated from the sum of CO and cholesterol. They do not include any values from the other lipids, which were too low to accurately quantify and include in this analysis.
Summed Lipid Deposition on Lenses
The values for CO and cholesterol were summed together for each participant. There was a significant difference determined between the two lens care solutions with respect to CO and cholesterol deposition on the senofilcon A lenses (34.1 vs. 21.7 μg/lens; p = 0.029), with those lenses disinfected with the care system preserved with Polyquad and Aldox depositing lower quantities of lipid. Approximately, 95% of the contribution to the total lipid value was from CO.
Cholesterol Oleate and Cholesterol Deposition on Lenses
CO deposition was significantly greater with the peroxide-based care system than with the care system preserved with Polyquad and Aldox (32.7 vs. 20.5 μg; p = 0.033). There was no difference between care products (1.4 vs. 1.3 μg/lens; p = 0.503) with respect to cholesterol deposition.
Oleic Acid Deposition on Lenses
OA was detectable in 14 of the 52 lenses examined (27%). The lower limit of reliable quantification was 0.5 μg/lens. Although OA was detectable in around one quarter of the lenses, one-half of these samples did not have sufficient quantities present for accurate determination. The values reported are extrapolated from beyond the standard curve, so are only given as estimates. Eight participants in the peroxide-based care system had OA ranging in values from about 0.1 to 2.5 μg/lens. Six participants in the arm using the care system preserved with Polyquad and Aldox showed OA between 0.3 and 3.2 μg/lens. Of these 14 samples, four participants had OA in both phases.
Triolein Deposition on Lenses
Triolein was less abundant on lenses than OA and occurred fewer times. It was found on only two lenses in the peroxide-based care system (0.2 to 0.3 μg/lens) and twice in the care system preserved with Polyquad and Aldox arm (0.3 to 0.6 μg /lens). The lower limit of reliably measuring triolein was 0.25 μg/lens.
Oleic Acid Methyl Ester Deposition on Lenses
None of the lenses analyzed had any measurable OAME. The lower limit to detect OAME was 0.25 μg/lens.
This study reports the deposition of major non-polar lipids on ex vivo senofilcon A lenses disinfected with either a care system preserved with Polyquad and Aldox or a peroxide-based care system, in participants symptomatic of dry eye. This study was able to analyze the major non-polar lipids that adsorbed to the study lenses. The results from our study (Table 4) demonstrate that senofilcon A lenses deposit 32.7 ± 27.9 μg/lens of CO with the peroxide-based care system used in this study (CIBA Vision ClearCare) and 20.5 ± 20.1 μg/lens when the lenses were disinfected with a care system preserved with Polyquad and Aldox (Alcon Opti-Free RepleniSH). To our knowledge, this is the first ex vivo study to have quantified CO deposited on senofilcon A SH contact lens material. An in vitro study by Pucker et al.34 reported that galyfilcon and lotrafilcon B lenses deposited 35 μg/lens and 65 μg/lens, respectively, after 3 days of incubation in 5.6 mg/ml of CO solution.
It is of interest to note that the care system preserved with Polyquad and Aldox was able to remove more CO, which makes up about 95% of the measured lipids, from senofilcon A lenses, than a peroxide-based care system (Table 4), which may be attributable to the composition of the care systems. The care system preserved with Polyquad and Aldox contains citrate, which is a negatively charged ion and a sequestering agent, which aids in the passive removal of protein.38–40 It is possible that citrate may also play a role in the removal of non-polar lipids from a non-surface modified SH contact lens material. In addition, the preserved system contains a complex mixture of surface-active, block copolymer agents that may have helped to remove a greater amount of lipids. Also, the surfactants are different between the two care solutions and could have different affinities to CO.
This study results are consistent with other ex vivo studies,20,35 which report that cholesterol is more abundant than OA and OAME on contact lenses. The data from this report shows lower levels of lipid adsorption compared with previous in vitro studies using OASYS or other group I lenses.5,33 The difference is likely explained by the absence of daily cleaning of lenses in an in vitro study. The cholesterol deposition values for the peroxide-based system in this study (1.4 ± 1.0 μg/lens) were similar to the study (1.2 ± 0.4 μg/lens) of Zhao et al.35 In addition, the care system preserved with Polyquad and Aldox also showed similar levels of cholesterol deposition in these two studies (1.3 ± 1.2 μg/lens vs. 1.4 ± 0.8 μg/lens). Another recent study which investigated cholesterol deposition on senofilcon lenses A when used in a care system with Polyquad- and Aldox-based system showed results similar to this study.41 Saville et al.36 reported higher amounts of cholesterol deposition on senofilcon A lenses (9.9 ± 2.2 μg/lens), which is likely due to differences in the techniques used. Saville et al.36 used mass spectrometry, as opposed to the HPLC method described in this study. Our laboratory has a well-established protocol for the analysis of lipids using HPLC. HPLC is a sensitive method for assessing lipids on contact lenses. It can measure more than one lipid at a given time point. HPLC also processes the samples in a consistent manner. Other techniques such as thin-layer chromatography can be labor intensive and the sensitivity and separation properties of thin-layer chromatography are low. Other ex vivo studies on lipid deposition such as those by Maziarz et al.20 and Pucker et al.34 did not study deposition in senofilcon A and did not use either of the care systems used in this study.
Deposition of CO on senofilcon A lenses was studied in vitro by Pucker et al.42 They found four- to sixfold less CO compared with this study. These differences can be attributed to the difference in the extraction procedure used in the studies. There was a slight difference in extraction solution (1 choloroform: 1 methanol vs. 2 chloroform:1 methanol, respectively) and a large difference in extraction times (25 s vs. 3 h, respectively).
This study has shown that lens care solutions can have a significant impact on the amount of lipid remaining on lenses after habitual care. The care system preserved with Polyquad and Aldox used in this study (Alcon Opti-Free RepleniSH) removed 37% more of the most abundant lipid (CO) found on the senofilcon A lens when compared with a peroxide-based system (CIBA Vision ClearCare). There was no difference between the two care systems for the amount of cholesterol found on the lens. In a previous ex vivo study examining lipid on SH lenses,20 six of eight (75%) of galyfilcon A wearers had no OAME on their lens, when compared with this study in which no OAME was found on any of the worn lenses.
In this study, participants were subjectively categorized as being symptomatic of dry eye based on a dry eye questionnaire. The meibomian gland secretions from the upper and lower lids could play a significant role in the amount of lipids deposited on the lens materials, and this is worthy of further study. Clinical signs such as meibomian gland evaluation, tear break-up time assessment, and tear collection for tear lipid analysis will provide valuable information on the status of lipids and hence its deposition in contact lens wearers. This study only analyzed the major non-polar lipids deposited on the lenses and further work on analyzing the polar lipids would provide a complete picture of lipid deposition on SH lenses and the role of care regimens in their removal. Previous studies in our laboratory have shown that protein deposition on lenses is primarily driven by lens material water content and ionicity and to a greater extent are not related to subjective differences.31 However, lipid deposition on lenses is related to many factors including material composition and intersubject differences in tear film components, blink factors, and environmental factors.31
Previous studies have clearly shown that a hydrophobic lens surface is more prone to lipid deposition,1,5,43 and this could potentially result in increased discomfort and dryness symptoms in contact lens wearers. The results from this study showed that a care system preserved with Polyquad and Aldox removed more non-polar lipids than a peroxide-based care system. Hence, the citrate containing Polyquad and Aldox care system may benefit patients who are heavy contact lens lipid depositors. In this study, Polyquad- and Aldox-based system when used as a “no rub, 5 s rinse only” format showed significantly lower amounts of lipid deposition. Future studies using Polyquad and Aldox system in a rub and rinse format may show a further reduction in the amounts of lipids deposited on to the lenses.
In conclusion, a care system preserved with Polyquad and Aldox removed more non-polar lipids from senofilcon A lenses than did a peroxide-based care system in a 2-week, cross-over study. Approximately, 95% of the measured lipid was CO, with cholesterol making up most of the rest. OA and triolein contributed <1% and no OAME was found on any of the lenses.
This study was sponsored by ALCON Research Ltd.
Centre for Contact Lens Research
School of Optometry
University of Waterloo
200 University Ave W
Waterloo, Ontario N2L3G1
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Keywords:© 2011 American Academy of Optometry
silicone hydrogel; contact lens; lens care solutions; lipids; cholesterol; dry eye; deposition