Mary-Ann Glasier

Lysozyme and lipid deposition on silicone hydrogel contact lens materials.

Purpose. We sought to determine whether there were differences in lysozyme (quantity and conformation) and lipid deposition on in vivo worn conventional (etafilcon) and silicone hydrogel (balafilcon and lotrafilcon) contact lenses. Methods. After extraction, lysozyme concentration in each extract was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Lysozyme activity was determined by the rate of lysis of Micrococcus lysodeikticus cells. Lipid deposition was determined by high-performance liquid chromatography. Results. Lysozyme deposition on etafilcon lenses was significantly greater than that measured on silicone hydrogel (SH) lenses (985 &mgr;g per lens versus 10 and 3 &mgr;g per lens for balafilcon and lotrafilcon materials, respectively;P <0.001). The degree to which lysozyme was denatured was influenced by the lens material, with the lowest degree of denaturation (22%) seen on the conventional lens material, as compared with 50% for balafilcon and 80% for lotrafilcon (P <0.001). Lipid deposition was greatest on the SH materials, with up to 600 &mgr;g per lens of certain lipid classes being deposited on balafilcon, as compared with 20 &mgr;g per lens on etafilcon (P <0.001). Conclusion. The quantity and conformation of lysozyme and the quantity of lipid deposited on hydrogel contact lenses is significantly influenced by the composition of the lens material. SH contact lens materials deposit low levels of lysozyme and high levels of lipid deposition compared with ionic contact lens materials. Although SH materials deposit only small amounts of lysozyme, the degree of lysozyme denaturation that occurs is higher relative to that seen on ionic lens materials.

Quantitative and conformational characterization of lysozyme deposited on balafilcon and etafilcon contact lens materials

Purpose. To determine whether differences in lysozyme deposition and/or activity exist on worn etafilcon and balafilcon contact lenses following care with a polyquaternium-based system (PQ) or a polyhexanide-based system (PHMB). Methods. Following acid-based deposit extraction, lysozyme concentration was determined via Western blotting and lysozyme activity was determined by a micrococcyl assay. Results. Lysozyme deposition on etafilcon lenses was greater following disinfection with the PHMB-based system (1551 ± 371µg/lens vs 935 ± 271µg/lens; p < 0.001). Deposition on balafilcon lenses was not influenced by the care regimen (10 ± 3.5µg/lens vs 10 ± 5µg/lens; p = 0.89). For both materials, the percentage of denatured lysozyme was greater when they were exposed to the PHMB-based system (28 vs 21%; p = 0.05 (etafilcon) and 57 vs 40%; p = 0.04 (balafilcon)). Conclusions. The quantity and conformation of lysozyme deposited on hydrogel contact lens materials is significantly influenced by both lens material and care regimen.

Kinetics of in vitro lysozyme deposition on silicone hydrogel, PMMA, and FDA groups I, II, and IV contact lens materials.

We sought to compare the kinetics of in vitro lysozyme deposition on silicone hydrogel (SH), polymethyl methacrylate (PMMA), and FDA groups I, II, and IV contact lenses. Lenses were incubated in 125I-labeled lysozyme for time periods ranging from 1 hr to 28 days, and radioactive counts were determined. SH lenses and PMMA deposited less lysozyme than conventional hydrogel lenses (p < 0.05). Lysozyme accumulation on group IV lenses reached a maximum on the seventh day and then plateaued, whereas on groups I, II, and SH lenses, deposition continued to increase across all time periods, reiterating that kinetics of lysozyme deposition is highly material dependent.

Quantity and conformation of lysozyme deposited on conventional and silicone hydrogel contact lens materials using an in vitro model.

Purpose. To determine the activity of hen egg lysozyme (HEL) deposited on conventional and silicone hydrogel contact lens materials by using an in vitro model. Methods. ACUVUE 2 (etafilcon A), PureVision (balafilcon A), ACUVUE Advance (galyfilcon A), Focus NIGHT & DAY (lotrafilcon A), O2 Optix (lotrafilcon B), Proclear (omafilcon A), and ACUVUE OASYS (senofilcon A) contact lenses were deposited in vitro in a phosphate-buffered solution (PBS) containing 2 mg/mL HEL. Lenses were briefly rinsed in PBS to remove unbound material and extracted in a mixture of acetonitrile and trifluoroacetic acid. After lyophilization, extracts were examined for lysozyme activity by micrococcal assay and total protein by Western blot. Results. In terms of total protein accumulation, ACUVUE 2 showed the most, with 1,800 &mgr;g per lens. Proclear was next, with 68 &mgr;g per lens, and Focus NIGHT & DAY showed the least, with 2 &mgr;g per lens. ACUVUE Advance, ACUVUE OASYS, and O2 Optix accumulated similar amounts of lysozyme, at approximately 6 &mgr;g per lens. Lysozyme deposited on ACUVUE 2 showed the greatest activity (91% ± 5%), and this result was statistically different from all other lens types (P<0.001). Lysozyme deposited on Focus NIGHT & DAY (24% ± 5%) and O2 Optix (23% ± 11%) showed the lowest activity. Lysozyme deposits on other lens materials showed intermediate activity (ACUVUE Advance, 60% ± 15%; ACUVUE OASYS, 51% ± 9%; PureVision, 58% ± 8%; and Proclear, 38% ± 3%). Conclusions. Silicone hydrogel lenses acquire less lysozyme deposit than conventional group II (Proclear) or group IV (ACUVUE 2) lenses do, and the levels of activity of the lysozyme are highly variable between materials.

Rewetting Drops Containing Surface Active Agents Improve the Clinical Performance of Silicone Hydrogel Contact Lenses

Purpose. The purpose of this study was to investigate the impact of using a rewetting drop (RWD) containing surface active agents (OPTI-FREE RepleniSH; Alcon, Fort Worth, TX) on the clinical performance and protein deposition when using a continuous-wear (CW) silicone hydrogel (SH) contact lens. Methods. Subjects wore lotrafilcon A SH lenses on a 30-day CW basis for two consecutive 1-month periods while inserting either 0.9% unpreserved unit-dose saline (control) or multidose OPTI-FREE® RepleniSH™ (test RWD). Subjective comfort and symptoms were assessed after 2 and 4 weeks with each product. After 1 month of wear with each product, lenses were collected and analyzed in the laboratory for total protein, total lysozyme, and percentage of denatured lysozyme. Results. Symptoms of dryness and comfort varied across the day regardless of drop type (p < 0.001) with dryness being maximal on waking, least in the middle of the day, and increased towards the evening. The test RWD provided greater comfort on insertion (p = 0.02), better visual quality (p < 0.01), and less mucous discharge on waking (p = 0.02) than the control product. Lysozyme deposition was significantly reduced after the use of the test RWD as compared to saline (0.73 ± 0.5 &mgr;g/lens vs. 1.14 ± 0.7 &mgr;g/lens; p < 0.001) as was total protein deposition (1.17 ± 0.7 &mgr;g/lens vs. 1.86 ± 0.8 &mgr;g/lens; p < 0.001). Lysozyme denaturation was also reduced with the use of the test RWD compared with the control (76 ± 10% vs. 85 ± 7%; p < 0.01). Conclusions. The use of a RWD containing surface active agents provided greater subjective satisfaction, reduced lysozyme and total protein deposition, and reduced denatured lysozyme than a RWD containing saline alone.

Confocal microscopy and albumin penetration into contact lenses.

Purpose. To develop a novel in vitro method to detect the depth of penetration of the tear film protein albumin into contact lens materials using confocal laser scanning microscopy (CLSM). Methods. A poly-HEMA-based hydrogel (etafilcon A) and a silicone hydrogel material (lotrafilcon B) were examined. In vitro, bovine serum albumin (BSA) was labeled with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein hydrochloride (DTAF). The lenses were incubated in this protein solution (0.5 mg/ml) at 37°C. After 1 and 7 days incubation, the lenses were examined using CLSM (Zeiss 510, config. META 18) and the location of the fluorescently labeled BSA was identified. Results. BSA adsorption on the surface and penetration into the lens matrix occurred at a higher concentration for etafilcon compared to lotrafilcon (p < 0.001). For both materials, BSA was detected on the surface after 1 day of incubation. Significant levels of BSA were detected within the matrix of etafilcon after as little as 1 day (p < 0.001), but no BSA was detected in the matrix of lotrafilcon at any time (p > 0.05). Conclusion. CLSM can be successfully used to examine the depth of penetration of fluorescently labeled proteins into various hydrogel polymers. Our results show that etafilcon lenses both adsorb BSA on the surface and absorb BSA within the matrix, whereas lotrafilcon B adsorbs small amounts of BSA on the surface only.

Protein deposition and clinical symptoms in daily wear of etafilcon lenses.

Purpose. To determine the relationship between clinical signs and symptoms and protein deposition over 8 h of wear of etafilcon 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 A lenses. In vivo wettability, non-invasive tear break-up time, and subjective symptoms (vision, comfort, and dryness) were assessed 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 the symptomatic group, there was a significant reduction in subjective comfort and dryness ratings at 6 and 8 h measurement with 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). Pearsons correlations between subjective symptoms and protein deposition showed poor correlations for total protein/lysozyme and any subjective factor (r < 0.3; p > 0.05), and only weak correlations between dryness and % active lysozyme (r = 0.3 to 0.5 for all time points). However, stronger correlations were found between active lysozyme and subjective comfort (r = 0.6 to 0.7; p < 0.001). Conclusions. In addition to investigating total protein deposited on contact lenses, it is of significant clinical relevance to determine the conformational state of the deposited protein.

Conformational and Quantitative Characterization of Lysozyme Extracted from Galyfilcon and Senofilcon Silicone Hydrogel Contact Lenses

Purpose: To compare two solvents for retrieval of lysozyme deposited on a silicone hydrogel (SH) contact lens material galyfilcon A (GA; Acuvue Advance). Methods: Two buffers used were 50:50 acetonitrile/0.02% trifluoroacetic acid (buffer 1) and 50:50 acetonitrile/50 mM NaOH (buffer 2). Results: Extraction efficiency from GA lenses was 74% (buffer 1) and 83% (buffer 2). Buffer 2 decreased lysozyme activity > buffer 1. Ex vivo GA lenses showed total protein deposition of 2–16 μ g/lens with total lysozyme deposition of 0.3–3.9 μg/lens. Conclusions: We have developed a low acid strength extraction buffer that can be used to efficiently extract active lysozyme protein from novel siloxane-based contact lens materials.

A Solid-Phase Assay for the Quantitation of Total Protein Eluted from Balafilcon, Lotrafilcon, and Etafilcon Contact Lenses

Purpose: To compare two variations of a membrane-based protein assay utilizing Amido black (AB) detection with a commercially available 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) assay for use in the quantitation of individual tear proteins, pooled human tear proteins, and protein extracted from ex vivo lotrafilcon A, balafilcon A, and etafilcon A contact lens materials. Methods: Ex vivo contact lens extracts, pooled human tears, and individual tear proteins (human serum albumin (HSA), bovine lactoferrin, human secretory immunoglobulin A (sIgA), human lysozyme) were subjected to three solid-phase assays: AB on polyvinylidene difluoride (AB on PVDF) and AB on nitrocellulose (AB on NC) and the CBQCA assay. Micro-bicinchonic acid (micro-BCA) assay was also employed with lens extracts to determine total protein concentration. Individual and pooled tear proteins were referenced to a micro version of the quantitative ninhydrin protein assay. Results: The CBQCA demonstrated the greatest overall sensitivity and lowest intra- and inter-assay variability. AB on NC demonstrated the most accurate ability to quantify total protein in pooled human tear samples, although it also displayed the greatest protein-to-protein variation using individual tear proteins. The CBQCA assay displayed the greatest cross-reactivity with unworn balafilcon and lotrafilcon lens extracts, whereas AB on NC demonstrated the least. AB on NC measured similar amounts of total protein in extracted ex vivo lenses as the CBQCA assay if background interference was subtracted from CBQCA values. AB on PVDF measured the lowest amount of deposited protein from ex vivo lenses. Conclusion: Both the AB on NC and CBQCA assays can be used to measure protein in extracts of lotrafilcon, balafilcon, and etafilcon lens materials.