Lysozyme and lipid deposition on silicone hydrogel contact lens materials.
ABSTRACT 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.
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 Micrococcis lysodeikticus cells. Lipid deposition was determined by high-performance liquid chromatography.
Lysozyme deposition on etafilcon lenses was significantly greater than that measured on silicone hydrogel (SH) lenses (985 microg per lens versus 10 and 3 microg 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 microg per lens of certain lipid classes being deposited on balafilcon, as compared with 20 microg per lens on etafilcon (P<0.001).
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.
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ABSTRACT: ABSTRACT Purpose: To optimize a fluorescence-based lysozyme activity assay to investigate the conformational state of lysozyme in solution and to determine the impact of extraction and evaporation procedures and the possible interference of contact lens materials on lysozyme activity. Methods: The fluorescence-based lysozyme activity assay, Enzchek (Molecular Probes Inc, Eugene, OR) which utilizes fluorescently quenched Micrococcus lysodeikticus, was compared to the gold standard, classical lysozyme turbidity assay, using four differently concentrated lysozyme samples (20, 10, 5.0 and 2.0 ng/µL). Furthermore, six differently concentrated lysozyme samples (2.0, 1.0, 0.5, 0.25, 0.125 and 0.01 µg/µL) were quantified using the fluorescence-based assay in the presence of extraction solvents consisting of 0.2% and 0.02% trifluroacetic acid/acetonitrile and following evaporation procedures. Results: A standard curve was generated by the fluorescence-based assay ranging from 2 to 150 ng. The total active lysozyme quantified in the four lysozyme samples was not significantly different between the two assays (p > 0.05) and the concordance correlation coefficient was determined to be 0.995. However an average discrepancy between the two assays was found to be 0.474 ng, with the turbidity assay typically reporting higher active lysozyme measurements. The sensitivity of the fluorescence-based assay was higher than the classical turbidity assay when quantifying 20 ng or less active lysozyme. Following the extraction and evaporation procedures and the addition of lens extracts, the total active lysozyme recovered was 95% or greater. Conclusions: In comparison to the classical turbidity assay, the fluorescence-based assay is a very sensitive method, making it a favorable technique, particularly when studying contact lens materials that deposit relatively low levels of lysozyme.Current eye research 02/2013; · 1.51 Impact Factor
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ABSTRACT: 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). Pearson's 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.Optometry and vision science: official publication of the American Academy of Optometry 10/2012; 89(10):1450-9. · 1.53 Impact Factor
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ABSTRACT: PURPOSE.: To analyze the impact of intermittent air exposure on the in vitro deposition of two radioactive lipids on various contact lens (CL) materials, using a custom-designed model blink cell. METHODS.: Six different CL materials (balafilcon A, lotrafilcon B, comfilcon A, senofilcon A, etafilcon A, and omafilcon A) were mounted on the model blink cell pistons, which cycled the lenses in and out of a complex artificial tear solution (ATS) that contained a trace amount of C-cholesterol or C-phosphatidylcholine. For the short-term experiment, air-exposed lenses were continuously cycled in and out of the ATS for 10 h. Longer term incubations for 6 days were tested with lotrafilcon B and balafilcon A materials incubated in C-cholesterol ATS. The air-exposed CLs were cycled for 14 h then submerged for 10 h each day. For both experiments, the control lenses were submerged for the entire test period. After incubation, lenses were processed, and deposited masses were quantified. RESULTS.: Exposure to air resulted in increased amounts of cholesterol deposited by 1.6 to 4.3 fold on omafilcon A, balafilcon A, comfilcon A, and senofilcon A (p ≤ 0.03) compared with submerged lenses. No differences in deposition were observed for etafilcon A and lotrafilcon B (p > 0.05). The longer term incubation of lotrafilcon B and balafilcon A showed statistically significant increases in cholesterol deposition for both air-exposed lens materials (p < 0.02) with the increase in deposition 1.8× and 2.8×, respectively. For phosphatidylcholine, all air-exposed lenses had increased masses of deposition. These deposits were statistically greater by 1.1 to 1.6 times for omafilcon A, comfilcon A, lotrafilcon B, and senofilcon A (p < 0.04), but not statistically different for etafilcon A or balafilcon A (p > 0.05). CONCLUSIONS.: This study found that lipid deposition profiles are CL material dependent and that intermittent air exposure can influence the mass of lipid deposited.Optometry and vision science: official publication of the American Academy of Optometry 10/2012; · 1.53 Impact Factor