Abstract
Objective: Amplify the absorbance signal related to the lysozyme extracted from contact lenses as measured by high-performance liquid chromatography (HPLC); examine the purity of lysozyme and how much thermal denaturation affects its elution profile. Methods: In trial chromatographic runs during which contact lens extracts had been injected into the system, the fractions collected between 4 and 5.5 minutes pointed to lysozyme as the eluting protein as indicated by a Western blot. Proteins were extracted from contact lenses in a 50:50 solution of 0.2% trifluoroacetic acid (TFA): acetonitrile (ACN). Each contact lens extract was separated into 2 aliquots. After vacuum evaporation, aliquots no 1 were dissolved into the initial mobile phase to produce an enrichment factor of 8. Aliquots no 2 were left untreated in the regular extraction solution. Calibration of the absorbance signal at 220 nm allowed measuring the lysozyme levels in chromatograms. Parallel injections of both aliquots into the HPLC allowed comparing their lysozyme content. Purity of the lysozyme extracted was evaluated by viewing its absorbance spectrum across peaks. Lysozyme solutions, previously heated to 80 and 100°C were injected and compared with control solutions. Differences in median lysozyme content between aliquots no 1 and 2 and in peak area of lysozyme heated compared to control were tested with non-parametric methods. Results: Median lysozyme level measured in enriched and regular extracts differed significantly (39.8 and 21.5 μg, respectively). However, once corrected for different injection volume and concentrating factor, the mean enriched lysozyme level (21.4 μg) was close to the one found in the regular extract. Observation of spectral absorbance suggests that eluting lysozyme is free from contaminants. Median ratios of peak areas of heated lysozyme over peak area of control lysozyme differed significantly from 1 at the temperature of 100°C (0.91), but not at the one of 80°C (0.95) with the signed rank test. Even when heated at 80°C, the elution profile of lysozyme appeared less symmetrical compared to control and presented an additional inflection point. These subtle changes were increased at 100°C. Conclusion: Enrichment of the lysozyme extracted from contact lenses and solubilized into the initial mobile phase improves the sensitivity of this chromatographic procedure. This chromatographic protocol coupled with UV absorbance spectroscopy can detect thermal denaturation at 80 and 100°C.
Highlights
Tear film components adsorb more or less to contact lens surfaces depending on their mutual characteristics, their affinity with the material of the lens, the specific characteristics of subject’s tears and glands as well as with the regimen of care used if any
The tear film is a complex mixture of numerous proteins, some of which interact with the contact lens material
The adsorption on contact lenses of high levels of protein has been associated in patients with a higher risk of adverse reactions such as giant papillary conjunctivitis (GPC) [12]
Summary
Tear film components adsorb more or less to contact lens surfaces depending on their mutual characteristics, their affinity with the material of the lens, the specific characteristics of subject’s tears and glands as well as with the regimen of care used if any. With its basic isoelectric point, lysozyme, a main tear film protein often used as a marker for protein deposition and lens usage [5], contains a predominance of positive charges at a physiological pH [6] It adsorbs to hydrogel lenses, especially those that contain negative charges due to their methacrylic acid content [7,8,9,10,11]. Patients, asked to wear their lenses longer than manufacturer’s recommendations on one eye, presented more severe upper conjunctival papillae, upper lid conjunctival hyperemia, as well as limbal congestion compared to the compliant control eye Extracts from their hydrogel lenses had higher protein content on the non-compliant side [13]. Lysozyme loosely bound to the surface of an ionic contact lens may desorb and re-enter solution [15]
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