| Author: | Ng, Sheung-shun Vincent |
| Title: | Human tear protein analysis, and factors affecting the concentrations of total and individual tear proteins |
| Degree: | Ph.D. |
| Year: | 2002 |
| Subject: | Hong Kong Polytechnic University -- Dissertations Tears -- Composition Proteins -- Analysis |
| Department: | Department of Optometry and Radiography |
| Pages: | xviii, 286 leaves : ill. ; 30 cm |
| Language: | English |
| Abstract: | OBJECTIVES The objectives of this study were to analyze human tear protein profiles, and to investigate the factors that could affect the total tear protein concentration (TTPC) and the individual tear protein fractions. Tear samples were collected (by capillary tubes) from young normal Hong Kong (HK)-Chinese and TTPC and/or individual protein concentrations were determined. In the first part, we investigated the repeatability of TTPC obtained using the commonly used colorimetric protein assays, Bio-Rad protein assay (Bradford method) and Bio-Rad protein DC assay (Lowry Method). We also evaluated the repeatability of individual tear protein analysis and determination using the technique of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning densitometry respectively. The physiological variations, including diurnal (daytime) and day-to-day variations, of human tear proteins were investigated. The variabilities of two tear sampling techniques (yawn method and eye-flush method) were also compared. In the main studies, we determined the tear protein levels of young HK-Chinese, and these results were used as baseline data for the later investigations. The results of TTPC obtained using different methods (Bradford or Lowry) and standards (bovine serum albumin (BSA) and bovine immunoglobulin G (IgG)) were compared, and their relationships were determined. We also investigated the accuracy of the Bradford and the Lowry methods for the determination of TTPC using known protein concentration mixtures, and developed a new standard for the total protein assays. In the last part of this study, tear protein profiles of non-contact lens wearers were compared with those of soft contact lens wearers. We also investigated the changes in tear protein levels before and after soft lens wear and rigid lens wear; and determined the tear protein deposits on worn soft lenses. RESULTS Repeatability of the techniques in the analysis of human tear proteins The Bradford and the Lowry methods were found to be repeatable in the determination of TTPC. The technique of SDS-PAGE was found to have clinically acceptable repeatability in the determination of lactoferrin, lysozyme and tear specific prealbumin (TSP). However, the variabilities of immunoglobulin A (sIgA) and human serum albumin (HSA) levels determined using this technique were of clinical significance. Therefore, caution should be taken when comparing these two proteins between subjects or between studies. Physiological variations in the tear protein profiles In general, there were no significant daytime and between-day variations in TTPC and the individual tear protein levels, except for the concentration of HSA. The eye-flush method consistently showed greater variability than the yawn method. Tear protein profiles of young normal HK-Chinese For the Bradford method using BSA and IgG as standards, the mean TTPC were 6.05+-1.58 and 11.48+-2.32 mg/ml respectively. For the Lowry method using BSA and IgG as standards, the mean TTPC were 9.66+-2.03 and 7.53+-1.80 mg/ml respectively. Using the technique of SDS-PAGE, five major tear proteins were consistently separated, i.e. sIgA, lactoferrin, HSA, TSP and lysozyme. The mean concentrations of sIgA and lactoferrin were 0.29+-0.10 and 2.73+-0.82 mg/ml respectively. The mean concentrations of HSA, TSP and lysozyme were 0.021+-0.028, 2.89+-0.88 and 2.46+-0.44 mg/ml respectively. Comparing the values of TTPC and the individual tear protein concentrations, we found significant correlations between TTPC and the concentrations of lactoferrin, TSP and lysozyme. Relationships between different colorimetric protein assays TTPC varied significantly with the methods and standards used. Although significantly different values of TTPC were obtained with different methods and standards used, their values were found correlated well with each other. Our results also indicated that there were good agreements between measured and calculated TTPC. Hence, using the appropriate equations, TTPC obtained by different methods/standards can be interchangeable. Validation of the colorimetric protein assays Using known concentration artificial tear solutions to simulate tears from normal and dry eye subjects, we found that the use of lactoferrin, BSA or HSA as standards in the Bradford method tended to underestimate TTPC. In contrast, IgG, IgA and lysozyme were found to overestimate TTPC. Our results indicated that the use of a new standard, comprising a mixture of 50% BSA and 50% IgG, would provide a more accurate and better estimation of TTPC than the other standards. For the Lowry method, using either IgG or IgA as standard would give a more accurate TTPC than using lactoferrin, BSA, HSA or lysozyme Contact lens wear on tear protein profiles We found no significant differences in any tear protein level between contact lens and non-contact lens wearers (cross-sectional study with relatively longer period of contact lens wear). Also, we did not find any significant changes in the tear protein levels before and after both soft (N = 7) and right lens wear (N = 7)(prospective study with relative shorter period of lens wear). Although there were trends for changes in the levels of sIgA and HSA after 15 minutes of rigid lens wear, these changes were not statistically significant. Protein deposition on soft contact lenses We confirmed that the FDA group IV lenses bound more proteins than the FDA group I lenses. The bound proteins on group IV lenses were mainly lysozyme and 30 kD protein (dimer of lysozyme). These findings were in agreement with those reported previously for Caucasian subjects. CONCLUSIONS To conclude, we have shown that the colorimeter protein assays (Bradford and Lowry methods) and SDS-PAGE have good and clinically acceptable repeatabilities in the determination of tear protein levels. Comparing the sampling methods, the yawn method was more reliable than the eye-flush method. The daytime and day-to-day variations of tear protein levels were small, except for the level of HSA. The tear protein levels of HK-Chinese appeared to be inconsistent with those reported for Caucasians. The discrepancy may be related to different methods and techniques used. Racial difference may be a possible reason as HK-Chinese has been reported to have lower tear volume and tear stability. TTPC varied significantly with different methods and standards used but their values were highly correlated and could be interchanged by applying appropriate equations. A new standard, consisting of a mixture of 50% BSA and 50% IgG, was suggested for the Bradford method in the determination of TTPC. In the Lowry method, the use of IgA or IgG as standard would give more accurate results of TTPC. We found no significant changes of tear protein levels due to normal and asymptomatic contact lens wear, in either soft or rigid lenses. However, in view of the limited sample sizes, further investigation in this area is warranted before firm conclusion can be drawn. For the investigations of contact lens deposit, we concluded that the characteristics of protein deposits for Chinese subjects were similar to those reported for Caucasian subjects. |
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| Access: | open access |
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