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Kristina M Haworth, Heather L Chandler; Oxidative Stress Measures and Correlations with Lipid and DNA Damage Assays for Human Tears and Conjunctival Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2769. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate feasibility, repeatability, and relationships between measures of ocular sun exposure (OSE), hexanoyl-lysine (HEL) ELISA, and 8-hydroxy-2’-deoxyguanosine (8OHdG) ELISA for in vivo human tear and conjunctival cell sample types.
Twenty healthy volunteers were seen for 2 visits 14±2 days apart. Collection of tear samples was by glass microcapillary tube from the inferior meniscus; conjunctival cells by cytology brush from the nasal and temporal regions. Ocular sun exposure was estimated over a two week time period by questionnaires. HEL and 8OHdG ELISAs were conducted for both tear and conjunctival cell samples. Four parameter logistic standard curves were generated for both ELISAs to quantify lipid and DNA damage. Repeatability for OSE and ELISAs was evaluated using Bland Altman plots. Pearson’s correlation analysis was conducted to evaluate relationships between measures.
Descriptive statistics (mean(±SD)) were generated for measures of OSE (n=20; 4.38(±3.02) hours), HEL ELISA for tears (n=20; 24794.86(±9256.71) nmol/L) and cells (n=7; 19.07(±8.20) nmol/L), and 8OHdG ELISA for tears (n=20; 66.72(±26.95) ng/mL). Using our methods, 8OHdG levels were below limits of detection for conjunctival cell samples. To obtain estimates of repeatability for each measure mean differences and 95% limits of agreement (LOA) were calculated: OSE (-0.04 hours, LOA -6.66 to 6.59); HEL tears (533.19 nmol/L , LOA -25799.39 to 26865.76); HEL cells (-3.70 nmol/L, LOA -21.32 to 13.92); and 8OHdG tears (-3.40 ng/mL, LOA -82.78 to 75.98). Positive correlations were found for ocular sun exposure with tear HEL levels (r=0.454;p=0.044) and for tear 8OHdG levels with cell HEL levels (r=0.903;p=0.005) and tear HEL levels (r=0.619;p=0.004).
We demonstrate feasibility and repeatability for previously unreported ELISA measurements of oxidative stress levels in human conjunctival tears (8OHdG) and cells (HEL). Our HEL ELISA findings for tears are notably higher than previous reports on human tears (Wakamatsu 2010, 2013), possibly due to experimental differences. Correlation analysis suggests that ocular sun exposure is associated with ocular surface lipid damage, and that lipid damage is associated with DNA damage. These methods may prove useful for future studies of UV induced oxidative stress on the ocular surface and other ocular surface disease.
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