The Impact of Diurnal Changes and Inter-Visit Variability on the Concentration of Insulin-Like Growth Factor-1 in Human Tears
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INTRODUCTION: There is a growing body of research focused on the use of tear film-derived proteins as biomarkers of disease. Previous studies have reported quantitative changes in tear-derived growth factors and related proteins associated with various systemic and ocular diseases. Major challenges when working with human tears however, includes sample volume limitation and the high potential for reflex tearing. One method of tear collection that is increasingly being reported involves the use of microcapillary tubes to draw tears from the inferior tear reservoir. The purpose of this study was to investigate the impact of diurnal changes and inter-visit variability on the concentration of a known growth factor present in human tears, the insulin-like growth factor-1 (IGF-1). METHODS: Nine healthy volunteers without any reported symptoms of dry eye were recruited for this study. At visit 1 (baseline), all participants underwent a standard dry eye examination to assess tear volume, tear film break up time (TFBUT), and tear production. Subjects were asked to return to clinic for an additional 5 visits (morning and afternoon on a total of 3 days). Tears were collected at the start of each visit from the inferior temporal tear meniscus of both eyes using 1 - 10 μl glass microcapillary tubes and frozen at -80C until use. Total protein was measured for each patient using a bicinchoninic assay. IGF-1 levels wear assessed using ELISAs. RESULTS: 8.8 ± 2.1 μg/μl of total protein was obtained from each subject. Total protein was unchanged at each visit. There was no difference in IGF-1 between morning and afternoon. Tear levels of IGF-1 did vary with visit, with the final visit showing a 2 fold-increase over baseline (p<0.05). Tear levels of IGF-1 were correlated with TFBUT (R=0.856, p=0.007). DISCUSSION: While diurnal variation did not affect basal levels of IGF-1 in tears, there was a visit-dependent increase. This increase was likely due to a reduction in reflex tearing during tear collection as patients became more comfortable with the technique. Similarly, the decrease in IGF-1 that corresponded with increased tear evaporation was likely due to changes in reflex tearing. Together, these findings suggest that low abundant proteins, such as IGF-1, are highly susceptible to changes in reflex tearing. These findings also suggest that a participant training phase may be required.