## 4 Discussion
The differences in tear metabolic protein concentrations across the studies may also be due to population differences, such as participant age, sex and inclusion criteria of the studies. In addition, the absolute concentrations of tear fluid metabolic proteins maybe sensitive to the tear collection techniques, tear sample volume and the time of the day samples were collected. For instance, in a study by [PERSON] et al. [31], a total of 5-10 \(\upmu\)L basal tears were collected with three different techniques, i.e., using a Whatman 3 MM filter paper disc, microcapillary tubes and Schirmer filter paper. While [PERSON] et al. [30] stimulated tears by a jet of pressurized air onto the cornea, which were then collected using disposable microcapillary tubes. In our study, we collected 1 \(\upmu\)L basal tear samples, a volume which has been previously shown to be sufficient for cytokine analysis [2; 4; 40; 53; 54] using microcapillary tubes. Regarding the inter-day variation, in our study we found that the metabolic protein concentrations of insulin, leptin, C-peptide and GIP measured did not change significantly over a period of 7 days. The exception to this was active Ghrelin, which was significantly different across the two time points measured (\(p\) = 0.02). However, although the active Ghrelin concentrations were determined to be higher than the MinDC for all samples, there were extremely low concentrations (under 35 pg/mL) found in 2 out of 17 samples collected in V1, and in 3 out of 17 samples collected in V2. Due to the low number of the participants (n = 17), these low values may have affected the calculation to determine inter-day variation. Therefore, further studies in a larger cohort are necessary to better address the true variation. We also calculated intra-subject variability for the tear fluid metabolic proteins to determine within- and between-subject variance components in the molecule levels in the two visits. The result of our study confirmed that intra-subject CV's of leptin, C-peptide and active ghrelin were within the manufacturer's values (<15%; Merck Millipore), which indicates that these biomarkers have a good intra-subject variability. Although insulin and GIP concentrations in tear fluids were found to have high intra-subject CV values, and therefore to be less reliable. Moreover, leptin had a high ICC value, and was classified as having a strong reproducibility rating. C-peptide and active ghrelin showed good intra-subject variability values, but they had low ICC values and were classified as a poor or low reproducibility rating. Additionally, the Bland-Altman graph analyses revealed that tear levels of these two molecules were significantly higher for each subject in V1 compared to V2. This may be due to the fact that the present study was carried out in healthy subjects. Therefore, further studies are necessary to confirm if there is variation of these metabolic proteins in ocular conditions, as well as in systemic health conditions. In addition, further studies including the comparison of selected/detected metabolic proteins in tears with their corresponding blood levels are warranted. Finally, in the current investigation, the concentrations of the studied metabolic proteins were not found to correlate with environmental conditions, such as room temperature and humidity. There were some limitations in this pilot study, such as sample size (n = 17 subjects). Therefore, further studies in a larger population, and also in other age groups, are warranted. In addition, the study results are limited to specific time points. Therefore, future studies of the diurnal variation of tear metabolic proteins may be important to determine how metabolic biomarker concentrations vary, depending on the time of day, i.e., early morning and evening.