## 4 Discussion  
This study confirmed that all five metabolic proteins included in the study can be detected in normal human tears. To date, this is the first study of these five metabolic proteins, simultaneously, in human tear fluid. Of particular interest, this study has measured, for the first time, active Ghrelin and GIP concentrations in human tears. Previous work in human plasma showed that the total Ghrelin level was 100-150 mmol/mL (equal to 44.5-66.7 pg/mL), and this was increased when fasting [47]. While a study by [PERSON] et al. [48] reported that the plasma concentrations of ghrelin and GIP in young adults were 1507 \(\pm\) 207 pg/mL and 16 \(\pm\) 2 pmol/L, respectively. In the present study, the C-peptide concentration in tears was 233.2 \(\pm\) 46.26 pg/mL (V1) and 211.9 \(\pm\) 64.18 pg/mL (V2). We [29] have previously shown the presence of C-peptide in human tears. To our knowledge, this is the only published data on this protein in tears. In our study, tear fluid C-peptide concentrations were found to be higher in patients with type 2 diabetes with DED, than in DED (without type 2 diabetes) and type 2 diabetic patients (without DED)  
Figure 2: The Bland-Altman plots for (**A**) active ghrelin and (**B**) C-peptide. and healthy controls, indicating a potential role for C-peptide as a tear fluid biomarker of type 2 diabetes-associated DED [29]. Recent C-peptide studies on urine and fasting serum have indicated this protein as a biomarker of type 2 diabetes [49; 50]. While another study showed serum C-peptide was inversely associated with retinal complications in type 2 diabetes [51]. The absolute concentrations of tear fluid insulin and leptin measured in this study were different from those in previous reports. For example, [PERSON] et al. [30] were the first to confirm the presence of insulin in normal human tears. Using a radioimmunoassay (RIA), they quantified the average insulin concentration as 0.404 \(\pm\) 0.129 ng/mL, while insulin concentrations in fed subjects (0.601 \(\pm\) 0.138 ng/mL) were significantly higher than in fasted subjects (\(p\) = 0.04; 0.204 \(\pm\) 0.055 ng/mL). In our study, the average insulin concentration was higher in both the V1 (1466.2 \(\pm\) 607.6 pg/mL) and the V2 (1336 \(\pm\) 408.6 pg/mL) tear samples. Tear fluid leptin concentrations were previously reported to be 8.90 \(\pm\) 1.81 pg/mL by [31] in healthy subjects, using a Human Leptin ELISA kit and were compared to patients with allergic conjunctivitis (8.68 \(\pm\) 1.70 pg/mL). In another study, tear fluid leptin concentrations were reported as 5.04 \(\pm\) 1.08 ug/L in subjects with DED and 5.14 \(\pm\) 1.12 ug/L in healthy controls using an ELISA kit as well [32]. In our present study, the average leptin concentration was much higher, 379.2 \(\pm\) 11.23 pg/mL (V1) and 381.5 \(\pm\) 7.87 pg/mL (V2). These differences in tear metabolic protein concentrations across studies may be due to the fact that different techniques and kits were used from different manufacturers. For example, [PERSON] et al. [52] compared the Simple PlexTM platform with colorimetric ELISA to detect plasma MCP-1/CCL2, VEGF-A, TNF-\(\alpha\) and IL-6 concentrations and reported that the Simple PlexTM may have potential for biomarker panel measurement. In addition, these differences may be due to the fact that the kits used across the studies had differing sensitivities. In an RIA study by [PERSON] et al. [30], the MinDC of insulin was 0.1 ng/mL (100 pg/mL), while it was 87 pg/mL in our study. Furthermore, the MinDC of Leptin in tear fluids was found to be 6 pg/mL using a Human Leptin ELISA kit [31], but it was 41 pg/mL in our multiplex study. The different sensitivities in the detection across kits can most likely be explained by the various antibodies and/or standards provided in different kits. In addition, no gold-standard guidelines yet exist for the multiplex magnetic bead immunoassay in tear fluid analysis, such as sample size, dilution factors, buffers used in analysis and tear collection techniques.