###### Abstract  
The concentrations of insulin, leptin, active ghrelin, C-peptide and gastric inhibitory polypeptide (GIP) and their inter-day variations were examined in normal human tears. In addition, correlations between the concentrations of these metabolic proteins and ocular surface parameters were determined. Subjects with healthy ocular surfaces attended three visits, with 7-day intervals. Tear evaporation rate (TER) and non-invasive tear break-up time (NITBUT) were assessed, and a total of 2 uL tears were collected from all subjects. Tear fluid concentrations of insulin, leptin, active ghrelin, C-peptide and GIP were measured by multiplex bead analysis. Insulin was the most highly expressed metabolic protein, followed by leptin, C-peptide, active ghrelin and GIP. Of these, only active ghrelin had a significant inter-day variation (\(p<0.05\)). There was no inter-day variation in the mean concentrations of the other metabolic proteins. Leptin had a strong intra-class reproducibility. No correlation was detected between tear metabolic protein concentrations and ocular surface parameters. This pilot study shows, for the first time, that active ghrelin and GIP are detectable in healthy tears. The strong intra-class reproducibility for leptin shows that it could be used as a potential tear fluid biomarker and, possibly, in determining the effects of metabolic disorders on the ocular surface. biomarker; ocular surface; metabolic protein; tear fluid; inter-day variation +
Footnote †: journal: Medical  
Given the global epidemic numbers of people affected by type 2 diabetes, the study of metabolomics is an emerging area to better understand the progression of metabolic diseases [23]. Moreover, as the metabolome offers an integrated perspective of cellular processes and the effects that environmental factors may have on the biological state, it may better represent the molecular phenotype than the genome, the transcriptome, or the proteome [24]. Therefore, the study and characterization of metabolites has been proposed to be a better prediction of the resulting phenotype than other -omics approaches [25]. Until recently, however, very few studies have assessed metabolic protein expression in human tear fluids. Those done so far have assessed free carnitine and acyl-carnitines, as well as amino acids and lysophospholipids [26; 27]. A recent study reported altered metabolomic profiles in tear fluids post-corneal crosslinking [28]. In addition, C-peptide [29], glucagon [29], insulin [29; 30] and leptin [29; 31; 32] have been identified in human tear fluids. Due to this lack of data in human tear fluids, validation of metabolic protein expression (including the determination of inter-day variation) is necessary for effective comparisons between healthy and diseased eyes, and in diseased eyes pre- and post-therapy. However, to our knowledge, there is no current data available about inter-day variation that may exist in tear fluid metabolic protein levels of healthy human eyes. In this study of normal healthy subjects, five metabolic proteins were selected for Luminex cytometric magnetic bead array analysis (multiplex), including insulin, leptin, active ghrelin, C-peptide and gastric inhibitory polypeptide (GIP). They were specifically chosen in this study due to the fact that most of the actions of these proteins could contribute to the metabolic syndrome. For example, Insulin plays crucial roles in regulating glucose homeostasis and metabolism [33]. Ghrelin and leptin are also opposing hormones. For example, ghrelin is termed a \"hunger hormone\", so it stimulates appetite, increases food intake and promotes fat storage, while leptin inhibits hunger and reduces fat storage in adipocytes. GIP is an \"incretin\" hormone, which binds to the glucagon-like-peptide receptor and releases insulin [34]. Moreover, C-peptide is produced from the pancreas, along with insulin in equal amounts and is the best indicator of endogenous insulin secretion in patients with diabetes [35].