1. Investigación

Eye fluids (aqueous humour and vitreous humour) may be helpful in estimating ante-mortem blood levels, since some parameters measured in these fluids have proved to be stable or to change in a predictable way after death. This would help in diagnosing the cause of death in some diseases or to evaluate ante-mortem blood levels in certain animals not easy to handle or with difficult access. In order to establish reference values of some parameters in blood and eye fluids (aqueous humour and vitreous humour), as well as the possible correlation among these three different fluids, various minerals and electrolytes (Ca, P, Mg, K, Na, Fe, Cr, Co, Ni, Cu, Zn, Se and Mo) were measured in 15 four to five year-old Lidia bulls, all dying after a period of significant stress and major exertion. Plasmatic values of Mg and P were much greater than reported in the literature. In general, mineral plasmatic values were greater than those found in ocular fluids (aqueous and vitreous), while Na, K and Cr were similar in the three fluids. We have verified the existence of correlations in P, Co and Mo among the three fluids measured, and between Se of plasma and vitreous humour. But the most marked correlations were observed in Mo (plasma -aqueous humour, r = 0.893, plasma-vitreous humour, r = 0.945, HA -HV, r = 0.849), in P (plasma-vitreous humour, r = 0.726) and in Co (plasma-vitreous humour, r = 879).

Aqueous humor is the transparent fluid found in the anterior chamber of the eye that provides the metabolic requirements to the avascular tissues surrounding it. Despite the fact that metabolomics could be a powerful tool in the characterization of this biofluid and in revealing metabolic signatures of common ocular diseases such as myopia, it has never to our knowledge previously been applied in humans. In this research a novel method for the analysis of aqueous humor is presented to show its application in the characterization of this biofluid using CE–MS. The method was extended to a dual platform method (CE–MS and LC–MS) in order to compare samples from patients with different severities of myopia in order to explore the disease from the metabolic phenotype point of view. With this method, a profound knowledge of the metabolites present in human aqueous humor has been obtained: over 40 metabolites were reproducibly and simultaneously identified from a low volume of sample by CE–MS, including among others, a vast number of amino acids and derivatives. When this method was extended to study groups of patients with high or low myopia in both CE–MS and LC–MS, it has been possible to identify over 20 significantly different metabolite and lipid signatures that distinguish patients based on the severity of myopia. Among these, the most notable higher abundant metabolites in high myopia were aminooctanoic acid, arginine, citrulline and sphinganine while features of low myopia were aminoundecanoic acid, dihydro-retinoic acid and cysteinylglycine disulfide. This dual platform approach offered complementarity such that different metabolites were detected in each technique. Together the experiments presented provide a whelm of valuable information about human aqueous humor and myopia, proving the utility of non-targeted metabolomics for the first time in analyzing this type of sample and the metabolic phenotype of this disease.