Abstract
About 5% of solar radiation comes from the ultraviolet (UV) region. It carries high energy and can do serious damage to living organisms. The eye of animals and humans is a critically exposed area and contains UV absorbing compounds like proteins, tryptophan, tyrosine, ascorbic acid and uric acid in the aqueous humour (AH) for protection. In addition, an unknown component (compound X) which causes red-shift in the UV absorbance spectrum at 254 nm has been observed in heavily exposed species like goose, which migrates at 10 000 m altitude. X is a major absorber. UV measurements confirmed the presence of a compound in geese which has higher absorption at 254 nm compared to other species. Compound X also has fluorescence activity that resembled indole-functionality. Aqueous humour samples from different species (goose, chicken, turkey) were measured by nuclear magnetic resonance spectroscopy (NMR). The proton NMR spectra did not show any specific high abundant compound in the goose eye at the aromatic region compared to the other species, thus NMR could not reveal what made the difference between in UV absorbance. Mass spectrometry with atmospheric pressure photoionization (APPI) has earlier showed that the molar mass of the compound X could be 149 g/mole, which suggests that compound X might be the indole: 5,6-dihydroxyindole (DHI). DHI has a central role in the biosynthesis of melanin which takes place in melanocyte cells. DHI tends to easily be polymerized and one of the results is a notable change in its solubility. It was not possible to obtain a suitable external standard which did not undergo auto-polymerization, mirroring the observations with the isolated compound.To further test the hypothesis of DHI being compound X, reversed phase (RP) high performance liquid chromatography (HPLC) and hydrophilic interaction liquid chromatography (HILIC) were performed. Ascorbic acid co-eluted with the most abundant peak (compound X) in RP-HPLC. In HILIC the most abundant peak was not ascorbic acid. Hence, compound X is not ascorbic acid, and subsequent experiments showed that it was neither the UV absorbing compounds tryptophan, tyrosine or uric acid. After using several analytical tools, the hypothesis that compound X is 5,6-dihydroxyindole was strengthened. However an external standard is still necessary to provide accurate information.