Application of high-resolution NMR spectroscopy in metabolic studies of the eye

Abstract

High-resolution NMR spectroscopy has, during the last two decades, had an increasing impact in biological and biochemical research. Rapid advances have led to improvements in sensitivity and dispersion of the spectra and have allowed more detailed assignment and monitoring of endogenous biochemical molecules. One of the latest implementations has been a technique known as high-resolution magic angle spinning (HR-MAS) NMR spectroscopy which has made it possible to obtain high-resolution proton spectra of intact tissue and cells. Simultaneous detection of a large number of metabolites by NMR spectroscopy has been successfully applied to investigate disordered metabolism for a numerous of diseases and toxic processes.

The objectives in the present work have been to evaluate different 1H NMR spectroscopy protocols as analytical tools in eye research, and further use these protocols to extract and interpret information on metabolic changes in the eye induced by external pathological stimuli. Special focus has been paid to changes in the lens and the development of cataracts.

The 1H NMR spectra of intact lenses and eye tissue extracts in present thesis showed an extensive picture of NMR detectable metabolites. In addition to the detailed analysis of extracts from cornea, lens and aqueous humour, this work has created a basis for implementation and interpretation of HR-MAS 1H NMR spectroscopy on intact lens tissue. Several significant changes in the metabolic content in cornea, aqueous humour, and lens after alkali-burns to the eye were detected and showed how careful 1H NMR spectroscopy analysis of tissue extracts provided new information (quantitative and qualitative) on the metabolic reaction pattern in the anterior eye segment in relation to eye alkali-burn injuries.

HR-MAS studies on lenses exposed in vivo to different ultraviolet-B doses did not reveal any dose-response relationship for the metabolic changes. However, significant concentration changes for most of the observed metabolites seven days post exposure demonstrated that closeto- threshold UVB radiation had great impact on the metabolites in the lens. Further time dependency studies of metabolic changes in rat lens after UVB radiation showed that significant changes in metabolite concentrations were subsequent to lens opacity development. Long-term steroid treatment (36 days) seemed to have greater impact on the metabolic changes compared to the UVB-induced changes 24 hours after UVB radiation. Even though no obvious cataract was detected after the combined treatment of steroids and UVB radiation, significant changes were observed for several metabolites.