Phosphatic metabolites of the intact cornea by phosphorus-31 nuclear magnetic resonance

The principal low molecular weight phosphatic metabolites of the intact cornea were identified and quantitated nondestructively by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy. As part of this analytical procedure, the intracorneal pH was approximated from the resonance shift position of inorganic orthophosphate. In addition the metabolic and pH stability of incubated corneas at 37 C in MK medium was evaluated during an 8-hr time course and compared to similar dynamic analyses performed on corneas with denuded endothelium. Perchloric acid extracts prepared from these same corneas were analyzed by P-31 NMR to verify the metabolite peak assignments and to quantitate the concentrations of minor corneal metabolites. The concentrations of phosphatic metabolites of the cornea, including three previously unidentified phosphorus-containing substances, were determined for freshly excised corneas. The initial corneal spectroscopic profile was not altered by removal of the endothelium. At 37 C the MK media-incubated intact whole corneas experienced a time-dependent decline in ATP levels with a concomitant rise in inorganic orthophosphate; however, the tissue levels of the other principal phosphatic metabolites were not altered by prolonged incubation. In contrast, removal of the endothelial layer of the cornea-induced progressive metabolic deterioration of intact corneas characterized, most prominantly, by time-dependent declines in ATP and glycerol 3-phosphorylcholine levels and concomitant increases in ADP and inorganic orthophosphate levels relative to intact whole corneas. This study has established the feasibility of monitoring the metabolic status of intact rabbit corneas nondestructively and noninvasively. As such, P-31 NMR spectroscopy offers a promising method that may enable analysis of the metabolic viability of intact human donor corneas to provide a basis for selecting donor corneas for transplantation.