The long term objective is to measure the energy demand of cultured human retinal pigment epithelium (RPE) and determine how this varies with different culture conditions or source of culture material.
The specific aims are to determine: 1) the glycolytic flux and respiratory activity of normal cultured RPE; 2) how this changes with the density of cells in culture i.e. from pre-confluent, to confluent, to post confluent cultures; 3) the effect of donor age, and of passage number on these activities; 4) what differences, if any, exist in the energy pathways of RPE cells cultured from eyes with diabetic retinopathy, age related macular degeneration, and retinitis pigmentosa as an indication of changes in energy production and energy demand; and 5) the relationship between the major organophosphate metabolites observable in the phosphorus-31 (P-31) nuclear magnetic resonance (NMR) spectrum of RPE cells to the glycolytic and respiratory activity of the cell. In order to achieve these goals RPE will be cultured from human donor eyes and expanded in culture. Morphology will be monitored by phase contrast light microscopy. The cells will also be characterized by cell doubling times. Quantitative measurements of glucose utilization and lactate production in RPE will be made with Carbon-13 (C-13) labeled glucose using C-13 NMR spectroscopy. Cells will be maintained in the NMR magnet by casting cells in agarose threads and perfusing medium past the cells. The organophosphate metabolites and stability of the preparation will be monitored using P-31 NMR. Oxygen consumption of the cells will be measured using an oxygen electrode in separate experiments. This data will increase our knowledge of RPE energy metabolism and determine if RPE from eyes with known retinal degenerations have altered energy production or energy demand. It is also hoped that the relationship between the major phosphorus metabolites and energy metabolism will provide specific metabolic markers which will be useful in the future development of techniques to examine RPE metabolism in vivo.
