Phosphorus-31 NMR spectroscopy of cultured human retinal pigmented epithelial cells

Cultured human retinal pigmented epithelial cells were studied using phosphorus-31 nuclear magnetic resonance spectroscopy (P-31 NMR). Retinal pigmented epithelial cells from normal human donors were isolated and expanded using roller-bottle culture. The P-31 NMR spectrum of the intact living cells was obtained at 121 MHz by casting the cells in agarose threads and perfusing the threads with culture medium. The cells remained viable at 37 degrees C in the NMR magnet for at least 24 hr, as determined by the stability of the phosphorus spectrum and by trypan blue dye exclusion at the end of the experiments. The intact cell spectrum showed resonances from phosphorylethanolamine, phosphorylcholine, inorganic phosphate, phosphodiesters, phosphocreatine, ATP, NAD+, and UDP-N-acetylglucosamine, with phosphorylethanolamine, phosphorylcholine, and ATP being the major metabolites present. The resonances were assigned by making a perchloric acid (PCA) extract of the intact cells and running under high-resolution conditions. The PCA extract spectrum also detected sugar phosphates, ADP and UTP, with the latter being approximately 20% of the nucleotide pool. These studies provide the basis for the study of normal and diseased human RPE cells by NMR spectroscopic methods.