The long term goal of this investigation is to first evaluate the effectiveness of 31-P and 13-C nuclear magnetic resonance (NMR) spectroscopy in the study of ocular tissues in cell culture, then assess the validity and accuracy of tissue culture models in reflecting their in vivo state and using this information to develop new means by which to detect, diagnose and study ocular malignancy. Specifically, the dynamic metabolic characteristics of well established cell lines will be obtained in the intact, viable state by 31-P and 13-C NMR. The following characteristics will be determined: (1) the type and relative concentration of specific phosphate metabolites (ATP, NAD, sugar phosphates), (2) intracellular pH, (3) the flux of glucose through certain cellular metabolic pathways and (4) cellular response as measured by (1) to (3) to elevated glucose levels (glucose challenge). The differences between malignant (retinoblastoma, melanoma) and control or 'normal' cell lines will be elucidated. Differences between cells in the 'suspended' and 'attached' states will be determined. 31-P and 13-C NMR spectroscopy is well established technique for the study of intact cells or organs. Cell lines (malignant and control) will be grown in microcarrier suspensions or in monolayers according to the previous established techniques. Cells in the monolayer state will be resuspended in either agar or liquid medium in standard NMR tubes and NMR spectra obtained with a Bruker WM 250 spectrometer. Cells grown in the microcarrier suspensions will be transferred to an NMR tube and spectra obtained. In this way, a comparison between attached and suspended cells can be made. PCA extracts of each sample will be obtained and re-examined by NMR. When using 13-C NMR spectroscopy, 13-C glucose labeled at the C-1 position will be used. Cells will be incubated with the labeled glucose (microcarrier state only) and after an appropriate period of time removed from the media and washed. Media without the labeled glucose will be added and spectra obtained. In a similar fashion, cells will be incubated with fluorophosphate, phosphite, or methylphosphonate for a designated period of time. After removal of media, washing, and addition of media without the phosphate analog, 31-P spectra will be obtained. In all experiments, cells will be counted before each spectroscopy study by a TV cell counting/sizing system or a hemocytometer.
