Laser microspectrofluorometry for the diagnosis of atherosclerosis
Fink, Tami Neal
Curl, Robert F., Jr.
Doctor of Philosophy thesis
Cardiovascular disease is a major cause of death in the U.S., and atherosclerosis, the principle cause of myocardial and cerebral infarctions, accounts for most of these deaths. In current clinical practice, cholesterol status, or atherosclerotic risk, is evaluated by fasting serum lipoprotein measurements. However, disease variation lies in the biologic responses of circulating blood cells and cells in the arterial wall, in the presence of given levels of plasma cholesterol. Lipid-laden monocytes or circulating macrophage 'foam cells' may be useful markers of atherosclerotic disease. Macrophage 'foam cells' can be identified by their massive accumulation of an atherogenic lipoprotein, oxidatively modified low-density lipoprotein (LDL). Oxidized LDL possesses unique fluorescence spectral characteristics that distinguish it from native (non-oxidized) LDL. Thus, fluorescence spectroscopy was chosen as a tool for identifying oxidized LDL accumulations in monocyte/macrophages, and may be useful for identifying a novel risk factor in the assessment of atherosclerosis. In cell suspension autofluorescence analysis, spectral characteristics of oxidized LDL were shown in cultured macrophages incubated with oxidized LDL preparations. Additionally, it was demonstrated that isolated peripheral blood monocytes may acquire spectroscopic signatures characteristic of oxidatively modified LDL. Identifying the percentage of oxidized LDL filled monocytes in a patient will be important in determining any correlation with atherosclerosis incidence or risk of atherosclerotic complications. Thus, more sophisticated analysis of the distribution of intracellular oxidized LDL content within a population of monocyte/macrophages required the development of instrumentation for spectroscopy at the microscopic level. A microspectrofluorometer was designed and constructed to detect intracellular oxidized LDL concentrations, based on autofluorescence signals, in individual cells smeared onto microscope slides. Good characterization of system losses improved the capability to quantify measurements in terms of absolute concentrations, a problem inherent in most fluorescence instruments. Using autofluorescence microspectrofluorometry, intracellular oxidized LDL concentrations could be identified within individual monocyte/macrophages incubated with oxidized LDL and calibrated with respect to oxidized LDL standards. Histologic comparisons using oil red O staining and transmission electron microscopy verified cultured macrophages were accumulating oxidized LDL. Increases in lipid droplets, cholesterol clefts, and secondary lysosomes correlated with increasing autofluorescence signals.