In situ investigation of the chemical composition of ceroid in human atherosclerosis by Raman spectroscopy

Fluorescent lipid peroxidation products (ceroid) form within the atheroma of an atherosclerotic lesion. At present, the initiation and propagation of such oxidative processes in situ are not well understood, in part because of the insolubility of the ceroid deposits. We investigated the chemical composition of ceroid in human atherosclerotic plaque by combining autofluorescence microscopy and Raman spectroscopy. In eight sectioned human atherosclerotic samples, we located granular and ring-shaped ceroid deposits by autofluorescence microscopy. Subsequently, two-dimensional Raman maps of the ceroid deposits and surrounding non-fluorescent atheroma were acquired at 1 μm resolution. These Raman spectra were subjected to a K-means clustering analysis. By assigning a different color to each cluster, pseudo-color Raman images of the tissue sections were obtained. From these images the clusters belonging to ceroid and the non-fluorescent surrounding tissue were selected. The averaged cluster spectra were analyzed by means of a least-squares fit with the Raman spectra of pure chemical compounds. In addition, the Raman spectra were compared with the Raman spectra of normal and oxidized lipids. Marked differences between the Raman spectra of ceroid and surrounding, non-fluorescent atheroma were observed, amidst large sample to sample variation in chemical composition. Compared with the non-fluorescent tissue, the changes in the Raman spectra of the ceroid deposits could best be explained by larger signal contributions from hemoglobin and cholesteryl esters. We conclude that there is a large heterogeneity in the chemical composition of ceroid. Its composition is largely dependent on the chemical constituents of the surrounding atheroma. Heme or heme degradation products are consistently present in the ceroid deposits. Our results support the hypothesis that iron and heme can form complexes with the intravascular lipoproteins, thereby stimulating oxidation and initiating the formation of ceroid. Copyright

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