BIOCHEMICAL-ANALYSIS AND MAPPING OF ATHEROSCLEROTIC HUMAN ARTERY USING FT-IR MICROSPECTROSCOPY

We report the application of FT-IR microspectroscopy for in situ spect roscopic characterization of molecular constituents of human atheroscl erotic lesions. Since water content in tissue affects conformation-sen sitive protein vibrational bands, tissue specimens were examined under moist conditions. In all measurements, vibrational bands from water w ere found to dominate the spectrum. By removing these water contributi ons, well resolved bands due to tissue components were readily observe d. Utilizing the high sensitivity and good spatial resolution of IR mi crospectroscopy, spectra from a sample volume of 40 x 40 x 4 mu m(3) w ere collected using unstained cryostat sections mounted on a BaF2 flat in neutral isotonic saline. Microstructures were confirmed histologic ally by light microscopy in stained serial sections. In the spectrum o f normal intima, major bands due to amide I (1656 cm(-1)), amide II (1 556 cm(-1)), and CH bending (1457 cm(-1)) vibrations of the proteins c ollagen and elastin were observed. In the spectrum of the intima of no ncalcified atherosclerotic plaque, major bands due to both proteins an d lipids were observed. The lipid bands at 1734, 1468, 1171 and 1058 c m(-1) were assigned to the C=O (ester) stretch, CH2 bend, C-O (ester) stretch and C-O stretch, respectively. At a more detailed level, bands specific to free cholesterol and cholesterol esters were identified. A plot of the integrated intensity ratio of these bands to the protein amide II mode versus depth from the luminal surface confirmed a heter ogeneous distribution of these constituents in the atheromatous core. In the spectra of calcified atherosclerotic plaque, bands were attribu ted to three types of biochemical microstructures: proteins (1657, 155 5, 1243 cm(-1)), lipids (1735, 1466, 1170, 1085, 1055 cm(-1)) and calc ium minerals such as hydroxyapatite (1094, 1040, 962 cm(-1)), and carb onated apatite (1463, 1412, 872 cm(-1)). The results demonstrate that IR microspectroscopy can be used for in situ characterization of molec ular constituents in human unstained arterial sections. The molecular information obtained from these studies could be important in understa nding the pathogenesis of atherosclerosis.