Quantification in situ of crystalline cholesterol and calcium phosphate hydroxyapatite in human atherosclerotic plaques by solid-state magic angle spinning NMR

Abstract

Because of renewed interest in the progression, stabilization, and regression of atherosclerotic plaques, it has become important to develop methods for characterizing structural features of plaques in situ and noninvasively. We present a nondestructive method for ex vivo quantification of 2 solid-phase components of plaques: crystalline cholesterol and calcium phosphate salts. Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of human carotid endarterectomy plaques revealed (13)C resonances of crystalline cholesterol monohydrate and a (31)P resonance of calcium phosphate hydroxyapatite (CPH). The spectra were obtained under conditions in which there was little or no interference from other chemical components and were suitable for quantification in situ of the crystalline cholesterol and CPH. Carotid atherosclerotic plaques showed a wide variation in their crystalline cholesterol content. The calculated molar ratio of liquid-crystalline cholesterol to phospholipid ranged from 1.1 to 1.7, demonstrating different capabilities of the phospholipids to reduce crystallization of cholesterol. The spectral properties of the phosphate groups in CPH in carotid plaques were identical to those of CPH in bone. (31)P MAS NMR is a simple, rapid method for quantification of calcium phosphate salts in tissue without extraction and time-consuming chemical analysis. Crystalline phases in intact atherosclerotic plaques (ex vivo) can be quantified accurately by solid-state (13)C and (31)P MAS NMR spectroscopy.

Figure 1

¹³C dephased CP MAS NMR spectrum of human carotid plaque. The spectrum was obtained with 28 000 scans and processed with 10-Hz line broadening. Selected resonances were identified as previously described,: protein carbonyl (C=O), fatty acid olefinic (C=C), terminal methyl (ωCH₃), and cholesterol resonances (C, followed by IUPAC carbon numbers).

Figure 2

CP MAS NMR spectra of varying amounts of CholM (C5 region only; left) obtained under the same experimental conditions as in Figure 1. Linear least-squares line fitting of integrated C5 peak intensity as a function of cholesterol mass (middle). Error bars represent experimental uncertainty for the integrated signal intensities (samples with >2 mg cholesterol) or for the peak height intensities (samples with <2 mg cholesterol) after repeated NMR experiments. Selected spectra (C5 regions; right) of carotid plaques, with compositions corresponding to those of sample Nos. 1, 2, 3, and 4 listed in Tables 1 and 2.

Figure 3

³¹P MAS NMR spectra of (A) synthetic CPH, (B) human carotid plaque, and (C) chicken bone powder. Each spectrum was obtained with 32 scans and processed without line broadening. A long pulse interval (50.0 seconds) was used for these ³¹P NMR experiments to achieve magnetization equilibrium because the spin-lattice relaxation time of ³¹P in CPH was close to 8.5 seconds (W.G. and J.A.H., unpublished results, 2000). X-ray diffraction powder patterns are schematic replicates of experimental results. Original films are available from the authors for short-term inspection.

Figure 4

Signal intensities of ³¹P spectra vs total phosphorus content as measured by chemical analysis of synthetic CPH (▲), chicken bone powder (○), and human plaques (●). The results for plaques show a close correspondence with results for bone (bottom) rather than synthetic CPH (top).