Elemental analysis with a proton microprobe of atherosclerotic lesions

  • N. Debernardi

Student thesis: Master


The present study explores whether calcium precipitations with a size in the order of micrometers or less (micro-calcifications) occur in atherosclerotic lesions in the mouse and human vascular wall. The presence of calcium deposits was determined with the proton microprobe, a combination of Particle Induced X-ray Emission (PIXE) and Rutherford Backscattering Spectroscopy (RBS) that can simultaneously measure concentrations of a variety of elements in the order of parts per million (?g/g dry weight) with sub-micrometer precision. To this end, specific regions of a carotid artery of an ApoE-/- mouse were exposed to low and high shear stress. The low shear stress area developed a severe form of atherosclerosis (type V lesion), where a number of micro-calcifications could be easily found. The high shear stress region remained unaffected and did not show calcium-rich precipitates. The calcium depositions in the low shear stress, atherosclerotic region showed calciumto- phosphorus mass ratios ranging from 0.3 to 4.8, indicating that the calcium hotspots were not composed only of hydroxyapatite, calcium carbonate or amorphous calcium phosphate, as proposed in literature, but most likely also with intermixing of another compound rich in calcium. Urate and oxalate are proposed as possible candidates to serve as alternative anions in the calcium-rich precipitate. Iron, zinc and bromine co-localized with the calcium-rich depositions in about 80% of the calcifications. The (patho-) physiological significance of this finding is at present incompletely understood. The presence of bromine was quite relevant, indicating that vascular tissue is able to accumulate bromine from the blood compartment. Micro-scale calcium precipitations in the early stages of atherosclerosis in human tissue were also determined with the proton microprobe. Samples of the coronary artery wall containing type I, II, and III atherosclerotic lesions from patients who died from noncardiac causes were subject of investigation. Here it is shown that calcium deposits at a micrometer scale can be observed in the thickened intima already in early stages of atherosclerosis (type I lesions). The calcium-to-phosphorus mass ratio, displayed a range of values from 0.2 to 2.2, strongly suggesting that the micro-calcifications consist of amorphous calcium phosphate or hydroxyapatite. In both kinds of tissues, the average concentrations of some biologically interesting elements are measured, as a reference, in different regions remote from the calcium precipitates of the wall vessel (intimal, medial and adventitial layer). Micro-calcifications in human and mouse tissue are well-defined spots where the calcium concentration increases very steep from normal background levels. The absence of a plateau having higher calcium concentration is confirmed in the close vicinity of micro-calcifications (up to 10 ?m). Further investigations are needed to disclose whether micro-calcifications are innocent bystanders or are playing an important role in the onset and progression of the atherosclerotic process.
Date of Award30 Jun 2008
Original languageEnglish
SupervisorPeter H.A. Mutsaers (Supervisor 1), G. van der Vusse (External coach) & R. Levi (External coach)

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