Ultrastructural Pathology, 2014; 38(3): 167–177 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2013.829149
ORIGINAL ARTICLE
Composition and Genesis of Calcium Deposits in Atheroma Plaques Marı´a Jesu´s Lara1, Eduardo Ros2, Manuel Sierra3, Carlos Dorronsoro3, and Jose´ Aguilar3 1
Department of Vascular Surgery, ‘‘Virgen de la Victoria’’ Hospital, Malaga University, Malaga, Spain, 2 Department of Vascular Surgery, ‘‘San Cecilio’’ Hospital, Granada University, Granada, Spain, and 3 Department of Soil Science, University of Granada, Granada, Spain
ABSTRACT The composition of atheromatous plaque determines its progression toward rupture or thrombosis. Although its histopathological structure has been widely studied, little attention has been paid to its structural and chemical composition and even less to its mineral component. Thirty-three atheromatous plaques were obtained by carotid thromboendarterectomy. Three types of materials were observed under polarized light microscopy: apatite crystals in the form of glomeruli (dark with plane polarized illumination and greensh with cross-polarized illumination); fibrous-like cholesterol (uncolored or grayish with plane-polarized illumination); and amorphous organic material as brownish deposits. SEM-EDX analysis showed an abundance of phosphorus and calcium in sufficient quantities to form calcium phosphates, and appreciably reduced levels of sodium. X-ray diffraction results differentiated samples into three groups: group I with predominance of hydroxyapatite-type crystals, group II with crystalline material containing an amorphous component, and group III with wholly amorphous material. The most abundant mineral in atheromatous plaque is hydroxyapatite, on which crystals of cholesterol and lipid nuclei are deposited, stratifying the plaque into layers that reflect the different stages of its formation. The difference in calcium and sodium concentrations between arteries with and without atheromata may indicate an important relationship in the pathophysiological development of calcium deposits. Keywords: Amorphous calcium phosphate, arterial plaque, atheroma mineralogy, cholesterol plaque, hydroxyapatite
The objective of this study was to determine the organic and mineral components of atheroma by means of different mineralogical methodologies, as a first step toward establishing the genesis of these deposits.
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Few studies of the compounds in atheromatous plaques have been carried out [1–3]. Schmid et al. [1] reported that the plaque was composed of apatite (71%), carbonates (9%), and a relatively high percentage of protein (15%). Marra et al. [3] applied electron probe analysis and demonstrated that the surface and interior of the mineral deposit had the same chemical composition. However, scanning electron microscopy revealed that the deposits were heterogeneous, consisting of five different structures [3]. Information about the composition and properties of calcified deposits helps to determine the risk associated with their presence, as pointed out by Marra et al. [3]
MATERIALS AND METHODS Thirty-three atheroma plaques were removed from the same number of patients by carotid endarterectomy at our hospital (Saint Cecil Hospital, Granada University, Spain) from May 2006 to June 2007.
Received 17 June 2013; Revised 17 July 2013; Accepted 23 July 2013; Published online 15 October 2013 Correspondence: M. Sierra, Department of Soil Science, University of Granada, Granada, Spain. E-mail: [email protected]
167
168 M. J. Lara et al. The 33 patients were predominantly male (29 males, 4 females), aged between 59 and 86, and had the following vascular risk factors: high blood pressure (55%), diabetes mellitus (65%), dyslipidemia (67%), and smoking (81%). All patients underwent a preoperative echo-Doppler study, and some also underwent arteriography. The indication for surgery was based on NASCET criteria (stenosis 460% in arteriography) and ultrasound assessment of blood flow velocity (stenosis 470%). A femoral artery obtained from the donated cadaver of a 32-year-old was also studied (healthy sample, 0). The thickness of the removed plaques ranged from 0.7 to 2 mm except for one 8-mm-thick sample. Plaques were dried in the laboratory at 35 C and prepared for the techniques used. Differential scanning calorimetry was performed in a Shimadzu TGA-50 thermobalance, which was also used for thermogravimetry (TGA) and ultravioletvisible near-infrared spectrophotometry (FTIR). Sample heating was carried out at 20 C min1 and in all cases reached a temperature of 950 C. A tablet consisting of powdered sample and potassium bromide (1:100) was prepared for infrared spectroscopy with a double-beam Beckman model IR 4240. Working conditions were velocity = 150 cm1 min1, gain = 2, slit width = 0.03 mm, and period = 2. X-ray diffraction was performed in a Philips PW 1710 diffractometer equipped with an automatic slit. The samples were ground with an agate molar and passed through a 0.05 mesh. The program published by Martin [4] was used to interpret the results. Thin sections (0.03 mm) of sample fragments, previously embedded in polyester resin by the Dorronsoro and Delgado method [5], were prepared for microscopic study. Optical study of the mineral was done with a Zeiss Axioplan POL transmitted light petrographic. The scanning electron microscopy/ energy dispersive using X-ray (SEM/EDX) study used a Carl Zeiss DSM 950 microscope coupled to an Oxford Link Analytical Pentafet model 6512 X-ray energy-dispersive microanalyzer.
RESULTS Table 1 shows the organic/mineral ratios obtained by thermal gravimetric analysis (TGA) in our samples. The mineral component ranged from 72.8% and 68.3% in samples 11 and 3, respectively, to 9.0% and 9.5% in samples 5 and 15. The mineral fraction was only 3.8% in the healthy artery sample (sample 0), a loss in weight of 96.2% and an organic/mineral ratio of approximately 96/4. This finding was confirmed when weight loss was plotted against temperature, with the sample virtually disappearing at 680 C.
TABLE 1. Unburned residues in the thermal gravimetric analysis for 16 samples. Sample Residue % Sample Residue % Sample Residue % 0 1 2 3 4 5
3.83 43.61 20.99 68.29 11.90 8.00
6 7 8 9 10
28.82 30.37 16.07 14.60 33.63
11 12 13 14 15
72.79 42.07 25.43 16.31 9.47
Derivative weight loss curves (TGA) of all samples revealed endothermic effects from 94 to 121 C corresponding to water loss; loss of carbon-bound –OH from 122 C to 230 C; carbonization with CO and CO2 production from around 250 C to around 400 C; decomposition of nitrogenized compounds from 400 to 550 C, consuming previously formed carbon and giving rise to the formation of inorganic oxides such as N2O and N2O5; and pyrolysis of phosphorus oxides from around 620 C. An endothermic effect was observed at around 730 C in samples 10, 11, 13, and 3, possibly due to whitlockite, at 890 C in samples 6 and 12, and at 826 C in sample due to a calcium orthophosphate (Figure 1A). All atheroma samples showed a high stable residual content, possibly corresponding to orthophosphate compounds (68.3 for the sample in Figure 1A). The healthy artery sample showed a slight endothermic effect at 908 C, which may be attributable to an orthophosphate compound (Figure 1B). FTIR spectra of effluent gases from atheroma samples 1–5 and from healthy artery (sample 0) showed combustions generating CO2 adsorption bands at (3735, 3628, 2366, 2330, and 675 cm1 wavelengths), CO adsorption bands at (2177 and 2116 cm1 wavelengths), H2O adsorption bands at (3857, 3740, 1698, and 1515 cm1 wavelengths), and nitrogen oxides adsorption bands at (2921, 2243, 2208, 1907, 1851, 1637, 1601, 1306, and 1271 cm1 wavelengths), and a low presence of isocyanic acid adsorption bands at (3547, 2284, and 2254 cm1 wavelengths) and ammonium. Nitric oxide and ammonium were present in the sample from the healthy artery but were virtually absent in the atheroma samples. Larger amounts of methane (3017 and 1306 cm1 wavelengths) were found in the healthy sample than in the atheromata (Figure 1C). Figure 2 shows the infrared absorption spectrograms for samples 1, 9, and 12, which are the most representative. The adsorption band between 3500 and 3250 cm1 corresponds to water and OH. Highintensity peaks at 2860, 2260, 1650, and 1380 cm1 correspond to cholesterol [6]. The peak at 1000–1100 corresponds to the C–OC bond, which indicates the presence of cholesterol esters. The peaks at 1500 and 1400 cm1 correspond to N–H and C–N St bonds, Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 169
FIGURE 1. Organic components. (A) Thermal gravimetric analysis of atheroma sample 3, showing the derivative weight loss versus temperature curve in the centre of the diagram. (B) Thermal gravimetric analysis of the healthy artery sample. (C) FTIR spectra of effluent gases obtained at representative intervals from atheroma sample 11 (above) and healthy artery sample (below).
respectively, indicating the presence of proteins. Hence, infrared spectroscopy confirms the differential calorimetry results, which are also consistent with previous reports [1–3].
X-ray Diffraction X-ray diffraction studies were performed on the 15 samples that underwent differential scanning calorimetry. Diffractograms obtained can be classified into four different groups, represented by sample numbers 0, 3, 5, and 6 (Figure 3). A defined diffraction was not obtained in the healthy artery sample, with a wavelength reaching maximum intensity ˚ region that corresponds to in the 4.41- to 2.85-A !
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amorphous components (organic materials and calcium phosphates). The diffractogram for sample 3 shows a large, well-crystallized inorganic component with diffraction maxima corresponding to the apatite mineral group, compatible with hydroxyapatite, although chlorapatite and fluorapatite may also be present. Sample 5 shows a broad maximun in the 6.38˚ region with a maximum at 4.57, probably to 2.83-A attributable to cholesterol. Finally, the diffractogram for sample 6 shows a mixture of inorganic (apatite) and organic (cholesterol) fractions. Table 2 summarizes the X-ray diffraction results, which partly confirm the finding by Tomazic [2] that hydroxyapatite is the most abundant mineral in atheromatous plaque. There was a good correspondence between X-ray diffraction and TGA results.
170 M. J. Lara et al.
FIGURE 2. Infra-red absorption spectrograms for samples 1, 9 and 12.
Samples with the highest residual values (samples 11 and 3) had the highest apatite content, while those with minimal residual content (samples 0, 5, and 15) had no apatite content or only trace values.
SEM/EDX SEM/EDX study of thin sections from the atheromatous plaque showed that the deposits started with the formation of small crystals of 1–5 mm with a chemical composition corresponding to hydroxyapatite [7] (Figure 4). These crystals form crystallization nuclei for other crystals, which coalesce to form different structures, some irregular and others that are more or less spherical nodules or glomeruli (Figures 4 and 5) and, most frequently, laminated structures (Figures 6 and 7). In the healthy artery, the chemical composition of inner/middle layers (A, B, and C of Figure 8) differed from that of the outer layers (samples D and E), with the innermost layers showing a higher sodium content (14 g/kg in A and 11 g/kg in B versus 3.9 g/ kg in D and 2.3 g/kg in E), chlorine content (14 and 15.1 g/kg versus 11.6 and 7.9 g/kg, respectively) and sulfur content (12 and 7.2 g/kg versus 5.4 and 4.6 g/ kg). Table 3 lists the differences in element
concentration ratios between inner and outer layers. Table 4 shows the significant differences in the presence of elements at different levels between atheromatous and healthy samples. Thus, the sodium content was reduced in the inner layers of the diseased arteries but elevated in the inner layers of the healthy artery. The Ca/Na ratio was as high as 22 in diseased arteries compared with 0.25 in the healthy artery.
DISCUSSION The available literature supports the hypothesis that the atheroma plaques from carotid arteries are partly organic and partly mineral. We mainly used differential scanning calorimetry and infrared spectroscopy to elucidate the composition of the organic component. In this study, elemental analysis of atheromatous plaque revealed a mineral component almost entirely made up of hydroxyapatite, in agreement with previous reports [1–3]. SEM revealed crystal formations that were nodular or glomerular and frequently laminar, similar to the structures described by Anderson et al. [8], Mohr [9], and Marra et al. [3] for three-dimensional samples. Crystal formation and the appearance of small hydroxyapatite plaques Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 171
FIGURE 3. X-ray diffractograms for samples 0, 3, 5 and 6. TABLE 2. Composition of 16 samples by X-ray diffraction. Sample 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Apatite
Cholesterol
n.d. þþ (þ) þþþ þþ n.d. þþ þþ þþ þþ (þ) þþþ þ þþ þ (þ)
n.d. n.d. þþþ n.d. þ þþþ þ þ þ þ þ n.d. (þ) (þ) þ þ
þþþ, very abundant; þþ, abundant; þ, not very abundant; (þ) low presence; n.d., not detected. !
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were also observed in a healthy artery sample. Development of the disease from this stage was characterized by progressive plaque growth, thickening of the artery, and consequent stenosis alongside a significant reduction in elasticity. In this study, the diseased arteries showed a higher thickness compared to the healthy artery (Table 5). Hydroxyapatite was sometimes observed on the inner wall of the atheroma, forming protrusions that would hinder the blood flow (Figure 6). Figure 7 depicts the progression of atheroma growth in the studied samples. Atherosclerotic samples showed a loss of sodium compared with the healthy sample and a high increase in concentrations of calcium and, to a lesser extent, of phosphorus. The much higher Ca/Na ratio found in the inner layers of diseased (up to 22) versus healthy artery (0.25) may be attributable to a process of metastasis due to the different solubility of Na and Ca compounds. Studies of endothelial cell cultures revealed a progressive loss of sodium in the cells as
172 M. J. Lara et al.
FIGURE 4. Cross-section of healthy artery. Backscattered electron image shows how small (1–5 mm) crystals (bright white colour) are beginning to form. These crystals can group together, forming nodules (nodule in image has diameter of 37 mm) at the inner surface of the artery. The thickness of the artery ranges from 420 to 270 mm (mean of 370 mm). The energy-dispersed spectrum of the white nodule (A) reveals calcium (Ca) and phosphorus (P) at a ratio of 2:22, according to the quantitative analysis, corresponding to hydroxyapatite (16). The small amount of chlorine (Cl) (Cl/P = 0.01) rules out chlorohydroxyapatite. The composition of the white matter (A) considerably differs from that of the rest of the artery (B). The artery is predominantly composed of sulphur (S), Cl and sodium (Na), whereas Ca and P concentrations in hydroxyapatite are in the order of hundreds of grams per kilogram. ND = not detected. Values expressed in g/kg. Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 173
FIGURE 5. Cross-section of artery with atheroma. The formation of atheroma formation can give rise to nodules or glomeruli that are visible in these petrographic microscopy images (Image 1, plane polarized; Image 2, plane polarized and cross-polarized). The energydispersed spectra (SEM/DEX) are shown below. The glomeruli (dark with plane polarized illumination and greenish with polarized and cross-polarized illumination) are composed of hydroxyapatite (point C), whereas the organic parts (points A, B and D) show high concentrations of Cl and S with small amounts of Ca and P.
they begin to lose viability and approach apoptosis or necrosis [10,11], and studies of arterial calcification demonstrated that calcium–phosphate deposits settle on accumulations of apoptotic and necrotic cells on the arterial wall [9]. !
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Increasing attention is being paid to the role of sodium in the development of certain lesions. Thus, Jia et al. [12] recently reported a significant relationship between angiographic coronary lesions and hyponatremia, finding more severe coronary lesions
174 M. J. Lara et al.
FIGURE 6. Back-scattered SEM images of cross-sections of healthy (1 and 2) and diseased (3) arteries. Image 1 shows the formation of a white, thin (4-mm) layer of hydroxyapatite immediately below the inner layer of the artery as well as scattered crystals approximately 1 mm in diameter. Image 2 shows the formation of a thin layer of hydroxyapatite whose growth appears to have deformed the inner artery wall, which has an irregular shape with indentations that might hinder the blood flow and favour the formation of deposits on the arterial wall. Image 3 shows a longitudinal section of an atheromatous artery with an inner accumulation of hydroxyapatite (white colour) in laminar arrangement, producing a thickening of the artery. Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 175
FIGURE 7. Possible sequence of the formation of an atheroma. Back-scattered SEM images. Hydroxyapatite deposits appear white.
in patients with reduced plasma sodium than in those with normal blood sodium levels. The present results suggest that calcification is a continuous process. The mineral component was practically absent in completely healthy arteries and gradually increased with the advance of the disease.
CONCLUSIONS Calcium deposits in arteries are composed of hydroxyapatite, most frequently in the form of laminae parallel to the blood flow and sometimes with a !
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glomerular or nodular crystalline structure. Hydroxyapatite formation (arterial calcification) may be present at very early stages of the atherosclerotic process. Once the initial hydroxyapatite nucleus has been formed, it accumulates more mineral components as well as organic compounds. The use of petrographic microscopy and X-ray diffraction allowed the organic fraction to be identified as cholesterol in this study. The petrographic microscopy and SEM/EDX studies show that apatite crystallization can develop on the vessel wall but is more frequently observed on the intima, producing an irregular and denticulate surface that may hinder
176 M. J. Lara et al.
FIGURE 8. Section of healthy artery. The inner surface of the artery is represented by sample 0C-A. Two layers are differentiated by their chemical composition: an inner/middle layer samples A, B and C) and an outer layer (samples D and E). The outer layer is characterised by high Na values (14 and 11 g/kg for A and B vs. 3.9 and 2.3 g/kg for D and E, respectively). The inner layer also contained more Cl (14 and 15.1 g/kg vs. 11.6 and 7.9 g/kg, respectively) and more S (12.7 and 13.6 vs. 5.4 and 4.6 g/kg, respectively). Values of chemical elements in g/kg.
TABLE 3. Ratios between concentrations of elements detected in the different layers of the healthy artery based on measurements in at least 10 sections. Sample 0 C-A 0 C-B 0 C-C 0 C-D 0 C-E
Ca/P
Ca/S
Ca/Cl
Ca/Fe
Ca/k
Ca/Na
Cl/S
Cl/Na
S/Na
1.00 0.71 1.25 2.00 5.44
0.24 0.26 0.28 0.30 1.07
0.21 0.23 0.13 0.14 0.62
5.80 5.83 4.00 16.00 6.13
5.80 5.83 3.33 4.00 16.33
0.25 0.31 0.18 0.41 2.13
1.17 1.11 2.07 2.15 1.72
1.23 1.35 1.34 2.97 3.43
1.05 1.21 0.65 1.38 2.00
Innermost layer, OC-A; outermost layer, OC-E.
Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 177 TABLE 4. Comparative elemental composition of arteries with and without atheromata based on measurements in at least 10 sections. Gray matter of
Ca
Whole healthy artery 3.0 Inner layers of 2.8 healthy artery Outer layers of 3.3 healthy artery Whole disease artery 19.3 Inner layers of 8.8 disease artery Outer layers of 11.7 disease artery White matter of 110.5 atheromata
P
S
Cl
Fe
K
Na
(1.6–4.9) (2.0–3.5)
2.2 (0.8–4.9) 3.1 (1.6–4.9)
8.6 (4.6–13.6) 12.7 (7.9–15.1) 0.5 (0.1–0.8) 0.5 (0.3–0.6) 8.0 (2.3–11.4) 10.9 (7.2–13.6) 14.7 (14.0–15.1) 0.5 (0.5–0.6) 0.6 (0.5–0.6) 11.2 (11.1–11.4)
(1.6–4.9)
0.9 (0.8–0.9)
5.0 (4.6–5.4)
(3.8–7.5) (2.3–15.3)
5.7 (3.2–92.6) 5.0 (3.2–6.8)
7.3 (3.4–8.6) 5.5 (2.9–8.1)
(11.0–12.4)
4.7 (3.9–5.4)
9.3 (8.6–1.0)
5.3 (3.9–6.6)
0.0 (0.0–0.0) 0.8 (0.4–1.1)
2.3 (1.4–3.2)
4.1 (0.0–7.4)
20.0 (1.6–42.1)
0.5 (0.0–1.4) 1.1 (0.5–1.7)
2.8 (0.2–4.5)
(16.2–315.4) 53.1 (3.7–143.0)
9.8 (7.9–11.6)
0.5 (0.1–0.8) 0.4 (0.3–0.4)
3.1 (2.3–3.9)
13.9 (3.0–41.6) 0.5 (0.0–3.3) 0.9 (0.0–1.6) 25.1 (12.2–38.0) 0.3 (0.0–0.5) 0.7 (0.5–0.9)
2.4 (0.4–4.9) 0.4 (0.4–0.4)
Besides distinguishing an inner and outer part, we differentiated in atheromatous arteries between areas with and without atheromata (white and gray on SEM). Means are shown in bold, with minimum and maximum values in parentheses. Values of elements are in g/kg.
TABLE 5. Thickness of arteries (10 sections measured in each sample). 2. Mean Minimum Maximum (mm) (mm) (mm) Healthy artery without hydroxyapatite accumulation Part of healthy artery with hydroxyapatite accumulation Arteries with atheromata
358
318
401
420
290
468
582
402
831
3.
4. 5.
laminar flow and facilitate the precipitation of calcium. All atherosclerotic samples showed a reduction in sodium levels in favor of an increase in calcium, which may be a decisive factor in the genesis of calcium deposits on atheromatous plaque.
DECLARATION OF INTEREST
6.
7. 8. 9.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
10.
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1. Schmid K, McSharry WO, Pameijer CH, Binette JP. Chemical and physiochemical studies on the mineral
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deposits of the human atherosclerotic aorta. Atherosclerosis 1980;37: 199–210. Tomazic BB. Characterization of mineral phases in cardio vascular calcification. In: Brown PW, Constanz B, eds. Hydroxy apatite and related materials. Boca Raton: CRC Press, 1994:93–115. Marra SP, Daghlian CP, Fillinger MF, Kennedy FE. Elemental composition, morphology and mechanical properties of calcified deposits obtained from abdominal aortic aneurysms. Acta Biomater 2006;2: 515–20. Martı´n JD. X Powder Version 2004.04.35 Pro. ISBN 84-6091497-6, 2004. Dorronsoro C, Delgado M. Iron compounds at the coarse sand fraction of some Spanish soils. In: Rutherford GK, ed. Soil Microscopy. Kingstone: Limestone Press, 1974: 682–93. Sa´enz de Buruaga A. Fitoquı´mica de Thimelaceas: Triterpenos y Esteroles con Hojas de Thymelea hirsuta L [Thesis]. Spain: Granada University, 1979. Deer A, Zussman J, Howie R. Rock-forming minerals. London: Longman, 1977. Anderson HC, Tanimura A, McGregor DH. Calcification in atherosclerosis. J Exp Pathol 1989;2: 261–73. Mohr W. Calcification of temporary artery: morphology and comparison of physiologie osteogenesys. Z Kardiol 2003;92: 60–72. Rodrı´guez Morata A. Microscopı´a Electro´nica Analı´tica de la Ce´lula Endotelial [Thesis]. Spain: Granada University, 2005. Zabitis E. Alteracio´n de la Homeostasis Io´nica Durante la Lesio´n Celular Inducida por Hipoxia Metabo´lica: Papel de los Iones Monovalentes [Thesis]. Spain: Granada University, 2002. Jia EZ, Yang ZJ, Zhu TB, et al. Serum sodium concentration is significantly associated with the angiographic characteristics of coronary atherosclerosis. Acta Pharmacol Sin 2007;28: 1136–42.
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ORIGINAL ARTICLE
Composition and Genesis of Calcium Deposits in Atheroma Plaques Marı´a Jesu´s Lara1, Eduardo Ros2, Manuel Sierra3, Carlos Dorronsoro3, and Jose´ Aguilar3 1
Department of Vascular Surgery, ‘‘Virgen de la Victoria’’ Hospital, Malaga University, Malaga, Spain, 2 Department of Vascular Surgery, ‘‘San Cecilio’’ Hospital, Granada University, Granada, Spain, and 3 Department of Soil Science, University of Granada, Granada, Spain
ABSTRACT The composition of atheromatous plaque determines its progression toward rupture or thrombosis. Although its histopathological structure has been widely studied, little attention has been paid to its structural and chemical composition and even less to its mineral component. Thirty-three atheromatous plaques were obtained by carotid thromboendarterectomy. Three types of materials were observed under polarized light microscopy: apatite crystals in the form of glomeruli (dark with plane polarized illumination and greensh with cross-polarized illumination); fibrous-like cholesterol (uncolored or grayish with plane-polarized illumination); and amorphous organic material as brownish deposits. SEM-EDX analysis showed an abundance of phosphorus and calcium in sufficient quantities to form calcium phosphates, and appreciably reduced levels of sodium. X-ray diffraction results differentiated samples into three groups: group I with predominance of hydroxyapatite-type crystals, group II with crystalline material containing an amorphous component, and group III with wholly amorphous material. The most abundant mineral in atheromatous plaque is hydroxyapatite, on which crystals of cholesterol and lipid nuclei are deposited, stratifying the plaque into layers that reflect the different stages of its formation. The difference in calcium and sodium concentrations between arteries with and without atheromata may indicate an important relationship in the pathophysiological development of calcium deposits. Keywords: Amorphous calcium phosphate, arterial plaque, atheroma mineralogy, cholesterol plaque, hydroxyapatite
The objective of this study was to determine the organic and mineral components of atheroma by means of different mineralogical methodologies, as a first step toward establishing the genesis of these deposits.
20 14
Few studies of the compounds in atheromatous plaques have been carried out [1–3]. Schmid et al. [1] reported that the plaque was composed of apatite (71%), carbonates (9%), and a relatively high percentage of protein (15%). Marra et al. [3] applied electron probe analysis and demonstrated that the surface and interior of the mineral deposit had the same chemical composition. However, scanning electron microscopy revealed that the deposits were heterogeneous, consisting of five different structures [3]. Information about the composition and properties of calcified deposits helps to determine the risk associated with their presence, as pointed out by Marra et al. [3]
MATERIALS AND METHODS Thirty-three atheroma plaques were removed from the same number of patients by carotid endarterectomy at our hospital (Saint Cecil Hospital, Granada University, Spain) from May 2006 to June 2007.
Received 17 June 2013; Revised 17 July 2013; Accepted 23 July 2013; Published online 15 October 2013 Correspondence: M. Sierra, Department of Soil Science, University of Granada, Granada, Spain. E-mail: [email protected]
167
168 M. J. Lara et al. The 33 patients were predominantly male (29 males, 4 females), aged between 59 and 86, and had the following vascular risk factors: high blood pressure (55%), diabetes mellitus (65%), dyslipidemia (67%), and smoking (81%). All patients underwent a preoperative echo-Doppler study, and some also underwent arteriography. The indication for surgery was based on NASCET criteria (stenosis 460% in arteriography) and ultrasound assessment of blood flow velocity (stenosis 470%). A femoral artery obtained from the donated cadaver of a 32-year-old was also studied (healthy sample, 0). The thickness of the removed plaques ranged from 0.7 to 2 mm except for one 8-mm-thick sample. Plaques were dried in the laboratory at 35 C and prepared for the techniques used. Differential scanning calorimetry was performed in a Shimadzu TGA-50 thermobalance, which was also used for thermogravimetry (TGA) and ultravioletvisible near-infrared spectrophotometry (FTIR). Sample heating was carried out at 20 C min1 and in all cases reached a temperature of 950 C. A tablet consisting of powdered sample and potassium bromide (1:100) was prepared for infrared spectroscopy with a double-beam Beckman model IR 4240. Working conditions were velocity = 150 cm1 min1, gain = 2, slit width = 0.03 mm, and period = 2. X-ray diffraction was performed in a Philips PW 1710 diffractometer equipped with an automatic slit. The samples were ground with an agate molar and passed through a 0.05 mesh. The program published by Martin [4] was used to interpret the results. Thin sections (0.03 mm) of sample fragments, previously embedded in polyester resin by the Dorronsoro and Delgado method [5], were prepared for microscopic study. Optical study of the mineral was done with a Zeiss Axioplan POL transmitted light petrographic. The scanning electron microscopy/ energy dispersive using X-ray (SEM/EDX) study used a Carl Zeiss DSM 950 microscope coupled to an Oxford Link Analytical Pentafet model 6512 X-ray energy-dispersive microanalyzer.
RESULTS Table 1 shows the organic/mineral ratios obtained by thermal gravimetric analysis (TGA) in our samples. The mineral component ranged from 72.8% and 68.3% in samples 11 and 3, respectively, to 9.0% and 9.5% in samples 5 and 15. The mineral fraction was only 3.8% in the healthy artery sample (sample 0), a loss in weight of 96.2% and an organic/mineral ratio of approximately 96/4. This finding was confirmed when weight loss was plotted against temperature, with the sample virtually disappearing at 680 C.
TABLE 1. Unburned residues in the thermal gravimetric analysis for 16 samples. Sample Residue % Sample Residue % Sample Residue % 0 1 2 3 4 5
3.83 43.61 20.99 68.29 11.90 8.00
6 7 8 9 10
28.82 30.37 16.07 14.60 33.63
11 12 13 14 15
72.79 42.07 25.43 16.31 9.47
Derivative weight loss curves (TGA) of all samples revealed endothermic effects from 94 to 121 C corresponding to water loss; loss of carbon-bound –OH from 122 C to 230 C; carbonization with CO and CO2 production from around 250 C to around 400 C; decomposition of nitrogenized compounds from 400 to 550 C, consuming previously formed carbon and giving rise to the formation of inorganic oxides such as N2O and N2O5; and pyrolysis of phosphorus oxides from around 620 C. An endothermic effect was observed at around 730 C in samples 10, 11, 13, and 3, possibly due to whitlockite, at 890 C in samples 6 and 12, and at 826 C in sample due to a calcium orthophosphate (Figure 1A). All atheroma samples showed a high stable residual content, possibly corresponding to orthophosphate compounds (68.3 for the sample in Figure 1A). The healthy artery sample showed a slight endothermic effect at 908 C, which may be attributable to an orthophosphate compound (Figure 1B). FTIR spectra of effluent gases from atheroma samples 1–5 and from healthy artery (sample 0) showed combustions generating CO2 adsorption bands at (3735, 3628, 2366, 2330, and 675 cm1 wavelengths), CO adsorption bands at (2177 and 2116 cm1 wavelengths), H2O adsorption bands at (3857, 3740, 1698, and 1515 cm1 wavelengths), and nitrogen oxides adsorption bands at (2921, 2243, 2208, 1907, 1851, 1637, 1601, 1306, and 1271 cm1 wavelengths), and a low presence of isocyanic acid adsorption bands at (3547, 2284, and 2254 cm1 wavelengths) and ammonium. Nitric oxide and ammonium were present in the sample from the healthy artery but were virtually absent in the atheroma samples. Larger amounts of methane (3017 and 1306 cm1 wavelengths) were found in the healthy sample than in the atheromata (Figure 1C). Figure 2 shows the infrared absorption spectrograms for samples 1, 9, and 12, which are the most representative. The adsorption band between 3500 and 3250 cm1 corresponds to water and OH. Highintensity peaks at 2860, 2260, 1650, and 1380 cm1 correspond to cholesterol [6]. The peak at 1000–1100 corresponds to the C–OC bond, which indicates the presence of cholesterol esters. The peaks at 1500 and 1400 cm1 correspond to N–H and C–N St bonds, Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 169
FIGURE 1. Organic components. (A) Thermal gravimetric analysis of atheroma sample 3, showing the derivative weight loss versus temperature curve in the centre of the diagram. (B) Thermal gravimetric analysis of the healthy artery sample. (C) FTIR spectra of effluent gases obtained at representative intervals from atheroma sample 11 (above) and healthy artery sample (below).
respectively, indicating the presence of proteins. Hence, infrared spectroscopy confirms the differential calorimetry results, which are also consistent with previous reports [1–3].
X-ray Diffraction X-ray diffraction studies were performed on the 15 samples that underwent differential scanning calorimetry. Diffractograms obtained can be classified into four different groups, represented by sample numbers 0, 3, 5, and 6 (Figure 3). A defined diffraction was not obtained in the healthy artery sample, with a wavelength reaching maximum intensity ˚ region that corresponds to in the 4.41- to 2.85-A !
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amorphous components (organic materials and calcium phosphates). The diffractogram for sample 3 shows a large, well-crystallized inorganic component with diffraction maxima corresponding to the apatite mineral group, compatible with hydroxyapatite, although chlorapatite and fluorapatite may also be present. Sample 5 shows a broad maximun in the 6.38˚ region with a maximum at 4.57, probably to 2.83-A attributable to cholesterol. Finally, the diffractogram for sample 6 shows a mixture of inorganic (apatite) and organic (cholesterol) fractions. Table 2 summarizes the X-ray diffraction results, which partly confirm the finding by Tomazic [2] that hydroxyapatite is the most abundant mineral in atheromatous plaque. There was a good correspondence between X-ray diffraction and TGA results.
170 M. J. Lara et al.
FIGURE 2. Infra-red absorption spectrograms for samples 1, 9 and 12.
Samples with the highest residual values (samples 11 and 3) had the highest apatite content, while those with minimal residual content (samples 0, 5, and 15) had no apatite content or only trace values.
SEM/EDX SEM/EDX study of thin sections from the atheromatous plaque showed that the deposits started with the formation of small crystals of 1–5 mm with a chemical composition corresponding to hydroxyapatite [7] (Figure 4). These crystals form crystallization nuclei for other crystals, which coalesce to form different structures, some irregular and others that are more or less spherical nodules or glomeruli (Figures 4 and 5) and, most frequently, laminated structures (Figures 6 and 7). In the healthy artery, the chemical composition of inner/middle layers (A, B, and C of Figure 8) differed from that of the outer layers (samples D and E), with the innermost layers showing a higher sodium content (14 g/kg in A and 11 g/kg in B versus 3.9 g/ kg in D and 2.3 g/kg in E), chlorine content (14 and 15.1 g/kg versus 11.6 and 7.9 g/kg, respectively) and sulfur content (12 and 7.2 g/kg versus 5.4 and 4.6 g/ kg). Table 3 lists the differences in element
concentration ratios between inner and outer layers. Table 4 shows the significant differences in the presence of elements at different levels between atheromatous and healthy samples. Thus, the sodium content was reduced in the inner layers of the diseased arteries but elevated in the inner layers of the healthy artery. The Ca/Na ratio was as high as 22 in diseased arteries compared with 0.25 in the healthy artery.
DISCUSSION The available literature supports the hypothesis that the atheroma plaques from carotid arteries are partly organic and partly mineral. We mainly used differential scanning calorimetry and infrared spectroscopy to elucidate the composition of the organic component. In this study, elemental analysis of atheromatous plaque revealed a mineral component almost entirely made up of hydroxyapatite, in agreement with previous reports [1–3]. SEM revealed crystal formations that were nodular or glomerular and frequently laminar, similar to the structures described by Anderson et al. [8], Mohr [9], and Marra et al. [3] for three-dimensional samples. Crystal formation and the appearance of small hydroxyapatite plaques Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 171
FIGURE 3. X-ray diffractograms for samples 0, 3, 5 and 6. TABLE 2. Composition of 16 samples by X-ray diffraction. Sample 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Apatite
Cholesterol
n.d. þþ (þ) þþþ þþ n.d. þþ þþ þþ þþ (þ) þþþ þ þþ þ (þ)
n.d. n.d. þþþ n.d. þ þþþ þ þ þ þ þ n.d. (þ) (þ) þ þ
þþþ, very abundant; þþ, abundant; þ, not very abundant; (þ) low presence; n.d., not detected. !
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were also observed in a healthy artery sample. Development of the disease from this stage was characterized by progressive plaque growth, thickening of the artery, and consequent stenosis alongside a significant reduction in elasticity. In this study, the diseased arteries showed a higher thickness compared to the healthy artery (Table 5). Hydroxyapatite was sometimes observed on the inner wall of the atheroma, forming protrusions that would hinder the blood flow (Figure 6). Figure 7 depicts the progression of atheroma growth in the studied samples. Atherosclerotic samples showed a loss of sodium compared with the healthy sample and a high increase in concentrations of calcium and, to a lesser extent, of phosphorus. The much higher Ca/Na ratio found in the inner layers of diseased (up to 22) versus healthy artery (0.25) may be attributable to a process of metastasis due to the different solubility of Na and Ca compounds. Studies of endothelial cell cultures revealed a progressive loss of sodium in the cells as
172 M. J. Lara et al.
FIGURE 4. Cross-section of healthy artery. Backscattered electron image shows how small (1–5 mm) crystals (bright white colour) are beginning to form. These crystals can group together, forming nodules (nodule in image has diameter of 37 mm) at the inner surface of the artery. The thickness of the artery ranges from 420 to 270 mm (mean of 370 mm). The energy-dispersed spectrum of the white nodule (A) reveals calcium (Ca) and phosphorus (P) at a ratio of 2:22, according to the quantitative analysis, corresponding to hydroxyapatite (16). The small amount of chlorine (Cl) (Cl/P = 0.01) rules out chlorohydroxyapatite. The composition of the white matter (A) considerably differs from that of the rest of the artery (B). The artery is predominantly composed of sulphur (S), Cl and sodium (Na), whereas Ca and P concentrations in hydroxyapatite are in the order of hundreds of grams per kilogram. ND = not detected. Values expressed in g/kg. Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 173
FIGURE 5. Cross-section of artery with atheroma. The formation of atheroma formation can give rise to nodules or glomeruli that are visible in these petrographic microscopy images (Image 1, plane polarized; Image 2, plane polarized and cross-polarized). The energydispersed spectra (SEM/DEX) are shown below. The glomeruli (dark with plane polarized illumination and greenish with polarized and cross-polarized illumination) are composed of hydroxyapatite (point C), whereas the organic parts (points A, B and D) show high concentrations of Cl and S with small amounts of Ca and P.
they begin to lose viability and approach apoptosis or necrosis [10,11], and studies of arterial calcification demonstrated that calcium–phosphate deposits settle on accumulations of apoptotic and necrotic cells on the arterial wall [9]. !
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Increasing attention is being paid to the role of sodium in the development of certain lesions. Thus, Jia et al. [12] recently reported a significant relationship between angiographic coronary lesions and hyponatremia, finding more severe coronary lesions
174 M. J. Lara et al.
FIGURE 6. Back-scattered SEM images of cross-sections of healthy (1 and 2) and diseased (3) arteries. Image 1 shows the formation of a white, thin (4-mm) layer of hydroxyapatite immediately below the inner layer of the artery as well as scattered crystals approximately 1 mm in diameter. Image 2 shows the formation of a thin layer of hydroxyapatite whose growth appears to have deformed the inner artery wall, which has an irregular shape with indentations that might hinder the blood flow and favour the formation of deposits on the arterial wall. Image 3 shows a longitudinal section of an atheromatous artery with an inner accumulation of hydroxyapatite (white colour) in laminar arrangement, producing a thickening of the artery. Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 175
FIGURE 7. Possible sequence of the formation of an atheroma. Back-scattered SEM images. Hydroxyapatite deposits appear white.
in patients with reduced plasma sodium than in those with normal blood sodium levels. The present results suggest that calcification is a continuous process. The mineral component was practically absent in completely healthy arteries and gradually increased with the advance of the disease.
CONCLUSIONS Calcium deposits in arteries are composed of hydroxyapatite, most frequently in the form of laminae parallel to the blood flow and sometimes with a !
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glomerular or nodular crystalline structure. Hydroxyapatite formation (arterial calcification) may be present at very early stages of the atherosclerotic process. Once the initial hydroxyapatite nucleus has been formed, it accumulates more mineral components as well as organic compounds. The use of petrographic microscopy and X-ray diffraction allowed the organic fraction to be identified as cholesterol in this study. The petrographic microscopy and SEM/EDX studies show that apatite crystallization can develop on the vessel wall but is more frequently observed on the intima, producing an irregular and denticulate surface that may hinder
176 M. J. Lara et al.
FIGURE 8. Section of healthy artery. The inner surface of the artery is represented by sample 0C-A. Two layers are differentiated by their chemical composition: an inner/middle layer samples A, B and C) and an outer layer (samples D and E). The outer layer is characterised by high Na values (14 and 11 g/kg for A and B vs. 3.9 and 2.3 g/kg for D and E, respectively). The inner layer also contained more Cl (14 and 15.1 g/kg vs. 11.6 and 7.9 g/kg, respectively) and more S (12.7 and 13.6 vs. 5.4 and 4.6 g/kg, respectively). Values of chemical elements in g/kg.
TABLE 3. Ratios between concentrations of elements detected in the different layers of the healthy artery based on measurements in at least 10 sections. Sample 0 C-A 0 C-B 0 C-C 0 C-D 0 C-E
Ca/P
Ca/S
Ca/Cl
Ca/Fe
Ca/k
Ca/Na
Cl/S
Cl/Na
S/Na
1.00 0.71 1.25 2.00 5.44
0.24 0.26 0.28 0.30 1.07
0.21 0.23 0.13 0.14 0.62
5.80 5.83 4.00 16.00 6.13
5.80 5.83 3.33 4.00 16.33
0.25 0.31 0.18 0.41 2.13
1.17 1.11 2.07 2.15 1.72
1.23 1.35 1.34 2.97 3.43
1.05 1.21 0.65 1.38 2.00
Innermost layer, OC-A; outermost layer, OC-E.
Ultrastructural Pathology
Calcium Deposits in Atheroma Plaques 177 TABLE 4. Comparative elemental composition of arteries with and without atheromata based on measurements in at least 10 sections. Gray matter of
Ca
Whole healthy artery 3.0 Inner layers of 2.8 healthy artery Outer layers of 3.3 healthy artery Whole disease artery 19.3 Inner layers of 8.8 disease artery Outer layers of 11.7 disease artery White matter of 110.5 atheromata
P
S
Cl
Fe
K
Na
(1.6–4.9) (2.0–3.5)
2.2 (0.8–4.9) 3.1 (1.6–4.9)
8.6 (4.6–13.6) 12.7 (7.9–15.1) 0.5 (0.1–0.8) 0.5 (0.3–0.6) 8.0 (2.3–11.4) 10.9 (7.2–13.6) 14.7 (14.0–15.1) 0.5 (0.5–0.6) 0.6 (0.5–0.6) 11.2 (11.1–11.4)
(1.6–4.9)
0.9 (0.8–0.9)
5.0 (4.6–5.4)
(3.8–7.5) (2.3–15.3)
5.7 (3.2–92.6) 5.0 (3.2–6.8)
7.3 (3.4–8.6) 5.5 (2.9–8.1)
(11.0–12.4)
4.7 (3.9–5.4)
9.3 (8.6–1.0)
5.3 (3.9–6.6)
0.0 (0.0–0.0) 0.8 (0.4–1.1)
2.3 (1.4–3.2)
4.1 (0.0–7.4)
20.0 (1.6–42.1)
0.5 (0.0–1.4) 1.1 (0.5–1.7)
2.8 (0.2–4.5)
(16.2–315.4) 53.1 (3.7–143.0)
9.8 (7.9–11.6)
0.5 (0.1–0.8) 0.4 (0.3–0.4)
3.1 (2.3–3.9)
13.9 (3.0–41.6) 0.5 (0.0–3.3) 0.9 (0.0–1.6) 25.1 (12.2–38.0) 0.3 (0.0–0.5) 0.7 (0.5–0.9)
2.4 (0.4–4.9) 0.4 (0.4–0.4)
Besides distinguishing an inner and outer part, we differentiated in atheromatous arteries between areas with and without atheromata (white and gray on SEM). Means are shown in bold, with minimum and maximum values in parentheses. Values of elements are in g/kg.
TABLE 5. Thickness of arteries (10 sections measured in each sample). 2. Mean Minimum Maximum (mm) (mm) (mm) Healthy artery without hydroxyapatite accumulation Part of healthy artery with hydroxyapatite accumulation Arteries with atheromata
358
318
401
420
290
468
582
402
831
3.
4. 5.
laminar flow and facilitate the precipitation of calcium. All atherosclerotic samples showed a reduction in sodium levels in favor of an increase in calcium, which may be a decisive factor in the genesis of calcium deposits on atheromatous plaque.
DECLARATION OF INTEREST
6.
7. 8. 9.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
10.
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