Calcification of aortic wall in cholesterol-fed rabbits

Atherosclerosis, 87 (1991) 183-193 0 1991 Elsevier Scientific Publishers ADONIS 0021915091000962

ATHERO

183 Ireland,

Ltd. 0021-9150/91/$03.50

04616

Calcification of aortic wall in cholesterol-fed rabbits E. Rokita

I, T. Cichocki

*, D. Heck 3, L. Jarczyk

’ and

A. Strzalkowski

’

’Institute of Physics Jagellonian University, Reymonta 4, PL-30059 Krakow (Poland), ’ Department of Histology Academy of Medicine, Kopernika 7, PL-31034 Krakow (Poland) and ’ Karlsruhe Nuclear Research Center, Institute of Nuclear Physics, P.0. Box 3640, D-7500 Karlsruhe (F.R.G.) (Received 16 March, 1990) (Revised, received 26 November, 1990) (Accepted 12 December, 1990)

Summary Development of the mineralization process in the course of atherogenesis was studied using the cholesterol-fed rabbit model. The aorta samples were investigated by means of proton and electron microprobes, infrared spectroscopy and X-ray diffraction as well as selected histochemical staining. Blood serum was analysed every 2 weeks to determine the content of cholesterol, triglycerides, inorganic phosphorus, ionized calcium, elemental composition as well as activity of alkaline phosphatase. It was found that the administered diet did not disturb the calcium and phosphorus homeostasis. Histochemical findings confirmed the formation of lipid-rich lesions blocking the lumen of the vessel. The dystrophic calcification was observed only in the atheroma, while in the tunica media a slight mineralization similar to that found in controls was observed after 210 days of the diet. In the atheroma the only phase detected was a defective hydroxyapatite. The perfection of the crystals, as well as the diameter of the deposits, increased during the course of the diet reaching about 2 pm after 210 days. The crystals were not contaminated with carbonate groups regardless of the duration of the diet.

Key words:

Atherosclerosis;

Calcification;

Rabbit

Introduction Calcification of the aortic most pronounced symptoms

wall is one of the of atherosclerosis

Correspondence to: Dr. E. Rokita, Institute of Physics, Jagellonian University, Reymonta 4, PL-30059 Krakow, Poland.

aorta;

Cholesterol

diet

commonly considered as an indicator of the advanced stage of the disease. Although the structural aspects as well as physicochemical properties of the minerals within the aortic wall were extensively investigated both in human autopsy samples [l-S] and in various animal models [9-141 the mechanism of inorganic deposit formation within the aortic wall still remains unclear. It is not even known how the mineralization of the vessel wall

184 develops in the course of atherogenesis. The other open question deals with the relationship between the development of the mineralization process in the tunica media and in the atherosclerotic plaque (atheroma). The focus of this paper is the calcification of the aortic wall during progression of atherosclerosis. The course of the mineralization process was investigated separately for the tunica media and for the atheroma using the cholesterol-fed rabbit model. Materials and methods The material used in the study was obtained from aortas of 14 rabbits. The animals were divided into 7 groups, 2 rabbits per group. One rabbit in each group was fed ad libitum a standard laboratory diet (LSK, manufactured by Central Laboratory of the Fodder Industry, Motycz, Poland) composed of: carbohydrates 59.1%, protein 19.6%, fibrin 10.5%, minerals 7.3%, and lipids 3.5%. The second animal in each group obtained a diet additionally supplemented with cholesterol (1.5%) and corn oil (3%). Every 2 weeks the levels of cholesterol [ 151, triglycerides [ 161, inorganic phosphorus [17], ionized calcium [18] and activity of alkaline phosphatase [19] were determined in blood serum samples. The concentrations of Na and Mg in these samples were measured by atomic absorption spectroscopy [20] and concentrations of P, S, Cl, K, Ca, Fe, Cu, Zn, Br and Sr by proton induced X-ray emission method [21]. After defined duration of the diet (0, 96, 118, 146, 167, 187 and 210 days) the rabbits were killed. The aortas were quickly dissected and divided into 2 parts. The first one, the ascending part of the aortic arch, was used for proton and electron microprobe (PMP and EMP, respectively) and histochemical studies. The samples were snap frozen in liquid nitrogen and cut in a cryomicrotome at - 25 o C. Serial sections were placed on glass slides, formvar backing foil or on a metal holder for the histochemical staining, PMP and EMP, respectively. Sections used for histochemical studies were stained with Mallory-azan method for routine examination, von Kossa’s method for inorganic

deposits, Sudan black B for lipids and Weigert’s method for elastin [22]. PMP measurements were performed using the proton microprobe of the 3.75 MV van de Graaff accelerator of the Karlsruhe Nuclear Research Center [23]. The X-rays induced by proton irradiation were analysed according to the procedure described elsewhere [24]. The measurements were registered in line scan mode, with the scanning line consisting of 128 or 256 equidistant points. The beam diameter was approximately 3 pm. Concentrations of the following elements were measured: P, S, Cl, K, Ca, Fe, Cu, Zn, Br and Sr. Additionally, the sample thickness was determined by measurements of the elastically back-scattered protons [24]. The presence of biominerals was determined by the peaks in the distribution of Ca concentrations. The distribution of P concentration was not taken into account due to a high content of this element in tissue, independent of the mineral content. Moreover, all peaks showing a Ca content less than 3 times the basal calcium level found in the surrounding tissue were omitted. The detailed histological verification of the reliability of PMP measurements in the studies on the localization of biominerals was presented elsewhere [25]. The inorganic deposits (ID) detected by PMP consist of a mixture of an organic matrix (OM) and inorganic material. In order to characterize the composition of the pure mineral phase, the OM contamination was subtracted in a following procedure: for each measured distribution of Ca concentration, 9 to 11 points at which the IDS did not appear (outside of the peaks in the distribution of the Ca concentration) were selected. These points were considered to be representative for OM only. The content of the OM at sites of IDS localization was determined by linear interpolation and, finally, subtracted from the measured Ca concentration. The identical subtraction procedure was used for the other elements. The EMP studies were carried out using an electron microprobe (Cameca MS 46) with wavelength dispersive system. Before element analysis, the samples were coated with carbon. A section was slowly moved (20 pm/mm) perpendicularly to the beam direction and X-ray counts were registered every 3 sec. To determine 3 elements (Mg, P, Ca), 2 runs along the same line were

185 carried out. The quantification of element concentrations was made using the method described elsewhere [26]. The second part of the aorta was used for infrared spectroscopy (IR) and X-ray diffraction studies (XRD). Moreover, the content of the inorganic phase in the sample was also determined. Immediately after dissection, the atheromas (if present) were separated, the samples were snap frozen in liquid nitrogen and transferred directly into the vacuum chamber. The material remained under vacuum for 48 h. The dried tissue was delipidized by soaking in several changes of chloroform/ methanol (3 : 1, v : v). Finally, the samples were deproteinized by immersion in several changes of hydrazine (after 1, 2, 4, 8 and 24 h) at 55 o C, washed in absolute ethanol and powderized. The IR spectra were measured in the region of 2000-400 cm-’ using a SPECORD M80 IR spectrometer (Carl Zeiss Jena). For the IR studies, KBr pellets were produced. The measured spectra were analyzed using the computer program [2’7,28]. X-ray powder diffraction patterns of rabbit aorta were obtained on DRON-3 diffractometer system (Burevestnik, USSR). Ni-filtered Cu K, radiation (X = 1.54 A) was used. The diffractograms were indexed by comparison with the American Society of Testing Materials (ASTM) index cards. For determination of the inorganic phase content, the dry samples were incinerated for 8 h at 450’ C in air atmosphere. The mass of the sample was measured before and after ashing. The mass of the ash, expressed as percent of dry mass, was considered as representative for the inorganic phase content. Student’s r-test (P < 0.05) was used for determination of statistical significance of the observed differences.

Results The analysis of blood serum from all animals before the beginning of cholesterol diet is presented in Table 1. In the course of the experiment, the values did not exhibit any statistically significant changes, except from the increase in concentration of cholesterol in cholesterol-fed rabbits

TABLE

1

COMPOSITION OF RABBIT BLOOD THE START OF THE CHOLESTEROL

SERUM DIET

BEFORE

Mean+S.D. Determined

parameter

Cholesterol a Triglycerides a Inorganic phosphorus Ionized calcium Alkaline phosphatase Na Mg P S Cl K Ca Fe CU Zn Br Sr

Concentration

’

(mg/l)

5.86& 1.22 1.23+ 0.28 43.8 f 4.4 64.7 i 14.1 3.29f 1.12 2820 +490 35.6 + 9.4 161 * 34 1250 f290 3060 +780 204 + 52 138 f 46 1.58* 0.40 1.04+ 0.17 1.29+ 0.26 4.58? 1.19 < 0.27

a mM/l. h IU/l.

to (47.6 + 11.6) mM/l (mean + SD.) after 2 weeks. This level remained constant afterwards. The cross section of rabbit aortic wall after 187 days of the diet is shown in Fig. 1. The atheroma occupied almost the entire lumen of the vessel. The results of histological staining obtained for the sample presented in Fig. 1 are illustrated in Fig. 2. The Mallory-azan staining showed well separated layers of the aortic wall: adventitia, media and very thick intima at the place of atherosclerotic plaque (atheroma). The atheroma contained large “foam cells”, while the media showed no morphological alterations. The von Kossa stain visualized the inorganic deposits in the form of dispersed grains or clusters. The clusters were localized outside the regions occupied by the “foam cells”. The Sudan black B stain showed lipids scattered irregularly within the atheroma (almost exclusively). The Weigert’s stain for elastin revealed that the elastic laminae of the media were well preserved. In the atheroma, however, the meshwork of fibers was also observed. The PMP results obtained for the rabbits fed cholesterol diet for 187 days is presented in Fig. 3.

Fig. 1. Rabbit aorta (X 25). Unstained specimen showing the structure of the vessel. The black strips show the normal wall structure while the remaining part of the sample represents the atheroma. The lumen of the vessel is visible at the bottom.

The section adjacent to that shown in Fig. 2 was irradiated. The distributions of element concentrations disclosed a close correlation between P, Ca and Sr. There were also elevated Fe and Zn levels at sites of Ca peaks. The mean concentrations of elements detected within inorganic deposits were collected in Table 2. It should be emphasized that in contrast to the previously reported results [l-3], the elemental composition of the inorganic deposits was determined in situ. For inorganic deposits detected in the aortic media of cholesterolfed rabbits only the mean concentrations of elements at the onset (0 day) and at the end (210 days) of the diet are included, since we did not observe any statistically significant differences for the intermediate durations of the diet. The same pertains to the control animals. In atheromas, the

concentrations are given for 4 durations of the diet, since the atheroma did not develop after duration of the diet shorter than 146 days. It should be pointed out that for inorganic deposits detected in the media, statistically significant differences in the element concentrations were not observed between cholesterol-fed rabbits and controls. In the course of experiment, only Ca and Sr levels were elevated after 210 days for both groups of animals. In the atheroma, the differences were observed also in Ca and Sr concentrations; additionally, the Cu content correlated with the duration of the diet. It should be stressed that the observed concentrations of elements were scattered within a broad range what caused that standard deviation values are very high (up to about 70%): It can be easily explained by different sizes of the detected deposits and by changes in elemental composition during deposit growth. On the other hand, the high values of standard deviations hamper the direct comparison of the mean values. It may be only stated that the massive mineralization was observed in atheromas and the concentrations of elements involved in the calcification process (P, Ca, Sr) increased during the diet reaching the highest values after 210 days. In contrast to atheroma, in the tunica media of both, cholesterol-fed rabbits and controls, only the initiation of the calcification process was observed after 210 days of the diet. The diameter of the individual deposits may be estimated on the basis of Ca concentration which ranged from 100 to 400 000 pg/g (40%). Assuming the average sample thickness 0.3 mg/cm2 and the beam cross section 9 pm2 we can calculate the absolute Ca content in the deposits as (2.7 X lo-l5 to 1.1 X 10-i’) g, i.e. (4.1 X lo7 to 1.6 X lOI’) Ca atoms. This means that the crystals composed of (4.1 X lo6 to 1.6 X 10”) unit cells of hydroxyapatite (HAP, Ca,,(PO,),(OH),) might be detected. Keeping in mind that the unit cell volume of HAP is equal to - 3 x 10-r’ pm3, the volume of the crystals would be equal to (1.2 x 10e3 to 4.8 X 10p3) pm3. It corresponds to deposit diameter of 0.13-2.1 pm (under the assumption of spherical shape). To get more information about the elements involved in the mineralization process of the atheromas, the correlation coefficient (r) was

187

Fig. 2. Histochemical staining of rabbit aorta sections obtained from animal fed cholesterol for 187 days. (a) Mallory-azan stain x 120) showing layered architecture of the vessel wall including tunica adventitia (TA), tunica media (TM) and atheroma (AT) with arrows indicating boundaries. The “foam cells” are indicated by arrowheads. (b) von Kossa stain ( x 110). The inorganic deposits are visible as black dispersed grains (G) or black clusters (C). (c) Sudan black B stain ( x 110). The lipids (black dots) are irregularly scattered within atheroma. (d) Weigert’s stain ( X 170). The elastic laminae of the media (arrowheads) are well preserved.

(

calculated. The values of r enable the division of all the detected elements into 3 groups. Group I consists of P, Ca, Cu and Sr (inorganic group) which always possess a strong correlation to each other (r > 0.61). Group II includes S, Cl, K and Br (organic group) which also have a high value of r ( > 0.68). Group III contains the other elements studied and may be characterized by the absence of apparent correlations. Assuming theoretically that the inorganic deposits in the atheroma consist of a single stoichiometric Ca-P salt, the chemical form of the Ca-P compound which forms the deposit may be recognized on the basis of its Ca/P ratio. For this purpose, the regression line based on the Ca and P concentrations was calculated for all deposits in the sample. The slope of the regression line repre-

sents the mean Ca/P ratio. The values of the slope obtained were 2.01 + 0.05, 2.00 + 0.07, 2.04 f 0.03 and 2.11 + 0.03 within atheroma of rabbits fed cholesterol diet for 146, 167, 187 and 210 days, respectively. Since the Ca/P ratio for defective hydroxyapatite (Ca,,_,(HPG,),(PG,),_x(OH),_,; 0
188 TABLE 2 ELEMENTAL COMPOSITION OF INORGANIC DEPOSITS DETECTED IN NORMAL AS IN THE WALL AND ATHEROMA OF CHOLESTEROL-FED ANIMAL

RABBIT AORTIC WALL AS WELL

Mean*S.D. Abbreviations: W = aortic wall of control animal, CW = aortic wall of cholesterol-fed (cholesterol-fed animals only). Concentrations are given in pg/g.

rabbit,

CA = atheroma

Element

W-l

W-7

cw-1

cw-7

CA-4

CA-5

CA-6

Ca-7

P f S dz Cl + K + Ca f Fe f cu + Zn + Br + Sr f

8380 1010 8620 760 10700 2 140 4770 730 1140 690 147 101 5.21 2.18 192 87 76.8 33.8 3.31 2.45

10200 2 500 8970 1120 11500 1400 4900 620 2030 1110 202 185 5.31 2.66 173 73 89.1 39.3 7.40 4.91

8970 2 160 8770 1340 11100 2000 4790 560 1150 620 258 220 5.28 2.30 171 68 69.1 43.0 3.63 2.91

9 560 2 730 8 560 1820 10800 2 520 4460 690 1740 1150 254 206 5.50 3.17 183 72 82.7 39.1 6.74 5.20

19500 13400 5 560 2270 9 500 2920 2980 1050 22500 17600 174 121 9.84 5.74 82.9 48.8 60.7 34.2 51.6 34.0

21900 15000 6110 2710 8740 2660 3060 1380 26400 20 700 251 190 14.3 8.8 86.8 53.9 57.3 31.3 59.3 43.5

22900 16500 5400 2460 7660 3420 2760 1190 30700 20900 216 184 16.6 7.9 76.5 49.9 55.2 31.4 78.5 44.3

28 800 19 800 4650 2200 6560 3610 2050 1110 39700 29 700 207 166 25.6 14.5 52.0 32.3 47.6 26.5 144 102

210

146

167

187

210

Duration of diet (days)

0

210

0

neous Ca distribution within the deposit. In Fig. 4, the plots for 4 atheroma samples (146, 167, 187 and 210 days of the diet) are presented. It is clearly visible that in the course of the experiment, the number of deposits with a larger diameter increased. The EMP studies revealed a close correlation between P and Ca (Fig. 5). The Mg concentration did not correlate, however, with either P or Ca. This speaks against the possibility that Mg might be incorporated into inorganic deposits during the aortic wall mineralization of hypercholesterolemic rabbits. The crystalline structure of the inorganic deposits in atheroma and aortic wall proper was investigated using IR and XRD techniques. Unfortunately, the amount of inorganic material in the latter samples was insufficient to perform IR or XRD studies. We used the simplest method to determine the content of inorganic material in an organic matrix, i.e. the measurement of the ash

content. The obtained results indicated that in the media and adventitia layers the amount of the ash was less than 0.2% of the dry mass, while in atheroma it amounted to, at least, 2.5%. The IR spectra obtained for 4 atheroma samples are presented in Fig. 6. In the case of rabbits fed cholesterol for 187 and 210 days, we observed the bands at 1093, 1057, 1035, 961, 604, 582 and 564 cm-‘as well as the band at 630 cm-’ characteristic for PO:and OH- groups, respectively, incorporated in the HAP lattice. For the samples obtained from rabbit fed cholesterol diet for 146 and 167 days the bands of PO:group were not separated, but the OH- band was visible. Moreover, in these samples we did not observe a splitting of the PO:bands at 560-580 cm-‘. It should be noted that the bands characteristic for the CO:group at 1400-1500 cm-’ and at 870880 cm-’ were not observed in the spectra from all the investigated samples. The same was true for the band of the P20;- group at 715 cm-’ and the

189 25

@g/cm4

5oo-

5000;~

r

I

10000

w

0’

146 d

167d

thickness

P

S Cl K Ca 3dCa concentration

Fe Zn

Fig. 4. Relative frequency polygons for inorganic observed in the atheroma of rabbits fed cholesterol 167,187 and 210 days.

deposits for 146,

Br Sr Fig. 3. Section of rabbit aorta irradiated by a proton microprobe. The points of measurements form the black line visible in the upper part of the micrograph. Data recorded along this line are presented below in the plots. They show the thickness of the section and concentrations of elements.

band of the HPOigroup at 870 cm-‘. The XRD diffraction patterns (Fig. 7) were indexed on the basis of the strongest reflections identified as a hexagonal hydroxyapatite structure in all atheroma samples. The following values of the interplanar lattice spacing (D) were determined: 3.41, 2.82, 2.79, 2.12 and 2.64 A for all samples ((hkl) values equal to (002), (211), (112), (300) and (202) respectively). The estimated relative intensities were 71, 100, 85, 78 and 83, respectively. Additionally, for the rabbits fed cholesterol for 210 days the values of D equal to 3.09, 2.00, 1.91 A (hkl) equal to ((210) (203) and (222), respectively), having the relative intensity of 38, 57 and 43 were determined. The observed interplanar

lattice spacing did not differ from sample to sample by more than 0.02 A and exhibited the excellent agreement with the HAP data (ASTM card 9-432). However, a careful examination of relative intensities of the detected peaks revealed some discrepancies both between samples and in comparison with HAP. The average crystal size was estimated from the broadening of the well sep-

0

600pm

Fig. 5. Electron microprobe measurements of the distributions of Mg, P and Ca concentrations in aorta of hypercholesterolemic rabbit. Note that Mg distribution does not mimic that of P and Ca.

Discussion

WAVE

NUMBER

(cm-1 1.

Fig. 6. The infrared absorption spectra of aorta from cholesterol-fed rabbits in the region 2000-400 cm-‘. Four different durations of the diet: 146 (A), 167 (B), 187 (C) and 210 days (D) are presented.

arated line (002). The values obtained ranged from 0.09 to 0.12 pm and correlated with the duration of the diet.

Fig. 7. X-ray diffraction patterns (Cu K, radiation) of the aorta from cholesterol-fed rabbit. Two different durations of the diet: 210 (1) and 146 days (2). as well as the model compound (synthetic hydroxyapatite) (3) are presented. The diffractogram of the hydroxyapatite was indexed.

These results confirm that cholesterol diet did not disturb the P and Ca homeostasis, i.e., the 2 elements very important for the calcification process. Therefore, the investigated model can be considered as a model of dystrophic calcification, i.e., calcification within altered or necrotic tissue at physiological CaxP ion product. The diet caused histochemically documented changes of the blood vessel morphology. It induced lipid-containing lesions of tunica intima, characteristic for atherogenesis. At the same time, the morphology of tunica media and its elemental composition were unchanged. The observed alterations could not be satisfactorily differentiated from changes associated with aging, observed in control animals. Therefore, the presented results indicate that the cholesterol-fed rabbit model may only be used to study atheroma mineralization. For the mineralization of the tunica media (Mbnckeberg’s sclerosis) [29], factors other than a disturbance of lipid homeostasis may be responsible. Two mechanisms are probably involved in the aortic wall mineralization during atherogenesis. The cholesterol-fed rabbit model may be used to study only one of them. The presented results allows us to follow the temporal development of the mineralization process in the atheroma. We found, in agreement with earlier results [30], that high content of lipids in a tissue facilitated mineralization. The initiation of the mineralization process might result from cell degeneration correlated with the formation of matrix vesicles 1311 as the nucleation sites. The process of hydroxyapatite deposition, however, is complex and may be regulated by many other factors such as proteolipids [32], calcium-acidic phospholipid-phosphate complexes [5], proteolytitally modified elastin [33] or proliferating collagenous connective tissue matrix [34]. Other factors may influence the growth of deposits. The IR and XRD spectra confirmed the presence of a single phase (HAP) within the deposits. This corresponds with the previously reported results that in a solution of low supersaturation and under physiological pH, defective HAP is precipitated without precursor phases [35,36]. The lack of a splitting of IR bands at 560-580 cm-’ and the inseparability

191 of the bands in the region 950-1150 cm-’ were observed for atheroma samples of rabbits fed cholesterol diet for 146 and 167 days. Probably, PO:- bands were not separated by IR-spectroscopy because of resolution effects associated with small crystal size and/or strain contents. In the course of the diet, the perfection of the crystals (distinct separation of IR bands) increases and the deposits acquire a larger diameter. The relative intensities of XRD lines differ from that of model compounds, regardless of the duration of the diet. The incorporation of other ions instead of Ca in the HAP lattice could account for this finding. With this information it is possible to construct the following model of deposit growth. The precipitation of inorganic phase is a permanent process, correlated with the atheroma development. The formed deposits grow in the course of atherogenesis. We can estimate linear growth rate of deposits on the base of the maximal deposit diameter observed (- lop2 pm/day). In comparison with the values obtained for other inorganic systems [37], the deposit growth is a slow process. Concerning the involvement of other elements in the process, the deposit growth is correlated with Sr and Cu accumulation. The higher Sr content simply results from elevated Ca concentration and chemical similarities between both elements (Sr/Ca ratio ranged from 2.62 x 10e3 to 3.12 x lo-‘). The most probable explanation of Cu accumulation in the course of the deposit growth is the adsorption of blood-derived Cu macromolecules onto the deposits surface. The high specific surface of hydroxyapatite equals to 100-200 m2/g [38]. It should be pointed out that Mg in contrast to Sr is not incorporated into the deposits. The latter observation may be considered as the proof that different mechanisms are involved in media and atheroma mineralization. The role of Mg ions in the calcification of the tunica media was described [3]. It is difficult to answer unequivocally the question why Mg ions, in contrast to Sr atoms, do not occupy Ca positions in the crystal lattice? One might postulate that different ionic radii of Ca2+, Mg2+ and Sr2+ (1.00, 0.72 and 1.18 A, respectively) are responsible for this phenomenon. Although the entry of Sr ions in the Ca position is accompanied by a structural distortion, the hydroxyapatite crystals can still exist, while in the

case of the replacement of Ca by Mg they cannot. Further studies are needed to explore the influence of Mg ions on the atheroma mineralization. A special comment should be devoted to the presence of the other phases in the atheroma especially for the 146 and 167 day of the diet. Although many Ca-P compounds were postulated to be involved in the mineralization process [35,39,40] the obtained results indicate that other crystalline phases (t&calcium phosphate, octacalcium phosphate, brushite) participate in atheroma mineralization in trace amounts (less than - 10%). It is also possible that we deal with the amorphous Ca-P salt (ACP, Ca,(PO,O,). The involvement of amorphous calcium phosphate as a precursor phase in the mineralization process was both postulated [35] and ruled out [41]. Unfortunately, the amorphous material does not influence the XRD lines, as well as the observed IR spectra may result both from a mixture of small crystalline and amorphous material and from imperfections of the crystallites. On the other hand, it was reported [42] that the presence of lipids seriously delayed the transformation of amorphous material to HAP. Therefore the presence of HAP as the only phase at the end of the experiment (210 days) does not confirm a substantial involvement of amorphous material in the atheroma mineralization. The concluding remarks deal with the problem of what can be learned from these results about human aortic wall mineralization. Our studies have demonstrated that atheromatous plaques (induced by the disturbance of the lipid homeostasis) undergo mineralization almost immediately after formation. At the same time the data do not confirm any influence of the disturbance of lipid homeostasis on the mineralization of tunical media. As revealed by the IR and XRD spectra and the Ca/P ratios, the only mineral observed was hydroxyapatite regardless of the duration of the atherogenesis. In the course of the atherosclerotic plaque development, the diameters of the inorganic deposits increase (- lop2 pm/day) and the crystals become more perfect. The observation that Ca and Sr concentrations within inorganic deposits correlate, whereas the Ca and Mg concentrations do not, is of special interest. It should

192 also be noted that the presented results differ from data reported for human autopsy samples: (1) the amount of ash in rabbit samples (maximum 6.5% of dry mass) is much lower than in the human material (even 57% was reported [l]); (2) carbonate groups are absent in rabbit in contradiction to the human material [1,2]; and (3) the Sr/Ca ratio is higher than that reported in human atheroma samples (3.46 X lop4 [7]). The first difference can be easily explained by different duration of the diet the second. one arises, probably, from differences in blood CO, content. The third discrepancy arises from different Sr content in the diet of humans and rabbits. Meat-eaters ingest lower amounts of Sr than do vegetarians, because Sr is accumulated only in bones but not in the soft tissue of vertebrates while it is homogenously distributed in plants. In our opinion, the above differences do not seriously influence the atheroma mineralization. Acknowledgement

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