Vol.2, No.1, 19-25 (2011)
doi:10.4236/jbpc.2011.21003
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
Journal of Biophysical Chemistry
Study on the structure and composition of aortic valve
calcific deposits: Etiological aspects
Rafael. M. Prieto1,2, I. Gomila1, O. Söhnel3, A. Cost a-Bauza1,2, O. Bonnin4, Felix Grases1,2
1Laboratory of Renal Lithiasis Research, University Institute of Health Sciences Research (IUNICS), University of Balearic Islands,
Palma of Mallorca, Spain;
2CIBER Fisiopatología Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, Madrid, Spain;
3University of J.E. Purkyne, Faculty of Environmental Studies, Usti n.L., Czech Republic;
4University Hospital Son Dureta, Palma of Mallorca, Spain;
Received 5 November 2010; revised 19 November 2010; accepted 25 November 2010.
ABSTRACT
The structures and chemical compositions of
valve calcific deposits were investigated. The
deposits was chosen arb itrarily and subjected to
chemical analysis, observation with scanning
microscope, semi-quantitative determination of
Ca, Mg, Na, K, P and C elements by energy dis-
persive X-ray, X-ray diffraction and Fourier
transform infra-red spectroscopy carried out.
These deposits were found to have non-uniform
internal structures composed of layers of a
structureless aspidinic inorganic material, sub-
stantial amounts of voluminous organic material
and in a few samples small spheres were also
present. Two groups of deposits with distinctly
different chemical compositions were identified:
one group with a low Ca/P molar ratio (1.59) and
the other group w ith a high (1.82) Ca/P molar ra-
tio. The deposits belonging to the group with a
low Ca/P molar ratio contain higher concentra-
tion of magnesium and consist of increased
amount of amorphous calcium phosphate. The
deposit s with a high Ca/P molar ratio contain low
concentration of magnesium and consist pre-
dominantly of carbonated hydroxyapatite. The
inorganic material was identified as a poorly
crystalline carbonate hydroxyapatite containing
molecular water of the average formula
Ca9.1Mg0.4(Na,K)(PO4)5.8(CO3)0.3(OH)2. The actual
chemical composit ion of the apatitic solid phase
varies not only from deposit to deposit but also
within the same deposit. The non-uniform inter-
nal structure of the deposits, the occasional
presence of spherical particles and the variable
point composition of the individual deposits in-
dicate that their format ion d id not pro ceed under
more or less constant conditions.
Keywords: Mitochondrial Swelling; Cytochrome C
Release; Calcium; Alloxan; Mitochondri al
Permeability Transition
1. INTRODUCTION
The formation and composition of calcific deposits in
living organisms have been intensively investigated with
different methods in both in vitro and in vivo studies.
This phenomenon is still not very well understood de-
spite all effort devoted to its unravelling. The principle
obstacle hindering conclusive elucidation of calcification
stems from the impossibility of observing the com-
mencement of formation of solid deposits in a living
organism and their subsequent development. In the ini-
tial stages of a calcific deposit, moreover, formation of
nanoparticles is undetectable under physiological condi-
tions with contemporary methods and thus even in vitro
studies cannot provide direct insight into the processes
taking place during this crucial period of solid phase
formation.
The fine structure, chemical composition and crystal-
linity of the deposits arising from the calcification of
human natural and bioprosthetic heart valve have been
investigated by using X-ray diffraction, Fourier trans-
form infrared (hereafter FTIR) and Raman spectra,
chemical and energy dispersive X-ray (hereafter EDX)
analysis and high-resolution transmission electron mi-
croscopy [1-7]. In these studies, however, only the total
compositions of the calcific deposits expressed as the
molar ratio Ca/P were determined.
The variation in the composition of calcific deposits
between different individuals has previously been stud-
ied. However, whether and to what extent the composi-
tion of the calcific deposits in the same individual can
vary is not known.
The object of this study was to determine the extent in
variation in the structure and chemical composition of

R. M. Prieto et al. / Journal of Biophysical Chemistry 2 (2011) 19-25
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
20
calcific deposits formed in explanted natural human aor-
tic valves of the same and different individuals.
2. MATERIALS AND METHODS
2.1. Sample Preparation
Calcified natural heart valves surgically removed from
10 patients were kept in 10% aqueous solution of for-
maldehyde containing 7.65 g of NaCl, 0.724 g of
Na2HPO4 and 0.21 g of KH2PO4 (pH 7.2) in 1000 ml.
From the numerous calcified objects formed within the
valve tissue of each patient a well developed rounded
concretion of size about 2 mm was chosen arbitrarily
and then removed, dried in ambient air, divided into four
parts of similar size and subjected to chemical analysis,
observation with scann ing microscope, semi-quantitative
determination of Ca, Mg, Na, K, P and C elements by
EDX (energy dispersive X-ray), X-ray diffraction and
FTIR (Fourier transform infra-red) spectroscopy carried
out in the reflective mode.
2.2. Electron Microscopy
The samples were glued to a metallic support and ob-
served using a Hitachi S 3400N scanning electron mi-
croscope and a Bruker EDX analyser. The samples were
not covered with gold as its spectral line interferes with
that of phosphorus and distorts the results of EDX
analyses. Several randomly selected sites on each sample
with areas of approximately 0.25 mm2 (hereafter referred
to as a large area) and several areas inside each large site
0.25 μm2 in size (hereafter referred to as small areas or
point measurements) were subjected to EDX analysis.
2.3. Chemical Analysis
Samples of approximately 0.01 g (the smaller samples)
and 0.1 g (the larger samples) from dry deposit were
dissolved separately in 12 M HCl at ambient temperature
for 24 h, diluted to 10 ml and filtered in order to remove
undissolved particles of organic origin. The organic
matter from the larger samples was dried and weighed.
Each solution was then 100 times diluted with deionized
water and used for the determination of calcium and
magnesium or diluted with 0.6 M H2SO4 for phosphoru s
determination. Calcium forms a blue purple complex
with Arzenazo III at neutral pH [8], magnesium forms a
red complex with Calmagite in an alkaline solution [9]
and phosphorus forms a green complex with malachite
green molybdate under acidic conditions [10]. The in-
tensity of solution discoloration was determined spec-
trophotometrically (Microplate Spectrophotometer Pow-
erWave XS, Biotek Instruments, Inc., Winoosk, VT) at
wave lengths of 650, 520 and 630 nm for Ca, Mg and P
respectively.
2.4. X-Ray and Ftir Analysis
A small piece from each concretion with a weight of
about 2 mg was pulverized and used for X-ray diffrac-
tion and then for FTIR reflective analysis. The X-ray
spectra were obtained using XRD Diffractometer (Bruker-
Siemens D5000, Bruker AX GmbH Karlsruhe, Germany)
and the FTIR spectra using a Bruker IFS66 infrared
spectroscope (Bruker AXS GmbH Karlsruhe, Germany).
3. RESULTS
3.1. Accuracy of the EDX Analyses
The accuracy of the calcific concretions chemical
composition deter minations with EDX was assessed for 6
different samples. A small area of approximatel y 0.25 μm2
in size was arbitrarily chosen from each of four samples
and analysed and a lar ge area of about 1 mm2 was selected
on each of two samp les. 12 succes siv e me asur emen ts w er e
carried out in each case. The arithmetical mean, standard
deviation and median were determined for the Ca/P and
Ca/Mg molar ratios in each series of measurements. The
results are shown in Table 1.
As can be seen, the magnitude of the standard devia-
tions are on average about 9 and 17% of the mean values
for the Ca/P and Ca/Mg molar ratios determined with
EDX respectively. The close coin cidence of the arithme-
tic mean and median values indicates that in each series
the distribution of individual measurements is nearly
symmetrical.
3.2. Fine Structure of the Deposits
All studied calcifications of natural human aortic heart
valve of approximately 2 mm in diameter were found to
have uneven compact surfaces, see Figure 1(a). The
interior of each calcification predominantly consists of
blocks of structureless aspidinic matter, see Figure 1(b).
Organic material including fibres was also observed. In
fact, not a single site was found without organic matter,
but its quantity varied significantly. The interiors of all
of the studied deposits are highly inhomogeneous with-
Table 1. Accu racy of the EDX analyses.
1 2 3 4 5∗ 6∗
Ca/P Mean 1.3421.525 1.361 1.476 1.3151.792
σ 0.0920.103 0.167 0.075 0.1490.279
Median1.331.505 1.405 1.484 1.271.78
Ca/MgMean 5.1331.12 7.96 9.33 8.2627.44
σ 0.964.62 1.17 1.10 1.138.03
Median5.0631.5 8.09 9.08 8.26 28.03
∗ large area analyzed.

R. M. Prieto et al. / Journal of Biophysical Chemistry 2 (2011) 19-25
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
21
Figure 1. Internal structure of the calcific deposits. (a) surface
of a deposit, (b) structureless aspidinic layer, (c)inhomoge-
neous interior, (d) small spheres in the cavity.
out a sign of any morphological order, see Figure 1(c).
Small spheres situated in cavities of deposits were in-
frequently observed, see Figure 1(d).
3.3. Chemical Composition of the Deposits
The chemical composition of the deposits was deter-
mined using EDX and expressed as molar percentage of
the constituting elements, specifically Ca, Mg, Na, K, P
and C. These data were converted into molar ratios Ca/P,
Ca/Mg, Ca/C and Ca/(K+Na). Potassium was always
present whereas sodium was missing in 11 out of the 87
data sets. Silicon, chlorine and sulphur were no t detected
by EDX in any of the studied deposits.
87 sets of data were obtained from the 15 samples. In
5 cases, specifically samples 6 through 10, two different
deposits were removed from the same aortic valve and
analyzed separately (the second denoted by an asterisk,
e.g. 6∗). 15 sets containing over 20 molar% of carbon
(obviously organic matter adhering to the analyzed site
surface) were excluded from further processing since a
high content of carbon significantly distorts the deter-
mination of other elements.
The chemical composition of 15 studied calcifications
determined by EDX analysis of areas of 0.25 mm2 and
0.25 μm2 in size are summarized in Tab le 2. Given val-
ues are the arithmetic means of all the data acquired for
each sample.
The calculated molar ratio Ca/P given in Tab le 1 var-
ies in rather wide limits, specifically between 1.29 and
2.55 with the overall arithmetic mean (all individual
values were taken into account) of 1.69 ± 0.21. There is
substantial variation of the Ca/P ratio not only between
the large scanned areas of the same sample but also
among point measurements within the same large area.
Magnesium was found to be present in approximately
70% of the sites; sites with and without magnesium was
detected on the same deposit and even within the same
large area. When present, magnesium is a minor com-
ponent amounting typically to around 5% of the calcium
content (from 0.7 to 10 mol%). The molar ratio Ca/Mg
varies in extremely wide limits, from 16 to about 100
and exceptionally to 200, even within the same sample.
The overall mean of the Ca/Mg molar ratio is 23.6 ±
11.5.
In contrast to magnesium, alkali metals were found to
be always present in all of the studied aortic deposits.
Sodium is absent from 15% of the measurements and
sodium is always accompanied by potassium. The alkali
metals are present at levels approximately 10 mol% (the
overall mean is 9.1 ± 3.3) of that of the main constituent
calcium.
EDX is least reliable for carbon of the measured ele-
ments due to the proximity of its peak to the first peak of
calcium. Carbon was detected at every investigated site,
for both large and small areas. Low carbon content (with
several exceptions that were excluded from further
processing) indicates that it is present predominantly as
inorganic carbon bound as carbonate in the apatitic
phase. The Ca/C molar ratio varies in the range 5-100,
with an overall m ean of 33. 8 ± 17 .7 .
The compositions of 15 calcific deposits were deter-
mined by chemical (wet) analysis and are given in Table
3. The first and second values in each column represent
are the results for the smaller and larger samples, respec-
tively; wt.% is the weight percentage of an organic mat-
ter in the larger sample.
3.4. Structure of the Deposits
The IR spectra of the 10 samples from different indi-
viduals are basically identical, see Figure 2. The spectra
contain a broad, mostly featureless, absorbance band
between 900 and 1200 cm–1 with an indistinct peak at
960 cm–1 and a high frequency shoulder typical of poorly
crystalline hydroxyapatite [11]. The weak absorbance
bands at 871cm–1 and the very week bands at 1430 and
1455 cm–1 signify the presence of carbonate [11,12]. No
band was detected at 864 cm–1 which if present would
indicate the substitution of carbonates for the hydroxyl
groups [13]. The broad band from 3700 to 2800 cm–1
and the weak band at 1645 cm–1 can be ascribed to
structural water in the molecular form [5]. Based on the
FTIR spectra all 10 studied deposits are composed of
poorly crystalline carbonated hydroxyapatite.
The X-ray diffraction spectra of the investigated calci-
fic deposits contain all the peaks characteristic of
stoichiometric HAP, see Figure 3, and can be divided
into a group with distinct merging of the 211 and 112
peaks, which is assumed to indicate the substitution of

R. M. Prieto et al. / Journal of Biophysical Chemistry 2 (2011) 19-25
Copyright © 2011 SciRes. http://www.scirp.org/journal/JBPC/Openly accessible at
22
Table 2. Composition of 15 calcifications determined by EDX.
Sample N Ca/P Ca/Mg Ca/(Na+K) Ca/C
1 5 2.00 ± 0.22
med 1.91 21.1 (1) 9.6 ± 1.8 med 9.6
49.8±29.3
med 35.3
2 4 1,78 ± 0.22
med 1.84
18.3 ± 3.0 (2) 9.5 ± 2.9
med 8.7
30.5 ± 12.4
med 26.9
3 4 1.55 ± 0.14
med 1.49
13.0 ± 9.4
med 15.0
6.9 ± 1.3
med 7.0
50.4 ± 31.9
med 44.6
4 4 1.67 ± 0.11
med 1.67 19.4 (1) 15.8 ± 11.0 med 7.3
29.2 ± 7.4
med 23.4
5 4 1.66 ± 0.29
med 1.76 20.3 (2) 11.4 ± 8.4 med 7.5
21.2 ± 14.9
med 21.0
6 5 1.61 ± 0.10
med 1.59
24.7 ± 14
med 23.3
9.4 ± 2.9
med 10.0
35.3 ± 15.2
med 30.6
6∗ 5 1.65 ± 0.13
med 1.68
19.0 ± 5.6
med 18.5
7.6 ± 1.9
med 7.6
32.4 ± 13.2
38.7
7 5 1.68 ± 0.27
med 1.71 18.8(2) 9.8 ± 4.3 med 8.0
26.0 ± 6.8
med 26.4
7∗ 4 1.61 ± 0.14
med 1.65
19.7 ± 7.1
med 21.9
7.6 ± 1.5
med 7.3
34.1 ± 12.8
med 34.5
8 5 1.86 ± 0.36
med 1.79
48.7 ± 21.8(3)
med 56.4
11.9 ± 6.0
med 8.8
44.3 ± 17.0
med 31.6
8∗ 5 1.71 ± 0.19
med 1.72
27.0 ± 13.0
med 24.4
9.1 ± 2.1
med 8.4
23.9 ± 12.6
med 20.6
9 6 1.64 ± 0.18
med 1.65
22.8 ± 4.8(4)
med 21.2
9.5 ± 1.2
med 9.6
38.6 ± 20.5
med 45.8
9∗ 5 1.71 ± 0.20
med 1.74
22.5 ± 8.9
med 24.7
8.7 ± 2.8
med 7.7
29.3 ± 13.9
32.4
10 6 1.64 ± 0.16
med.1.63
26.6 ± 11.8
med 21.2
7.3 ± 1.6
med 7.4
36.3 ± 16.2
med 31.0
10∗ 5 1.57 ± 0.11
med 1.54
23.7 ± 9.0
med 24.7
6.5 ± 0.71
med 5.7
24.7 ± 10.5
med 21.4
med – median; N – number of measurements; number in parentheses – number of determinations if different from N; ∗ - 2nd deposit from the same individual.
this phenomenon is not observed. Samples 2, 4, 5, 7 and
10 belong to the former group. The comparison of the
phosphate with carbonate [12], and a group for which
Ta ble 3. Composition of 15 calcifications expressed as molar
ratios determined by chemical analysis. peaks and the background intensities according to [5]
indicates that a significant fraction of amorphous phos-
Sample 1 2 3 4 5
Ca/P 1.25;1.40
∅1.32
1.79;2.11
∅1.95
1.45;1.59
∅1.52
1.61;1.68
∅1.64
1.58;1.82
∅1.70
Ca/Mg 35;18.2
∅26.6 ̶ ;11.4 26;6.7 ∅16.3
30;6.9
∅18.4
37;6.5
∅21.7
wt.% 15.5 13.8 18.1 11.5 17.2
Sample 6 7 8 9 10
Ca/P 1.62;1.83
∅1.72
1.60;1.57
∅1.58
1.52;1.82
∅1.67
1.55;1.49
∅1.52
1.48;1.67
∅1.57
Ca/P ∗ 2.05 1.64 1.74 1.67 1.62
Ca/Mg 35;18.2
∅26.6
26;10.6
∅18.3
30;9.5
∅19.7
28;17.5
∅22.7
27;26.9
∅27
Ca/Mg∗ 10.4 6.8 12.6 6.9 18.9
wt.% 18.9 9.0 16.4 23.3 15.0
Figure 2. FTIR spectra of the c alcific deposits.
∗ - 2nd deposit (a smaller sample) from the same individual

R. M. Prieto et al. / Journal of Biophysical Chemistry 2 (2011) 19-25
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
23
Figure 3. X-ray diffraction spectrum of sample No. 5.
phate is present in all studied samples.
4. DISSCUSION
The 15 studied natural human aortic calcific deposits
consist of amorphous and poorly crystalline carbonated
hydroxyapatite containing molecular water in which
carbonate is substituted for phosphate and not hydroxyl
groups as shown unambiguously by the X-ray and FTIR
spectra. All deposits also contain between 9 and 23 wt.%
of organic matter.
The chemical composition of the calcifications varies
widely not only among individual samples but also
within the same deposit; ev en the composition of no t too
distant small areas (point composition) are dissimilar.
Different deposits from the same individual do not have
identical composition, but the deviations are not as pro-
found as the variations among different individuals.
The composition of our deposits differs from that re-
ported elsewhere [5,7] namely in the levels of potassium,
magnesium and silicon. Our deposits contained non-
negligible amount of potassium and none silicon in con-
trast to virtually none potassium and about 3 mol% of
silicon or the “substantial in corporation of Si” [4,5].
When expressed in terms of the Ca/P molar ratios of
the smaller and larger samples, the chemical composi-
tions of the same deposit are reasonably close. The Ca/P
ratio as determined by wet chemistry was found to vary
between 1.25 and 2.11 with an overall mean of 1.61 ±
0.19. The Ca/P molar ratio determined by EDX varied
between 1.29 and 2.25 with an overall mean of 1.71 ±
0.14. Both values are below the average 1.83 ± 0.03
(spread 1.62-2.13) and 1.91 ± 0.02 (spread 1.87-1.93)
reported for natural aortic deposits in [3] and [5] respec-
tively, but are close to the value of 1.676 reported in [4].
The determined Ca/P ratio corresponds to that of hy-
droxyapatite (1.67 ) wh ich has also been found in mature
arteriosclerotic plaque [1].
The Ca/P ratios determined by EDX and wet chemis-
try are in good agreement with the exception of those of
sample 1. Taking into account the accuracy of the
chemical composition determination and the fact that the
median is less influenced by a remote value than the
arithmetical mean and is thus a better quantity for com-
parison, it seems that two different groups of deposits
can be distinguished based on the Ca/P ratio: 1) A group
with high Ca/P ratio—specifically samples 1, 2, 5 and 8
and 2) A group with low Ca/P ratio, n amely samples 3, 9
and 10. The remaining three samples 4, 6 and 7 exhibit
composition that due to the uncertainty in the data can-
not be unequivocally assigned to the abovementioned
groups or identified as a third grou p with an intermediate
composition. The other ratios, i.e. Ca/Mg, Ca/(Na + K)
and Ca/C, vary within the same limits in both groups. It
should be stressed that the same classification transpires
from both EDX and wet chemistry with the ex ception of
sample 1 for which the difference between the analytical
results is considerable.
The average Ca/P ratios of the three distinct groups
are 1.82, 1.66 and 1.59 using the arithmetic mean of
medians or 1.82, 1.65 and 1.61 using EDX means and
1.80, 1.65 and 1.54 using wet analysis means. Thus it
seems that the deposits belonging to the group with the
high Ca/P ratio consist of carbonated hydroxyapatite
(theoretical Ca/P > 1.67) whereas in deposits of the
group with the low Ca/P ratio amorphous calcium phos-
phate (theoretical Ca/P = 1.5) prevails.
The Ca/Mg molar ratio determined by wet chemistry

R. M. Prieto et al. / Journal of Biophysical Chemistry 2 (2011) 19-25
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
24
seems to be less reliable than the Ca/P ratio considering
the significant differences between the smaller and larger
samples shown in Ta bl e 3 . The overall arithmetic mean
is 21.4 ± 10.4. The Ca/Mg ratio determined by EDX
varies between 11.4 and 65.7 with an overall arithmetic
mean 23.4 ± 9.6 which is, however, in a good agreement
with the wet chemistry results. The amount of magne-
sium in our deposits is con siderably high er than found in
[5] which reported a value for the Ca/Mg ratio of ap-
proximately 120.
These results for the Ca/Mg molar ratio indicate that
magnesium is a minor component, if present at all, of the
studied deposits; the magnesium content is on average
only 5% of that of the major component, calcium. The
content of magnesium varies not only among individual
deposits but also among discrete parts of the same ob-
served large area; some sites in a large area can contain
magnesium even though the others do not.
Wet chemistry provides the composition averaged
over a relatively large part of a deposit and so it reflects
the total compositio n of the entire depos it rather than the
variation of the composition within a deposit. EDX is the
only currently available method for determining the
chemical composition of selected small areas of deposits.
However the accuracy of this method is rather limited, as
can be seen in Table 1, and thus provides only rough
insight into the in homogeneities of deposit composition.
The data in Table 2 and specifically the comparison of
the standard deviations and medians show the wide
variations in the composition of the solid phase on the
deposits. The data spread is narrower in the case of the
large area measurements than in the case of the small
area measurements. However, the average values of the
Ca/P and Ca/Mg ratios determined for the small and
large scanned areas of the same sample are reasonably
close. The substantial difference b etween po int composi-
tion of the phosphatic phase, particularly for deposits 1,
2, 5, 7, 8, 9 and 10 and the large fluctu ations of the mag-
nesium concentration in nearly all samples are signifi-
cant even when the uncertainties of ± 9% and ± 17%
(the estimated magnitudes of standard deviation ex-
pressed as percentages of the mean) in the Ca/P and
Ca/Mg ratios respectively are consid ered.
With only one exception, all the studied sites con-
tained potassium, more frequently in combination with
sodium, in proportions around 10 mol % of the major
component calcium. Potassium is in the minority if both
elements are present. Potassium is a minor component of
plasma, 4.5 mmol/L of potassium compared to 140
mmol/L of sodium. The presence of potassium in the
deposits is not an artefact created during the storage of
the valves in formaldehyde solution containing also po-
tassium because 1) The surfaces of the deposits are com-
pact and thus resistant to penetration of solution into
their interiors during storage, 2) The sites analyzed with
EDX were situated inside each deposit, 3) The amount
of potassium varies significantly between individual
points situated close to each other and 4) The Ca/(Na+K)
ratio is fairly similar for different deposits and even
throughout each deposit. Neither chlorine nor sulphur
was detected in deposits which indicates that the alkali
metals are not present in the form of chlorides or sul-
phates, so these elements are likely be incorporated in
the HAP lattice replacing calcium.
EDX is least reliable for quantitative determinatio n of
carbon because carbon is a light element and also pro-
duces only one peak that is close to one of the two peaks
of calcium. No site was without carbon. The average
Ca/C molar ratio was found to be around 30 (with sev-
eral exceptions), i.e. the amount of carbon present is
around 3 mol% (a similar content of carbon was repor ted
in [5]) which indicates that the carbon present is pre-
dominantly “inorganic” and therefore bound in the
structure of phosphate. However, a substantial amount of
organic matter which is not incorporated in the solid
phase (between 9 and 23%, see Table 3) indicates its
important r ol e i n the formation of calcific deposits.
The composition of the solid phase, i.e. the Ca, P, K,
Na, Mg and C contents vary between the measured
points in all studied deposits. Taking into account the
randomness of the selection of analyzed sites we conclude
that the composition of each deposit varies throughout
its whole volume.
Based on the determined average molar ratios, Ca/P =
1.71, Ca/Mg = 23, Ca/(Na+K) = 9.3 and Ca/C = 34, the
average total composition of the calcific deposits is
generally Ca10-5 x- yMgy(Na,K)10x(PO4)6-2x(CO3)3x(OH)2
where in our case x = 0.1 and y = 0.4 tj.
Ca9.1Mg0.4(Na,K)(PO4)5.8(CO3)0.3(OH)2. The respective
molar ratios calculated from this formula, Ca/P = 1.57,
Ca/Mg = 22.7, Ca/(Na+K) = 9.1 and Ca/C = 30.3, cor-
respond well to the experimentally determined
values. This formula is close to the formula
Ca8.66Mg0.22Na0.32H0.14(PO4)5(CO3)1.22(OH,F,Cl) 0.80
suggested in [14]. The actual composition of the solid
phase at various sites deviates from the formula given
above.
5. SUMMARY
The studied human aortic valve calcific deposits con-
tain poorly crystalline solid phase and can be divided
into two distin ct grou ps, one consisting predominantly of
carbonated hydroxyapatite with high Ca/P molar ratio
and the other of amorphous calcium phosphate with
lower Ca/P molar ratio. The non-uniform internal fine
structure of these deposits, the occasional appearance of
spherical particles and the variable spatial point compo-

R. M. Prieto et al. / Journal of Biophysical Chemistry 2 (2011) 19-25
Copyright © 2011 SciRes. http://www.scirp.org/journal/JBPC/Openly accessible at
25
sition of the individual deposits indicate that their for-
mation does not proceed under constant conditions.
6. ACKNOWLEDGMENTS
This work was supported by Fundació Barceló (Ref. 1458/2007)
and project grant CTF 2010-18271 from the Ministerio de ciencia e
innovación del Gobierno de España. I.G. expresses her appreciation to
the Conselleria d’Innovació i Energia del Govern de les Illes Balears
(Spain) for a fellowship supporting her work. O.S. is grateful to the
University of Balearic Islands for grant which made this cooperation
possible.
REFERENCES
[1] Tomazic, B.B., Brown, W.E. and Schoen, F.J. (1994)
Physicochemical properties of calcific deposits isolated
from porcine bioprosthetic heart valves removed from
patients following 2-13 years function. Journal of Bio-
medical Materials Research Part A, 28(1), 35-47.
doi:10.1002/jbm.820280106
[2] Tomazic, B.B., Edwards, W.D. and Schoen, F.J. (1995)
Physicochemical characterization of natural and biopros-
thetic heart valve calcific deposits: implications for pre-
vention. The Annals of Thoracic Surgery, 60(Suppl. 2),
S322-S327.
doi:10.1016/0003-4975(95)00205-Y
[3] Mikroulis, D., Mavrilas, D., Kapolos, J., Koutsoukos P.G.
and Lolas, C. (2002) Physicochemical and microscopical
study of calcific deposits from natural and bioprosthetic
heart valves. Comparison and implications for minerali-
zation mechanism. Journal of Materials Science: Mate-
rials in Medicine, 13(9), 885-889.
doi:10.1023/A:1016556514203
[4] Delonge, C., Lawford, P.V., Habesch, S.M. and Carolan,
V.A. (2007) Char acterization of the calcification of cardiac
valve bioprostheses by environmental scanning electron
microscopy and vibrational spectroscopy. Journal of Mi-
croscopy, 228(1), 62-77.
doi:10.1111/j.1 365-2818.2007.01824.x
[5] Gilinskaya, L.G., Grigorieva, T.N., Okuneva, G.N. and
Vlasov, Yu.A. (2003) Investigation of phatogenic miner-
alization on human heart valves. I. Chemical and phase
composition. Journal of Structural Chemistry, 44(4),
622-631.
doi:10.1023/B:JORY. 0000017938.42883.9f
[6] Gilinskaya, L.G., Okuneva, G.N. and Vlasov, Yu.A. (2003)
Investigation of pathogenic mineralization on human
heart valves. II. ESR spectroscopy. Journal of Structural
Chemistry, 44(5), 813-820.
doi:10.1023/B:JORY. 0000029819.16581.8a
[7] Gilinskaya, L.G., Rudina, N.A., Okuneva, G.N. and Vlasov,
Yu.A. (2003) Pathogenic mineralization on human heart
valves. III. Electron microscopy. Journal of Structural
Chemistry, 44(6), 1038-1045.
doi:10.1023/B:JORY. 0000034811.28903.9b
[8] Michaylova, V. and Ilkova, P. (1971) Photometric deter-
mination of micro amounts of calcium with arsenazo III.
Analytica Chimica Acta, 53(1), 194-198.
doi:10.1016/S0003-2670(01)80088-X
[9] Chauman, U.P.S. and Ray Sarkar, B.C. (1969) Use of
calmagite for the determination of traces of magnesium
in biological materials. Analytical Biochemistry, 13(1),
70-80.
doi:10.1016/0003-2697(69)90105-5
[10] Martin, M., Celi, L. and Barberis, E. (1999) Determina-
tion of low concentrations of organic phosphorus in soil
solution. Communications in soil science and plant
analysis, 30(13-14), 1909-1917.
doi:10.1080/00103629909370341
[11] Pleshko, N., Boskey, A. and Mendelsohn, R. (1991) Novel
infrared spectroscopic method for the determination of
crystallinity of hydroxyapatite minerals. Biophysical Jour-
nal, 60(4), 786-793.
doi:10.1016/S0006-3495(91)82113-0
[12] Kapolos, J. and Koutsoukos, P.G. (1999) Formation of
calcium phosphate in aqueous solutions in the presence
of carbonate ions. Langmuir, 15(19), 6557-6562.
doi:10.1021/la981285k
[13] Ito, A., Maekawa, K., Tsutsumi, S., Ikazaki, F. and Tatei-
shi, T. (1997) Solubility product of OH-carbonated hy-
droxyapatite. Journal of Biomedical Materials Research
Part A, 36(4), 522-528.
doi:10.1002/(SICI)1097-4636(19970915)36:4<522::AID
-JBM10>3.0.CO;2-C
[14] Tomazic, B.B. (2001) Physicochemical principles of
cardiovascular calcification. Clinical Research in Cardi-
ology, 90(Suppl. 2), 1168-1180.