Download The role of bivalent metals in hydroxyapatite structures as revealed

The role of bivalent metals in hydroxyapatite structures as
revealed by molecular modeling with the HyperChem
software
Izabela Gutowska,1 Zygmunt Machoy,1 Bogusław Machaliński2
Department of Biochemistry and Chemistry, Pomeranian Medical University, Szczecin, Poland.
2
Department of General Pathology, Pomeranian Medical University, Szczecin, Poland
1
Received 22 March 2005; accepted 20 April 2005
Published online 1 September 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30511
Abstract: Hydroxyapatite, the major component of bone,
demonstrates significant reactivity with metals. Knowledge
of spatial structure and energy data of the molecule helps
understand the binding of metals by hydroxyapatite and
elucidate the chemical and physical properties of such complexes. We used HyperChem software (Hypercube Inc.) to
analyze the structure of hydroxyapatite when the central
calcium atom is replaced by one of the metal ions (Mg, Cu,
Zn, Fe, Cr, Mn) marked by us in bone. Our results show that
hetero-ionic exchange affects composition and leads to deformation of hydroxyapatite crystals. Replacement was ac-
companied by changes in bond lengths between oxygen and
calcium atoms in the hydroxyapatite molecule and by displacement of groups of atoms surrounding the central calcium atom. The use of molecular modeling as a computational tool enabled a preliminary and theoretical
understanding of chemical structure without the need for
laboratory tests. © 2005 Wiley Periodicals, Inc. J Biomed
Mater Res 75A: 788 –793, 2005
INTRODUCTION
Ca10(PO4)6(OH)2 or 3Ca3(PO4)3 䡠 Ca(OH)2. Spatially,
hydroxyapatite is a triangular structure made up of a
central Ca(OH)2 and three surrounding Ca3(PO4)3
groups.2 Hydroxyapatite crystals are encased in an
aqueous envelope that sticks to the crystals thanks to
polarized and hydratized calcium and phosphate ions.
This envelope plays an important role during exchange of ions between the hydroxyapatite crystal and
extracellular fluid. Ion exchange depends on ion type,
size, and site in bone structure. Potassium and chlorine ions of the aqueous envelope undergo rapid and
total displacement by ions present in the extracellular
space. Exchange is slower for magnesium, sodium,
and carbonate and is slowest in the case of calcium
and phosphate.3 In comparison to ions located in the
crystal core, those in external layers are much more
predisposed to substitution.3,4 Much less is known
about substitution of the central Ca2⫹ atom. Okazaki5
is of the opinion that calcium may be substituted by
magnesium and sodium, phosphate by carbonate and
citrate and hydroxyl groups by fluoride and chloride.
As molecular structure and function can nowadays be
studied through model building and computation
known as molecular modeling,6,7 we devised a theoretical study to elucidate replacement of the calcium
atom by another bivalent metal. An important advan-
Pollution of the natural environment with heavy
metals is among the most concern-raising problems in
contemporary ecology. Metal toxicity in human and
animal organisms develops during accumulation,
which principally targets hard tissues. On the molecular level, incorporation of metals into bones depends
on interactions with hydroxyapatite, their chief structural component. Bone hydroxyapatite is a natural
compound exhibiting significant reactivity to metals.
This property is important in view of the role of bone
in living organisms. Hexagonal cells located in outermost layers of hydroxyapatite and capable of binding
other ions are called the “valency surplus” of bone.
Additional valency is used not only to bind ions in the
hydroxyapatite lattice but also for uptake of exogenous metals.1 The immense surface of hydroxyapatite
predisposes its chemical components to displacement
by other atoms with a similar atomic radius. The
chemical formula of hydroxyapatite has been approximated in the middle of the 20th century as
Correspondence to: Zygmunt Machoy; e-mail: IzaGut@
poczta.onet.pl
© 2005 Wiley Periodicals, Inc.
Key words: hydroxyapatite; structure spatial; bivalent metals substitution; molecular modeling
HYDROXYAPATITE STRUCTURE MOLECULAR MODELING
789
Figure 1. Spatial model of the hydroxyapatite molecule with calcium as the central atom (Ca, big white; O, small black; P,
small white; H, smallest black).
tage of molecular modeling is that it respects the basic
physical and chemical laws of the micro-world of
molecules. Molecular modeling is the science and art
of studying molecular structure and function through
model building and computation.6
configuration. The next step is to minimize the chair structure by performing a molecular mechanics optimization.
The optimization method is selected from a palette of ab
initio and eight semi-empirical calculations.10 We chose ETH
method developed by Hückl12 as a special case of the molecular orbital method with pi-electron approximation.10
Now we compare the structural properties of the minimized
system with those of the model-built structure.
MATERIALS AND METHODS
Samples (10 mg) of powdered deer mandibular bone were
dissolved in 65% nitric acid and the content of Mg, Ca, Cu,
Zn, and Fe were determined with atomic absorption spectroscopy, whereas Cr and Mn were determined with inductively coupled plasma–atomic emission spectrometry.
We used the HyperChem software8 –10 to determine the
properties of molecules by analyzing energy parameters at
various energies of the molecule and estimating differences
between energy states of the system. This program can
perform specific actions on selected atoms, residues, and
molecules. HyperChem also enables conformational studies
of the molecule through computations of the energy surface
in relation to two spatial angles. For this purpose, HyperChem performs the so-called limited optimization to render conformations of the molecule, depending on the distribution of electron and spin densities, electrostatic potentials,
electrostatic maps, and energy diagrams.11 An important
feature of HyperChem is the ability to manipulate and compute properties of macromolecules, such as proteins and
nucleic acids. With HyperChem, you can mix classical and
quantum mechanical calculations in the same molecular system.8,9 The first step involved drawing of the molecule’s
structural formula. Before we build the structure and perform a molecular mechanics optimization, we should choose
a molecular mechanics force field provided with HyperChem. A force field contains atom types and parameters
that must be assigned to the molecule before molecular
mechanics calculation. We measure the structural properties
of the model-built structure and compare this with geometry
measurements from the optimized structure and perform
the calculation to obtain the total energy of the unoptimized
RESULTS
Figures 1–3 display theoretical models of the bone
hydroxyapatite molecule obtained by substituting the
central calcium atom with other bivalent metals. Such
substitution between calcium and magnesium in bone
and tooth take place in living organisms indeed.13
Regardless of the substituting metal, however,
Ca3(PO4)2 groups of natural hydroxyapatite remain
intact.
Symmetric distribution of calcium atoms around the
central one was found by us only in the outermost
layer of the hydroxyapatite molecule (Fig. 1), in accordance with the hydroxyapatite molecule model of Aurich.2 Substitution of the central calcium atom with
another metal (Figs. 2 and 3) resulted in asymmetry,
with hydroxyl groups positioned on one side of the
central atom and the six external calcium atoms displaced contralaterally.
Table I presents computed bond lengths between
the central atom (Ca, Mg, Cu, Zn, Fe, Cr, Mn) and
surrounding oxygens. Bond length was greatest in the
case of natural hydroxyapatite (Fig. 1). Replacement of
the central calcium atom by any of the six metals
previously detected by us in bone hydroxyapatite produced shortening of the bond and spatial shrinking of
the model.
Information on chemical stability can be obtained
790
GUTOWSKA, MACHOY, AND MACHALIŃSKI
Figure 2.
Model of the hydroxyapatite molecule. Central Ca atom replaced by (a) Mg; )b) Cu; (c) Zn.
by looking at molecular energy data. The lower is the
total energy of the molecule (greater negative energy),
the more stable is the hydroxyapatite complex. Table
II shows that natural hydroxyapatite with calcium as
the central atom is least stable among the hydroxyapatite complexes studied by us. This finding appears to
be relevant to bone metabolism. One may expect that
increasing chemical stability of hydroxyapatite will
impair bone turnover and reduce the availability of
calcium from its stores in the bone. Such an increase
accompanies displacement of the central calcium atom
by any of the six bivalent metals studied by us, stability being greatest in the case of Cu-hydroxyapatite.
Theoretically, metals are incorporated into natural hy-
droxyapatite by substitution of the central calcium atom.
We assessed molecular stability by determining bond
energy of the metal in the hydroxyapatite structure.
Bond energies are shown in Table III. The greatest negative bond energy (⫺1447.4 kcal/mol) was obtained for
zinc, whereas the positive bond energy of 1585.2 kcal/
mol calculated for manganese prohibits bond formation
by this metal with the hydroxyapatite molecule.
DISCUSSION
There is a spate of biologically active compounds
that contain metals. Often, metals form complexes and
HYDROXYAPATITE STRUCTURE MOLECULAR MODELING
Figure 3.
791
Model of the hydroxyapatite molecule. Central Ca atom replaced by (a) Cr; (b) Fe; (c) Mn.
are positioned centrally, as in the case of heme with
iron or chlorophyll with magnesium.14,15 Seven bivalent metals (Ca, Mg, Cu, Zn, Fe, Cr, Mn) were marked
by us in mandibular bone of deer using mentioned
analytical methods, and only these metals were included to the further investigation. HyperChem has
already been applied to experimental studies of
enamel crystals with the electron microscope,16,17 in
research on synthetic hydroxyapatite with reduced
calcium content,18 and in theoretical investigations on
changes in properties of heme and chlorophyll in-
duced by replacement of the central atom with another metal.14,15 We have now decided to use HyperChem for modeling of hydroxyapatite molecules
containing bivalent metals previously detected by us
among mineral components of bone.
Calcium atoms of hydroxyapatite may be substituted by magnesium, lead, strontium, sodium,3 iron,
copper, or manganese,19 whereas phosphate may be
replaced by carbonate or hydroxyl anions. This property makes bone an ideal ion exchanger and a reservoir for many ions.3,4,20
792
GUTOWSKA, MACHOY, AND MACHALIŃSKI
TABLE I
Comparison of Bond Length between the Central Atom and Surrounding Oxygen Atoms
in the Hydroxyapatite Molecule
Bond length [Å]
No. of
atoms
MgCuZnFeCrMnHydroxyapatite hydroxyapatite hydroxyapatite hydroxyapatite hydroxyapatite hydroxyapatite hydroxyapatite
*(1)–O(2)
*(1)–O(3)
*(1)–O(4)
*(1)–O(5)
*(1)–O(6)
*(1)–O(7)
*(1)–O(8)
from OH
*(1)–O(9)
from OH
2.4242
2.4259
2.4198
2.4244
2.4315
2.4297
2.0832
2.0788
2.1049
2.0845
2.1152
2.0990
1.9297
1.9154
2.0077
1.9419
2.0382
1.9702
1.9897
1.9758
2.0273
2.0028
2.0370
2.0181
1.9282
1.9136
2.0103
1.9413
2.0364
1.9705
1.9351
1.9213
2.0099
1.9481
2.0399
1.9746
1.8642
1.8956
1.9139
1.9468
1.9238
1.9378
2.4085
2.0362
1.8648
1.9437
1.8653
1.8744
1.8957
2.4111
2.0426
1.8789
1.9897
1.8781
1.8862
1.9276
*Central atom.
Hetero-ionic exchange results in composition
changes and may lead to spatial deformation of the
hydroxyapatite crystal.1,3,21 Replacement of calcium
by another metal (ion) produces deformation and alteration of bond lengths between calcium and oxygen
atoms in the hydroxyapatite molecule (Table I)4,19 or
displacement of atom groups surrounding the central
atom (Figs. 1–3). Perhaps this property explains why
hydroxyapatite of bones and teeth is susceptible to
structural and crystallographic changes described in
the literature.1 The symmetrical hexagonal structure of
bone hydroxyapatite crystals may be subjected to
stretching vibrations and it is presumed that the surface of expansion exerts a destructive effect on the
symmetry and structure of growing crystals.22 Expansion takes place near the surface of the polarized layer
containing deformed channels with OH⫺ groups and
deformed tetrahedral molecules of phosphate.22
Chemical stability of compounds may be judged
according to molecular energy data. Hydroxyapatites
with lower total energy, i.e., those with greater negative energy, are more stable. Data in Table II show that
replacement of calcium with any of the six metal ions
studied by us increases stability, with copper forming
the most stable complex. Substitution by magnesium
is accompanied by a minimal change in the molecule’s
energy. This finding confirms the key role of magnesium in stabilizing amorphous hydroxyapatite23 and
preventing transition to the crystalline form. It also
explains why calcium ions reduce the inhibitory effect
of magnesium ions on growth of enamel crystals.24
These interactions were observed in human teeth.13,25
Admassu and Breese26 investigated the possibility
of applying fish apatite to remove heavy metals from
aqueous solutions. Binding to the hydroxyapatite matrix decreased in the following order: Pb, Zn, Cu, Cd,
Ni, Mg. Out of the six metals in the present study, the
zinc bond was found to possess the greatest negative
energy. By displacing calcium, zinc appears to modify
the rate of crystal growth.18 Calcium, copper, chromium, iron, and magnesium follow zinc in bond
strength. Unlike previous reports,3,4,20 our calculations
show that manganium does not form chemical bonds
with the hydroxyapatite molecule. We speculate that
manganium is attached in a different manner to the
crystal surface and is not incorporated into its structure.
Displacement of calcium by another ion (Mg, Zn, Fe,
Cu, Mn, Cr) produces changes in bond lengths, deforms the crystalline structure, and affects chemical
TABLE II
Total Energy of the Complex (Complex Stability)a
TABLE III
Bond Energy of the Metal in the Complexa
Complex
Total Energy of the
Complex [kcal/mol]
Complex
Bond Energy of the Metal
in the Complex [kcal/mol]
Hydroxyapatite
Mg-hydroxyapatite
Cu-hydroxyapatite
Zn-hydroxyapatite
Fe-hydroxyapatite
Cr-hydroxyapatite
Mn-hydroxyapatite
ⴚ95159
⫺95175
⫺98219
⫺96759
⫺97266
⫺96616
⫺96761
Hydroxyapatite
Mg-hydroxyapatite
Cu-hydroxyapatite
Zn-hydroxyapatite
Fe-hydroxyapatite
Cr-hydroxyapatite
Mn-hydroxyapatite
⫺366.6
⫺59.1
⫺260.3
⫺1447.4
⫺59.4
⫺178.4
1585.2
a
The greater is the negative value of total energy, the more
stable is the complex.
a
The greater is the negative value of total energy, the
stronger is the bond energy of the metal in the complex.
HYDROXYAPATITE STRUCTURE MOLECULAR MODELING
stability of hydroxyapatite. Application of molecular
modeling as a computation tool is useful for preliminary theoretical analysis of chemical compounds without resorting to laboratory testing.
12.
13.
14.
The authors thank Department of Physical Chemistry,
Technical University in Szczecin, for facilities of HyperChem
molecular modeling program.
15.
16.
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