Formation of Spherical Granules of Calcium Pyrophosphate Ombretta Masala, Paul O’Brien,* and Georgios Rafeletos Department of Chemistry and Manchester Materials Science Centre, The University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom
CRYSTAL GROWTH & DESIGN 2003 VOL. 3, NO. 3 431-434
Received November 11, 2002
ABSTRACT: The synthesis of calcium pyrophosphate, doped with zinc(II) ions at different [Zn]/[Ca] molar ratios, showed that the zinc ions partially substitute Ca2+ ions. The undoped particles are observed to form small crystals, which become amorphous spherical particles as the zinc content increases. The detoxification of heavy metals by barnacles in part involves the formation of insoluble granules, which may derive from calcium pyrophosphate.1-4 The deposits are close to spherical, amorphous, and with diameters ranging between 0.1 and 10 µm (Figure 1). In addition to pyrophosphate and calcium, metals such as aluminium, copper, iron, manganese, and zinc are found in these materials.5,6 Barnacles have been shown to accumulate heavy metals in body concentrations proportional to those of the surrounding seawater and are considered suitable for monitoring the pollutants in the marine environments.7,8 The use of this method raises the need to understand the mechanism of metal bioaccumulation in such granules. On the basis of various observations,9 it has been proposed that the deposits of calcium pyrophosphate in invertebrates act as traps for foreign metal ions. The amorphous structure of the deposits seems to facilitate the movement of ions from the solid phase to solution and to allow the incorporation of metal ions, i.e., it enables the granules to act as labile sinks for exogenous potentially toxic metal ions. The granules are always intracellular, suggesting that their formation may be the results of a process that takes place within the cell. However, the factors that control their specific composition and morphology and the mechanism involved in their formation are poorly understood. The majority of the studies conducted to date have concentrated on the characterization of the chemical composition of the pyrophosphate deposits; very few have focused on granule formation. Investigations have been carried out to study the control of a wide range of additives such as small organic molecules, polymers, and metal ions on the nucleation, growth and resulting morphology of inorganic crystals of calcium carbonate and calcium orthophosphate.10 For example, Mann and co-workers recently described the formation of spherical particles (with diameters up to 1000 µm) of octacalcium phosphate from aqueous solutions of calcium acetate and sodium orthophosphate containing small amounts of macromolecules such as polyaspartate or polyacrilate.11 The influence of additives such as organic molecules or metals on calcium pyrophosphates is poorly documented in the literature. * To whom correspondence should be addressed. E-mail: paul.obrien@ man.ac.uk.
Figure 1. TEM micrograph of spherical granules isolated from the barnacle Tetraclita squamosa.
The studies on the behavior of these salts in aqueous solution have been mostly inspired by the occurrence of calcium pyrophosphate dihydrate as pathological crystals in joints of the human body. These crystals, when released from cartilage into the synovial fluid, cause an inflammatory disease termed pseudogout.12,13 This condition affects various tissues, especially articular cartilages and ligaments of knee joints, causing acute pain. Although it is a very common disease, little is known about the formation mechanism of the crystals in the affected tissues. Calcium pyrophosphate dihydrate crystal formation is also related to the CoffinLowry syndrome (CLS),14,15 a heritable disorder characterized by pronounced mental retardation and vertebrae abnormalities. Crystal deposits are associated with marked degeneration of collagen fibrils of the vertebrae.16 Deformity of the skeletal structure may be caused by the disruption of collagen fibrils. However, the pathogenesis of the CLS has not been elucidated. Since the discovery of these diseases, many attempts have been made to synthesize pure hydrated calcium pyrophosphate crystals for in vitro studies.17-20 However, very few studies have focused on the influence of additives such as metal ions on the nucleation, growth,
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Table 1. Conditions Used to Precipitate Samplesa Zn/Ca molar ratio (%)
Na4P2O7‚10H2Ob
CaCl2‚2H2Oc
ZnCl2c
0 2 5 10
10 10 10 10
10 9.8 9.5 9.0
0 0.2 0.5 1.0
a Concentrations are in 10-3 mol dm-3. b Solution inside the membrane. c Solution outside the membrane.
and morphology of calcium pyrophosphates.21,22 No investigations on the role of zinc ions have been reported in the literature. Zinc has a smaller ionic radius than calcium and might be thought to replace calcium in calcium pyrophosphate with no dramatic effects on the structure, a suggestion supported by predictions based on interatomic potential studies.23 In this paper, we show that zinc ions have a remarkable influence on the morphology of precipitates of calcium pyrophosphates. We studied the formation of calcium pyrophosphate and zinc-doped calcium pyrophosphate by regulating the diffusion of aqueous solutions of CaCl2·2H2O and ZnCl2 into an aqueous solution of Na4P2O7·10H2O across a tubular membrane (CelluSep T3 cellulose, semipermeable membrane of 20 µm wall thickness and 2.5 nm pore diameter). The experiments were carried out at 25 °C, using different molar ratios of zinc/calcium and the initial pH of all the solutions was adjusted to 7 (see Table 1 for details of experimental conditions). The solution on the outside of the membrane was stirred continuously to assure a flux of the ions between the membrane and solution. A white precipitate formed from the solution within the membrane. The precipitate formed slowly and the reaction was in general allowed to proceed for 3 days. The final pH at the end of the reaction was typically 4.0. The solid products from the reaction were characterized by X-ray powder diffraction (XRPD), transmission electron microscopy (TEM), X-ray energy disperse analysis (EDAX), infrared spectroscopy (IR), and thermogravimetric analysis (TGA). We found that when only calcium and pyrophosphate ions were present, the solid product is crystalline and consists of a mixture of Ca2P2O7·2H2O and Ca2P2O7· 4H2O. The product is mostly made of Ca2P2O7·4H2O as shown by XRPD (Figure 2). TEM showed that the undoped material consists of discrete orthorhombic plates, with characteristic lengths ranging from 1 to 10 µm (Figure 3A). The addition of small molar percentages of zinc to the reaction mixture (from 2 to 10%) affects the extent of crystallinity, as indicated by XRPD (Figure 2). In particular, the sample doped with 10% of zinc is predominantly amorphous. However, the compound doped with 5% molar ratio presents new peaks in the XRPD pattern which suggests a change in structure. The effect on the crystallinity is remarkable. However, the effects on morphology are even more striking. The doped samples are characterized by the predominance of agglomerates of spherical particles of diameter ranging between 100 and 500 nm (Figure 3B). The particle size is not affected by the amount of dopant present as found by EDAX. The particles are amorphous to the electron diffraction. EDAX showed that the spherical particles are composed mostly of
Figure 2. XRPD patterns of undoped and doped materials. For clarity, only the main peaks are indexed in the pattern of Ca2P2O7‚2H2O. Indexing is not available for Ca2P2O7‚4H2O.
calcium, oxygen, and phosphorus with a small percentage of zinc. The percentage of zinc incorporated into a single particle quantitatively reflects the metal percentage in the starting solutions (Figure 4). As revealed by XRPD all doped samples contain a variable amount of crystalline material, which decreases as the zinc content increases. This crystalline material appears as discrete plates similar in form to the undoped compound with typical lengths of the order of 1 µm and has an approximate composition CaZnP2O7 as suggested by EDAX. The IR analysis of both the undoped and doped materials showed two bands at 932 and 738 cm-1 that are typical of the pyrophosphate group.24 All bands around 1100 cm-1, that are very sharp in crystalline pyrophosphate salts, lose resolution as the zinc content in the doped material increases, suggesting a gradual loss of crystallinity (Figure 5). The spectra also showed a broad band at 3300 cm-1, due to the presence of water molecules, as confirmed by TGA. The material doped with 10% of zinc contains two water molecules of crystallization as shown by TGA. Our results show that the doping of calcium pyrophosphate with only 10% of zinc produces material amorphous to X-ray and electron diffraction that possesses a very different morphology from the undoped calcium pyrophosphate. Such a dramatic transformation in crystallinity and morphology is significant, considering the small amount of dopant added. A possible explanation may lie in the relative stability of the complexes formed by zinc and calcium with pyrophosphate. The binding constants for the metal ions in aqueous solution are 6.8 for calcium and 8.7 for zinc.25 Thus, in the presence of pyrophosphate zinc can effectively compete with calcium for binding pyrophosphate even when present at a concentration 10-fold lower than that of calcium.
Formation of Spherical Granules of Calcium Pyrophosphate
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Figure 3. TEM micrographs of (A) undoped compound and (B) compound doped with 10% of Zn.
Figure 4. EDAX spectrum of spherical particles in the material doped with Zn% ) 2.
phous to X-ray and electron diffraction, they have spherical shape with diameters ranging between 0.1 and 0.5 µm and are composed of pyrophosphate and calcium with traces of zinc. Thus, particles that resemble biological granules in morphology and composition can be synthesized in vitro by using experiments in which ion diffusion is controlled by a membrane. Our experiments have been performed in a different environment than the original biological system since the role of the organic component has not been taken into account. Very few studies have been carried out to investigate the origin of biological deposits in barnacles, and very little is known on how their structure depends on composition. Moreover, many aspects of the chemistry of the metal pyrophosphates are entirely unknown. Our observations may appear very straightforward, but we believe that an understanding of the basic properties of pyrophosphate salts might provide a foundation for the comprehension of complex biological processes in which these compounds are involved. Experimental Procedures XRPD was carried out using Philips X’Pert MPD X-ray powder diffractometer with Cu-KR radiation. Samples for TEM were prepared as suspensions in acetone and deposited on carbon coated copper grids. Unstained samples were viewed in an EM 400T Philips electron microscope with associate link EDAX. Infrared analysis was carried out on nondiluted samples using an Excalibur BioRad FT-IR spectrometer equipped with a Golden Gate attenuated total reflectance accessory. TGA was carried out using DSC-2 Perkin Elmer calorimeter at a heating rate of 5 °C/min in air. All chemicals were used as purchased from Aldrich Ltd. without any further purification.
Figure 5. IR spectra of undoped and doped materials.
When the doped samples are prepared by rapid mixing of reagents without the aid of the membrane, an amorphous compound still forms, but it is made of particles of irregular shape and small size (less than 30 nm).26 If the same preparation is carried out by regulating the ion flux with a membrane, an amorphous compound is produced but with well formed spherical particles. The spherical particles synthesized by doping calcium pyrophosphate with zinc ions are similar in morphology and composition to the deposits in barnacles. Like the biological deposits, the zinc-doped particles are amor-
Acknowledgment. The authors are grateful to Professor Philip Rainbow (Natural History Museum, London) for helpful discussions on marine invertebrate biochemistry and for critically reading the manuscript. O.M. is funded by La Regione Autonoma della Sardegna, Italy. P.O.B. is the Sumitomo Visiting Professor of Materials Chemistry at Imperial College of Science, Technology and Medicine, London. References (1) Pullen, J. S. H.; Rainbow, P. S. J. Exp. Mar. Biol. Ecol. 1991, 150, 249-266. (2) Walker, G.; Rainbow, P. S.; Foster, P.; Holland, D. L. Mar. Biol. 1975, 33, 161-166.
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(3) Barber, S.; Trefry, J. H. Bull. Environ. Contam. Toxicol. 1981, 27, 654-659. (4) Nott, J. A.; Nicolaidou, A. J. Mar. Biol. Assoc. U. K. 1990, 70, 905-912. (5) Simkiss, K. Symp. Soc. Exp. Biol. 1976, 30, 423-444. (6) Walker, G.; Rainbow, P. S.; Foster, P.; Crisp, D. J. Mar. Biol. 1975, 30, 57-65. (7) Phillips, D. J. H.; Rainbow, P. S. Biomonitoring of Trace Aquatic Contaminants; 2nd ed.; Chapman and Hall: London, 1994. (8) Fialkowski, W.; Newman, W. A. Mar. Pollut. Bull. 1998, 36, 138-143. (9) Rainbow, P. S. J. Mar. Biol. Assoc. U. K. 1997, 77, 195210. (10) Dujardin E; Mann S. Adv. Eng. Mater. 2002, 4, 461-474. (11) Bigi A.; Boanini E.; Walsh D.; Mann S. Angew. Chem. Int. Ed. 2002, 41, 2163-2166. (12) Cheng, P. T.; Pritzker, K. P. H.; Adams, M. E.; Nyburg, S. C.; Omar, S. A. J. Rheumatol. 1980, 7, 609-616. (13) McCarty, Jr., D. J.; Kohn, N. N.; Faires, J. S. Ann. Intern. Med. 1962, 56, 711-737. (14) Temtamy S. A.; Miller J. D.; Dorst J. P.; Hussels-Maumenee I.; Salinas C.; Lacassie Y.; Kenyon K. R. Birth Defects, Orig. Art. Ser. 1975, 11, 133-152. (15) Young I. D. J. Med. Genet. 1988, 25, 344-348.
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