Langmuir 1992,8, 676-679
676
Influence of Citrate and Phosphocitrate on the Crystallization of Octacalcium Phosphate V. K. Sharma,t M. Johnsson,*p§J. D. Sallis,l and G. H. Nancollas*J9§ Chemistry Department, Brookhaven National Laboratory, Upton, New York 111 73, Departments of Chemistry and Biomaterials, State University of New York at Buffalo, Buffalo,New York 14214, and Department of Biochemistry, University of Tasmania, Hobart, TAS 7001, Australia Received September 12,1991. In Final Form: October 11, 1991 The crystallization of octacalcium phosphate (OCP) in the presence of citrate and phosphocitrate ions has been studied using a constant codposition method. The adsorption of these ions and their influence on the particle size distribution and t potentials of OCP crystals were also investigated. Although citrate and phosphocitrate showed similar adsorption affinities, the OCP crystal growth inhibition of phosphocitrate was considerably more pronounced. When OCP surfaces were exposed to both citrate and phosphocitrate, the uptake of the latter was markedly increased in the range of citrate concentration, (1.6-10.0) x 10-4moi L-1. -
Introduction Biochemical agents capable of inhibiting the crystallization of calcium phosphate are found in most body fluids such as serum, saliva, and The inhibitory agents are generally phosphorylated proteins such as statherin4 and the proline-rich proteins5 or compounds such as pyHowardg detected, in urine, a rophosphate or very active inhibitor of calcium phosphate and calcium oxalate precipitation which he suggested to be a phosphorylated citrate, phosphocitrate. This substance was shown to be an efficient inhibitor of the seeded growth of both hydroxyapatite (HAP) and calcium oxalate monohydrate (COM).lGl5 The affinity of phosphocitrate for HAP surfaces and its ability to inhibit HAP crystal growth are considerably greater than those of citrate.12 Furthermore, the adsorption of phosphocitrate to HAP is greatly enhanced if citrate is present a t the surface during the adsorption,14 and mixtures of the two additives are
* To whom correspondenceshouldbe addressed at the Department of Chemistry, SUNY at Buffalo, Buffalo, NY 14214. + Brookhaven National Laboratory. t Department of Chemistry, SUNY at Buffalo. 0 Department of Biomaterials, SUNY at Buffalo. 1 University of Tasmania. (1)Howard, J. E.; Thomas, W. C. Trans. Am. Clin. Climatol. Assoc. 1958, 70, 94.
(2) Eidelman, N.; Chow, L. C.; Brown, W. C. Calcif. Tissue Int. 1987,
40, 78.
(3)Eidelman, N.; Chow, L. C.; Brown, W. C. Calcif. Tissue Int. 1987,
41, 18.
(4)Schlesinger, D. H.; Hay, D. I. J. Biol. Chem. 1977,252, 168. (5)Moreno, E. C.;Varughese, K.; Hay, D. I. Calcif. Tissue Int. 1979, 28, 7. (6) Fleisch, H.; Russell, R. G. G.; Bisaz, S.; Termine, J. D.; Posner, A. S. Calcif. Tissue Res. 1968, 2, 49. (7)Felix, R.; Fleisch, H. Calcif. Tissue Res. 1976, 22, 1. (8)Posner, A.S.; Betta, F.; Blumenthal, N. C. Calcif. Tissue Res. 1977, 22, 208.
(9)Howard, J. E. Johns Hopkins Med. J . 1976, 139, 239. (10)Williams, G.; Sallis, J. D. Biochem. J. 1979, 184, 181. (11)Williams, G.;Holdsworth, E. S.; Sallis,J. D.Pharm.Physiol.1980, 7,42. (12)Tew, W. P.; Mahle, C.; Benavides, J.; Howard, J. E.; Lehninger, A. E. Biochemistry 1980,19, 1983. (13)Tew,W. P.;Malis,C. D.;Lehninger,A.L.; Walker,G.W.; Howard, J. E. In Urolithiasis, Clinical and Basic Research; Smith, L. H., Robertson, W., Finlayson, B., Eds.; Plenum Press: New York, 1981;p 559. (14)Johnsson, M.; Richardson, C. F.; Sallis, J. D.; Nancollas, G. H. Calcif. Tissue Znt. 1991, 49, 134. (15)Richardson, C. F.; Johnsson, M.; Bangash, F. K.; Sharma, V. K.; Sallis,J. D.; Nancollas, G. H. In Material Synthesis Utilizing Biological Processes; Rieke, P. C., Calvert, M., Alper, Eds.; Plenum Press: New York, 1990,p 174.
considerably more efficient in inhibiting HAP crystal growth than are the additive effects of each.13-17 Since the crystallization of HAP in vivo is suggested to take place through an octacalcium phosphate (OCP) precursor phase,lg20 it is of special interest to investigate the ability of phosphocitrate to influence the growth of this phase. In this work, the adsorption of citrate (Na&~H507), phosphocitrate ( N ~ ~ C ~ H ~ Oand ~ Otheir P ) , mixtures onto OCP has been investigated. The effects of these substances on the particle size distribution and ( potentials of OCP crystals and on their growth rates in supersaturated solutions have been determined using the constant composition (CC) method.21
Experimental Section Analytical reagents were used in all experiments. Phosphocitrate was synthesized and characterized as described previCalcium chloride solutions were analyzed by atomic absorption (Perkin-Elmer Model 503) and by ion exchange on a Dowex 50W8 cation exchange column in the hydrogen form. The released hydrogen ions were titrated with standardized potassium hydroxide solutions. Potassium dihydrogenphosphate solutions were analyzed spectrophotometricallyat 420nm aa the molybdovanadium phosphate complex as well as by titration against standardized potassium hydroxide solutions. OCP crystals (calciumlphosphate molar ratio 1.33 f 0.031, prepared using a CC methodF3 were stored dry to prevent transformation to HAP.X-ray analysis (Nicolet/Nicwith a Stoe attachment)showed a characteristicOCP spectrum with a strong 100reflection at 4.27O. The specific surface area, determined by Brunauer-Emmett-Teller (BET)nitrogen adsorption (30/70Nd He, Quantasorb, Quantachrome), was 24.1 0.2 m2 gl. The adsorption of phosphocitrate and citrate (Fisher Lot. No. 731362) onto OCP was determined by equilibrating OCP crystallites in polycarbonate tubes at 25 "C with 2.5 X 10-3 L of phosphocitrate and/or citrate solutions in 0.15 mol L-l NaC1.
*
(16) Williams, G.; Sallis, J. D. In Uriolithiasis, Clinical and Basic Research; Smith, L. H., Robertson, W. G., Finlayson, B., Eds.; Plenum Press: New York, 1981; p 569. (17)Williams, G.; Sallis, J. D. Calcif. Tissue Znt. 1982, 34, 169. (18)Brown, W. E.; Eidelman, N.; Tomazic, B. Ado. Dent. Res. 1987. 1,306.
(19)Tomazic,B.B.;Tung,M.S.;Gregory,T.M.;Brown,W.E.Scanning Microsc. 1989, 3, 119. (BO!LeGeros, R. 2.;Daculsi, G.; Orly, I.; Abergas, T.; Torres, W. Scanning Microsc. 1989, 3, 129. (21)Tomson, M. B.; Nancollas, G. H. Science 1978,200, 1059. (22)Williams, G.; Sallis, J. D. Anal. Biochem. 1980,102,365. (23)Salimi, M. H.; Heughebaert, J. C.; Nancollas, G. H. Langmuir 1985, 1, 119.
0743-746319212408-0676$03.00/0 0 1992 American Chemical Society
Crystallization of Octacalcium Phosphate Although adsorption was shown to be completed within 30 min, a 4-h adsorption period was chosen to ensure equilibration. Following this time, the OCP crystals were separated from the solution by centrifugation at 250g for 10 min and the concentrations of additives in the solutions were determined by ion chromatography, HPIC (DionexQIC analyzer). Phosphocitrate was hydrolyzed to citrate and phosphate with alkaline phosphatase (Sigma Lot Number 98F-8145)at 37 'C for 1h prior to analysis. In combined studies, citrate concentrations were determined before and after alkaline phosphatase hydrolysis. Solutions, supersaturated with respect to OCP in 0.15 mol L-l NaCl, were prepared in double-walled Pyrex glass vessels thermostated at 37 O C by mixing stock solutions of calcium chloride, potassium dihydrogen phosphate, and sodium chloride to give concentrations of 1.18 X W3,8.85 X lo-', and 0.15 mol L-l, respectively. Potassium hydroxide solution was then slowly added to raise the pH to 7.4. The supersaturation, u = 1.24, with respect to OCP was calculated from eq 1,where the ionicproduct, IP, is given by eq 2 and Kspis the solubility product of OCP.
Langmuir, Vol. 8, No. 2, 1992 677
9.0
6.0
3.0
0.0 0.0
1.0
2.0
3.0
Equil. Conc.
/
5.0
4.0
6.0
mol L-l
Figure 1. Adsorption onto OCP of citrate, Cit 0 ( 0 ) phospho; citrate, Pcit 0 ( 0 ) ;phosphocitrate with citrate, Pcit 1 (A,0.5 X lo-' mol L-l Cit), Pcit 2 (0,1.6 X lo4 mol L-l Cit), Pcit 3 (v,
(2.0-10.0) X lo4 mol L-' Cit);and citrate with preadsorbed phosphocitrate, Cit 1 (X, 2.0 X lo4 mol L-l Pcit). 100
Crystal growth was initiated by the addition of seed suspensions g of OCP with or without adsorbed inhibitors (usually 1.0 X crystalsin 1.0 X lO-3L)to 6.00 X 1C2L of supersaturated solutions. Supersaturation was maintained constant during mineralization by the potentiometrically controlled addition of titrant solutions containing calcium chloride with sodium chloride and potassium phosphate with potassium hydroxide.14v21The effective titrant mol L-l with respect to concentration was usually 7.62 X OCP. Duringthe mineralizationexperiments,samplesofreaction mixtures were withdrawn, filtered (Millipore 2.2 X lo-' m), and analyzedfor calcium and phosphate in order to confiim constancy of the supersaturation. The moles of titrant added after correction for dilution gave the experimental growth rate which was normalized with respect to the surface area of the added seed crystals. The growth rate obtained in the presence of additive was compared to that observed in the absence of citrate or phosphocitrate, and the precentage decrease was calculated for the first 100 min of reaction. All experiments were performed at least three times with a reproducibility of growth rates to within 3%.
The particle size of OCP aggregates in the presence of citrate or phosphocitrate in stirred solutions, saturated with respect to OCP, was measured using a Malvern 3600E particle sizer (633nm He/Ne laser). Measurements were continued for 1-2 h after the addition of adsorbate. Electrophoretic mobility of OCP crystals suspended in citrate and phosphocitrate solutions saturated with respect to OCP at 37 OC was measured using a Malvern Zetasizer IIC with 28-V applied voltage. A standard electrophoresis cell with electrodes consisting of cylindrical platinum sheets mounted in a poly(tetrafluoroethy1ene)(PTFE) threaded sleeve was used.
Results The results of the adsorption experiments of citrate, phosphocitrate, and mixtures of these two additives onto OCP are shown in Figure 1asplots of the adsorbed amount, Q (mol m-9, as a function of the equilibrium concentration, C (mol L-I). It can be seen that, in the concentration range studied, the adsorptions of citrate and phosphocitrate were similar. I n a competitive adsorption study, the amount of adsorbed phosphocitrate increased by more than 100% in the presence of (1.6-10.0) X mol L-' citrate. Under similar conditions, no adsorption of citrate onto the OCP particles was detected. Adsorption onto OCP surfaces has been interpreted in terms of a Langmuir model given by eq 3.24 In eq 3, K is (24) Kremk, M.; Moreno, E. C.; Zahradnik,R. T.;Hay, D.I. J.Colloid Interface Sci. 1977,59, 283.
I
'
I
'
l
'
I
*
l
.
Pcit 1 -
~
. -..0 Cit 0
0 \ 0
0
Equil. Conc.
/
mol L - l
Figure 2. Langmuir isotherms of citrate, Cit 0, phosphocitrate, Pcit 0, and phosphocitrate with citrate, Pcit 1(0.5 X 1o-L mol L-1 mol L-' Cit), Pcit 3 ((2.0-10.0) X lo-' mol Cit), Pcit 2 (1.6 X
L-l Cit).
Table I. Affinity Coefficient ( K ) and Maximum Concentration of Adsorption Sites (N) onto OCP Surfaces Calculated from a Langmuir Model, C/Q = 1/KN + C/N, for Citrate, Phosphocitrate, and Phosphocitrate with Citrate
adsorbate citrate phosphocitrate phosphocitrate with citrate 0.5 X 10-4mol L-l 1.6 X lo4 mol L-l (2.0-10.0) X 10"' mol L-' Taken from ref 14.
K / W L mol-' OCP HAPa 0.24 13.0 0.13 26.9
N / W mol m-2 OCP HAPa 1.42 15.0 1.03 26.0
0.27 2.38 5.21
1.05 0.94 1.28
the adsorption affinity coefficient (L mol-') and N is the maximum concentration of adsorption sites (mol m-2). CIQ = 1IKN + CIN
(3)
Plots of C/Q against C for citrate, phosphocitrate, and mixtures of these additives are given in Figure 2. The calculated parameters of eq 3 are given in Table I which includes the data for HAP surfaces14 for comparison. It can be seen that the affinities for OCP are considerably lower than those for HAP. Furthermore, citrate had a somewhat higher affinity for OCP than phosphocitrate.
Sharma et al.
678 Langmuir, Vol. 8, No. 2, 1992 100
I
'
I
'
1
.
1
'
I
'
al
Y
e
5
3
$ u
Y
u)
* 0 U
t
0
$
a
40
/
mol m-* Figure 3. Effects of adsorbed citrate, Cit 0 (O),phosphocitrate, Pcit 0 (o),and both molecules, Pcit 1 (A)(shown aa a function of adsorbed phosphocitrate), on the crystal growth of OCP. Adsorbed amount
The uptake of citrate and phosphocitrate on OCP is similar and considerably lower than on HAP (Table I). During a competitive OCP adsorption study, it was found that, in the presence of (0.16-1.0) X mol L-l citrate, the initial adsorption of phosphocitrate was increased with a higher affinity coefficient, K , compared to conditions in the absence of citrate while no significant change in the maximum number of adsorption sites, N , was observed. However, the lower concentration of citrate, 0.05 X mol L-l, had no effect on the phosphocitrate uptake. In transformation experiments, OCP was equilibrated in a phosphate-buffered solution a t pH 7.4 for up to 12 h. After 30 min, transformation to HAP was detected by X-ray diffraction as a decreased intensity of the sharp peak a t 4.75". In similar equilibration experiments in the presence of citrate or phosphocitrate ((0.5-10.0) X mol L-l), no decreased intensity in the peak at 4.75O was observed even after 12 h of equilibration. The results of OCP crystal growth experiments are given in Figure 3. The crystallization was strongly inhibited even a t low surface concentrations of phosphocitrate. At an additive concentration of 0.2 X 10" mol m-2,the reaction rate decreased by 8096, while with increasing phosphocitrate concentration, an inhibition of 99 % was observed a t 0.55 X 10* mol m-2 additive. In the case of citrate, the rate of OCP crystallization was reduced by 18% a t 0.2 X 10" mol m-2surface concentration and reached a maximum inhibition of 40% a t a coverage of 1.40 X lo* mol m-2. In experiments in which both additives were present in supersaturated solutions, there was no difference in the extent of inhibition of OCP crystal growth as compared to reactions in the presence only of phosphocitrate. The particle size distributions (PSD) of OCP particles suspended in a saturated solution are shown in Figure 4. In the presence of citrate (or phosphocitrate), the average PSD decreased, indicating a marked dispersion of the OCP aggregates. Citrate was a more efficient dispersant as compared to phosphocitrate, but in both cases, a maximum dispersion was found to occur a t a surface concentration of 1 X lo* mol m-2, considerably lower than the concentrations for maximum surface coverage. The influence of citrate and phosphocitrate on the { potential of OCP crystallites in saturated solution is shown in Figure 5. A positive {potential (9 mV) in the absence of additives decreased to about -24 and -36 mV in the presence of citrate and phosphocitrate, respectively. The { potential with phosphocitrate was more negative than that of citrate. For citrate and phosphocitrate adsorption,
0
3
2
1
5
4
6
Adsorbed amount / mol m-2 Figure 4. Particle size distribution of OCP crystals with ador phosphocitrate (0). sorbed citrate (0)
-40
1
0
I
2
, 4
I
I
6
8
10
Adsorbed amount / mol m-2 Figure 5. {potentials of OCP crystals with adsorbed citrate (0) or phosphocitrate (0). the { potential became zero a t adsorbed amounts of 0.67 X 10" and 0.17 X lo4 mol m-2, respectively.
Discussion A suggested requirement for strong adsorption to HAP is the presence of a t least two charged groups in close proximity to each other, with one being a phosphate moiety.17 This is exemplified in a comparative HAP adsorption study where phosphocitrate was found to bind to almost twice the extent of citrate.14 In the present work with OCP surfaces, the difference between citrate and phosphocitrate was much smaller and the uptake of citrate was actually larger than that of phosphocitrate (Figure 1). This suggests that the factors controlling the binding of phosphorylated compounds a t HAP surfaces may be less important in the case of OCP. The low OCP adsorption affinity of citrate and phosphocitrate indicates that adsorption onto OCP occurs mainly by electrostatic interactions to a more positively charged surface as well as increased hydrogen bonding to the protonated phosphates a t the OCP surface. The higher adsorption of phosphocitrate during a competitive study a t certain concentrations of citrate suggests that the mechanism of phosphocitrate adsorption in the presence of citrate is different from that of phosphocitrate alone. This can also be seen in Figure 1 in which there is a steep increase of phosphocitrate adsorption as compared to adsorption in the absence of citrate. Neutralization of the positive surface by citrate may enable phosphocitrate to recognize calcium sites on the surface and thereby show high affinity.
Crystallization of Octacalcium Phosphate The lower ability of phosphocitrate to disperse aggregates of OCP as compared with citrate (Figure 4) suggests that the highly charged phosphate groups may be adsorbed to the surface. The binding of this group together with one of the carboxyl groups may give this molecule high affinity for the adsorption sites. This suggestion is supported by the {potential results (Figure 5 ) in which phosphocitrate neutralizes the positively charged OCP surface more effectively than does the citrate ion. The two major processes, either a dissolution of OCP followed by precipitation of HAP or direct hydrolysis, result in the formation of HAP from OCP. The presence of additives such as citrate and pyrophosphate has been shown to retard these processes.25 The present results show that phosphocitrate also inhibits HAP formation from OCP. The strong inhibition of OCP crystal growth with adsorbed phosphocitrate suggests that preferential adsorption occurred a t active growth sites on the surfaces. Meyer and N a n ~ o l l a spostulated ~ ~ ~ ~ ~ that a chelation of (25) LeGeros, R. Z.;Daculsi, G. I.; Orly, T.; Abergat, .; Torres, W. Scanning Microsc. 1989,3,129. (26) Nancollas, G. H.International Symposium on Urolithiasis Research; Plenum Press: New York, 1976; p 5. (27) Meyer, J . L.; Nancollas, G. H. Calcif. Tissue Res. 1973, 13, 295.
Langmuir, Vol. 8, No. 2, 1992 679 surface calcium is responsible for the strong inhibiting properties of some diphosphonates. A similar surface adsorption may also occur for phosphocitrate which would enable this molecule to strongly inhibit crystal growth. Although both additives showed similar adsorption, the crystallization-inhibiting properties of citrate were considerably lower than those of phosphocitrate. If the high citrate adsorption was due to electrostatic interactions to the surface, this may result in binding also to growth sites that are less active in the crystallization reactions. The inhibition of crystallization would consequently be lower. In conclusion, phosphocitrate adsorbs strongly to OCP and inhibits the growth of this calcium phosphate phase. The results indicate that both citrate and phosphocitrate retard transformation of OCP to HAP. Therefore, the marked ability of phosphocitrate to inhibit growth of both HAP and OCP suggests that it may act as an in vivo agent inhibiting the formation of an important apatite precursor.
Acknowledgment. We thank the National Institute of Dental Research for a grant (DE03223) in support of this work. Registry No. OCP, 13767-12-9; C O H ~ O , ~ 126-44-3; -, CsHiOl$”, 137300-27-7.
