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Effect of concentrations of lecithin, calcium and oxalate on crystal growth of calcium oxalate in vesicles
Colloids and Surfaces A: Physicochem. Eng. Aspects 257–258 (2005) 215–220
Effect of concentrations of lecithin, calcium and oxalate on crystal growth of calcium oxalate in vesicles Jian-Ming Ouyang∗ , Feng Deng, Li Duan Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632, PR China Available online 24 November 2004
Abstract The crystal growth of calcium oxalate (CaOxa) in lecithin (PC)–water vesicles has been studied by X-ray powder diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), and dynamic laser scattering (DLS). The molar ratio of calcium to oxalate (Ca2+ /Oxa2− ), the original concentration of CaOxa [c(CaOxa)], the concentrations of PC and additive potassium citrate (K3 cit) can influence the morphology and phase composition of CaOxa crystals. The diameter of CaOxa crystals obtained in vesicles (80–150 nm) is smaller than that in bulk solutions (about 1500 nm). When the ratio of Ca2+ /Oxa2− is nearly 1.0, the percentage of COD in CaOxa crystals reaches the maximum. When c(CaOxa) increases, the percentage of COM decreases and that of COD increases. The content of COD reaches a constant percentage when c(CaOxa) > 0.10 mol/L. COT was obtained only at a very low CaOxa concentration of less than 0.01 mol/L. The concentration of PC greatly affects crystal planes of CaOxa crystals. When PC concentration is above critical vesicle concentration (CVC, 1.875 mg/mL), vesicles were formed. In vesicle system, the (0 2 0) crystal plane of CaOxa was preferentially grown. K3 cit inhibits the pre-critical nuclei of COM and favors COD. © 2004 Elsevier B.V. All rights reserved. Keywords: Calcium oxalate; Lecithin; Vesicles; Potassium citrate
1. Introduction Urolithiasis constitutes a serious healthy problem that affects a significant section of mankind. It is very common and high recurrent disease in the whole world area. Calcium oxalate (CaOxa) is the most frequent crystalline phase in human urinary stones and occurs in more than 80% [1]. Three types of hydrated calcium oxalate – the monohydrate (COM), dihydrate (COD) and trihydrate (COT) – are found in urinary stones. Thermodynamically stable COM is much more common in stones and is not easy to be expelled out from body [2]. While the thermodynamically unstable COD is more easy to be expelled out than COM [3]. Thermodynamically metastable trihydrate (COT) can be a precursor in CaOxa stone formation [4]. If the nucleation, growth or aggregation of COM can be inhibited and/or more COD and
∗
Corresponding author. Tel.: +86 20 85223353; fax: +86 20 85223353. E-mail address: [email protected] (J.-M. Ouyang).
0927-7757/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2004.10.049
COT can be induced, or the transformation of COM to COD and/or COT is promoted, it will be helpful for the prevention of CaOxa stones. Hence, an understanding of crystallization processes of various types of hydrated CaOxa is essential if urologists hope to develop therapeutic agents [4]. There are many reports about the crystallization of CaOxa in aqueous solutions, diluted urines, and artificial urines [2–4]. However, common aqueous solutions are much different from those in biological systems. Urinary stones are usually formed within membrane-bound microspace, and the nucleation and growth of urinary stones are regulated by organic matrix [5,6]. So in the recent years, some ordered systems were designed to mimic the formation of CaOxa stones especially with Langmuir monolayer as a model system [1,7,8]. Khan et al. [8] have investigated the crystallization of CaOxa at Langmuir monolayers of dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylsome glycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG) and dimyristoylphosphatidyl-glycerol
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(DMPG). For each monolayer CaOx crystals were predominantly monohydrate, and most often grew singly with the calcium rich (1¯ 0 1) face toward the monolayer. The number of crystals/mm2 decreased in the order DPPG > DPPS > DPPC and was inversely proportional to surface pressure and mean molecular area/molecule. Talham and co-workers [7] reported the precipitation of COM beneath Langmuir monolayers of four different phosphatidylglycerol lipids. The influence of the density of headgroup packing and monolayer compressibility on heterogeneous precipitation was investigated. The results suggest that the compressibility of the monolayer and the potential of the monolayer to achieve a small area per molecule are more important characteristics that lead to heterogeneous crystallization of COM than is providing a prearranged template. We [1] have examined the nucleation and growth of COM crystals beneath stearic acid (SA) monolayers in the presence of citric acid (H3 cit). The (1¯ 0 1) faces of COM crystals were remarkably stabilized independent of either the change of H3 cit concentration from 0.01 to 0.30 mmol/L or the change of the surface pressure of SA monolayer from 1 to 20 mN/m. This result was due to that there is a strong interaction between the Ca2+ -rich (1¯ 0 1) face of COM and the negatively charged carboxylic groups in the headgroups of SA monolayers and H3 cit. Vesicles have the advantage of providing confined microspace and organic matrix. The crystallization of CaOxa in vesicles can mimic the formation conditions of CaOxa stones. We once reported the deposition behavior of CaOxa in lecithin–water vesicles [5]. The vesicle membranes play an important role in inducing COD formation. We [6] also reported the effects of different kinds of potassium carboxylates on the phase compositions of CaOxa crystals grown in vesicles. The investigated potassium carboxylates included: monocarboxylate potassium acetate (KAc), dicarboxylate potassium tartrate (K2 tart), tricarboxylate potassium citrate (K3 cit), and tetracarboxylate dipotassium of ethylenediaminetetraacetic acid (K2 edta). KAc only induce COM. COT was induced when concentrations of K2 tart > 1.0 mmol/L. K3 cit and K2 edta can induce COM, COD and COT crystals depending on their concentrations. The ability to induce COD follows the order: K3 cit > K2 edta K2 tart ∼ KAc, and the ability to induce COT follows: K2 tart K3 cit > K2 edta KAc. In this paper, the effect of the concentration of PC, the original concentration of calcium oxalate, as well as the ratio of calcium to oxalate (Ca2+ /Oxa2− ) on the crystal growth of CaOxa was studied in PC–H2 O vesicle system.
Fluka or Sigma. Pure water was obtained from a Millipore-Q system: the resistance was 18.2 M cm. 2.2. Apparatus X-ray powder diffraction (XRD) was carried out by a D/max-␥A X-ray diffractometer (Rigaku, Japan), Ni-filtered ˚ Philips Tecnai-10 transmission Cu K␣ radiation (λ = 1.54 A). electron microscopy (TEM). Bruker IFS25 Fourier transform infrared (FT-IR) spectrometer (Bruker Spectrospin, Wissembourg, France). 2.3. Effect of calcium and oxalate concentrations A certain amount of PC was first dissolved in chloroform. After the organic solvent was volatilized at room temperature, 30.0 mL CaCl2 with different original concentration was added. The final concentration of PC in the CaCl2 solution was 5 mg/mL. Sometimes, 1.0 mL aqueous 0.2 mol/L K3 cit was also added together with CaCl2 . The solution was sonicated for 20 min in order to form stable vesicles. Dynamic laser scattering showed the diameter of the vesicles to be about 80–100 nm and the vesicles were stable. Then 30.0 mL K2 Ox solution was added under stirring. The molar ratios of calcium to oxalate are shown in Table 1. After 10 min of reaction, a drop of the suspension was examined by TEM to obtain the size, shape, and appearance of the particles. The rest solution was allowed to age for 2 h at room temperature without stirring. Then the product was centrifuged, washed with chloroform, and dried under vacuum. The phase compositions of the products were analyzed by XRD and FT-IR. The relative percentages of three kinds of hydrated phases of CaOxa crystals, the monohydrate (COM), dihydrate (COD) and trihydrate (COT), was calculated according to K value methods [5]. Blank groups without the presence of lecithin and K3 cit were carried out simultaneously. 2.4. Effect of PC concentration Different amounts of PC were first dissolved in chloroform. After the organic solvent was volatilized at room temperature, 30.0 mL aqueous 0.02 mol/L CaCl2 in the absence or presence of 3.3 mmol/L K3 cit were added. The final Table 1 Mass fraction (W%) of calcium oxalate hydrates grown in vesicles at different molar ratios of Ca2+ /Oxa2− [c(Ca2+ ) = c(Oxa2− ) = 0.10 mol/L] [Ca2+ ]/[Oxa2− ]
2. Materials and methods 2.1. Materials Egg (yolk) lecithin (PC) and all the other chemicals such as calcium chloride, potassium oxalate, potassium citrate and chloroform were of analytical purity and purchased from
With K3 cit COM
9:1 4:1 2:1 1:1 1:2 1:4 1:9
84 38
76 100
Without K3 cit COD
COM
COD
16 62 100 100 100 24
100 61 40 50 66 86 100
39 60 50 34 14
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concentration of PC in the solution was showed in Table 3. Then CaOxa was precipitated and characterized as described above.
3. Results and discussion 3.1. The size of vesicle and the precipitation of CaOxa crystals in vesicles Fig. 1 shows the dynamic laser scattering data of blank vesicles. It can be seen that the diameter of blank vesicles ranges from 80 to 120 nm. However, TEM measurement shows the size of CaOxa crystals precipitated in vesicles is about 80–150 nm, which is lightly larger than that of the blank vesicles. It indicated that the deposition of CaOxa crystals occurs within the vesicles and at membrane surfaces outside the vesicles. The formation of CaOxa crystals in vesicles are much different from that in control solution. The size of CaOxa crystals grown in pure water is about 1500 nm, which is much larger than that (80–150 nm) in vesicles. This is because the nucleation and growth of CaOxa crystals in vesicles was controlled at the ordered vesicle membrane interfaces (inside and outside). So the structure, morphology, and particle size of CaOxa obtained are much different from that obtained from bulk solution.
COM was obtained when the ratio is more than 4 or less than 0.25 in the absence of K3 cit. In a clinic experiment, it is also founded that a much high dietary intake of calcium can decrease the risk of urinary stone. Messa et al. [9] explained this might be due to increasing binding of oxalate by calcium in the gastrointestinal tract, leading to a decrease in urinary oxalate excretion. That is, urinary oxalate is more important than urinary calcium in determining stone formation. In our experiment, oxalate plays more important role in inducing COD growth, which is favorable for preventing stone formation [2].
3.2. Effect of the ratio of Ca2+ /Oxa2− in vesicles
3.3. Effect of potassium citrate
When the molar ratios of Ca2+ /Oxa2− in vesicles vary from 9:1 to 1:9, the relative percentages of different hydrated CaOxa crystals changed as shown in Table 1. When the original concentrations of both Ca2+ and Oxa2− are 0.050 mol/L, namely the molar ratio of Ca2+ /Oxa2− is 1.0, the percentage of dihydrate COD reaches the maximum, nearly 50 and 100% in the absence (Fig. 2a) and presence of K3 cit (Fig. 2b), respectively. If the ratio of Ca2+ /Oxa2− deviated away from 1, less COD and more COM are grown. For example, only
Addition of potassium citrate (K3 cit) can induce much more dihydrated COD formation in spite of the molar ratio of calcium to oxalate (Table 1), the original concentration of CaOxa (Table 2 and Fig. 3) and the PC concentration (Table 3). This effect may be ascribed to the following factors: At first, there is a good structural match between the tricarboxylic K3 cit and calcium spacing in the crystal lattices of COM crystals [3], so K3 cit can inhibit the pre-critical nuclei of COM and favor COD. Second, the surface absorption
Fig. 1. Dynamic laser scattering data for the PC–water vesicles.
Fig. 2. Distribution of COM and COD crystals in vesicle at different molar ratios of Ca2+ /Oxa2− : (a) without K3 cit and (b) with 3.3 mmol/L K3 cit.
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Table 2 FT-IR data of CaOxa crystals grown in vesicles in different original concentrations of CaOxa c(CaOxa) (mol/L)
νas /COM or COT νas /COD or COT νs (COO− ) 948/COM or 914/COD 880/COM 668/COM or 611/COD 550/COT
0.004
0.01
0.05
0.1
0.5
1.0
1670 1618 1326.5
Shoulder 1620.1 1322.2 947 885 669
1646.3 1622.2 1324.3
1647 Shoulder 1328.8 914
1647.3
1646.3
1328.1 914.2
1327.6 917
617.8
615.2
616
885 668
550
Fig. 3. Effect of original concentration of CaOxa on the distribution of COM, COD and COT crystals in vesicles.
of K3 cit on COM is higher than that on COD [10]. That is to say, K3 cit inhibit crystal growth of COM stronger than that of COD, thus it increases the percentage of COD. Third, K3 cit can inhibit the phase transformation from thermodynamically unstable COD to thermodynamically stable COM, therefore, much COD was obtained. Since COD is easy to be expelled out from human body, it is hoped that if more COD can be obtained, it is favorable for preventing and curing urolithiasis. So citrate are often used as drugs for the therapy of urinary stones.
supersaturation. The results are shown in Table 2 and Fig. 3. When the original concentration of CaOxa [c(CaOxa)] is above 0.05 mol/L, the dihydrate COD was dominant. When c(CaOxa) is decreased to 0.03 mol/L, only COM crystals was grown. COT was grown when c(CaOxa) is less than 0.01 mol/L. Fig. 4 shows the FT-IR spectra of the CaOxa crystals obtained in different original concentration of CaOxa. When c(CaOxa) > 0.1 mol/L, no apparent effect was observed as changing the concentration from 0.1 to 2.0 mol/L. The main antisymmetric carbonyl stretching band νas (COO− ) of CaOxa and the secondary symmetric carbonyl stretching band νs (COO− ) are located at about 1647 and 1328 cm−1 , respectively. It indicates COD was dominant (Fig. 4a). Two peaks at about 914 and 614 cm−1 in the fingerprint regions further attest it (Table 2). When 0.005 ≤ c(CaOxa) < 0.10 M, νas (COO− ) split to two peaks at about 1646 and 1621 cm−1 (Fig. 4b and c). The peak at 1646 cm−1 become weak and appears as a shoulder at last as decreasing c(CaOxa) in this concentration region. Theνs (COO− ) also shifted from 1328.8 cm−1 to 1324.3, 1322.2 and 1321.1 cm−1 , respectively, when c(CaOxa) decreases from 0.10 to 0.05, 0.01 and 0.005 mol/L. It indicated that a mixture of COD and COM was formed and the percentage of COM increases with decreasing the CaOxa concentration.
3.4. Effect of original concentration of CaOxa The effect of CaOxa relative supersaturation on the crystallization of CaOxa in vesicles was investigated. The morphology of CaOxa was closely related to relative Table 3 The ratios of the intensity of main crystal faces of CaOxa hydrates grown in the presence of various concentrations of PC [c(CaOxa) = 0.02 mol/L] PC (mg/mL)
Without K3 cit, I1¯ 0 1 /I0 2 0
With K3 cit, I2 0 0∗ /I4 1 1∗
0 0.02 0.2 0.4 2 5 15
91/100 92/100 55/100 41/100 12/100 10/100 5/100
61/100 57/100 44/100 46/100 43/100 40/100 34/100
The crystal faces with asterisk indicate COD and that without asterisk COM.
Fig. 4. FT-IR spectra of CaOxa precipitates grown at different original CaOxa concentrations: (a) 0.50, (b) 0.05, (c) 0.02, (d) 0.01 and (e) 0.003 mol/L.
J.-M. Ouyang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 257–258 (2005) 215–220
Fig. 5. XRD patterns of COM crystals obtained in the presence of various concentrations of PC without K3 cit. c(CaOxa) = 0.02 mol/L. c(PC) (mg/L): (a) 0, (b) 0.2, (c) 0.4 and (d) 2.0.
However when c(CaOxa) < 0.005 mol/L, 1645 cm−1 splits to two peaks at about 1670 and 1618 cm−1 (Fig. 4d). It indicates COT is dominant in this case. 3.5. Effect of PC concentration on the crystal planes of CaOxa crystals The XRD patterns of CaOxa crystals grown in the presence of various concentration of PC with and without K3 cit were shown in Figs. 5 and 6, respectively. The ratios of the intensity of their main crystal faces are listed in Table 3. When the concentration of PC increased from 0 to 15 mg/mL, the ratio of I1¯ 0 1 /I0 2 0 (I1¯ 0 1 and I0 2 0 : the peak intensity of 1¯ 0 1 and 0 2 0 crystal faces of COM crystals) gradually decreased from 0.91 to 0.05. The reaction in vesicles is different from that in aqueous solution, which is similar to biological systems. Both specific interactions between the matrix forming material
219
and the crystal planes of the growing nuclei and the curved bilayer surface can affect the structure and morphology of CaOxa crystals. It is known that when the concentration of PC is below critical vesicle concentration (CVC), namely 1.875 mg/mL, vesicles have not yet formed. While PC concentration is above CVC, vesicles were formed. In the latter case, a strong interaction occurs between the negatively charged surface of the vesicle membrane and the positively charged (1¯ 0 1) crystal faces of COM [11]. It results in the inhibition of (1¯ 0 1) face and the preferential growth of (0 2 0) crystal faces (Fig. 5). The addition of K3 cit not only induces COD formation, but the ratio of I2 0 0∗ /I4 1 1∗ (I2 0 0∗ and I4 1 1∗ : the peak intensity of 2 0 0 and 4 1 1 faces of COD crystals) gradually decreased as increasing the PC concentration. As the concentration of PC increases, more and more Ca2+ ions are bound in the form of lipid complexes since the Ca2+ ion binding constant with egg PC is 20 M−1 at 25 ◦ C [12]. That is, more and more Oxa2− ions react with the complexed Ca2+ ions at the membrane–water interface. Since the complexed Ca2+ ions have certain orientation, only CaOxa crystals with a certain crystal planes can be preferentially grown. That is, some crystal faces of CaOxa were inhibited and other crystal faces were preferentially grown.
4. Conclusions The presence of the vesicles can influence the morphology and phase compositions of CaOxa precipitation. The diameter of CaOxa crystals obtained in vesicles (80–150 nm) is smaller than that in bulk solutions (about 1500 nm). In vesicle systems CaOxa precipitated both intravesicular and extravesicular. The original concentration of CaOxa crystal and the ratio of calcium to oxalate also influence the crystallization of CaOxa. With increasing CaOxa concentration in vesicles, the percentage of COM decreases and that of COD increases. When c(CaOxa) > 0.10 mol/L, COD reaches a constant percentage. COT was obtained only at very low CaOxa concentrations. When the ratio of Ca2+ /Oxa2− is nearly 1.0, the percentage of COD reaches the maximum. If the ratio deviated away from 1, less COD and more monohydrate COM are grown. Since COD is easy to be expelled out from human body, it is hoped that if more COD can be obtained, it is favorable for preventing and curing urolithiasis in this systems.
Acknowledgements
Fig. 6. XRD patterns of CaOxa crystals obtained in the presence of various concentrations of PC with 3.3 mmol/L K3 cit. c(CaOxa) = 0.02 mol/L. c(PC) (mg/L): (a) 0.02, (b) 0.2, (c) 4.0 and (d) 8.0. The crystal faces with asterisk indicate COD.
This research work was granted by the Key project of Natural Science Foundation of China (20031010), the Key project of Guangzhou city (2001-Z-123-01), the Key project of Natural Science Foundation of Guangdong Province (013202) and the Key Project of Guangdong Province (2001C31401).
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References [1] J.-M. Ouyang, S.-P. Deng, Dalton Trans. (2003) 2846. [2] J.-M. Ouyang, X.-Q. Yao, Z.-X. Su, F.-Z. Cui, Sci. China, Ser. B 33 (2003) 14. [3] M. Yuzawa, K. Tozuka, A. Tokue, Urol. Res. 26 (1998) 83. [4] T. Bretherton, A. Rodgers, J. Cryst. Growth 192 (1998) 448. [5] J.-M. Ouyang, L. Duan, J.-H. He, B. Tieke, Chem. Lett. 32 (2003) 268. [6] J.-M. Ouyang, L. Duan, J.-H. He, Acta Chim. Sinica 61 (2003) 1597.
[7] R. Backov, C.M. Lee, S.R. Khan, C. Mingotaud, G.E. Fanucci, D.R. Talham, Langmuir 16 (2000) 6013. [8] S.R. Khan, P.A. Glenton, R. Backov, D.R. Talham, Kidney Int. 62 (2002) 2062. [9] P. Messa, M. Marangella, L. Paganin, M. Codardini, A. Curciatti, D. Turrin, Z. Filiberto, G. Mioni, Clin. Sci. 93 (1997) 257. [10] L. Tunik, H. Fueredi-Milhofer, N. Garti, Langmuir 14 (1998) 3351. [11] L. Tunik, N. Garti, J. Cryst. Growth 167 (1996) 748. [12] D. March, CRC Handbook of Lipid Bilayers, CRC Press, Boca Raton, FL, 1990, p. 125.
Effect of concentrations of lecithin, calcium and oxalate on crystal growth of calcium oxalate in vesicles Jian-Ming Ouyang∗ , Feng Deng, Li Duan Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632, PR China Available online 24 November 2004
Abstract The crystal growth of calcium oxalate (CaOxa) in lecithin (PC)–water vesicles has been studied by X-ray powder diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), and dynamic laser scattering (DLS). The molar ratio of calcium to oxalate (Ca2+ /Oxa2− ), the original concentration of CaOxa [c(CaOxa)], the concentrations of PC and additive potassium citrate (K3 cit) can influence the morphology and phase composition of CaOxa crystals. The diameter of CaOxa crystals obtained in vesicles (80–150 nm) is smaller than that in bulk solutions (about 1500 nm). When the ratio of Ca2+ /Oxa2− is nearly 1.0, the percentage of COD in CaOxa crystals reaches the maximum. When c(CaOxa) increases, the percentage of COM decreases and that of COD increases. The content of COD reaches a constant percentage when c(CaOxa) > 0.10 mol/L. COT was obtained only at a very low CaOxa concentration of less than 0.01 mol/L. The concentration of PC greatly affects crystal planes of CaOxa crystals. When PC concentration is above critical vesicle concentration (CVC, 1.875 mg/mL), vesicles were formed. In vesicle system, the (0 2 0) crystal plane of CaOxa was preferentially grown. K3 cit inhibits the pre-critical nuclei of COM and favors COD. © 2004 Elsevier B.V. All rights reserved. Keywords: Calcium oxalate; Lecithin; Vesicles; Potassium citrate
1. Introduction Urolithiasis constitutes a serious healthy problem that affects a significant section of mankind. It is very common and high recurrent disease in the whole world area. Calcium oxalate (CaOxa) is the most frequent crystalline phase in human urinary stones and occurs in more than 80% [1]. Three types of hydrated calcium oxalate – the monohydrate (COM), dihydrate (COD) and trihydrate (COT) – are found in urinary stones. Thermodynamically stable COM is much more common in stones and is not easy to be expelled out from body [2]. While the thermodynamically unstable COD is more easy to be expelled out than COM [3]. Thermodynamically metastable trihydrate (COT) can be a precursor in CaOxa stone formation [4]. If the nucleation, growth or aggregation of COM can be inhibited and/or more COD and
∗
Corresponding author. Tel.: +86 20 85223353; fax: +86 20 85223353. E-mail address: [email protected] (J.-M. Ouyang).
0927-7757/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2004.10.049
COT can be induced, or the transformation of COM to COD and/or COT is promoted, it will be helpful for the prevention of CaOxa stones. Hence, an understanding of crystallization processes of various types of hydrated CaOxa is essential if urologists hope to develop therapeutic agents [4]. There are many reports about the crystallization of CaOxa in aqueous solutions, diluted urines, and artificial urines [2–4]. However, common aqueous solutions are much different from those in biological systems. Urinary stones are usually formed within membrane-bound microspace, and the nucleation and growth of urinary stones are regulated by organic matrix [5,6]. So in the recent years, some ordered systems were designed to mimic the formation of CaOxa stones especially with Langmuir monolayer as a model system [1,7,8]. Khan et al. [8] have investigated the crystallization of CaOxa at Langmuir monolayers of dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylsome glycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG) and dimyristoylphosphatidyl-glycerol
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(DMPG). For each monolayer CaOx crystals were predominantly monohydrate, and most often grew singly with the calcium rich (1¯ 0 1) face toward the monolayer. The number of crystals/mm2 decreased in the order DPPG > DPPS > DPPC and was inversely proportional to surface pressure and mean molecular area/molecule. Talham and co-workers [7] reported the precipitation of COM beneath Langmuir monolayers of four different phosphatidylglycerol lipids. The influence of the density of headgroup packing and monolayer compressibility on heterogeneous precipitation was investigated. The results suggest that the compressibility of the monolayer and the potential of the monolayer to achieve a small area per molecule are more important characteristics that lead to heterogeneous crystallization of COM than is providing a prearranged template. We [1] have examined the nucleation and growth of COM crystals beneath stearic acid (SA) monolayers in the presence of citric acid (H3 cit). The (1¯ 0 1) faces of COM crystals were remarkably stabilized independent of either the change of H3 cit concentration from 0.01 to 0.30 mmol/L or the change of the surface pressure of SA monolayer from 1 to 20 mN/m. This result was due to that there is a strong interaction between the Ca2+ -rich (1¯ 0 1) face of COM and the negatively charged carboxylic groups in the headgroups of SA monolayers and H3 cit. Vesicles have the advantage of providing confined microspace and organic matrix. The crystallization of CaOxa in vesicles can mimic the formation conditions of CaOxa stones. We once reported the deposition behavior of CaOxa in lecithin–water vesicles [5]. The vesicle membranes play an important role in inducing COD formation. We [6] also reported the effects of different kinds of potassium carboxylates on the phase compositions of CaOxa crystals grown in vesicles. The investigated potassium carboxylates included: monocarboxylate potassium acetate (KAc), dicarboxylate potassium tartrate (K2 tart), tricarboxylate potassium citrate (K3 cit), and tetracarboxylate dipotassium of ethylenediaminetetraacetic acid (K2 edta). KAc only induce COM. COT was induced when concentrations of K2 tart > 1.0 mmol/L. K3 cit and K2 edta can induce COM, COD and COT crystals depending on their concentrations. The ability to induce COD follows the order: K3 cit > K2 edta K2 tart ∼ KAc, and the ability to induce COT follows: K2 tart K3 cit > K2 edta KAc. In this paper, the effect of the concentration of PC, the original concentration of calcium oxalate, as well as the ratio of calcium to oxalate (Ca2+ /Oxa2− ) on the crystal growth of CaOxa was studied in PC–H2 O vesicle system.
Fluka or Sigma. Pure water was obtained from a Millipore-Q system: the resistance was 18.2 M cm. 2.2. Apparatus X-ray powder diffraction (XRD) was carried out by a D/max-␥A X-ray diffractometer (Rigaku, Japan), Ni-filtered ˚ Philips Tecnai-10 transmission Cu K␣ radiation (λ = 1.54 A). electron microscopy (TEM). Bruker IFS25 Fourier transform infrared (FT-IR) spectrometer (Bruker Spectrospin, Wissembourg, France). 2.3. Effect of calcium and oxalate concentrations A certain amount of PC was first dissolved in chloroform. After the organic solvent was volatilized at room temperature, 30.0 mL CaCl2 with different original concentration was added. The final concentration of PC in the CaCl2 solution was 5 mg/mL. Sometimes, 1.0 mL aqueous 0.2 mol/L K3 cit was also added together with CaCl2 . The solution was sonicated for 20 min in order to form stable vesicles. Dynamic laser scattering showed the diameter of the vesicles to be about 80–100 nm and the vesicles were stable. Then 30.0 mL K2 Ox solution was added under stirring. The molar ratios of calcium to oxalate are shown in Table 1. After 10 min of reaction, a drop of the suspension was examined by TEM to obtain the size, shape, and appearance of the particles. The rest solution was allowed to age for 2 h at room temperature without stirring. Then the product was centrifuged, washed with chloroform, and dried under vacuum. The phase compositions of the products were analyzed by XRD and FT-IR. The relative percentages of three kinds of hydrated phases of CaOxa crystals, the monohydrate (COM), dihydrate (COD) and trihydrate (COT), was calculated according to K value methods [5]. Blank groups without the presence of lecithin and K3 cit were carried out simultaneously. 2.4. Effect of PC concentration Different amounts of PC were first dissolved in chloroform. After the organic solvent was volatilized at room temperature, 30.0 mL aqueous 0.02 mol/L CaCl2 in the absence or presence of 3.3 mmol/L K3 cit were added. The final Table 1 Mass fraction (W%) of calcium oxalate hydrates grown in vesicles at different molar ratios of Ca2+ /Oxa2− [c(Ca2+ ) = c(Oxa2− ) = 0.10 mol/L] [Ca2+ ]/[Oxa2− ]
2. Materials and methods 2.1. Materials Egg (yolk) lecithin (PC) and all the other chemicals such as calcium chloride, potassium oxalate, potassium citrate and chloroform were of analytical purity and purchased from
With K3 cit COM
9:1 4:1 2:1 1:1 1:2 1:4 1:9
84 38
76 100
Without K3 cit COD
COM
COD
16 62 100 100 100 24
100 61 40 50 66 86 100
39 60 50 34 14
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concentration of PC in the solution was showed in Table 3. Then CaOxa was precipitated and characterized as described above.
3. Results and discussion 3.1. The size of vesicle and the precipitation of CaOxa crystals in vesicles Fig. 1 shows the dynamic laser scattering data of blank vesicles. It can be seen that the diameter of blank vesicles ranges from 80 to 120 nm. However, TEM measurement shows the size of CaOxa crystals precipitated in vesicles is about 80–150 nm, which is lightly larger than that of the blank vesicles. It indicated that the deposition of CaOxa crystals occurs within the vesicles and at membrane surfaces outside the vesicles. The formation of CaOxa crystals in vesicles are much different from that in control solution. The size of CaOxa crystals grown in pure water is about 1500 nm, which is much larger than that (80–150 nm) in vesicles. This is because the nucleation and growth of CaOxa crystals in vesicles was controlled at the ordered vesicle membrane interfaces (inside and outside). So the structure, morphology, and particle size of CaOxa obtained are much different from that obtained from bulk solution.
COM was obtained when the ratio is more than 4 or less than 0.25 in the absence of K3 cit. In a clinic experiment, it is also founded that a much high dietary intake of calcium can decrease the risk of urinary stone. Messa et al. [9] explained this might be due to increasing binding of oxalate by calcium in the gastrointestinal tract, leading to a decrease in urinary oxalate excretion. That is, urinary oxalate is more important than urinary calcium in determining stone formation. In our experiment, oxalate plays more important role in inducing COD growth, which is favorable for preventing stone formation [2].
3.2. Effect of the ratio of Ca2+ /Oxa2− in vesicles
3.3. Effect of potassium citrate
When the molar ratios of Ca2+ /Oxa2− in vesicles vary from 9:1 to 1:9, the relative percentages of different hydrated CaOxa crystals changed as shown in Table 1. When the original concentrations of both Ca2+ and Oxa2− are 0.050 mol/L, namely the molar ratio of Ca2+ /Oxa2− is 1.0, the percentage of dihydrate COD reaches the maximum, nearly 50 and 100% in the absence (Fig. 2a) and presence of K3 cit (Fig. 2b), respectively. If the ratio of Ca2+ /Oxa2− deviated away from 1, less COD and more COM are grown. For example, only
Addition of potassium citrate (K3 cit) can induce much more dihydrated COD formation in spite of the molar ratio of calcium to oxalate (Table 1), the original concentration of CaOxa (Table 2 and Fig. 3) and the PC concentration (Table 3). This effect may be ascribed to the following factors: At first, there is a good structural match between the tricarboxylic K3 cit and calcium spacing in the crystal lattices of COM crystals [3], so K3 cit can inhibit the pre-critical nuclei of COM and favor COD. Second, the surface absorption
Fig. 1. Dynamic laser scattering data for the PC–water vesicles.
Fig. 2. Distribution of COM and COD crystals in vesicle at different molar ratios of Ca2+ /Oxa2− : (a) without K3 cit and (b) with 3.3 mmol/L K3 cit.
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Table 2 FT-IR data of CaOxa crystals grown in vesicles in different original concentrations of CaOxa c(CaOxa) (mol/L)
νas /COM or COT νas /COD or COT νs (COO− ) 948/COM or 914/COD 880/COM 668/COM or 611/COD 550/COT
0.004
0.01
0.05
0.1
0.5
1.0
1670 1618 1326.5
Shoulder 1620.1 1322.2 947 885 669
1646.3 1622.2 1324.3
1647 Shoulder 1328.8 914
1647.3
1646.3
1328.1 914.2
1327.6 917
617.8
615.2
616
885 668
550
Fig. 3. Effect of original concentration of CaOxa on the distribution of COM, COD and COT crystals in vesicles.
of K3 cit on COM is higher than that on COD [10]. That is to say, K3 cit inhibit crystal growth of COM stronger than that of COD, thus it increases the percentage of COD. Third, K3 cit can inhibit the phase transformation from thermodynamically unstable COD to thermodynamically stable COM, therefore, much COD was obtained. Since COD is easy to be expelled out from human body, it is hoped that if more COD can be obtained, it is favorable for preventing and curing urolithiasis. So citrate are often used as drugs for the therapy of urinary stones.
supersaturation. The results are shown in Table 2 and Fig. 3. When the original concentration of CaOxa [c(CaOxa)] is above 0.05 mol/L, the dihydrate COD was dominant. When c(CaOxa) is decreased to 0.03 mol/L, only COM crystals was grown. COT was grown when c(CaOxa) is less than 0.01 mol/L. Fig. 4 shows the FT-IR spectra of the CaOxa crystals obtained in different original concentration of CaOxa. When c(CaOxa) > 0.1 mol/L, no apparent effect was observed as changing the concentration from 0.1 to 2.0 mol/L. The main antisymmetric carbonyl stretching band νas (COO− ) of CaOxa and the secondary symmetric carbonyl stretching band νs (COO− ) are located at about 1647 and 1328 cm−1 , respectively. It indicates COD was dominant (Fig. 4a). Two peaks at about 914 and 614 cm−1 in the fingerprint regions further attest it (Table 2). When 0.005 ≤ c(CaOxa) < 0.10 M, νas (COO− ) split to two peaks at about 1646 and 1621 cm−1 (Fig. 4b and c). The peak at 1646 cm−1 become weak and appears as a shoulder at last as decreasing c(CaOxa) in this concentration region. Theνs (COO− ) also shifted from 1328.8 cm−1 to 1324.3, 1322.2 and 1321.1 cm−1 , respectively, when c(CaOxa) decreases from 0.10 to 0.05, 0.01 and 0.005 mol/L. It indicated that a mixture of COD and COM was formed and the percentage of COM increases with decreasing the CaOxa concentration.
3.4. Effect of original concentration of CaOxa The effect of CaOxa relative supersaturation on the crystallization of CaOxa in vesicles was investigated. The morphology of CaOxa was closely related to relative Table 3 The ratios of the intensity of main crystal faces of CaOxa hydrates grown in the presence of various concentrations of PC [c(CaOxa) = 0.02 mol/L] PC (mg/mL)
Without K3 cit, I1¯ 0 1 /I0 2 0
With K3 cit, I2 0 0∗ /I4 1 1∗
0 0.02 0.2 0.4 2 5 15
91/100 92/100 55/100 41/100 12/100 10/100 5/100
61/100 57/100 44/100 46/100 43/100 40/100 34/100
The crystal faces with asterisk indicate COD and that without asterisk COM.
Fig. 4. FT-IR spectra of CaOxa precipitates grown at different original CaOxa concentrations: (a) 0.50, (b) 0.05, (c) 0.02, (d) 0.01 and (e) 0.003 mol/L.
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Fig. 5. XRD patterns of COM crystals obtained in the presence of various concentrations of PC without K3 cit. c(CaOxa) = 0.02 mol/L. c(PC) (mg/L): (a) 0, (b) 0.2, (c) 0.4 and (d) 2.0.
However when c(CaOxa) < 0.005 mol/L, 1645 cm−1 splits to two peaks at about 1670 and 1618 cm−1 (Fig. 4d). It indicates COT is dominant in this case. 3.5. Effect of PC concentration on the crystal planes of CaOxa crystals The XRD patterns of CaOxa crystals grown in the presence of various concentration of PC with and without K3 cit were shown in Figs. 5 and 6, respectively. The ratios of the intensity of their main crystal faces are listed in Table 3. When the concentration of PC increased from 0 to 15 mg/mL, the ratio of I1¯ 0 1 /I0 2 0 (I1¯ 0 1 and I0 2 0 : the peak intensity of 1¯ 0 1 and 0 2 0 crystal faces of COM crystals) gradually decreased from 0.91 to 0.05. The reaction in vesicles is different from that in aqueous solution, which is similar to biological systems. Both specific interactions between the matrix forming material
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and the crystal planes of the growing nuclei and the curved bilayer surface can affect the structure and morphology of CaOxa crystals. It is known that when the concentration of PC is below critical vesicle concentration (CVC), namely 1.875 mg/mL, vesicles have not yet formed. While PC concentration is above CVC, vesicles were formed. In the latter case, a strong interaction occurs between the negatively charged surface of the vesicle membrane and the positively charged (1¯ 0 1) crystal faces of COM [11]. It results in the inhibition of (1¯ 0 1) face and the preferential growth of (0 2 0) crystal faces (Fig. 5). The addition of K3 cit not only induces COD formation, but the ratio of I2 0 0∗ /I4 1 1∗ (I2 0 0∗ and I4 1 1∗ : the peak intensity of 2 0 0 and 4 1 1 faces of COD crystals) gradually decreased as increasing the PC concentration. As the concentration of PC increases, more and more Ca2+ ions are bound in the form of lipid complexes since the Ca2+ ion binding constant with egg PC is 20 M−1 at 25 ◦ C [12]. That is, more and more Oxa2− ions react with the complexed Ca2+ ions at the membrane–water interface. Since the complexed Ca2+ ions have certain orientation, only CaOxa crystals with a certain crystal planes can be preferentially grown. That is, some crystal faces of CaOxa were inhibited and other crystal faces were preferentially grown.
4. Conclusions The presence of the vesicles can influence the morphology and phase compositions of CaOxa precipitation. The diameter of CaOxa crystals obtained in vesicles (80–150 nm) is smaller than that in bulk solutions (about 1500 nm). In vesicle systems CaOxa precipitated both intravesicular and extravesicular. The original concentration of CaOxa crystal and the ratio of calcium to oxalate also influence the crystallization of CaOxa. With increasing CaOxa concentration in vesicles, the percentage of COM decreases and that of COD increases. When c(CaOxa) > 0.10 mol/L, COD reaches a constant percentage. COT was obtained only at very low CaOxa concentrations. When the ratio of Ca2+ /Oxa2− is nearly 1.0, the percentage of COD reaches the maximum. If the ratio deviated away from 1, less COD and more monohydrate COM are grown. Since COD is easy to be expelled out from human body, it is hoped that if more COD can be obtained, it is favorable for preventing and curing urolithiasis in this systems.
Acknowledgements
Fig. 6. XRD patterns of CaOxa crystals obtained in the presence of various concentrations of PC with 3.3 mmol/L K3 cit. c(CaOxa) = 0.02 mol/L. c(PC) (mg/L): (a) 0.02, (b) 0.2, (c) 4.0 and (d) 8.0. The crystal faces with asterisk indicate COD.
This research work was granted by the Key project of Natural Science Foundation of China (20031010), the Key project of Guangzhou city (2001-Z-123-01), the Key project of Natural Science Foundation of Guangdong Province (013202) and the Key Project of Guangdong Province (2001C31401).
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References [1] J.-M. Ouyang, S.-P. Deng, Dalton Trans. (2003) 2846. [2] J.-M. Ouyang, X.-Q. Yao, Z.-X. Su, F.-Z. Cui, Sci. China, Ser. B 33 (2003) 14. [3] M. Yuzawa, K. Tozuka, A. Tokue, Urol. Res. 26 (1998) 83. [4] T. Bretherton, A. Rodgers, J. Cryst. Growth 192 (1998) 448. [5] J.-M. Ouyang, L. Duan, J.-H. He, B. Tieke, Chem. Lett. 32 (2003) 268. [6] J.-M. Ouyang, L. Duan, J.-H. He, Acta Chim. Sinica 61 (2003) 1597.
[7] R. Backov, C.M. Lee, S.R. Khan, C. Mingotaud, G.E. Fanucci, D.R. Talham, Langmuir 16 (2000) 6013. [8] S.R. Khan, P.A. Glenton, R. Backov, D.R. Talham, Kidney Int. 62 (2002) 2062. [9] P. Messa, M. Marangella, L. Paganin, M. Codardini, A. Curciatti, D. Turrin, Z. Filiberto, G. Mioni, Clin. Sci. 93 (1997) 257. [10] L. Tunik, H. Fueredi-Milhofer, N. Garti, Langmuir 14 (1998) 3351. [11] L. Tunik, N. Garti, J. Cryst. Growth 167 (1996) 748. [12] D. March, CRC Handbook of Lipid Bilayers, CRC Press, Boca Raton, FL, 1990, p. 125.