J. Phys. Chem. B 2009, 113, 9547–9550
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The Complexon-Renal Stone Interaction: Solubility and Electronic Microscopy Studies Andrey V. Kustov,* Boris D. Berezin, and Vyacheslav N. Trostin Institute of Solution Chemistry of Russian Academy of Sciences, IVanoVo, Russian Federation, Akademicheskaya Street, 1, 153045, IVanoVo State UniVersity of Chemistry and Technology, IVanoVo, Russian Federation, F. Engels AVenue, 7, 153460 ReceiVed: February 18, 2009; ReVised Manuscript ReceiVed: May 25, 2009
We have studied how complex formation between calcium and ethylenediaminetetraacetate or citrate ions influences the surface texture and the size of passed oxalate-phosphate renal stones. The four hour concrement treatment by sodium citrate or ethylenediaminetetraacetate aqueous solutions strongly affects the stone texture and provides a mass loss of 6-15%. We have found a significant decrease of the calcium and phosphor content on a concrement surface and formation of appreciable cracks. Our results do indicate that the Ca-complexon interaction can be effectively applied for disrupting some types of renal stones and, especially, residual concrements, which frequently occurs after a surgical operation or an extracorporeal shock-wave lithotripsy. This study provides an additional quantitative physicochemical basis for this slightly invasive therapy. I. Introduction Urolithiasis is a frequent disorder affecting at least 10% of the population of Europe and the United States, more than 70% of renal stones being hydrates of calcium oxalate and phosphates or usually its mixture.1-3 It is not surprising that significant efforts are directed towards urolithiasis to search for effective inhibitors of the insoluble calcium salt formation in vivo.3-12 In particular, it has been recognized long ago that citrate excreting with human urine is an inhibitor of stone-forming calcium and magnesium salts due to the favorable ion-citrate interaction (see refs 3-5 and references therein). Moreover, citrates affect both model calcium oxalate monohydrate crystals (COM) and renal stones in vitro,4,7,9,10 which defines the wide use of magnesium, potassium, and sodium citrates (Na3Cit) in the conservative therapy of the renal stone disease.3-5 Several studies10-12 have shown that some proteins can also affect oxalate crystallization and, therefore, prevent oxalate stone formation in vivo. In particular, it has been found11 that bovine serum albumin inhibits the nucleation of COM and promotes crystallization of calcium oxalate dihydrate (COD). We have obtained similar results studying the interaction between Ca2+ and C2O42- ions in an aqueous solution of cationic surfactantcetyltrimethylammonium bromide, where the hydrophobic electrolyte addition to water delays sedimentation and produces loose crystallohydrates.13 Complexon solutions should provide a more effective inhibition. In fact, we have found13 that the addition of sodium ethylenediamintetraacetate (Na2H2EDTA) to an aqueous solution containing sodium oxalate and calcium chloride prevents COM formation even in the case when the concentration of Ca2+ and C2O42- ions exceeds the solubility product more than one thousand times. Since the Na2H2EDTA therapy is used in medical clinics for treating and preventing cardiovascular disease, atherosclerosis, coronary heart disease, etc.,14,15 it is obvious that complexons may help to improve the conservative urolithiasis therapy.16 Nevertheless, up to this date, the stone-Na2H2EDTA interaction is not studied well enough, and therefore, such chelation therapy does not have sufficient * Corresponding author. E-mail:
[email protected].
Figure 1. Structural formulas of Na2H2EDTA and Na3Cit. Both chelating agents dissolve in an aqueous solution of multicharged organic anions and sodium cations. pH of complexon aqueous solutions used was 4.7 and 8.6 for Na2H2EDTA and Na3Cit, respectively.
experimental basis, although several attempts have been described in the literature.14-18 The present study focuses on the influence of Na2H2EDTA and Na3Cit aqueous solutions on the surface texture and the size of oxalate-phosphate kidney stones in vitro. The main goal of our research is to obtain qualitative information about the concrement-complexon interaction in vitro and extend the physicochemical basis for the urolithiasis chelation therapy. II. Experimental Section Na2H2EDTA (>99%, Chemapol, Chezh. Rep.) and Na3Cit (>99%, Panreac, Spain) were dried under reduced pressure at 353 K for 72 h and used without further purification. The molecular structures of both chelation agents are given in Figure 1. Water was distilled twice. Complexon aqueous solutions containing 5 mass % of the solute were prepared by weight. Renal oxalate-phosphate stones (m ) 250 - 500 mg) were removed by the uretero- or pyelolithotomia in the urological clinic of Ivanovo State Medical Academy. The concrement type17 was determined in the clinical biochemical laboratory. Before the experiment, all the stones were washed by distilled water and dried at first in forced air circulating at 318 K and then at room temperature. For the evaluation of the complexon influence on renal stones, we have chosen a simple but
10.1021/jp901493x CCC: $40.75 2009 American Chemical Society Published on Web 06/18/2009
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Kustov et al. TABLE 1: Solubility (Mass %) of Oxalate-Phosphate Renal Stones in Aqueous Solutions of Na2H2EDTA and Na3Cit at 310 Ka group
distilled water
Na3Cit
I II
0.25 ( 0.1 (2) 0.35 ( 0.1 (2)
7 ( 1.4 (5) 9.1 ( 1.2 (5)
Na2H2EDTA b
6.4 ( 1 (13) 15.4 ( 1.5 (10)
a Molalities of 5 mass % solutions are equal to ∼0.16 mol/kg and 0.21 mol/kg for Na2H2EDTA and Na3Cit, respectively. b Uncertainties represent twice the standard deviation, digits in brackets show the number of experiments performed.
Figure 2. Flow cell for treating renal stones. (1) a platinum thermometer connected with the standard temperature measuring instrument; (2) and (3) steel pipes for input/output of thermostatted complexon solutions; (4) and (5) holes for water input/output for the cell to be thermostatted; (6) a renal stone in a glass capsule; and (7) a magnetic stirrer.
informative method based on the analysis of the sample mass loss during the complexon treatment. The stone was put into the glass cylindrical capsule equipped with holes for input and output of a complexon solution and weighed with the “OHAUS” analytical balances with the sensitivity of 0.01 mg. The capsule was fastened in the 50 mL glass thermostatted cell with distilled water (see Figure 2) and then the system was stirred until thermal equilibrium was reached. The flow rate was adjusted to ensure constant complexon concentration in the reaction cell. The experiment duration was 4 h, and the feed rate of the thermostatted solution provided by the “Zalimp” peristaltic pump (Poland) was chosen to be equal 2.5 mL/min. The temperature of the cell measured by the standard temperature measuring instrument19,20 was 310 ( 0.1 K during the experiment. Within 4 h, the peristaltic pump and the stirrer were cut off and the capsule with the stone was washed three times by distilled water, dried in forced air at 318 K, and then at room temperature up to a constant weight. The stone mass loss (%), which equals in this study its solubility, can be easily estimated from the difference between the capsule mass before and after each experiment and the empty capsule mass. It is obvious that the method applied is not appropriate to determine solubility of a solid concrement in a fixed amount of solution, as it usually occurs in chemical thermodynamics, but it is rather directed to obtain the maximum destruction of the sample within the minimum time under conditions approximated to clinical ones. An electronic microscopy study was performed with the LEO1455 VP microscope (Carl Zeiss) equipped with the SiLi semiconductor detector (Ro¨ntec) for the X-ray spectral microanalysis. The quantitative determination of a chemical element content in a surface layer was performed with the program package enclosed by means of the analysis of the braking radiation level and the comparison of the radiation intensity observed with the etalons provided. For analyzing the composition of the samples selected (see Results and Discussion Section) elemental analysis (Flash EA 1112) and spectrophotometry were employed. III. Results and Discussion The results of solubility measurements are given in Table 1. As can be seen from the results obtained, the 4 h treatment with distilled water did not affect the stone size since the mass loss only slightly exceeds zero. Solubility in complexon solutions varies from 6 to 15 mass % depending on a stone and complexon nature. Table 1 shows that maximum solubility value is observed in the Na2H2EDTA aqueous solution. Apparently, it results from the fact that the stability constant for the [CaEDTA]2- complex
in an aqueous solution is about six orders larger than that for the [CaCit]- complex.18 Table 1 illustrates some more interesting features which are worth noting. It is obvious that the samples studied reveal the dual behavior which is more pronounced in the Na2H2EDTA solution. On the basis of the solubility data in aqueous Na2H2EDTA, we may divide our samples studied into two groups. For the first group, labeled as I in this work, the solubility is found to be rather small, and the mean value equals about 6 mass %. For the second one, labeled as II, the solubility is higher and and the mean value exceeds 15 mass %. Thus, the experimental data appear to indicate that the stone surface composition and texture, where the complexon-calcium interaction occurs, should differ for the groups selected. To test this idea and establish how this interaction affects the stone surface, we have chosen one characteristic sample from both groups and sawed each of them accurately into two identical parts. One half has been treated with the Na2H2EDTA aqueous solution for 4 h. Solubility values obtained are equal to 6.8 and 15.7 mass %, which is in a good agreement with the data given in Table 1. Then the cut sides of the raw and etched halves have been analyzed with electronic microscopy and X-ray spectral microanalysis. Figures 3 and 4 show that both stones reveal heterogeneous structure that appears to arise due to variable crystallization in a human kidney.21 The image represented in Figure 3a allows to register at least two characteristic regions in the sample studied, which we have called the C and P phases. X-ray microanalysis shows that the C phase contains mainly Ca, C, and O atoms. It indicates that it is formed mainly by COM and COD as they are usually observed in many extracted oxalate stones.21,22 The texture of the P phase containing Ca, P, O, and C atoms is associated in turn with a mixture of calcium phosphates and calcium oxalate mono- and dihydrates (see Table 2). Nearly the same picture is observed for the sample belonging to group II (see Figure 4a and Table 3). However, the mean phosphor content at the surface in this case appears to be significantly higher. We have performed the elemental analysis of the raw halves and found that the oxalate-to-phosphate mol ratio is equal to 5.5 and 2.7 for the first and second samples, respectively. Therefore, the phosphate content is higher both at the surface and inside of the raw stones belonging to group II. We believe that this fact just defines higher solubility values due to the favorable H2EDTA2--calcium phosphate interaction. The results of the X-ray microanalysis of the etched halves given in Tables 2 and 3 provide additional support to this idea. The complexon treatment is seen to result in a decrease of the Ca and P content at the surface for both samples. Nevertheless, this effect is much more pronounced for group II, where Na2H2EDTA provides almost complete phosphate removal from the surface (see Table 3). One more important result is that the treatment performed strongly affects the stone texture causing the formation of appreciable cracks (see Figure 4 b).
Complexon-Renal Stone Interaction
Figure 3. Images of the oxalate-phosphate renal stone from group I for (a) raw and (b) etched samples. Symbols C and P denote the phases with the relatively high carbon and phosphor content on a concrement surface, respectively. The scale bars on both photos are 100 µm.
As for citrate, it is obvious that, despite sufficiently large uncertainties of the experimental data, the stone solubility appears to increase with the rise of phosphate content in the sample. This effect, however, is insignificant in comparison to a Na2H2EDTA solution. Table 1 shows that solubility of oxalatephosphate stones, belonging to group I with the higher oxalateto-phosphate mol ratio, appears to be larger in a citrate solution than in a Na2H2EDTA one. Therefore, we may draw an interesting conclusion that Na2H2EDTA will strongly influence phosphate stones, while citrate would be more effective for treating oxalate concrements. In fact, we have found that tablets with the mass of 300 mg pressed from freshly prepared COM are better dissolved in a Na3Cit solution. The sample mass loss in this case is 6 mass % within 4 h, which is much larger than the mass loss in a Na2H2EDTA solution being lower than 3 mass %. This important result is rather surprising from the thermodynamic point of view, since, as has been mentioned above, ethylenediamintetraacetate-ion forms a much stronger complex with the Ca2+ ion than the citrate ion.18 Apparently, this rather weak ability of H2EDTA2- to bind with calcium at a stone surface arises due to steric problems. In particular, geometric factors are found to play an important role in the energetics of the citrate binding with the oxalate crystal surface. Molecular modeling has shown that citrate ions bind strongly to an acute single [101] step on the (-101) face of the COM crystal, but not to a [-100] step on the (010) face.9 Such citrate behavior arises from electrostatic repulsion between carboxylic groups of the citrate molecule and the dicarboxylic residues.9 Figure 1 shows that the H2EDTA2- ion is larger than citrate and contains one more carboxyl group. Therefore, we may state that the geometric and electrostatic factors should affect the Na2H2EDTA-oxalate stone interactions stronger than in a citrate
J. Phys. Chem. B, Vol. 113, No. 28, 2009 9549
Figure 4. Images of the oxalate-phosphate renal stone from group II for (a) raw and (b) etched samples. Symbols C and P denote the phases with the relatively high carbon and phosphor content on a concrement surface, respectively. The scale bars are 10 and 100 µm for photos (a) and (b), respectively.
TABLE 2: The Chemical Element Content (at. wt %) on the Renal Stone Surface from Group I as Depicted in Figure 3 chemical element C O Ca P Na Mg Al Si
mean content Ra 25.6 55.9 13.8 4.4 0.4 -
b
C phase
P phase
Ea
R
E
R
E
32.6 55.5 8.4 3.0 0.4
10.6 54.3 35.0 0.1 -
26.1 58.9 13.3 1.7 -
7.6 56.4 20.9 13.7 1.1 0.3 -
16.1 58.0 17.4 7.3 1.0 0.1 -
0.2 0.4
a Symbols R and E denote the raw and etched halves, respectively. b Uncertainty of the chemical element content is within 10-20 at. wt %.
solution, which, in our opinion, results in larger repulsion and weaker binding to the stone surface. We believe that Monte Carlo or molecular dynamics simulations of the H2EDTA2--COM crystal interaction in water with appropriate potentials may help to better understand this phenomenon. Conclusions Our results do indicate that citrate and ethylenediamintetraacetate may be recommended for both preventing the stone formation13 and removing concrements from a kidney. It is obvious that as phosphate content in a stone is increased, the
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TABLE 3: The Chemical Element Content (at. wt %) on the Renal Stone Surface from Group II as Depicted in Figure 4 chemical element C O Ca P Na Mg Si a
mean content R
a
19.8 54.2 15.6 9.5 0.5 0.1 0.2
C phase
P phase
E
R
E
R
E
53.2 42.3 3.1 0.9 0.6 0.2
31.8 58.2 9.5 0.6 -
45.8 48.5 4.5 0.4 0.7 -
9.5 62.4 18.2 8.4 1.5 -
42.1 48.5 7.6 0.7 0.8 0.3
Symbols are identical to those in Table 2.
complexon-stone interaction becomes more and more intensive. The Na2H2EDTA therapy appears to be very effective for removing phosphate-oxalate and probably phosphate-oxalateurate stones but not for pure oxalate concrements. We have found that some advantages of citrates, which peroral administration to calcium stone formers is frequently used to provide physiological and physicochemical correction and inhibit new stone formation.3-5 However, solubility values obtained are not high enough to provide a rapid removal of such concrements from a human kidney (see Table 1). We believe that it is more appropriate to join the peroral citrates therapy and intravenous Na2H2EDTA infusions14,15 with the extracorporeal shock-wave lithotripsy, since, as can be seen from Figure 3 b, the complexon-stone interaction should reduce the resistance to shock even for presumably oxalate concrements. One more possible scope of this research consists of possibility improving the results of the uretero- and pyelolithotomia. Thermostatted complexon solutions with the peristaltic pump mentioned above may be simply used in this case for removing residual concrements through catheters installed in a kidney. This extension of the chelation therapy, however, appears to require additional studies dealing with the effects of complexon solutions on the kidney. Acknowledgment. The authors are grateful for Drs. L.I. Ivanova and M.A. Krestyaninov for significant help in the preparation of the manuscript. This work was supported by the Russian Foundation for Basic Researches (Grant N 08-0312053-ofr).
References and Notes (1) Marangella, M.; Vitale, C.; Bagnis, C.; Bruno, M.; Ramello, A. Nephron 1999, 81, 38–44. (2) Prie´, D.; Ravery, V.; Boccon-Gibod, L.; Friedlander, G. Kidney Int. 2001, 60, 272–276. (3) Baumann, J. M. Urol. Research 1998, 26, 77–81. (4) Ettinger, B.; Pak, C. Y. C.; Citron, J. T.; Thomas, C.; AdamsHuet, B.; Vangessel, A.; Preminger, G. M. J. Urol. 1997, 158, 2069–2073. (5) Caudarella, R.; Vescini, F.; Buffa, A.; Stefoni, S. Front. Biosci. 2003, 8, 1084–1106. (6) Nakagawa, Y.; Abram, V.; Kezdy, F. J.; Kaiser, E. T.; Coe, F. L. J. Biol. Chem. 1983, 258, 12594–12600. (7) Shiraga, H.; Min, W.; Van Dusen, W. J.; Clayman, M. D.; Miner, D.; Terrell, C. H.; Sherbotie, J. R.; Foreman, J. W.; Przysiecki, C.; Neilson, E. G.; Hoyer, J. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 426–430. (8) El’nikov, V. Yu; Rosseeva, E. V.; Golovanova, O. A.; FrankKamenetskaya, O. V. Russ. J. Inorg. Chem. 2007, 52, 150–157. (9) Qiu, S. R.; Wierzbicki, A.; Orme, C. A.; Cody, A. M.; Hoyer, J. R.; Nancollas, G. H.; Zepeda, S.; De Yoreo, J. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1811–1815. (10) El-Shall, H.; Jeon, J.-H.; Abdel-Aal, E. A.; Khan, S.; Gower, L.; Rabinovich, Y. Cryst. Res. Technol. 2004, 39, 577–585. (11) Junfeng, L.; Huaidong, J.; Xiang-Yang, L. J. Phys. Chem. B 2006, 110, 9085–9089. (12) Wang, L.; Guan, X.; Tang, R.; Hoyer, J. R.; Wierzbicki, A.; De Yoreo, J. J.; Nancollas, G. H. J. Phys. Chem. B 2008, 112, 9151–9157. (13) Kustov, A. V.; Berezin, B. D.; Strel’nikov, A. I.; Shevyrin, A. A.; Berezin, M. B.; Korolev, V. P. Dokl. Chem. 2006, 410, 150–153. (14) Walker, M. The Chelation Answer; M. Evans & Co: New York, 1982. (15) Cranton, E. M.; Frackelton, J. P. Scientific Rationale for EDTA Chelation Therapy in Treatment of Atherosclerosis and Diseases Therapy. In EDTA Chelation Therapy; Cranton, E. M., Ed.; Hampton Roads Publishing Co., Inc.: Charlottesville, VA, 2001, pp 3-61. (16) Tiktinskii, O. L., Alexandrov, V. P. Urolithic disease; S. Petersburg, 2000 (in Russian). (17) Shevyrin, A. A. Ph.D. Thesis, MONIKI, Moscow, 2008 (in Russian). (18) Dyatlova, N. M.; Temkina, V.Ya.; Kolpakova, I. D. Complexons and complexonates of metals; Chemistry: Moscow, 1988 (in Russian). (19) Kustov, A. V.; Emely`anov, A. A.; Syshchenko, A. F.; Kresty`aninov, M. A.; Zheleznyak, N. I.; Korolev, V. P. Russ. J. Phys. Chem. 2006, 80, 1532–1536. (20) Kustov, A. V.; Korolev, V. P. J. Phys. Chem. B 2008, 112, 2040– 2044. (21) Sokol, E.; Nigmatulina, E.; Maksimova, N.; Chiglintsev, A. Eur. J. Miner. 2005, 17, 285–295. (22) Palc`hik, N. A.; Moroz, T. N.; Maksimova, N. V.; Darı`n, A. V. Russ. J. Inorg. Chem. 2006, 51, 1098–1105.
JP901493X
