Investigation on Dissolution Mechanism of Calcium Oxalate Crystals Using Various Chelating Agents

Transcription

1 Investigation on Dissolution Mechanism of Calcium Oxalate Crystals Using Various Chelating Agents ANNUAL REPORT OF MINOR RESEARCH PROJECT FOR THE PERIOD OF Submitted to UNIVERSITY GRANTS COMMISSION GOVERNMENT OF INDIA Principal Investigator Shanthil M. Department of Chemistry Government Victoria College Palakkad Kerala, India

2 Investigation on Dissolution Mechanism of Calcium Oxalate Crystals Using Various Chelating Agents CONTENTS SI. no. Pages 1. Introduction 3 2. Experimental Section 4 3. Results and Discussion 6 4. Conclusions References Outcome of the project 19

3 1.0 INTRODUCTION Chemolysis or dissolution therapy using drug molecules brings more attention due to the simplicity and safety in the procedure. 1 Among the other treatment options in kidney stone, such as surgery, extracorporeal shock wave lithotripsy (ESWL) etc. patient prefer dissolution therapy due to painless and uncomplicated procedure. 1, 2 However in modern medicine doesn t have an effective drug for dissolution therapy for kidney stone. Most of the dissolution agents have shown the toxicity (for eg. EDTA) and have significant side effects as a drug. 1-3 Another important challenge in the treatment of kidneys tone was its high recurrence rate as approximately 50% of the stone formers have recurrent stones within 10 years after the stone removal. 4,5 Thus surgery is not favorable treatment for recurrence stone formers. The new and simpler method of treatment of kidney stone removal was needed either by chemical aid or mechanical aid. Chemical composition analysis suggests that urinal or renal calculi mainly contains calcium oxalate (CaOx) crystals. Thus the treatment of kidney stone involves the chemistry of dissolution, a proper understanding is required to design the drug for dissolution therapy. Since the main content of stone is CaOx calcium chelating molecules can serve as dissolution agent. Large number of chelating agents are available, a well known calcium chelating agent is EDTA, had been previously shown to have dissolution activity on COM stone in vitro. However, the toxicity of these agents limits its application in vivo. 4-6 One of the main drug was used to prevent the recurrence rate of kidney stone was potassium citrates. However the use of such drug for chemolysis of calcium oxalate stone was not commonly employed due to the poor activity in the case of bigger sized stone. 5,6 Our previous investigations results point out that sodium citrate can be a better dissolution agent for calcium oxalate crystals. The role of citrate was well investigated and found to have a major role in the dissolution process of CaOx. However the function of counter cation was still unclear. Hence we carried out the detailed experiment to understand the role of number of sodium ions in citrate in dissolution activity. It showed an increase in the dissolution activity if the number of sodium ions increases. 11 Therefore, understanding the dissolution mechanism of citrate has a significant scientific and practical application for the prevention and treatment of kidney stone. In the present investigation there for aimed to evaluate and exploring the mechanism of dissolution activity of citrate on calcium oxalate crystals.

4 2.0 EXPERIMENTAL SECTION All of the chemicals and reagents (Calcium oxalate, citric acid, Trisodium citrate, etc.) were purchased from Nice chemicals, merck and sigma Aldrich and used were as such. All the glass wares was washed and cleaned with distilled water. 2.1 Turbidity and conductivity measurements Turbidity measurements of the sample are done using an ELICO-PE-138 water quality analyzer with filter in the range of Conductance measurements are done using Systronic conductivity meter Optical microscope studies Optical microscope measurements were carried out in Olympus CH20i with transmission mode. The digital camera was inserted one of the eyepiece and images were recorded with the help of a computer. The calcium oxalate crystal is placed in the petridish and immersed in the sample solution such as K-Citrate, Na-Citrate etc. Using 100 x magnifications, we focussed the crystal of CaOx and recorded the photographs at various time and video of dissolution. 2.3 SEM Investigations 0.1 M K-citrate prepared by adding KOH in citricacid. About g of CaOx was accurately weighed in an analytical balance and transferred to three separate beakers. To the beaker-1, about 7ml of 0.1M Na-Cit, K-Cit solution and 43ml of distilled water is added. The same quantity of reagents is added to beaker-2 and beaker-3. Then the suspension in the beaker-1, beaker-2 and beaker-3 is allowed to stand for 1hr, 2hr and 3hr respectively. After 1 hr, decant the clear solution in the beaker-1 through a quantitative ashless (Whatman No: 40) filter paper. Wash the precipitate left in the beaker and the glass rod with minimum quantity of distilled water (approximately 15ml), allow the precipitate to settle and decant the clear solution through the same filter paper. Repeat this process several times. Now transfer the precipitate completely to the filter paper. Remove any precipitate sticking to the sides of the beaker and glass rod carefully using a rubber-tipped glass rod and transfer it to the filter cone. Allow the filter paper to become dry. The sample is send for SEM analysis.

5 2.4 Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES) ICP-AES analysis of calcium in CaOx treated samples were done from the Sophisticated Test and Instrumentation Centre (STIC) cochin, Kerala. 2.5 Gravimetry CaOx dissolution was quantified by gravimetric measurements by weighing the undissolved CaOx remaining in the beaker after the treatment of sample solution (trisodium citrate, citric acid etc.) The details of experiment methods were illustrated in figure 2. About g of CaOx was accurately weighed and transferred into a Beakers. 50 ml of sample solution in distilled water is added.then the 2 beakers are sonicated for 3 minutes using a fast clean ultrasonic cleaner.then the suspension was incubated for 2 hours, so as to allow the precipitate of CaOx to settle for about 2 hours. Decant the clear solution through a quantitative ashless (whatman No: 40) filter paper. Wash the precipitate left in the beaker and glass rod with cold water with minimum quantity of distilled water (approximately 15 ml), allow the precipitate to settle and decant the clear solution through the same filter paper. Repeat this process several times. Now transfer the precipitate completely to the filter paper. Remove any precipitate sticking to the sides of the beaker and glass rod carefully using a rubber- tipped glass rod and transfer it to the filter cone. Allow the filter paper to become dry. Fold the moist filter paper carefully around the precipitate and place it in a silica crucible which has been previously ignited to constant weight. Cover the crucible loosely with its lid, place it on a clay pipe triangle kept on a tripod and gently heat it in a small non-luminous flame so that the filter paper first dries and then chars slowly without catching fire. When the charring is complete, increase the burner flame and heat the crucible at dull redness with free access of air so that carbon is completely burnt off. Heat the crucible at red heat for about 30 minutes and then allow it to cool. Transfer it to a desiccator, cool and weigh. Repeat heating, cooling, and weighing till the weight becomes constant. 2.6 Complexometric titration method The dissolution of CaOx was analyzed by estimating the Ca 2+ concentration in filterate by EDTA titrations using Eriochrome Black T as indicator.

6 10 ml of this filterate solution is pipette out into a conical flask. 2 ml of buffer solution of P H 10 and 6 drops of Eriochrome Black T indicator are added. The solution is shaken well and titrated against the EDTA solution which is set in the burette, until the color of the solution changes from wine red to blue at the endpoint. The titration is repeated for concordant titre values. 3.0 RESULTS AND DISCUSSIONS Dissolution therapy has been widely accepted as an effective treatment for kidney stone disease. Dissolution agents may be used as primary treatment to dissolve the preformed stones. EDTA, a well-known Calcium-chelating agent, had been previously shown to have dissolution activity on COM stone in vitro. However, the toxicity of this agent limits its application in vivo. Potassium Citrate (K 3Cit) is one of the main drugs for treatment and prevention of renal stones. As a clinical drug, K 3Cit is widely used for its advantages such as biological nontoxicity, low price, few side effects, and potential for long-term use. However, their dissolution effects on COM crystals, which are the major crystalline compositions of kidney stones, remained unclear and under investigated. Therefore understanding the mechanism of Citrate has a significant scientific and practical application for the prevention and treatment of renal stones. Our previous studies showed that Na-Cit is a better candidate for chemolysis of CaOx crystal which is a main component in kidney stone. The role of citrate in the mechanism of dissolution of CaOx is well investigated and found to be great effect on CaOx dissolution process. Our studies suggest that the mechanism of dissolution occurs as follows. (1) Binding of citrate on the exposed calcium in the CaOx crystal surface, (2) dissolution process occurred through equilibrium and (3) Time has a significant effect on this dissolution process. In the case of sodium citrate driven calcium oxalate chemolysis, we found a difference in the amount of dissolved calcium which was analysed through gravimetry and complexometry. Estimated amount of calcium from the complexometry is found to be greater than by

7 Scheme 1. Scheme illustrating the investigation of dissolution of CaOx crystal. gravimetric estimation. In principle, gravimetry estimates the undissolved CaOx and adsorbed sodium on the surface of CaOx where as complexometry gives the direct quantification of dissolved Ca. In order to understand this discrepancy in analysis, we carried out a detailed investigation on the CaOx dissolution by Na3-Cit in comparison with K3-Cit. 3.1 Binding of Citrate on the surface of CaOx crystals The dissolution of CaOx mainly involves two steps: (1) binding of Citrate with Ca 2+ ions on the surface of solid CaOx crystals and (2) Dissolving the Ca 2+ ions from the CaOx crystal surface. We carried out Zeta Potential Analysis to understand the binding of citrate on calcium oxalate crystals. Zeta potential measurements directly determine the surface charge of the particles is in nano or micrometer size. From the analysis, we observed a Zeta Potential of -18 mv in the case of CaOx in distilled water. This value clearly indicates that the dispersed CaOx crystals are unstable, and therefore may tend to coalesce to form large aggregates. Then we measured the Zeta Potential of dispersed CaOx in the presence of trisodium citrate. The result showed an increase in the magnitude of surface charge to -38 mv that infers the binding of citrate on the solid CaOx surface. On increasing the sodium citrate concentration, surface charge of CaOx increases. This infers the citrate bound on the surface CaOx and increases the magnitude of negative surface charge. Obvious increase in the ph was observed at higher concentration of sodium citrate. Increase in Zeta potential of CaOx crystals after CitNa3 addition inhibits the aggregation of CaOx crystals by increase in repulsive force between

8 crystals. It is important to note that the presence of citrate molecules in the urine, may prevent the stone formation even calcium oxalate crystals are present in the urine. We carried out detailed investigations of time dependent dynamic light scattering measurements of calcium oxalate crystals in distilled water, calcium oxalate in citric acid and sodium citrate solution. Figure 1 shows the DLS analysis of aggregation of CaOx in distilled water. Initially the average hydrodynamic diameter of crystals was 900 nm and it was increased to 1200 nm after 10 min. After 30 min the extent of aggregation was high the size has become 2000 nm. The stability of calcium oxalate crystals were very poor in water and it tend to aggregate to form bigger stones and it support the zeta potential data. But in the case of citrate treated CaOx, the increase in size was negligible even after one hour (Figure 1). This experiment reveals the citrate prevents the aggregation of CaOx by increasing the surface charge. Further understanding the agglomeration, we studied the sedimentation of calcium oxalate using turbidity measurements. Turbidity measurements were carried out as same like DLS analysis. Here, the turbidity changes are measured as a function of time and shown in the figure 1. With in 3 hour of time CaOx in distilled water and citric acid solution nm 30 minutes 1500 nm A h 900 nm 1100 nm B Intensity, % Intensity, % Size, d h Size, d h Turbidity Change in % C A Sodium Citrate DI water Citric acid Time, h D B DI water CitH3 CitNa3 Figure 1. Dynamic Light Scattering measurement of aggregation of calcium oxalate powder in distilled water(a) and in the presence of trisodium citrate (B). (C) Turbidity changes in percentage with time, (D) turbidity difference is shown in the photograph.

9 sediments at a higher rate, however in sodium citrate solution sediments slowly. We have taken the photographs after 3hr of dispersed calcium oxalate in distilled water, citric acid and sodium citrate solution. Among the three solution sodium citrate treated sample (Figure 1, right side) retained the turbidity even after 3 h where as others solution becomes relatively clear. Thus it was proved that the citrates electrostatically binds on the surface of CaOx crystals and prevent the aggregation for a period of time. The above results conclusively prove the citrate binding on the surface of calcium oxalate and this may be the first step of dissolution process. 3.2 Effect of cation(na+ / K+)in citrate on dissolution of CaOx It is well known that sodium citrates show better dissolution activity towards calcium oxalate crystals. However the efficiency of dissolution of calcium oxalate crystals by various citrates derivatives like mono, di, tri sodium derivatives is not well understood. Calcium oxalate crystals of 0.3 g were weighed accurately and transferred in a beaker having (A) distilled water, (B) Citric acid, (C) mono sodium, (D) di sodium, (E) tri sodium derivative of citric acid and incubated at room temperature for 3 hour. Sodium derivatives of citric acid were prepared by reacting citric acid with equivalent amount of sodium hydroxide. In order to balance the sodium concentration added respective amount of sodium chloride in each solution. After the addition of solution into the calcium oxalate, the solution becomes turbid and major parts of the calcium oxalate remain undissolved. Three hour incubation settles the undissolved calcium oxalate and filtered the clear solution and it is shown in scheme. We estimated the calcium content in filtrate by complexometric titrations using EDTA as complexing reagent and weight of the undissolved CaOx determined by Gravimetric method. Initially analyzed the ph of the solution A, B, C, D, E and found to be increasing alkalinity when number of sodium increases. Further we observed that calcium oxalate slightly lowering the ph of distilled water. The control experiment with distilled water was carried for the estimating the role of water alone on dissolution of calcium oxalate crystals. Gravimetric experiment results were plotted and shown in the figure 5. Trisodium citrate shows 26 % better performance of dissolution of CaOx crystals as compared to control. The number of sodium ions increases in citric acid that increases the efficiency of dissolution, for eg. di sodium citrate and mono sodium citrate shows 10% and 5 % dissolution efficiency less than the trisodium citrate. This may be due to the following reasons (1) increase in the alkalinity (2) increase in efficacy of complexation of calcium. In order to understand it clearly, we carried out complexometric titrations in which

10 EDTA is used as complexing agent. EDTA strongly binds calcium as compared to the oxalate and citrate thus it directly determines the concentration of soluble calcium in solution. Generally sparingly soluble salts exist in equilibrium with undissolved and dissolved parts as shown in the figure 2. Dissolved Calcium strongly bound with Citrate by a proportion and the ability of binding with calcium determined by the ease of formation of carboxylate anion. Dissolved calcium estimated from the filterate of the samples A, B, C, D, E. by EDTA titrations and it showed, 4 times increase in the dissolved calcium concentration as compared to the control when trisodium citrate treated sample. Amount of Calcium (g) ph -5 ph -1 ph -4 ph -6 ph -8 0 Figure 2. Amount of undissolved calcium determined using gravimetry after the dissolution of Calcium oxalate crystals using various Citrate derivatives and distilled water Results shows an increasing trend of calcium concentration as a function of number of sodium ions in citrates and the observed trend are in good agreement with gravimetric results. 3.3 Effect of time and concentration of citrates on dissolution of calcium oxalate We carried out gravimetric analysis. Calcium oxalate crystals of 0.3 g were weighed accurately and transferred in a beaker having (A) distilled water, (B) tri sodium derivative of citric acid and incubated at room temperature for 2 hour. Sodium derivatives of citric acid were prepared by reacting citric acid with equivalent amount of sodium hydroxide. In order After the addition of solution into the calcium oxalate, the solution becomes turbid and major parts of the calcium oxalate remain undissolved. Three hour incubation settles the undissolved calcium oxalate and filtered the clear solution. In gravimetric experiment, the amount of undissolved CaOx after

11 the treatment with citrate is determined. The result shows that Na 3Cit shows a better dissolution effect than distilled water (Figure 3). We found that the time has a crucial role on quantity of dissolution. We increased the incubation time to 24 h and the quantified the calcium ions by gravimetry. Results (Figure 3) clearly indicate the enhancement of dissolution for longer time on citrate treated samples. Increased the incubation time from 2 hour to 24 hour and each time compared the amount of dissolution in sodium citrate treated samples with control. Relative dissolution was enhanced by two times in longer period of 24 h incubation in sodium citrate treated samples. Required amount of sodium chloride was added to maintain the equal sodium concentration in the samples. Figure 3. (A) Dissolution efficacy of citrate at various treatment period( 2 and 24 h) with respect to the control. (B) Gravimetric analysis showing time dependent increase in the dissolution of CaOx by sodium citrate. (C) Gravimetric analysis showing time dependent decrease in the dissolution of CaOx by potassium citrate. (D) A comparative plot of percentage of dissolution by K-Cit and of % of dissolution by Na-Cit.

12 We have investigated the time dependent dissolution by conductivity analysis, which can directly measure the dissolved calcium ions. The observed conductance of solution is directly relates to the amount of soluble positive and negative ions. Any change in the conductivity with respect to time can be correlated to the dissolution of calcium oxalate with time. It was evident from the Figure 4, citrates shows slight increase in the conductance as compared to the control and citric acid. The time dependent changes observed in the plot directly attribute to the increase in the dissolution of calcium oxalate. Sodium citrate showed an increasing trend of dissolution of CaOx with increase in time and it follows a different dissolution mechanism from the potassium citrate. Citrate is common component in both samples, where the only difference is in their counter cation. In the case of sodium citrate, dissolution is not only driven by ph change (sodium citrate make alkaline) but also an alternative mechanism plays a crucial role in dissolution process. A comparative plot is shown in the figure 3. At nearly 3 hour the dissolved calcium amount were same for K-Cit and Na-Cit. This indicates that a different mechanism plays in the dissolution process of CaOx by Na-Cit. 0.6 A A Conc. Of Ca 2+, mm H-Conc L-Conc Dissolved calcium, % B Dissolved Ca in moles C Weight of CaOxin gm Concentration of Na-Cit Figure 4. (B) Gravimetric analysis showing the effect of concentration of CaOx on dissolution by Na-Cit and ratio of Na-Cit to CaOx.

13 This result suggests the action of citrate on calcium oxalate can be following reversible mechanism. Concentration of citrate is another parameter which can alter the dissolution of CaOx. We increased the citrate concentration to 6 times and quantified the dissolved calcium in the solution using ICP-AES. The measured values were plotted in Figure 10, shows an enhancement of dissolved calcium in the case of higher concentration. About 15 times enhancement of dissolution were observed if the concentration of citrate increases by 6 times. Concentration and time have a crucial role in the dissolution efficacy of citrate. 3.4 Effect of concentration of CaOx on dissolution by Sodium citrate It is important to note that the molar solubility of CaOx remains as same and it is not affected by the amount of increase in weight of the CaOx. Hence increase in weight of the CaOx does not have any influence on the amount of dissolved CaOx in water. The above observation can be explained; undissolved CaOx exists in the Na-Cit solution as in solid form. Our previous experiments found that the undissolved CaOx is bound by Na-Cit. This bounded citrate helps to remove calcium from the surface of CaOx. Number of exposed surfaces increases with the amount of CaOx. This leads to an increase in the citrate bounded surface of CaOx. As a result the amount of dissolved calcium increases with the help of bounded citrate. The figure 8 shows the CaOx dissolution becomes maximum after 1.2 g. At higher amount of CaOx, surface coverage of citrate becomes less as compared to the amount of undissolved CaOx. Dissolved CaOx is proportional to the number of bounded citrates. We calculated the ratio between sodium citrate and CaOx and is plotted against the dissolution of calcium oxalate in percentage and is shown in the Figure 8. The result showed an increase in the dissolution with increase in the Na-Cit: CaOx ratio. However it becomes constant after 0.08 showing a negligible change in the dissolution of CaOx. All these experiment were carried out at 3 h incubation time. 3.5 Effect of concentration of sodium citrate on CaOx dissolution We carried out CaOx dissolution by keeping weight of CaOx (0.3) constant and varying the Na-Cit concentration from M. Estimated the dissolved calcium directly by Inductively Coupled Plasma Atomic Emission Spectrometer from the filtrates shown in scheme 1. Figure 4 showed an increase in the calcium dissolution when the citrate concentration increases. We found that a linear relationship between calcium dissolution and Na-Cit

14 concentration. This observation further supports that in the above mechanism the dissolution occurred through binding of sodium citrate on CaOx surface and solubilizes the Ca 2+ ions in solution. Hence it was expected that, the concentration of sodium citrate increases dissolution of Ca 2+ from CaOx also increases. Direct evidence of dissolution effect of Na-Cit and K-Cit solution on CaOx crystal is obtained from optical microscope analysis. We have used transmission mode to illuminate the sample and the CaOx was dispersed in the Na-Cit and K-Cit solution. The images were recorded at different times and presented in the figure 5. Sodium citrate and potassium citrate treated CaOx crystal dissolves initially but in the course of time K-Cit has no effect on dissolution. In the case of Na-Cit treated sample, crystal size decreases with time with a morphological change. The surface of the CaOx crystal showed the formation of pore due to the etching of Ca 2+ by Na-Cit and size of CaOx crystal with respect to time. It was evidenced from the image of Na-Cit treated CaOx crystal which showed a surface roughness and it increases with time. Citrate may bind on the surface of the CaOx, due to this binding Ca 2+ gets detached from the lattice of CaOx crystal and dissolves in the solution as calcium citrate. This calcium citrate formation was thermodynamically favorable due to the high association constant. In the case of sodium citrate, sodium may substitute the vacant lattice position of Ca 2+ in CaOx crystal due to the size matching of Na + (size in pm) and Ca 2+ (size in pm). This process hinders the reversible binding of Ca 2+ on the CaOx crystal and favours the dissolution process. Since the above process is time dependent, dissolution process also increases with time. This observation suggests that Na + and citrate combination have a cooperative effect on dissolution process of CaOx crystal. However, in the case of K-Cit, K + ion cannot substitute and occupy on the vacant lattice position of Ca 2+ (size in pm) due to the mismatch in the size.

15 Figure 5. Optical microscopic images of small CaOx crystal in Na-Cit. solution. In order to verify the above mechanism, we carried out SEM investigation which is presented in the Figure 5. The Figure 5 showed the CaOx crystal treated with water, K-Citrate, Na-Citrate treated samples and as compared to the water size of crystal reduced in K-Citrate, Na-Citrate treated samples. This indicates the dissolution is occurring effectively when K-Citrate and Na- Citrate treated. The Figure 5 clearly showed a numerous pores are shown in the Na-Citrate treated sample. The surface structure are similar to the results obtained in optical microscopic studies. Qiu et al have proved the presence of citrate modulate growth kinetics and morphology of steps on the CaOx crystallization. The crystal surface of CaOx formed is roughened. This evidence suggest and support our mechanism but in addition to this sodium plays a role on the morphological changes happened in the CaOx crystal. This observation suggest the NaCitrate is the better choic of treatment for larger CaOx stones.

16 4.0 CONCLUSIONS In conclusion, the dissolution mechanism of CaOx by Na-Cit is entirely different from any other citrate. The Na + ion and citrate have a very crucial role on the dissolution mechanism of CaOx by Na-Cit. The first step in the dissolution process is the binding of citrate on the exposed Ca 2+ of CaOx crystal surface and the dissolved Ca 2+ which is in equilibrium with undissolved Ca 2+ ions. This disturbs the equilibrium and increases the forward reaction due to the high association constant of Ca-Cit as compared to CaOx. On the other hand vacant lattice site was created due to detachment of Ca 2+ by citrate. The vacant sites were occupied by Na + ions due to the charge similarity and matching of the size. The cation exchange is a time dependent process, thus sodium citrate dissolution enhances with time. Optical microscopic studies provide direct evidence that Na + and citrate combination have a cooperative effect on dissolution process of CaOx crystal. The time dependent gravimetric and complexometric analysis supports this exchange mechanism. ICPAES results shows that the concentration of citrate have a tremendous effect on the dissolution process. Thus the dissolution mechanism of CaOx by K-Cit follows leaching out mechanism, which by Na-Cit follows exchange mechanism. This exchange mechanism is responsible for the higher efficacy of Na-Cit in the dissolution of CaOx than K-Cit. In summary, our present investigation is a direct evidence for the exchange mechanism of the dissolution of CaOx crystal by Na3- Cit. Our studies suggest that Na3-Cit is more effective agent for the dissolution of CaOx. These investigations may help to design the drug and course of treatment for the CaOx kidney stone diseases. 5.0 REFERENCES 1. Worcester, E.M.; Coe, F.L. Nephrolithiasis.Prim.Care 2008, 35, Borghi, L.; Schianchi,T.; Meschi,T.; Guerra,A.; Allegri,F.; Maggiore,U.; Novarini,A. N.Engl.J.Med. 2002, 346, M. Y. M. Fazil and A. Salim, Urological Research 2009, 37, J. C. Williams and J. A. McAter, Journal of Nephrology 2013, 26, Jeffrey A Wesson, Elaine M Worcester, John H Wiessner, Neil S Mandel and Jack G Kleinman Kidney International 1998, 53,

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