Formation of poorly crystalline and amorphous precipitate, a

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Formation of poorly crystalline and amorphous precipitate, a component of infectious urinary stones - role of tetrasodium pyrophosphate Jolanta Prywer, and Ewa Mielniczek-Brzoska Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b01581 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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Crystal Growth & Design

Formation of poorly crystalline and amorphous precipitate, a component of infectious urinary stones role of tetrasodium pyrophosphate Jolanta Prywer a,*, Ewa Mielniczek- Brzóska b aInstitute

of Physics, Lodz University of Technology, ul. Wólczańska 219, 93-005 Łódź, Poland

bInstitute

of Chemistry, Health & Food Sciences, Jan Długosz University in Częstochowa, ul. Armii

Krajowej 13/15, 42-200 Częstochowa, Poland Abstract The present study concerns an important topic regarding the search for new, improved and effective methods of preventing the development of infectious urinary stones. In this article the effect of tetrasodium pyrophosphate (TSPP) on the formation of poorly crystalline and amorphous precipitate in artificial urine in the context of infectious urinary stone formation is studied. The spectrophotometric results suggest that TSPP presence shifts the formation of poorly crystalline and amorphous precipitate (PCaAP) towards lower pH, which means that PCaAP is formed earlier. In other words, TSPP promotes the formation of PCaAP. Additionally, TSPP causes the formation of calcium pyrophosphate. These results are confirmed by X-ray diffraction and Energy Dispersive X-ray studies. The experimental results obtained are explained on the basis of theoretical chemical speciation analysis of chemical complexes formed in artificial urine. 1. Introduction Infectious urinary stones make from 10% [1] to 30% [2] of all human urinary stones and arise as a result of infection of the urinary tract with urease-producing bacteria. Urease is a bacterial enzyme that breaks down urea, naturally occurring in the urine of a healthy person, into ammonia and carbon dioxide [3]. * Corresponding author: phone: +48 042 6313653, fax: +48 042 6313639, E-mail: [email protected]

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Further chemical reactions occurring in urine lead to an increase in pH and appearance of NH4+ and CO32ions in urine [4,5,6]. These ions together with magnesium (Mg2+) and calcium (Ca2+) ions, normally present in human urine, lead to the formation of poorly crystalline and amorphous precipitate (PCaAP) and crystalline struvite (MgNH4PO4∙6H2O) [7,8]. The first stage of the processes of infectious urinary stones formation is the appearance of PCaAP. This process begins when pH of 6.8 is achieved. Literature data [7,8] indicate that the poorly crystalline phases include carbonate apatite, Ca10(PO4)6CO3, (CA) and hydroxylapatite Ca10(PO4)6(OH)2, (HAP). It should be noted that the Ca2+, CO32-, OH- and PO43- ions in CA and HAP may be substituted with various ions, thus providing non-stoichiometric forms of CA and HAP. Such non-stoichiometric forms can be, for example, calcium sodium phosphate carbonate hydroxide, Ca9Na0.5(PO4)4.5(CO3)1.5(OH)2, and chlorapatite, Ca10(PO4)6Cl2 [8]. In addition to the poorly crystalline phases, infectious urinary stones may contain also amorphous phases, which can include: amorphous calcium carbonate (ACC), amorphous calcium phosphate (ACP), and/or amorphous carbonated calcium phosphate (ACCP) [9,10,11]. All of these phases are called poorly crystalline and amorphous precipitate (PCaAP). The formation of PCaAP is accompanied by struvite crystallization which starts later than PCaAP formation, when pH exceeds 7.2. In contrast to PCaAP, struvite has a highly crystalline form and gives sharp diffraction peaks. Struvite is a major component of infectious urinary stones, which together with PCaAP and bacteria can form aggregates leading to the formation of stones of considerable size that cannot be removed from urinary tract in physiological way. In recent years, the most frequently used treatment procedure against urinary stones is extracorporeal shock wave lithotripsy (ESWL). The essence of this method is breaking stones into small pieces that can be removed from the urinary tract with the urine stream. However, in the case of infectious urinary stones this method does not give the fully positive clinical results as bacteria can build into the structure of the stone during its formation. This means that when the stone is broken, the bacteria are released, causing the infection to recur. That is why, the recurrence of infectious urinary stones after treatment is on a level of 50 % [12]. Additionally, the lack of implementation of appropriate treatment procedure can lead to the loss of kidney [13,14]. For the above reasons the infectious urinary stones are still a serious medical problem and


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the search for methods or substances which may improve the effectiveness of the current treatment procedures is continued. In this paper we focus on the influence of tetrasodium pyrophosphate (Na4P2O7, abbreviated as TSPP) on the formation of PCaAP in the context of arising of infectious urinary stones. The effect of TSPP on urinary stone formation has been studied many times. The results presented in Ref. [15] show that, in the concentration range from 6 to 60 mg/l, this substance causes inhibition of struvite formation. The experiments performed in artificial urine, in the presence and absence of bacteria, have proved that the inhibition action of TSPP is not related with the inhibition of urease activity or bacteria viability, but it is caused by the disruption of ionic and hydrogen bonding which are formed in the process of struvite nucleation and growth. Additionally, the authors of Ref. [15] suggest that for the TSPP in concentration from 6 mg/l to 60mg/l, the inhibition of crystallization does not change markedly. It means that in this concentration range the action of TSPP is not related to formation of chemical complexes with Mg2+ ion. However, the mechanism of TSPP action has not been explained yet. The studies presented in Ref. [16] have been also focused on the influence of sodium pyrophosphate on the formation of CA and struvite. They were performed in the presence of bacteria and the tested concentrations of TSPP were equal to 0.5, 2.5 and 5 mg/ml. According to the results, the presence of sodium pyrophosphate does not change the urease activity and bacteria growth. However, it has been found that for the TSPP concentrations equal to 2.5 and 5 mg/ml the nucleation and growth of struvite is completely stopped and after 24 h only CA is formed [16]. The effect of TSPP on struvite nucleation and growth has been also considered in Ref. [17]. The experiments were performed in artificial urine and concentrations of TSPP were equal to 0.25, 0.5, 1.0 and 2.5 mg/ml. The results obtained show that increasing concentration of TSPP causes a delay of struvite nucleation. TSPP in the highest tested concentration totally inhibits the crystallization processes [17]. Additionally, it has been found that the presence of TSPP in artificial urine leads to the reduction of the total number of struvite crystals. The results obtained are explained on the basis of theoretical chemical speciation analysis of chemical complexes formed in artificial urine. It appears that the inhibitory action of TSPP –

against struvite formation is related to the formation of the MgP2O7 complex, so that magnesium needed to 3

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form struvite does not occur in the form of free ions in artificial urine, but is bound in this complex. In this way, the concentration of Mg2+ ions is reduced, which can be involved in the processes of nucleation and growth of struvite [17]. The presence of TSPP causes also changes in the habit and morphology of struvite crystals formed in artificial urine. The typical habit of struvite in the presence of the tested substance is rocket-like habit, often with homoepitaxic overgrowth [17]. Literature review presented indicates that TSPP has been studied in the context of crystallization of struvite and formation of CA. However, the mechanism of action of TSPP has been explained only regarding the struvite crystallization [17]. For this reason, the presented study has been undertaken to determine the effect of TSPP on the formation of PCaAP and to determine its mechanism of action. The motivation for undertaking the presented study is the search for new, improved and effective methods of preventing the development of infectious urinary stones. 2. Materials and methods The crystallization processes in artificial urine was studied. The composition of urine corresponds to the mean values of concentrations of mineral components found in human urine in a 24 h period and it is widely accepted in literature [18,19]. The artificial urine usually used for experiments is made of the following components, with their concentrations (g/l) given in brackets: CaCl2·2H2O (0.651), MgCl2·6H2O (0.651), NaCl (4.6), Na2SO4 (2.3), KH2PO4 (2.8), KCl (1.6), NH4Cl (1.0), Na3C6H5O7 (0.65), Na2C2O4 (0.023), CH4N2O (25.0), C4H9N3O2 (1.1).The artificial urine was prepared by dissolving chemicals (Sigma Aldrich) of reagent-grade purity in distilled water. Then, the solution was filtered through a membrane filter with the pore size of 0.2 μm. The artificial urine was stored for a maximum 48 h at 4 C. In this study we are focused on the effect of TSPP on the formation of PCaAP. Taking this into account we modified the above mentioned composition of artificial urine in such a way that the magnesium chloride hexahydrate (MgCl2·6H2O) was not added. This modification means that crystallization of struvite is not possible, because there is no magnesium needed to create struvite; only PCaAP is formed. In the actual infection of urinary tract, the crystallization processes are induced by the activity of bacterial urease which catalyses urea splitting and in this way leads to the increase in pH of urine, 4 ACS Paragon Plus Environment

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crystallization of struvite and formation of PCaAP. This study was performed without bacteria producing urease, while the activity of this enzyme was simulated by the consecutive addition of aqueous ammonia solution (1.2 M). Such an addition increases the pH and causes a cascade of reactions, which consequently lead to the crystallization of struvite and the formation of PCaAP, as in the actual infection of the urinary tract. The pH of the solution of artificial urine was screened during the experiments using a digital pH-meter (Elmetron CPC-401). Using this equipment the pH measurements are made with accuracy of 0.01. In the paper, the average results with accuracy of 0.1 are given. The experiments were conducted under thermostated conditions at 37 ± 0.5C. The temperature was kept constant by circulating water from a constant temperature water bath. The course of the formation of PCaAP was monitored by the spectrophotometric method by measuring the absorbance of light of optimized wavelength for different pH. The optimized wavelength was estimated to be 550 nm. The spectrophotometric measurements were performed using a spectrophotometer SPEKOL 11 (Carl Zeiss) and glass cuvettes with path length of 10 mm. During all growth experiments the samples were collected at regular intervals (at different pH) and observed under an optical microscopy OptaTech MN 800. X-ray diffraction (XRD), Energy Dispersive X-ray (EDX) and Scanning Electron Microscopy (SEM) were also performed within this study. In order to separate the PCaAP formed from artificial urine, the samples were centrifuged (a centrifuge Eppendorf 5702, 12 000 rpm for 5 min). Then, the resultant precipitates were rinsed in distilled water three times and each time centrifuged again. So prepared precipitates were dried at room temperature for 24 h and examined directly after drying. XRD was performed using X’pert PRO MPD (PANalytical) diffractometer. The Cu K radiation monochromatized by nickel filter was applied. Measurements were carried out in the range of 2θ angles from 5 to 90°. A continuous scan was used (step 0.0167°), the measurement time of one step was 25 seconds. The X’pert High Score Plus (PANalytical) software was used for indexing peaks in the XRD pattern. The SEM micrographs were taken using an FEI Quanta 200F microscope. The EDX analysis was performed using Oxford Instruments X-Max (50 mm2).



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In order to explain the experimental results, the theoretical speciation analysis of chemical complexes formed in artificial urine was performed. The calculations were carried out by using computer code HySS (Hyperquad Simulation and Speciation) [20] and indicated the concentrations of chemical complexes formed in the artificial urine during formation of PCaAP. On the basis of the stability constants and the initial concentrations of various complexes (see Table 1 and 2), the concentrations of complexes formed in the artificial urine within a given pH range were calculated. The appearance of these complexes has a direct impact on formation of PCaAP. Chemical interaction of TSPP with different complexes present in the artificial urine changes the chemical balance between them. As a consequence, it can lead to inhibition or facilitation of precipitation of a given phase. The stability constants used in the HySS program were calculated with the aid of computer code EQUIL [21] or taken from literature. The calculations were carried out for the temperature equal to 37 oC. 3. Results 3.1. Influence of TSPP on the formation of PCaAP In order to evaluate the effect of TSPP on formation of PCaAP, the spectrophotometric measurements were performed. The obtained results are presented in Fig. 1. According to this figure (data 1), in control test (without TSPP) the initial value of absorbance equals to 0 and remains constant until pH of 7.0 is achieved. Then, a sudden increase in absorbance of artificial urine is observed (Fig. 1, data 1). This means that from this pH the formation of PCaAP begins. The increase of absorbance is related to increasing amount of solid phases. The absorbance increases in the range of pH from 7.0 to 9.0 and then the maximum value is reached (Fig. 1, data 1). On the basis of these results it can be concluded that for pH = 9.0 the maximum amount of PCaAP in artificial urine is achieved. In the range of pH from 9.0 to 9.5 the absorbance of artificial urine does not change (Fig. 1, data 1). A different situation can be observed in the presence of TSPP. For the smallest tested TSPP concentration (0.25 mg/ml) the initial value of absorbance also equals to 0, but with increasing pH, absorbance also increases (Fig. 1; data 2). This observation means that for the concentration of TSPP equal to 0.25 mg/ml, the formation of solid phases starts earlier (at lower pH) than in the control test. However, it should be also noted that, in the presence of TSPP, the maximum value of absorbance (the state of 6 ACS Paragon Plus Environment

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saturation) is not achieved. Additionally, at the end of the experiment (pH = 9.5) the value of absorbance is by about 45 % lower than in the control test. In the TSPP concentration of 0.5 mg/ml the course of precipitation processes also differs from that in the control test (Fig. 1; data 3). The initial value of absorbance is by about 20 % higher than in the control test, which means that quite a large amount of solid phases is formed at the beginning of the experiment. Then, with increasing pH, absorbance increases, but similarly to the case of TSPP concentration of 0.25 mg/ml, the maximum value of absorbance (the state of saturation) is not achieved. For TSPP concentration of 0.5 mg/ml, the absorbance at pH = 9.5 is by about 55 % lower than that in the control test (Fig. 1; data 3). The changes in the course of precipitation processes are also observed for the TSPP concentration of 1.0 mg/ml. From the data presented in Fig.1 (data 4) it follows, that in this case, the initial value of absorbance is by about 40 % higher than in the control test (without TSPP). This means that the presence of TSPP at this concentration causes the precipitation of solid phases even at pH = 6.1. Similarly to the previous cases, with increasing pH, the absorbance of artificial urine increases but again, the maximum value (the state of saturation) is not observed. The final value of absorbance (for pH = 9.5) is by about 30 % lower than in the control test, but by about 15 % and 25 % higher than in the experiments with TSPP in concentrations of 0.25 and 0.5 mg/ml, respectively (Fig. 1). In the presence of TSPP at the highest tested concentration, the initial value of absorbance is also by about 70 % higher than in the control test. For TSPP in concentration of 2.5 mg/ml, an increase in pH also causes an increase in absorbance and at the end of the experiment (pH = 9.5) it reaches a value by about 20 % higher than in the control test. It should be also noted that with increasing concentration of TSPP, the initial pH of artificial urine also increases (Fig. 1). For example, the initial pH of the artificial urine for the control test is 5.8 (this point is not shown in Fig. 1), while for the highest TSSP concentration the initial pH value is 6.33 (Fig. 1). These results are in line with our previous studies [17], in which we investigated the effect of TSPP on struvite crystallization. We have also observed that increasing concentration of TSPP leads to an increase in the initial pH. The explanation of this effect is presented in Ref. [17]. The presented results clearly show that the presence of TSPP in artificial urine causes the increase in the initial value of absorbance. This means that this substance leads to the formation of solid phases at lower values of pH than in the control test. In order to verify these results, microscopic observations were 7 ACS Paragon Plus Environment


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performed at different stages of precipitation processes. The obtained results are presented in Fig. 2 and demonstrate that in the control test (without TSPP) the first solid phases are observed at pH of about 7.5 (Fig. 2; panel a2, arrow). At this stage of experiment, PCaAP appears in the form of very small deposits. Then, with increasing pH the amount of this solid phase increases (Fig. 2; panels a3 - a5). It should be also noticed that an increase in pH also leads to the aggregation of PCaAP and for the highest value of pH the sizes of its particles are greater. In the presence of TSPP the situation is different (Fig. 2; columns b-e). At all tested TSSP concentrations, the solid phases are observed even at pH of 6.3, which means that in the presence of TSPP, the solid phases appear much earlier (at lower pH) than in the control test. Additionally, as follows from the microscopic observations, with increasing concentration of TSPP the amount of solid phases at the beginning of the experiments also increases. These results are in good agreement with spectrophotometric results which demonstrate that with increasing TSPP concentration the initial value of absorbance also increases (Fig. 1). In face of the above results, the question arises whether the resulting deposit is PCaAP, or whether its chemical composition is different. To answer this question we carried out additional research. First of all, we checked the SEM images of the resulting precipitate, presented in Fig. 3. On the basis of this figure it can be claimed that the structure of solid phases formed in the presence and absence of TSPP is different. It implies that in the presence of TSPP in artificial urine a different precipitate is formed than in the control test. In order to check this possibility, a speciation analysis of chemical complexes formed in artificial urine was carried out. 3.2. Theoretical chemical speciation analysis In order to explain the changes in the course of solid phases formation, the theoretical chemical speciation analysis was performed. In the first step we checked the influence of TSPP on the formation of Ca10(PO4)6CO3 (CA) and Ca10(PO4)6(OH)2 (HAP) complexes. The obtained results are presented in Fig. 4. According to them, in the control test (without TSPP) a sudden increase in the concentration of Ca10(PO4)6CO3 complex is observed at pH = 8.1. Then, with increasing pH, the concentration of this complex increases and its maximum value is achieved at pH of 9.0. In the presence of TSPP in the smallest 8 ACS Paragon Plus Environment

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tested concentration (0.25 mg/ml) the changes in the concentration of Ca10(PO4)6CO3 complex are different. On the basis of Fig. 4 it is seen that in this case the increase in concentration of Ca10(PO4)6CO3 complex appears when the pH = 6.5 is reached. This means that the TSPP of concentration 0.25 mg/ml causes the formation of Ca10(PO4)6CO3 (CA) complex earlier (at lower pH) than in the control test. In the presence of TSPP in concentrations of 0.5, 1.0 and 2.5 mg/ml, the increase in concentration of CA complex is observed at pH equal to 6.5, 6.7, 7.0, respectively. It means that the increasing concentration of TSPP causes a shift in the formation of CA complex towards higher pH. The maximum amounts of CA are formed in different pH ranges. In the control test, the maximum amount of CA is formed at a higher pH (around 9), and in the presence of TSPP at a lower pH. However, it should be noted that the maximum amount of CA in the presence of TSPP occurs at the lowest TSPP concentration (0.25 mg / ml). For higher TSPP concentrations, the maximum amount of CA decreases and is the lowest for the highest TSPP concentration (2.5 mg / ml). At the same time, it should be noted that the maximum amount of CA in the presence of TSPP not only decreases with increasing TSPP concentration, but also shifts towards higher pH. Additionally, on the basis of Fig. 4 it can be concluded that in the range of higher pH values (pH > 8.0) also Ca10(PO4)6(OH)2 (HAP) complex can be formed. In the control test the precipitation of HAP complex starts when pH of 8.3 is achieved. However, increasing concentration of TSPP leads to a shift of formation of HAP complex towards higher pH (Fig. 4). It means that TSPP delays the formation of HAP complex compared to the control test. It can also be seen from Fig. 4 that the concentration of HAP complex does not reach saturation in the pH range of interest (up to pH 9.5) and with increasing concentration of TSPP the amount of HAP complex at pH = 9.5 decreases. The results presented in Fig. 4 suggest that the solid phase/phases formed at low pH (about 6.5) may be CA, however there is one fact that raises doubts. Namely, the results of the chemical speciation analysis indicate that the concentration of CA complex decreases with increasing TSPP concentration (Fig. 4), while the experimental spectrophotometric results (Fig. 1) indicate that with increasing TSPP concentration, the amount of precipitate formed at low pH (about 6.5) increases. This result suggests that in addition to CA, at a low pH, there is another solid phase in the system.



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In order to check this hypothesis we performed calculations of other chemical complexes which can be formed in artificial urine in the absence and presence of TSPP. First of all, the complexes with PO43- ion were analysed. The results obtained are presented in Fig. 5. As follows from Fig. 5a the presence of TSPP in artificial urine causes changes in the concentration of CaHPO4 complex. At the smallest tested concentration of TSPP (0.25 mg/ml) the concentration of CaHPO4 complex is higher than in the control test. However, this effect is reduced with increasing concentration of TSPP, i.e. with increasing TSPP concentration, the maximum concentration of CaHPO4 complex decreases. It would seem that this complex could form a precipitate that appears at low pH in accordance with the experimental observations shown in Figure 1. However, it should be noted that the concentration of this complex decreases with increasing TSSP concentration (Fig. 5a), which is inconsistent with experimental observations (Fig. 1). The PO43- ion can also form CaH2PO4+ complex (Fig. 5b). From Fig. 5b it can be concluded that the presence of TSPP in artificial urine has very small effect on the formation of CaH2PO4+ complex. The maximum concentration of this complex is lower in the presence of TSPP compared to the control test. Additionally, the presence of TSSP causes a small increase in the concentration of this complex in the pH range from 5 to 7. However, the obtained differences between the control test and the experiments with the presence of TSPP are so small that they cannot play important role in the formation of solid phases. The results presented in Fig. 5c demonstrate also that TSSP does not change the concentration of the complex HnPO4(n-3), where n = 0, 1, 2, 3 (Fig. 5c). For all tested concentrations of TSPP, only small changes in concentrations of these complexes are observed compared to the control test. Fig. 5d shows that the presence of TSPP shifts the formation of CaPO4- complex towards lower pH. For all concentrations of TSPP, this shift is the same. It can be concluded that the results presented in Fig. 5 indicate that some complexes (CaHPO4, CaH2PO4+, CaPO4-) formed with PO43- may have an impact on the formation of solid phases, but it is not significant. The formation of HnPO4(n-3) complex has no effect on the precipitation of solid phases. The formation of solid phases, in particular CA, is also influenced by CO32- ion. Therefore, speciation analysis of chemical complexes formed with the participation of this ion was performed. The results are presented in Fig. 6, which implies that the presence of TSPP causes a reduction of the maximum concentration of CaCO3 complex. In the control test the formation of this complex starts when pH = 6 is 10 ACS Paragon Plus Environment

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achieved. From this pH up to 9.7 the concentration of CaCO3 complex increases and at pH = 9.7 the maximum value is achieved. On the basis of Fig. 6 it can be concluded that at pH = 9.7, about 21 % of Ca2+ ion appears in the form of CaCO3 complex. However, in the presence of TSPP in all tested concentrations, the maximum concentration of the CaCO3 complex is reduced to a level of 8 – 9 %. This means that only 8 – 9 % of Ca2+ ions present in artificial urine is used to form CaCO3 complex. This result is very important from the point of view of precipitation of solid phases because it means that in the TSSP presence more Ca2+ ion can take part in the formation of CA and/or other solid phases in the pH range from 6 to 9.5. This conclusion may explain the results presented in Fig. 4 showing clearly that in the presence of TSPP, formation of CA complex starts at lower pH compared to the control test. Literature data [22] indicate that trisodium citrate, normally present in urine, has an inhibitory effect on the formation of CA. From this point of view, it is necessary to check the influence of TSPP on the complexes which can be formed with citrate (C6H5O73-) ion. The results of this analysis are presented in Fig. 7 and they prove that the presence of TSPP in artificial urine causes changes in the concentration of CaCitcomplex (the abbreviation Cit in the names of complexes stands for citrate group, C6H5O7). The formation of this complex starts at pH of 3.8 (Fig. 7). In the control test, the concentration of CaCit- complex increases with increasing pH and the maximum value is reached at pH ≈ 7.0. At this point 40 % of the total amount of Ca2+ ion in artificial urine appears in the form of CaCit- complex. A different situation can be observed in the presence of TSPP in the concentration of 0.25 mg/ml. The formation of CaCit- complex also starts at pH = 3.8 but the maximum concentration of this complex is by about 10% higher compared to the control test (Fig. 7). With increasing concentration of TSPP, the maximum concentration of the analysed complex decreases when compared to that at the TSPP concentration of 0.25 mg/ml (Fig. 7). In general, it can be said that the presence of TSPP promotes the formation of CaCit- complex. Additionally, as follows from Fig. 7 TSPP at all tested concentrations does not change the concentration of CaHCit complex. As a part of the speciation analysis, we also focused on the chemical complexes made by TSPP. The results obtained are presented in Fig. 8. On the basis of Fig. 8a it can be claimed that in the control test, as expected, the Ca2P2O7 complex is not formed in the whole range of pH. A different situation is observed in the presence of TSPP. Then, the formation of Ca2P2O7 complex starts at pH = 5.0 and with increasing pH, 11 ACS Paragon Plus Environment


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the concentration of this complex also increases. For the smallest concentration of TSPP (0.25 mg/ml) the maximum concentration of the Ca2P2O7 complex is achieved at pH ≈ 6.8. With increasing TSPP concentration, the

maximum concentration of Ca2P2O7 complex increases, however, with increasing

concentration of TSPP the pH at which the maximum concentration of Ca2P2O7 complex is observed is shifted towards lower pH. In the presence of TSPP at the highest tested concentration, the maximum appears at pH ≈ 6.3. These results may explain the experimental results in which we observed the formation of solid phases at lower pH values compared to the control test. The spectrophotometric image (Fig. 1) also indicates that increasing TSPP concentrations causes an increase in the total amount of solid phases formed in artificial urine. Calcium pyrophosphate (Ca2P2O7, abbreviated as CPP) is a stable chemical compound, an insoluble in water calcium salt containing the pyrophosphate anion and, as indicated by speciation analysis, CPP can be a solid phase that arises in our experiment. On the basis of Fig. 8b it is also clearly seen that CPP may exist in ionized form, specifically in the form of CaP2O72- and CaHP2O7- ion. The maximum concentration of CaP2O72- ion is achieved at the same pH as that of Ca2P2O7 complex and also increases with increasing concentration of TSPP. It should be emphasized that at the highest TSPP concentration tested, at pH ≈ 7.2, approximately 40% of all Ca2+ ions in artificial urine occur in the form of CaP2O72- ion. This result may mean that the formation of CaP2O72- ion in artificial urine plays an important role in the formation of solid phases in the pH range from 6.5 to 7. This fact can have great impact on the formation of CA in the low pH range from 6.5 to 7, because at such pH values, 40% free Ca2+ ions are used to form CaP2O72- ion. In conclusion, chemical speciation analysis indicates that TSPP strongly influences the formation of CA complex, shifting its start towards low pH from 6.5 to 7. TSPP also causes the formation of CPP complex and its ionized form. Therefore, the speciation analysis shows that the solid phases, which, as shown by spectrophotometric analysis, are formed at low pH from 6.5 to 7 can be CA and CPP. To verify the results of theoretical speciation analysis, EDX tests of the obtained samples were carried out. This method allows us to define the atomic composition of the resulting precipitate. 3.3. EDX Analysis From the analysis carried out so far it appears that CA and CPP (CPP also in ionized form, specifically in the form of CaP2O72- and CaHP2O7- ions) are present in the analysed precipitates at low pH (about 6.5). In 12 ACS Paragon Plus Environment

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order to check the atomic composition and the percentage of elements in the precipitates formed in Mg-free artificial urine we performed EDX measurements. The tested samples were obtained in Mg-free artificial urine at pH of 6.55 and for the concentration of TSPP equal to 2.5 mg/ml. The obtained EDX results are presented in Table 3 (experimental results). The experimental results are mean values from several measurements. At the beginning it should be noted that the amount of carbon is overestimated for two reasons. Firstly, the samples were tested on a carbon substrate and secondly, the collimator of the device is made of carbon which can give an additional signal. In order to determine which chemical compounds can form a precipitate of a specific atomic composition, theoretical calculations were performed. The calculation procedure includes: (1) calculation of the starting composition of the sample on the basis of the above mentioned experimental results, (2) calculation of the percentage content of each element in the starting composition, and (3) comparison of the results with those obtained with the use of the EDX method. The results of calculations are presented in Table 3; cases 1 and 2. In Case 1 it is assumed that CPP and non-stoichiometric CPP: NH4NaCaP2O7 ∙ 3H2O (ammonium sodium calcium phosphate trihydrate) and non-stoichiometric carbonate apatite Ca10-x(PO4)6-yCO3 are formed at the molar ratio of 1:1:1. The non-stoichiometric form of CA suggests that it is formed with a deficit of calcium ions, Ca2+. If we assume that x = 5, it means that Ca2+ ion deficiency is compensated by K+0.3, Na+0.2 and H+9.5 ions being incorporated into the structure of CA. Finally, in this case, it is assumed that the following three compounds are formed: Ca5K0.3Na0.2H9.5(PO4)6CO3, Ca2P2O7 and NH4NaCaP2O7∙ 3H2O. Adopting such a composition of the resulting precipitate gives good compatibility with the EDX experimental results (see Table 3) for most of the elements except oxygen. One can see that in this case the content of oxygen (O) is higher than that given by the experimental data. Therefore, in Case 2 (Table 3) it is assumed that in the non-stoichiometric carbonate apatite, CO32- ions are partly replaced by the ions of Cl-. This in turn means that besides the compounds mentioned for Case 1 the precipitate also contains chlorapatite Ca10-x(PO4)6Cl2 (where x is the same as for Case1). The results of the contents of the individual elements of

this mixture, i.e. Ca10-x(PO4)6CO3 and Ca10-x(PO4)6Cl2 and

Ca2P2O7, and also

NH4NaCaP2O7∙3H2O are shown in the ratio 1:2:1:1, respectively, in Table 3. As can be seen from the Table 3, case 2 gives a good agreement with the experimental EDX analysis. 13 ACS Paragon Plus Environment


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The presence of chlorapatite agrees well with literature data. For example in Ref. [23] chlorapatite is found as a component of infectious urinary stones. Its presence has also been confirmed in Ref. [8]. The occurrence of CPP in the non-stoichiometric form: NH4NaCaP2O7 ∙ 3H2O is confirmed by the XRD study presented in Fig. 9. However, it should be noted that the XRD results were obtained for the sample at pH of 9.5, while the EDX analysis for the sample at pH = 6.55 (Table 3). Both the XRD and EDX studies were performed for the highest tested concentration of TSPP of 25 mg/ml. From Fig. 9 it is seen that the obtained peaks match well the peaks corresponding to non-stoichiometric CPP: NH4NaCaP2O7∙3H2O (ammonium sodium calcium phosphate trihydrate). This result shows that the calcium ion is replaced by NH4+ and Na+ ions in the CPP structure. It should be noted that the speciation analysis of the chemical complexes (Fig. 8b) clearly indicates that at pH = 9.5, 25 % of calcium ions are bound in the CaP2O72- complex. Consequently, this complex can create non-stoichiometric CPP, whose presence is confirmed by the XRD study. It should be noted that at this pH, there may be no other solid phase, or there is very little other solid phase, as indicated in Fig. 4. The figure shows that at pH of 9.5 at the highest concentration of TSPP (25 mg/ml) only 0.6 % of calcium ions form the HAP complex. Such a small amount may be below the detection threshold of XRD and is therefore invisible in Fig. 9. In conclusion, it should be noted that in the presence of TSPP, at low pH (6.5), CA, possibly non-stoichiometric CA and CPP, or non-stoichiometric CPP, are formed. However, CPP is formed in much greater amounts than CA. At high pH (9.5), CPP dominates, possibly nonstoichiometric CPP, HAP may occur in small amounts. Conclusions The presented research work was undertaken to search for substances that may inhibit the formation of PCaAP, which is a component of infectious urinary stones. TSSP was chosen because we expected it to have an inhibitory effect on PCaAP formation. Unfortunately, TSPP, in artificial urine, causes the formation of PCaAP to be shifted towards lower pH, which means that this precipitate is formed earlier. Moreover, TSPP causes the formation of a crystalline phase of CPP in addition to the known PCaAP. Both the results of the theoretical speciation analysis and the XRD study indicate that CPP may exist in non-stoichiometric form. The results of the speciation analysis indicate that in the pH range we are interested in, from 6 to 9.5, the


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amount of CPP formed is greater than the amount of the known PCaAP. In conclusion, the results obtained indicate that TSSP is not a sufficiently good therapeutic substance. Acknowledgement This work has been partially supported by Ministry of Science and Higher Education (Poland), Grant No. I3/501/17-3-1-727. Jolanta Prywer thanks the University of Florida, Department of Pathology, Immunology and Laboratory Medicine for the computer program EQUIL. Jolanta Prywer wishes to thank Mr. Marcin Olszynski, for spectrophotometric measurements and technical assistance in presentation of results. Author information The authors declare no competing financial interest. Authors’ Contributions: Jolanta Prywer's contribution consisted in formulating the problem presented in the article and developing the concept of its solving. Jolanta Prywer coordinated the XRD, EDX and spectrophotometric measurements; analysed the results obtained; managed the research project covering the research described in this article; she wrote the manuscript, interpreted the obtained results and formulated conclusions. Ewa Mielniczek Brzóska made chemical speciation analysis, helped to interpret the results of this analysis; calculated the theoretical cases 1 and 2 presented in Table 2 and described them in the article; she was also involved in the formulation of conclusions.



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Tables and Figures Table 1. The initial concentrations, c, of ions in the artificial urine taking into account in the theoretical speciation analysis (HySS calculation).

Species

Ca2+

Mg2+

PO43-

C2O42-

Cit3-

SO42-

c[mM]

4.43

0a

20.6

0.2

2.5

16.2

a

Na+ 126.3b 130.0 b 133.8 b 141.3 b 163.9 b

P2O720b 0.940 b 1.879 b 3.761 b 9.402 b

K+ 42.0

CO32- NH4+ 400c

800c

In the present study we used artificial urine with modified composition – without magnesium chloride

hexahydrate which causes that initial concentration of the Mg2+ equal to 0. b

These five concentrations concern the concentrations of TSSP equal to 0, 0.25, 0.5, 1.0 and 2.5 mg/ml,

respectively. c

This concentration follows from the assumption that all accessible urea (25 g/l) is decomposed into CO2

and NH3 as described in Ref. [28].


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Table 2 Stability constants –logβ of complexes which are taken into account in the analysis. The presented stability constants are calculated using the computer code EQUIL at 37oC. Complex HPO42H2PO4H3PO4 NaHPO4KHPO4CaPO4CaHPO4 CaH2PO4+ NH4HPO4-

- log 12.6 19.3 21.56 13.2 13.3 6.52 14.99 20.77 13.16

NH3 · H2O CaCitCaHCit CaH2Cit+ NaCit2KCit2HCit2H2CitH3Cit NH4Cit2HC2O4H2C2O4 HCO3H2CO3 HP2O73H2 P2O72H3P2O7-

6.73 4.75 9.28 12.26 1.35 1.24 6.46 11.16 14.49 0.085 4.32 5.68 10.1a 16.68a 8.5c 14.6c 17.1c

Complex CaC2O4 Ca2C2O42+ Ca(C2O4)22CaHC2O4+ CaH2(C2O4)2 NH4C2O4NaC2O4CaOHCa5(PO4)3OH ( 2) = Ca10(PO4)6CO3 KOH NaSO4KSO4NH4SO4HSO4KC2O4CaCO3 CaHCO3+ NaCO3NaHCO3 CaSO4 KP2O73NaP2O73CaP2O72CaHP2O7Ca2P2O7 H4P2O7


- log 3.44 5.29 4.68 6.17 11.1 1.11 0.995 1.38 38.97b 1.5 0.61 0.997 0.67 2.14 1.2 3.3a 11.6a 1.27a 10.08a 2.23 2.3c 2.3c 5.0c 10.8c 2.8d 18.1c


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aThis

stability constant is taken from Ref.[24]. stability constant is taken from Ref. [25]. cThis stability constant is taken from Ref.[26]. dThis stability constant is taken from Ref.[27]. bThis

Table 3. Results of EDX analysis for the sample obtained in the presence of TSPP in concentration 2.5 mg/ml at pH = 6.55 (experimental results) and theoretical calculations based on stoichiometry (Cases 1 - 2). Weight [%] Element C O P Ca K Na H N Cl Total

Experimental results

Theoretical results

(mass fraction of elements %)

11.22 41.83 22.06 23.36 1.07 0.46 100

Case 1

Case 2

0.85 49.60 21.82 22.59 0.83 1.94 1.38 0.99 100

0.78 44.69 22.21 23.51 1.91 0.75 1.07 0.46 4.62 100


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Fig. 1. Changes in absorbance of artificial urine versus pH for different TSPP concentrations given in the inset.


Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Fig. 2. Course of formation of solid phases in artificial urine for different TSPP concentrations (columns a – e). The arrow on a2 indicates the first solid phases. Scale bar: 50 μm. The images’ quality is enhanced by the contrast and brightness to enable the identification of solid phases. 20 ACS Paragon Plus Environment

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Fig. 3. The precipitate in Mg-free artificial urine; SEM micrographs in (a) control test (without TSPP) and (b) and in presence of TSPP of concentration 2.5 mg/ml and at pH = 6.55.



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Fig. 4. Percentage contents of Ca10(PO4)6CO3 and Ca10(PO4)6(OH)2 complexes versus pH of the Mg-free artificial urine for different TSPP concentrations given in the inset. These percentage contents are given with respect to the initial concentration of Ca2+ ion.


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Fig. 5. Percentage contents of different complexes formed with PO43- ion versus pH for different concentrations of TSPP given in the insets; (a) CaHPO4 complex, (b) CaH2PO4+ complex, (c) HnPO4(n-3) complexes (n = 0, 1, 2, 3), (d) CaPO4- complex. These percentage contents are given with respect to the initial concentration of Ca2+ ion. The curves presented in b−d overlap each other for different TSPP concentrations.



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Fig. 6. Percentage contents of CaCO3 complex versus pH for different TSPP concentrationsgiven in the inset. These percentage contents are given with respect to the initial concentration of Ca2+ ion.


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Fig. 7. Percentage contents of CaHCit and CaCit-complexes versus pH for different TSPP concentrations given in the inset. These percentage contents are given with respect to the initial concentration of Ca2+ ion.



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Fig. 8. Percentage contents of different complexes versus pH for different TSPP concentrations given in the insets; (a) Ca2P2O7 complex, (b) CaHP2O7- and CaP2O72-complexes. These percentage contents are given with respect to the initial concentration of Ca2+ ion.


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Fig. 9. XRD pattern of the sample in the case of the presence of TSPP at concentration of 2.5 mg/ml and pH = 9.5. This spectrum corresponds to ammonium sodium calcium phosphate trihydrate according to The International Centre for Diffraction Data (ICDD PDF-2 ver. 2009) card no. 00-0311274. The black line passing under the peaks is the measurement background.



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References [1] K. P. Aggarwal, S. Narula, M. Kakkar, Ch. Tandon, Nephrolithiasis: Molecular Mechanism of Renal Stone Formation and the Critical Role Played by Modulators. Biomed Res. Int. 2013, Article ID 292953. [2] Ch. K. Chauhan, M. J. Joshi, In vitro crystallization, characterization and growth-inhibition study of urinary type struvite crystals. J. Cryst. Growth 362 (2013) 330–337. [3] J. Prywer, A. Torzewska, Bacterially induced struvite growth from synthetic urine - experimental and theoretical characterization of crystal morphology, Cryst. Growth Des., 9 (2009) 3538 - 3543. [4] K. H. Bichler, E. Eipper, K. Naber, V. Braun, R. Zimmermann, S. Lahme, Urinary infection stones. Int. J. Antimicrob. Ag. 19 (2002) 488-498. [5] R.J.C. McLean, J.C. Nickel, K.J. Cheng, J.W. Costerton, The ecology and pathogenicity of ureaseproducing bacteria in the urinary tract. Crit. Rev. Microbiol. 16 (1988) 37–79. [6] J. Prywer, M. Olszynski, E. Mielniczek - Brzóska, Inhibition of precipitation of carbonate apatite by trisodium citrate analysed in base of the formation of chemical complexes in growth solution. J. Solid State Chem. 231 (2015) 80 -86. [7] F. Grases, O. Söhnel, A. I. Vilacampa, J. G. March, Phosphates precipitating from artificial urine and fine structure of phosphate renal calculi, Clin. Chim. Acta 244 (1996) 45-67. [8] J. Prywer, M. Kozanecki, E. Mielniczek-Brzóska and A. Torzewska, Solid Phases Precipitating in Artificial Urine in the Absence and Presence of Bacteria Proteus mirabilis−A Contribution to the Understanding of Infectious Urinary Stone Formation, Crystals 8 (2018) 164. [9] X. Carpentier, M. Daudon, O. Traxer, P. Jungers, A. Mazouyes, G. Matzen, E. Véron, and D. Bazin, Relationships Between Carbonation Rate of Carbapatite and Morphologic Characteristics of Calcium Phosphate Stones and Etiology, Urology 73 (2009) 968-75. [10] T. Tsuji, K. Onuma, A. Yamamoto, M. Iijima, and K. Shiba, Direct transformation from amorphous to crystalline calcium phosphate facilitated by motif-programmed artificial proteins, PNAS 105 (2008) 16866–16870. [11] S. Weiner, I. Sagi, L. Addadi, Structural biology. Choosing the crystallization path less traveled, Science 309 (2005) 1027-8. [12] L. Benramdane, M. Bouatia, M.O.B. Idrissi, M. Draoui, Infrared analysis of urinary stones, using a single reflection accessory and a KBr pellet transmission. Spectrosc. Lett. 41 (2008) 72–80. [13] M. Singh, R. Chapman, G.C. Tresidder, Y.J. Bland, The fate of the unoperated staghorn calculus, Brit. J. Urol. 45 (1973) 581–585. [14] A. Wojewski, T. Zajączkowski, The treatment of bilateral staghorn calculi of the kidneys, Int. Urol. Nephrol. 5 (1974) 249–260. [15] R.J.C. McLean, J. Downey, L. Clapham, J.W.L. Wilson, J.C. Nickel, Pyrophosphate inhibition of Proteus mirabilis-inducted crystallization in vitro. Clin. Chim. Acta 200 (1991) 107-118. [16] A. Torzewska, A. Rozalski, Inhibition of crystallization caused by Proteus mirabilis during the development of infectious urolithiasis by various phenolic substances. Microbiol. Res. 169 (2014) 579– 584. [17] M. Olszynski, J. Prywer, E. Mielniczek- Brzóska, Inhibition of Struvite Crystallization by Tetrasodium Pyrophosphate in Artificial Urine: Chemical and Physical Aspects of Nucleation and Growth, Crystal Growth & Design, 16 (2016), 3519–3529. [18] A. Torzewska, A. Rozalski, Various intensity of Proteus mirabilis-induced crystallization resulting from the changes in the mineral composition of urine, Acta Biochimica Polonica 62, (2015) 127–132. [19] D. P. Griffith, D. M. Musher, C. Itin, Urease. The primary cause of infection-induced urinary stones, Invest Urol. 13 (1976) 346–50. 28 ACS Paragon Plus Environment

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[20] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species, Coordin. Chem. Rev.184 (1999) 311–318. [21] C.M. Brown, D.K. Ackermann, D.L. Purich, EQUIL 93: a tool for experimental and clinical urolithiasis, Urol. Res. 22 (1994) 119-126. [22] J. Prywer, M. Olszynski, E. Mielniczek - Brzóska, Inhibition of precipitation of carbonate apatite by trisodium citrate analysed in base of the formation of chemical complexes in growth solution. J. Solid State Chem. 231 (2015) 80 -86. [23] A. Kubala-Kukuś, M. Arabski, I. Stabrawa, D. Banaś, W. Różański, M. Lipiński, U. Majewska, J. Wudarczyk-Moćko, J. Braziewicz, M. Pajek and S. Góźdź, Application of TXRF and XRPD techniques for analysis of elemental and chemical composition of human kidney stones, X-Ray Spectrometry 46 (2017) 412–420. [24] Yi-Pin Lin, P.C.Singer, Inhibition of calcite precipitation by orthophosphate: Speciation and thermodynamic considerations. Geochim. Cosmochim. Acta 70 (2006)2530–2539. [25] Å. Bengtsson, A. Lindegren, S. Sjöberg, P. Persson, Dissolution, adsorption and phase transformation in the fluorapatite–goethite system. Appl. Geochem. 22 (2007) 2016–2028. [26] J. Inczédy, Równowagi Kompleksowania w Chemii Analitycznej (Complexing Equilibria in Analytical Chemistry); Państwowe Wydawnictwo Naukowe: Warszawa, 1979; page 249. [27] V.W. Leung, B.W. Darvell, Artificial salivas for in vitro studies of dental materials. J. Dent. 25 (1997) 475−484. [28] J. Prywer, E. Mielniczek-Brzóska, Chemical equilibria of complexes in urine. A contribution to the physicochemistry of infectious urinary stone formation. Fluid Phase Equilib. 425 (2016) 282−288.



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FOR TABLE OF CONTENTS USE ONLY

Manuscript title: Formation of poorly crystalline and amorphous precipitate, a component of infectious urinary stones - role of tetrasodium pyrophosphate

Authors: Jolanta Prywer, Ewa Mielniczek-Brzóska,

TOC GRAPHIC

The effect of tetrasodium pyrophosphate (TSPP) on the formation of poorly crystalline and amorphous precipitate (PCaAP) in artificial urine in the context of infectious urinary stone formation is studied. The results indicate that TSPP promotes the formation of PCaAP and causes the formation of calcium pyrophosphate. These results are explained on the basis of theoretical speciation analysis of chemical complexes.


Formation of poorly crystalline and amorphous precipitate, a

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