Dual Roles of Melamine in the Formation of Calcium Oxalate Stones

May 22, 2019 - Dual Roles of Melamine in the Formation of Calcium Oxalate Stones .... S7: The reaction rate constant k of the crystallization of CaOx ...
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Article Cite This: Cryst. Growth Des. 2019, 19, 3998−4007

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Dual Roles of Melamine in the Formation of Calcium Oxalate Stones Wenya Dong and Qingsheng Wu* School of Chemical Science and Engineering, Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai 200092, People’s Republic of China

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ABSTRACT: Melamine (Mel) is widespread in food and the environment. The effects of Mel on calcium oxalate (CaOx) crystallization and phase transformation were studied in aqueous solution and synthetic urine systems. The mechanism of Mel affecting the crystallization process was analyzed from the perspective of thermodynamics and kinetics. The morphology, structure, and composition of CaOx crystals were characterized; the ion selective electrode method was used to study the crystallization rate. For the first time, it was found that in synthetic urine, Mel could stabilize thermodynamically unstable calcium oxalate trihydrate (COT) and inhibit its conversion to calcium oxalate monohydrate (COM). Because Mel enriched calcium ions and oxalate on its surface through the dual effects of electrostatic adsorption and hydrogen bonding, the local supersaturation increased. High supersaturation is conducive to the stability of COT, thus inhibiting the formation of stones. Kinetic analysis showed that Mel could accelerate the crystallization rate of COM in aqueous solution by reducing the average activation energy, thus increasing the risk of stone formation. This discovery allows us to fully utilize the stabilizing effect of Mel on COT to prevent the formation of stones and to find the treatment of stones by understanding the process of Mel promoting COM crystallization.

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components of this urinary stones were Mel and uric acid,19 so Mel not only induced but also participated in the formation of uric acid stones. After the outbreak of this milk powder incident, Mel became a new kind of stone inducement, which attracted worldwide attention.20 Through structural calculation, Steed et al. showed that Mel and uric acid can form an insoluble supramolecule by hydrogen bonding, which leads to the formation of stones.21 Wu et al. found that environmental low-dose Mel exposure could cause early renal damage, which was associated with urolithiasis risk in adults, not just in infants.22 Lu et al. demonstrated that Mel would promote the formation of largesized CaOx crystals while suppressing the total crystal weight.23 Bandyopadhyay et al. discovered that Mel promoted the formation of CaP/CaOx crystals by stabilizing the calcium crystals and preventing their dissolution.24 Zhao et al. found that in the system where Mel and Escherichia coliform secretions coexist, the Escherichia coliform secretion could change the crystal form of CaOx while Mel could accelerate the crystallization process of CaOx.25 Therefore, Mel is closely related to the formation of stones. However, whether Mel has an influence on the phase transformation of CaOx crystals is rarely reported in the literature, and few people have studied the mechanism of Mel affecting the crystallization process from the perspective of

INTRODUCTION Urinary stones are a common and frequently occurring disease worldwide, and their incidence has been on the rise in recent years.1,2 Urinary stones are composed of inorganic minerals and an organic matrix. According to the different components of inorganic minerals, the stones can be classified into the following categories: CaOx stones, calcium phosphate (CaP) stones, uric acid stones, cystine stones, magnesium ammonium phosphate stones, and so on.3 About 80% of urinary stones are mainly composed of CaOx. CaOx crystals exist in three forms: COM (Ca2C2O4·H2O, whewellite), monoclinic system;4,5 calcium oxalate dihydrate (COD, Ca2C2O4·2H2O, weddellite), tetragonal system;6 COT (Ca2C2O4·3H2O, caoxite), triclinic system.7,8 Among them, COM is the most stable in thermodynamics and accounts for the largest proportion in CaOx stones. The thermodynamically metastable phases COD and COT are easily excreted with urine.9 On the other hand, COD and COT usually act as precursors in the formation of CaOx stones in the human body.10 Mel (2,4,6-triamino-1,3,5-triazine, C3H6N6), a nitrogencontaining heterocyclic organic compound, has a wide variety of applications in industry.11,12 Since the nitrogen content of Mel is as high as 66%, some illegal traders adulterated it in food to create the illusion of high protein content, which led to the pet food contamination incidents in Asia (2004) and North America (2007), as well as the Chinese infant milk powder contamination incident (2008).13−17 Of the infants who consumed Mel-contaminated milk powder, 294 000 were diagnosed with urinary stones and at least six died.18 The main © 2019 American Chemical Society

Received: March 23, 2019 Revised: May 15, 2019 Published: May 22, 2019 3998

DOI: 10.1021/acs.cgd.9b00389 Cryst. Growth Des. 2019, 19, 3998−4007

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percentage of each crystal phase of CaOx was calculated according to the K-value method. A is the phase to be measured, and S is the reference (α-Al2O3). S and pure phase A were mixed at a mass ratio of 1:1 (mass percentages were WS and WA), and the diffraction intensities (take the strongest diffraction peaks) IA and IS of the two phases in the mixture were determined. IA/IS is the reference intensity K of phase A. A known amount of reference phase S is added to the sample to be tested, and the intensity of the diffraction peaks of phase A and phase S are measured. According to the formula:

thermodynamics and kinetics. Although Mel did not cause an explosive disease incident associated with CaOx stones, it does not mean that it does not pose a risk of stone formation. In this work, we explored the influence of Mel on CaOx crystallization in aqueous solution and synthetic urine systems. It was found that CaOx existed in the form of COM in an aqueous solution system and COD in a synthetic urine system; citrate in synthetic urine played an important role in crystal phase transformation. In addition, we discovered for the first time that Mel could induce the formation of thermodynamically unstable COT, due to the increase of the local supersaturation. Thermodynamic and kinetic measurements showed that Mel could accelerate the crystallization rate of CaOx by reducing the average activation energy of the reaction. Therefore, Mel plays dual roles in the crystallization of CaOx. On the one hand, it accelerates the crystallization rate of COM and promotes the formation of stones. On the other hand, it stabilizes the unstable crystal phase COT and inhibits the formation of stones.

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WA =

WSIA KIS

the relative content WA of A phase in the mixed sample can be obtained. Then, according to the formula:

wA = WA /(1 − WS) the content wA of phase A in the sample to be tested can be further determined. Thermodynamic Studies. All solutions were prepared with 150 mM NaCl solution to maintain the total ionic strength.27 A total of 10 mL of 16 mM CaCl2 solution and 180 mL of 150 mM NaCl solution were mixed, then 10 mL of 16 mM Na2C2O4 solution was added dropwise with constant stirring. The 200 mL reaction solution was placed in a constant-temperature water bath at 25 °C. After the system was stabilized, the E value of Ca2+ in the solution was measured, and the concentration of calcium ions ([Ca2+]) in the solution was calculated by linear regression equation. The oxalate concentration ([C2O42−]) is equal to [Ca2+]. According to the equations:

EXPERIMENTAL SECTION

Materials. The following reagents were purchased from Shanghai Chemical Reagents Co., Ltd. and without further purification: Mel (C3H6N6, ≥99.0%), sodium hydroxide (NaOH, ≥96.0%), calcium chloride (CaCl2, ≥96.0%), sodium oxalate (Na2C2O4, ≥99.8%), sodium chloride (NaCl, ≥99.5%), sodium dihydrogen phosphate (NaH2PO4, ≥99.0%), sodium citrate (Na3Cit, Na3C6H5O7, ≥98.0%), magnesium sulfate heptahydrate (MgSO4·7H2O, ≥99.0%), sodium sulfate (Na2SO4, ≥99.0%), potassium chloride (KCl, ≥99.5%), ammonium chloride (NH4Cl, ≥99.5%), aluminum oxide (α-Al2O3, 0.20 μm, 99.99% metals basis), urea (H2NCONH2, ≥99.0%), LHistidine (C6H9N3O2, [α]20D= −38.0 ∼ −42.0° m2/kg). A model 232-01 saturated KCl calomel reference electrode and PCa-1-01 calcium ion selective electrode were all purchased from Shanghai Yidian Scientific Instrument Co., Ltd. A PHS-3C type pH meter was purchased from Shanghai Kuosi Electronics Co., Ltd. The calcium ion selective electrode was first immersed in a 1 mM calcium standard solution for 24 h and then cleaned with deionized water. Effect of [Ca2+]/[C2O42−] Ratio on CaOx Crystallization. Stock solutions of CaCl2 (5 mM, 50 mM, 100 mM) and Na2C2O4 (5 mM) were prepared by dissolving an appropriate quantity of each reagent in deionized water or synthetic urine. The following composition of synthetic urine was used: 16.95 mM Na2SO4, 3.85 mM MgSO4·7H2O, 45.5 mM NH4Cl, 63.7 mM KCl, 105.5 mM NaCl, 32.3 mM NaH2PO4, and 3.21 mM Na3Cit.26 A total of 80 mL of CaCl2, 40 mL of H2O, and 80 mL of Na2C2O4 solutions were added to a 500 mL conical flask in turn under magnetic stirring. CaOx crystallization in the presence of modifiers (Mel or Na3Cit) was performed by adding an appropriate amount of modifiers to achieve the desired concentrations. Modifiers were added to the solution prior to the addition of Na2C2O4. The conical flask was then placed in a constanttemperature water bath at 37 °C for 24 h. All products obtained here were washed thoroughly three times with deionized water and anhydrous ethanol, respectively. Characterization of products was operated after drying at room temperature. Characterization. The size, structure, and morphology of the products were characterized by scanning electron microscope (SEM, Hitachi S-4800, Japan). The chemical components and crystal phase of the obtained products were confirmed by X-ray diffraction (XRD, Bruker D8, Germany) using Cu Kα radiation with a two-theta scope from 10 to 80° and Fourier transform infrared spectroscopy (FTIR, Nicolet iS10, America). The zeta potential (ζ) was measured with a dynamic light scattering instrument (Malvern Zetasize Nano ZS90, England). A pH meter (PHS-3C, China) was used to access the electrode potential (E) value of free calcium ions in the solution. Quantitative Analysis of Crystal Phase Composition of CaOx. Quantitative analysis was conducted by XRD, and the

K sp⊖ = [Ca 2 +][C2O4 2 −]

[Ca 2 +] = [C2O4 2 −] K ⊖ = 1/K sp⊖ ΔG⊖ = − RT ln K ⊖

where the universal gas constant (R) = 8.314 J·mol−1·K−1 and temperature (T) = 298.15 K. The reaction equilibrium constant (K⊖), solubility product constant (Ksp⊖), and the change of Gibbs free energy (ΔG⊖) of the CaOx crystallization reaction were calculated according to the above equations. Kinetic Studies. All solutions were prepared with a 150 mM NaCl solution to maintain the total ionic strength. The kinetics of the COM crystallization process were measured using a calcium ion-selective electrode (ISE). COM growth was analyzed at 24 ± 1 °C using a mixture solution of 1 mM CaCl2 and 1 mM Na2C2O4 with continuous stirring. Mel was incorporated into the solution after the addition of CaCl2 and prior to the addition of Na2C2O4. The electrode was placed in the solution immediately after adding Na2C2O4. The E value was recorded per minute and tested for 30 min; three parallel experiments were conducted under each condition. Prior to each ISE measurement, the electrode was calibrated with calcium standards prepared by first diluting a commercial calcium solution (0.1 M) in deionized water to four different concentrations (10−2, 10−3, 10−4, and 10−5 M), and linear regression equations were drawn at different temperatures (273.15, 298.15, and 310.15 K). In the crystallization process of COM, the reaction rate (r) equations are as follows:

Ca 2 + + C2O4 2 − = CaC2O4 r = kc α = k(a − x)α r=−

d(a − x) dx = dt dt

i dx y lg r = lg jjj zzz = lg k + α lg(a − x) k dt {

The logarithm is taken on both sides:

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DOI: 10.1021/acs.cgd.9b00389 Cryst. Growth Des. 2019, 19, 3998−4007

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Figure 1. Effect of [Ca2+]/[C2O42−] ratio on CaOx crystallization in an aqueous solution system. (A−C) SEM images of the products at a [Ca2+]/ [C2O42−] ratio = 1:1 (A), 10:1 (B), 20:1 (C). The XRD diagram (D) and FTIR spectra (E) of the products (scale bar: 10 μm). where a and c are the free [Ca2+] (mM) in the solution at the initial (t = 0) and the moment of t, x is the consumption of [Ca2+] in the solution at the moment of t (mM), α is the reaction order, and k is the reaction rate constant. There is a linear relationship between lg r and lg(a − x). The slope of the line is α; the intercept is lg k. Then, the reaction rate constant k can be obtained. In order to obtain the average activation energy Ea required for the reaction, we measured the k of the COM crystallization process at different temperatures (T). According to the Arrhenius equation:

0231). The detected diffraction peaks of (2θ) at 14.93°, 15.29°, 24.37°, 30.11°, 35.98° and 38.18° correspond to the (−101), (−110), (020), (−202), (112), and (130) crystal faces.30 No diffraction peaks of other impurities were observed, indicating that the product was of high purity. It can be seen from the FTIR spectra (Figure 1E) that there were five stretching vibration peaks of the O−H bond of crystal water at 3491−3058 cm−1, which were the characteristic peaks of COM. The asymmetric stretching (νas) and symmetric stretching (νs) vibrations of the −COO in COM were at approximately 1624 and 1319 cm−1, respectively. In the fingerprint region, the absorption peaks of COM occurred at about 947 cm−1 (C−O stretching vibration), 781 cm−1 (C−C stretching vibration), and 663 cm−1 (O−C−O plane bending vibration).31 The results of FTIR and XRD analysis showed that the prepared COM crystals were pure products. According to the calculation, when the [Ca2+]/[C2O42−] ratio was 1:1, 10:1, and 20:1, the yield of product COM was 30.5%, 43.2%, and 43.0%, respectively (Table S1). This indicated that increasing the [Ca2+]/[C2O42−] ratio could improve the yield of the reaction. When the ratio was 10:1, the product yield had reached the maximum. The effect of Mel on the crystallization of CaOx was asessed with a [Ca2+]/[C2O42−] ratio of 1:1. As the concentration of Mel increased from 0 to 4 mM, the morphology of the product remained a twinned hexagonal structure, with no obvious changes (Figure S1A−E). XRD analysis also showed that the products were all COM (Figure S1F). As the concentration of Mel increased, the yield of product COM was 30.5%, 33.4%, 37.2%, 42.8%, and 51.2%, respectively (Table S2). These results showed that in the pure water system, the most stable form of the CaOx crystal was COM. The addition of Mel hardly changed the morphology and composition of CaOx, but

k = A e−Ea / RT The logarithm is taken on both sides: ln k = −

Ea + ln A RT

The average Ea of the reaction was calculated by plotting the ln k ∼ 1/ T curve.

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RESULTS AND DISCUSSION Bulk Crystallization of CaOx in an Aqueous Solution System. The formation of CaOx stones is closely related to [C2O42−] and [Ca2+] in urine.28,29 Bulk crystallization in an aqueous solution system was used to study the effect of [Ca2+]/[C2O42−] ratio on CaOx crystallization. When the [Ca2+]/[C2O42−] ratio was 1:1, the product exhibited a hexagonal morphology, which was about 20 μm long and 10 μm wide. We also found that an appreciable fraction of crystals was twinned (Figure 1A). When the [Ca2+]/[C2O42−] ratio was 10:1 and 20:1, the product showed a variety of irregular polyhedral shapes with a narrow size distribution (Figure 1B,C). XRD patterns of CaOx crystals obtained in the aqueous solution system at different [Ca2+]/[C2O42−] ratios were shown in Figure 1D. All three groups of products were consistent with standard data for COM (JCPDS, No. 204000

DOI: 10.1021/acs.cgd.9b00389 Cryst. Growth Des. 2019, 19, 3998−4007

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rate is slow, small crystals are easily excreted with urine and do not cause stones. When the rate is fast, small crystals grow and aggregate rapidly, blocking the renal tubules, resulting in pathological stones. Therefore, we tested the kinetic parameters of the COM crystallization process, the change of [Ca2+] and the pH value in the CaOx crystallization process were detected in real time. There was no significant difference in pH between the two systems with and without Mel, and the fluctuation was weak (Table S3), so the effect of pH change on the reaction was very small. Figure 2A and B show the variation of free [Ca2+] with reaction time (t) in three parallel experiments. In the system with Mel, the initial [Ca2+] was lower than that without Mel, possibly because Mel adsorbed Ca2+ and reduced the free calcium ions in the solution. The differential method was used to plot the reaction time (t) with the consumption of calcium ions. The slope at any point on the curve (derived from Origin data analysis software) was the reaction rate (r). Then, lg r was plotted against lg[Ca2+] (Figure 2C,D). The slope of the obtained straight line was the reaction order (α), and the reaction rate constant (k) could be obtained through the intercept (lg k). All results are shown in Table 2 and Figures S3 and S4. After the addition of Mel into the system, the average k increased from (2.54 ± 1.39) × 107 to (7.39 ± 1.92) × 108, and the average α increased from 3.93 ± 0.05 to 4.44 ± 0.03. The value of k directly reflects the rate of the reaction and is related to the reaction process. Therefore, the addition of Mel accelerated the crystallization rate of COM and changed the reaction process. Since the main component of CaOx stones is COM, and Mel can accelerate the crystallization rate of COM, from this perspective, Mel has the risk of inducing CaOx stone formation. Bulk Crystallization of CaOx in a Synthetic Urine System. In order to get closer to the environment of stone

it could increase the yield of the product, indicating that Mel promoted the formation of COM. Thermodynamic Analysis of CaOx Formation Process. In order to study the thermodynamic and kinetic processes of COM crystallization in aqueous solution, and to calculate the changes of physicochemical parameters in the process, we used a calcium ion selective electrode to measure the electrode potential E at different times in the crystallization process in real time. In the concentration range of 0.01−100 mM, the calcium ion electrode has an ideal Nernst response and good reproducibility to calcium ions. We measured the standard curves of the calcium ion at different temperatures (Figure S2). At 298.15 K, the linear regression equation is E = 27.5 lg[Ca2+] + 52.5 (Figure S2B). The thermodynamic parameters of the CaOx crystallization process were tested and calculated under standard conditions (Table 1). It was found that when there was no Mel, the ΔG⊖ Table 1. ΔG⊖, K⊖ of the Reaction between CaCl2 and NaC2O4 at 298.15 K no.

[Ca2+] (mol/L)

K⊖

ΔG⊖ (kJ/mol)

without Mel with Mel

1.233 × 10−4 1.134 × 10−4

6.579 × 107 7.779 × 107

−44.624 −45.039

of the reaction was −44.624 kJ/mol, indicating that the process of forming CaOx had a large reaction tendency. When Mel was added to the system, the ΔG⊖ of the reaction did not change significantly, which was −45.039 kJ/mol. It suggested that Mel had no obvious influence on the crystallization process of COM in thermodynamics. Kinetic Analysis of CaOx Formation Process. In addition to the thermodynamic factors, the formation of stones is closely related to the rate of crystallization. When the

Figure 2. Kinetic analysis of CaOx formation process. (A, B) Curves of the calcium ion concentration ([Ca2+]) with time (t) in three parallel experiments (without Mel A, with Mel B). (C, D) Relationship between lg r and lg[Ca2+] (a representative group of three parallel experiments, without Mel C, with Mel D). 4001

DOI: 10.1021/acs.cgd.9b00389 Cryst. Growth Des. 2019, 19, 3998−4007

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Table 2. Reaction Order α and Rate Constant k of CaOx Crystallization Process without and with Mel parameter

no. 1

no. 2

no. 3

α (without Mel) α (with Mel) k (without Mel) k (with Mel)

3.88 4.42 1.15 × 107 5.47 × 108

3.99 4.42 2.20 × 107 6.38 × 108

3.97 4.47 3.93 × 107 9.31 × 108

average 3.93 4.44 (2.54 (7.39

± ± ± ±

0.05 0.03 1.39) × 107 1.92) × 108

Figure 3. Effect of [Ca2+]/[C2O42−] ratio on CaOx crystallization in a synthetic urine system. (A−C) SEM images of the products at a [Ca2+]/ [C2O42−] ratio = 1:1 (A), 10:1 (B), 20:1 (C). The XRD diagram (D) and FTIR spectra (E) of the products (scale bar: 10 μm)

[Ca2+]/[C2O42−] was 10:1 or 20:1, the crystals obtained were completely COD (JCPDS, No. 17-0541). Obviously, an increase in [Ca2+] resulted in greater inhibition of the growth rate of {100} faces along the direction (arrows in Figure 3C), thereby promoting the formation of elongated COD. This observation is generally consistent with the crystal growth principle, which illustrates the situation that a growth crystal (due to the gradual displacement of the fast growing faces) is dominated by the slowest growing faces. Figure 3E showed the FTIR spectra of COM and COD crystals. COD crystals only had a single broad absorption peak of the O−H bond at 3480 cm−1; this is obviously different from the characteristic absorption peak in COM. The asymmetric (νas) and symmetric (νs) stretching vibration peaks belonging to −COO in COD appeared at 1647 and 1326 cm−1, respectively. In the fingerprint region, the absorption peaks of COD occurred at about 916, 781, and 609 cm−1.33 According to the calculation, when the [Ca2+]/ [C2O42−] ratio was 1:1, 10:1, and 20:1, the yield of the product was 33.0%, 72.1%, and 70.3%, respectively (Table S4). This result indicated that the yield of COD was significantly higher than that of COM in the synthetic urine system. In order to explore the effect of Mel on the formation of CaOx in a synthetic urine system, we studied the crystallization process under the condition of a [Ca2+]/[C2O42−] ratio = 1:1. It was found that Mel had no obvious effect on the crystal

formation in the human body, we used a synthetic urine system to simulate the crystallization process of CaOx.26 When the [Ca2+]/[C2O42−] ratio was 1:1, the product showed irregular polyhedron morphology, and part of the product presented a layered stacking structure (Figure 3A). When the [Ca2+]/ [C2O42−] ratio became 10:1, most of the products exhibited a uniform tetragonal bipyramidal structure with a size of about 3−5 μm (Figure 3B). In addition, some flower-like assembly structures also appeared in the products. When the [Ca2+]/ [C2O42−] ratio increased to 20:1, the tetragonal bipyramidal structure changed into elongated tetragonal bipyramids, but no significant change in size was observed (Figure 3C). XRD analysis (Figure 3D) showed that the product of the layered stacking structure was still COM, while the tetragonal bipyramidal structure product was COD. COM usually exists in the form of a hexagonal morphology; the COM of this layered stacking structure may be converted from COT. COT and COM are very similar in structure; this structural similarity facilitates the direct conversion of COT to COM.32 Therefore, thermodynamically unstable COT is preferentially formed in synthetic urine and then further converted to COM. A high [Ca2+]/[C2O42−] ratio favored the formation of COD. Typical characteristic peaks of COD were different from those of COM, and the five strongest peaks were (200), (211), (400), (411), and (213). There was no characteristic peak of COM in the XRD diagram of COD, indicating that when the ratio of 4002

DOI: 10.1021/acs.cgd.9b00389 Cryst. Growth Des. 2019, 19, 3998−4007

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Figure 4. Effect of different [Mel] on CaOx crystallization in a synthetic urine system when [Ca2+]/[C2O42−] ratio = 10:1. (A−E) SEM images of the products at different [Mel]: 0 mM (A), 1 mM (B), 2 mM (C), 3 mM (D), 4 mM (E). (F) The XRD diagram of the products (scale bar: 10 μm).

phase and morphology of CaOx (Figure S5). Since the concentration of urinary calcium ions in normal humans is 10− 20 times that of urinary oxalate, we also selected a [Ca2+]/ [C2O42−] ratio of 10:1 to study the effect of Mel on CaOx crystallization. In the absence of Mel, the product obtained was 3−5 μm tetragonal bipyramidal COD (Figure 4A). When the concentration of Mel ([Mel]) was 1−3 mM, the morphology of the product was consistent with that without Mel (Figure 4B−D). However, the size of the products obtained in the presence of 2 mM Mel was 10−20 μm, significantly larger than that of the other groups (Figure 4C). When [Mel] continued to increase to 4 mM, except the tetragonal bipyramidal COD, a large number of rod-like structures appeared in the product, with a length of about 15 μm (Figure 4E). After further analysis by XRD (Figure 4F), the product of the rod-like structure was calcium hydrogen phosphate dihydrate (CaHPO4·2H2O, DCPD). We suspected that Mel could adsorb calcium ions by electrostatic interaction, thereby increasing the local concentration of calcium ions. High concentrations of calcium ions could easily combine with phosphate ions in synthetic urine to form DCPD crystals. Therefore, Mel also has the risk of inducing the formation of calcium phosphate stones. In addition, the diffraction peak intensity of the large-sized COD was higher, indicating that the larger the size, the better the crystallinity. Zeta potential (ζ) is a parameter to evaluate the intensity of mutual repulsion or attraction among particles. When the particle has a higher surface charge density (high absolute value of ζ), it is not easy to gather because of its high electrostatic repulsion and stability, and vice versa.34 The zeta potential of COM and COD obtained under different [Mel] were shown in Figure 5 and Table S5. It could be seen that the zeta potential of COD was much more negative than that of COM, so the electrostatic repulsion between COD crystals was larger; it is not easy to aggregate to form stones. With the increase of [Mel], the surface charges of both COM and COD

Figure 5. Zeta potential (ζ) of COM and COD obtained with different [Mel].

decreased. This indicated that the electrostatic repulsion between crystals became weaker; it was easier to grow and aggregate into larger crystals, so the addition of Mel is more likely to cause the formation of stones. In addition, two molecules with similar properties to Mel (urea and histidine (His)) were selected as the modifiers to study their effects on the zeta potential of CaOx crystals. The results (Table S6) showed that urea slightly increased the surface charge of CaOx crystals, indicating that urea had the opposite effect with Mel, which could inhibit the aggregation of CaOx crystals. However, His reduced the surface charge of CaOx crystals, but the charge reduction was much less than that caused by Mel, indicating that Mel was more likely to cause crystal aggregation and induce the formation of CaOx stones. Effects of Mel on CaOx Crystallization in Synthetic Urine without Na3Cit. Citrate is an effective inhibitor of calcium-containing stones,35−37 so we hypothesized that the stability of COD in synthetic urine was not only related to the high [Ca2+]/[C2O42−] ratio but also to Na3Cit. Therefore, we used synthetic urine without Na3Cit to study the crystallization 4003

DOI: 10.1021/acs.cgd.9b00389 Cryst. Growth Des. 2019, 19, 3998−4007

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Figure 6. Effect of different [Mel] on CaOx crystallization in synthetic urine without Na3Cit. (A−E) SEM images of the products at different [Mel]: 0 mM (A), 1 mM (B), 2 mM (C), 3 mM (D), 4 mM (E). (F) The XRD diagram of the products (scale bar: 5 μm).

of CaOx at a [Ca2+]/[C2O42−] ratio of 10:1, and further explored the influence of Mel on the crystallization process. After removing the Na3Cit component from the synthetic urine, the product formed was an aggregate of irregular layered stacking structure (Figure 6A), and the XRD analysis showed that the composition was COM. This phenomenon suggested that the formation of COD in synthetic urine was also closely related to Na3Cit, which could stabilize COD and inhibit the formation of COM. Therefore, CaOx stones are more likely to form in humans with low citrate concentration. On this basis, we explored the effect of Mel on the CaOx crystallization process. When [Mel] was 1 mM, COT with a large sheet structure appeared in the product in addition to COM, and the surface of COT was rough (Figure 6B). When [Mel] increased to 2 mM, almost all the products became sheet-like COT (Figure 6C); the intensity of the COM diffraction peak in the XRD diagram was very weak. When the [Mel] continued to increase to 3 and 4 mM, COM almost disappeared, and the product was a mixture of COT and DCPD (Figure 6D,E). This phenomenon indicated that the addition of Mel changed the reaction process of CaOx crystallization. COT was preferentially formed in synthetic urine and then converted to COM as a precursor. In addition, with the increase of [Mel], the content of COT in CaOx crystals was getting higher and higher. When [Mel] was 3 and 4 mM, the content of COT reached 100% (Table 3, Figure S6). This result suggested that Mel could stabilize the thermodynamically unstable COT crystal phase and inhibit its conversion to COM. There were five distinct diffraction peaks in the XRD pattern of COT (Figure 6F), corresponding to the (010), (001), (110), (−101), and (−102) crystal faces,

which are consistent with the standard data of COT (JCPDS, No. 20−0232). The {100} crystal face family appeared in the standard card information, indicating that the COT is a typical layered material with a layer spacing of 0.79 nm. In the aqueous solution system without Na3Cit, Mel did not change the crystal phase of CaOx. However, in the synthetic urine system without Na3Cit, Mel induced the formation of COT. We hypothesized that Mel and MgSO4 in the synthetic urine played a synergistic role in stabilizing the COT crystal phase. Therefore, we explored the effect of Mel on CaOx crystallization in synthetic urine without Na3Cit and MgSO4. Through the analysis of SEM and XRD (Figure S7), it was found that the presence or absence of MgSO4 had no effect on the composition of CaOx, indicating that the stabilization effect of Mel on COT was not related to the existence of MgSO4. Therefore, we suspected that this stabilization was due to the fact that the addition of Mel increased the supersaturation of the reaction system, which was beneficial to the formation of the COT crystal phase. From this perspective, Mel can inhibit the formation of CaOx stones. In addition, in order to further simulate the growth process of stones in renal tubules, we combined the synthetic urine and membrane systems to study the crystallization process of CaOx and obtained various CaOx crystals with different morphologies and structures (Figures S8 and S9). Mechanism Analysis. In order to study the mechanism by which Mel accelerated the crystallization rate of COM, we measured the reaction rate constant k of the COM crystallization process at different temperatures (Table S7) and then calculated the average activation energy Ea required for the reaction according to the Arrhenius equation. The data in Figure 7 showed that when there was no Mel in the system, the average Ea for the reaction was 127.0 kJ·mol−1. After adding Mel, the average Ea was greatly reduced to 31.9 kJ· mol−1. According to these data, we concluded that the promotion effect of Mel on the crystallization process of COM was controlled by kinetics. Mel could significantly reduce the average Ea required for the reaction, thus accelerating the crystallization rate of COM. Therefore, when Mel enters the human body from the environment or food, the crystallization rate of COM is much greater than its excretion rate, which in turn leads to the formation of stones. However,

Table 3. Distribution of CaOx Crystal Phase in the Products Formed under Different [Mel] [Mel] (mM)

COM (%)

COT (%)

0 1 2 3 4

100 62 47 0 0

0 38 53 100 100 4004

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with the increase of [Ca2+] or [Na3Cit], the (100) crystal face of the tetragonal bipyramidal COD became more and more obvious and changed into an elongated tetragonal bipyramidal COD. This morphological change is due to the fact that a high concentration of citrate or calcium ions can effectively inhibit the growth of the (100) crystal face of COD crystals. In the synthetic urine containing Mel (Figure 8C), the formation tendency of the COT crystal phase with the greatest instability and solubility was obviously enhanced. COT was a hexagonal layered stacking structure with a layer thickness of about 2 μm (Figure S10G−I), which was consistent with the layered information reflected by XRD. This layered structure of COT can exist stably in the presence of Mel, which can lay a foundation for the study of intercalated materials in the future. Mel is a six-membered heterocyclic organic compound. The sp2 hybridized nitrogen atoms of the triazine ring provide three unshared pairs of electrons,38 showing the negative charge property, which can adsorb positively charged calcium ions through electrostatic interaction and increase the local concentration of calcium ions.39 Meanwhile, the six hydrogen atoms on the three unsubstituted −NH2 outside the ring can be used as hydrogen bond donors to combine with the oxygen atoms on CO in the oxalate. In this way, Mel enriches calcium ions and oxalate ions on its surface through the dual effects of electrostatic adsorption and hydrogen bonding, thereby the calcium ions and oxalate reached a local supersaturation state. At this point, the average Ea required for the combination of calcium ions and oxalates is greatly reduced, thus accelerating the rate of the reaction. In addition, when the relative supersaturation is higher, all three crystal phases of CaOx were likely to be formed; the formation of a highly dissolved COT crystal phase was most kinetically advantageous.40,41 Therefore, Mel can stabilize thermodynami-

Figure 7. Average activation energy Ea of CaOx crystallization in the presence or absence of Mel.

in the synthetic urine system, Mel could also accelerate the crystallization rate by reducing the Ea of the reaction. But at this time, COT was formed instead of COM; COT is easily excreted with urine, which does not easily cause the formation of stones. Mel could stabilize the COT crystal phase and inhibit its conversion to COM, thereby inhibiting the formation of stones. In an aqueous solution system (Figure 8A), CaOx crystals were predominantly in the form of COM, which was the thermodynamically most stable crystal phase. The COM obtained by bulk crystallization was a variety of irregular polyhedral morphology, while a hexagonal and twinned hexagonal structure with a large (100) surface area was obtained at low concentration growth (Figure S10A−C). In a synthetic urine system (Figure 8B), CaOx crystals mainly existed in the form of metastable tetragonal bipyramidal COD with sharp edges and corners (Figure S10D−F). In addition,

Figure 8. Crystallization of CaOx under different conditions. (A) Aqueous solution system. (B) Synthetic urine system. (C) Mel system. 4005

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cally unstable crystal phase COT, which is easily excreted with urine, reducing the risk of stone formation.

under low concentration conditions. Table S1: Yield of product obtained under different [Ca2+]/[C2O42−] ratios in an aqueous solution system. Table S2: Yield of the product obtained in the presence of different concentrations of Mel in aqueous solution when [Ca2+]/ [C2O42−] ratio = 1:1. Table S3: pH changes in the process of CaOx crystallization. Table S4: Yield of product obtained under different [Ca2+]/[C2O42−] ratios in a synthetic urine system. Table S5: Zeta potential (ζ) of the products COM and COD obtained with different concentrations of Mel. Table S6: Zeta potential (ζ) of the products COM and COD obtained with different modifiers. Table S7: The reaction rate constant k of the crystallization of CaOx with or without Mel at different temperatures. (PDF)

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CONCLUSION Mel caused the outbreak of infant urinary stones, which shocked the whole scientific and medical fields, making Mel a new environmental pollutant. In this work, the effect of Mel on CaOx crystallization was studied by simulating the formation process of CaOx stones in vitro. In an aqueous solution system, CaOx was mainly present in the form of COM. Through the calculation of the reaction order α and reaction rate constant k in the crystallization process of COM, it was found that Mel could significantly accelerate the reaction rate by reducing the average activation energy Ea, so Mel could easily induce the formation of CaOx stones. However, in the synthetic urine system, COD was the most stable crystal phase because of the presence of Na3Cit and a high [Ca2+]/[C2O42−] ratio. COD is easily excreted with urine, so it is not easy to form CaOx stones in normal humans. In addition, we found for the first time that the addition of Mel could stabilize the COT crystal phase in synthetic urine. Because Mel could enrich calcium ions and oxalate on its surface through the dual effects of electrostatic adsorption and hydrogen bonding, increasing the local concentration of calcium ions and oxalate. In solutions with high relative supersaturation, the thermodynamically unstable crystal phase COT is easily formed. COT is easily excreted in the urine, so the presence of Mel could inhibit the formation of CaOx stones. In conclusion, Mel plays two opposite roles in the crystallization process of CaOx. On the one hand, Mel can accelerate the crystallization rate of COM and increase the risk of stone formation. On the other hand, Mel can stabilize the thermodynamically unstable COT crystal phase, thus inhibiting the formation of stones. This work clarified the inhibition and promotion effect of Mel on the formation of CaOx stones, laying a scientific foundation for the prevention and treatment of Mel-related CaOx stones.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 21 65982620. E-mail: [email protected]. ORCID

Qingsheng Wu: 0000-0002-2371-5805 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grants 51771138, 91122025) and the State Major Research Plan (973) of China (grant 2011CB932404).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.9b00389. Supporting Information and methods for CaOx crystal growth under low concentration and in the membrane system, respectively. Figure S1: Effect of different concentrations of Mel on CaOx crystallization in an aqueous solution system. Figure S2: The standard curve of the calcium ion and the linear regression equation at different temperatures. Figure S3: Relationship between lg r and lg[Ca2+] in the system without Mel. Figure S4: Relationship between lg r and lg[Ca2+] in the system with Mel. Figure S5: Effect of different concentrations of Mel on CaOx crystallization in a synthetic urine system when [Ca 2+ ]/[C 2 O 4 2− ] ratio = 1:1. Figure S6: Quantitative analysis of COM crystal phase in CaOx by K-value method. Figure S7: Effect of different concentrations of Mel on CaOx crystallization in synthetic urine without Na3Cit and MgSO4. Figure S8: SEM images of CaOx crystals controlled by different membrane systems. Figure S9: XRD diagram of CaOx crystals controlled by different membrane systems. Figure S10: SEM images of CaOx crystals grown 4006

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DOI: 10.1021/acs.cgd.9b00389 Cryst. Growth Des. 2019, 19, 3998−4007