Formation Mechanism of Magnesium Ammonium Phosphate Stones : A Component Analysis of Urinary Nanocrystallites

The components of urinary nanocrystallites in patients with magnesium ammonium phosphate (MAP) stones were analyzed by X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectrometer, high-resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), fast Fourier transformation (FFT), and energy-dispersive X-ray spectroscopy (EDS). The main components of the stones were MAP hexahydrate (MAP⋅6H 2 O), magnesium hydrogen phosphate trihydrate (MgHPO 4 ⋅3H 2 O), and a small amount of calcium phosphate (CaP), while the main components of urinary nanocrystallites were MgHPO 4 ⋅3H 2 O, CaP, and MAP monohydrate (MAP⋅H 2 O). MAP⋅H 2 O induced the formation of MAP stones as seed crystals. MgHPO 4 ⋅3H 2 O was accompanied by the appearance of MAP⋅6H 2 O. The formation mechanism of MAP stones and influencing factors were discussed on the basis of the components of urine nanocrystallites. A model diagram of MAP stone formation was also put forward based on the results. Formation of MAP stones was closely related to the presence of high amounts of MAP crystallites in urine. Urinary crystallite condition and changes in urine components could indicate the activity of stone diseases.


Introduction
Urinary stones can be divided into acidic (e.g., uric acid and cystine), neutral (e.g., calcium oxalate (CaOx)), and alkaline (e.g., magnesium ammonium phosphate (MAP)) stones based on their chemical properties.However, indices that can be used to predict the formation and recurrence risk of renal stones are unavailable.Moreover, the same methods and drugs are used in uroliths without distinction.Consequently, curative effects and lower cured rates differ [1,2].MAP, the most common component of alkaline stones, contains MAP hexahydrate (struvite, MgNH 4 PO 4 ⋅6H 2 O) and magnesium hydrogen phosphate trihydrate (newberyite, MgHPO 4 ⋅3H 2 O).MAP accounts for approximately 10% to 15% of all stone components [3].Other alkaline stones comprise carbapatite (Ca 10 (PO 4 ) 6 CO 3 ), hydroxyapatite, and monoammonium urate.MAP is defined as infective stones associated with urinary tract infection.Although the morbidity of infective stones constantly decreases, the recurrent rate of infective stones is higher than that of other stones, and female morbidity is higher than male morbidity [4].
The conditions of urinary crystals are closely related to stone formation [1,2,[5][6][7][8][9][10].On the one hand, the disappearance of specific types of urinary crystallites in urine (e.g., cystine and struvite) demonstrates a recurring trend of reduction in stone formation.On the other hand, the continuous or new appearance of these urinary crystals in urine indicates a continuous risk of stone formation or lithiasis relapse.Detecting crystallite components can help predict renal stone formation and even the formation of different types of stones, thereby providing information on appropriate remedies and personalized treatments.
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The first step in the formation of urinary stones is the nucleation of urinary minerals from supersaturated urine.The formed nucleus (generally less than 10 nm) grows and/or aggregates to a pathological size (several tens of microns).These crystallites are then retained in the urinary tract or fixed by urinary tract organization, forming urinary stones (millimeters to several centimeters) [20].Therefore, morphological characteristics, composition, and crystal structures of urinary nanocrystallites are important factors of stone formation [21,22].To further investigate the relationship between urinary nanocrystallite properties and magnesium ammonium phosphate (MAP) stone formation, we analyzed the chemical constituents of urinary nanocrystallites in six patients with MAP stones through high-resolution transmission electron microscopy (HRTEM), fast Fourier transformation (FFT), selected area electron diffraction (SAED), and energy-dispersive X-ray spectroscopy (EDS).The relationship between urinary crystallite components and MAP stone formation was discussed.Our results can provide insights into the exploration of preventing renal calculi formation from a chemical perspective.

Reagents and Instruments.
Absolute ethanol and sodium azide (NaN 3 ) were of analytical purity.Glass vessels were cleaned with double-distilled water.
TEM was conducted on a HRTEM (JOEL 2100F) with a maximum acceleration voltage of 200 kV and lattice resolution of 0.19.To determine the morphology, component, element, and crystal structure of urinary nanocrystallites, we performed HRTEM, fast Fourier transformation (FFT), SEAD, and EDS of the HRTEM.FFT analysis in the Digital Micrograph software was also conducted to obtain the patterns.X-ray diffraction (XRD) results were recorded using a D/max 2400 X-ray diffractometer (Rigaku, Tokyo, Japan) with Ni-filtered Cu Ka radiation ( = 1.54 Å) at a scanning rate of 2 ∘ min −1 (40 kV, 30 mA).The divergence and scattering slit was 1 ∘ for the range of 5 ∘ < 2 < 60 ∘ .A Nicolet 6700 Fouriertransform infrared (FT-IR) spectrometer (Nicolet Company, USA) was also used.

Collection and Treatment and Component Detection of
Stones.MAP stones were collected from six patients (2 men and 4 women; mean age = 35.6 years; range = 23-51 years) with stones after surgery, disinfected with 75% alcohol, cleared with distilled water, and placed in a dustfree incubator at 45 ∘ C to dry.The urinary stones were then ground to powder by an agate mortar for XRD and FT-IR characterization.It was shown that the main components of these six stones were MAP.

Collection and Treatment of Urine.
Fasting morning urine samples from the patients with MAP stones were collected.pH was detected and 2% NaN 3 solution (10 mL/L urine sample) as antiseptic was added to these urine samples.Afterward, 20 mL of anhydrous alcohol was added to 30 mL of urine sample to denature the proteins.The solution was stirred for 3 min, left undisturbed for 30 min, and centrifuged at 4000 r/m for 15 min to remove the proteins and cell debris from the urine sample.The treated urine was then filtered using a microporous membrane with a pore size of 1.2 m to eliminate the effect of large urinary crystallites.The filtered urine was stored in clean glassware for examination.

HRTEM Detection of Urinary Nanocrystallites.
In the preparation of samples for HRTEM detection, the urine sample was first subjected to ultrasound treatment for 5 min, approximately 5 L of urine was submerged in a copper mesh by a microsyringe, and the urine was preliminarily dried using an absorbent paper from the back of the mesh so as to remove most of the water in urine.After such a treatment, most of the soluble salts (such as NaCl and urea) in urine were sucked off with the urine by the paper.Then the mesh was stored in a desiccator for 2 d prior to HRTEM, SAED, and EDS analyses.

Detection of Magnesium and Phosphate in Urine.
The Mg 2+ and phosphate concentrations in the urine of healthy control and patients with MAP stones were, respectively, detected using an atomic absorption spectrophotometer (TAS-990) and colorimetric method (molybdate) using spectrum automated analyzer.

Component Analysis of MAP Stones.
The components of six MAP stones were characterized by XRD and FT-IR spectra.The results are shown in Table 1.The stones mainly comprised MAP⋅6H 2 O and small amounts of MgHPO 4 ⋅3H 2 O, CaP, and COM.

SAED Analysis of Urinary
Nanocrystallites.SAED analysis was conducted to further characterize the components of some urinary nanocrystallites (Figure 2).The appearance of a series of diffraction points or diffraction rings demonstrated that the sample is monocrystal or polycrystal [23,24].The diffraction data were indexed and compared with the PDF standard card [25].Figure 2

Component Comparison of Urinary Nanocrystallites and Urine between Patients with MAP Stones and Healthy
Subjects.Table 1 shows the statistical analysis results of Urine pH and phosphate concentration in patients with MAP stones (6.5 ± 0.4, 550 ± 72 mg/L) were higher than those of healthy subjects (6.0 ± 0.3, 441 ± 59 mg/L), respectively.However, Mg 2+ concentration (58.8 ± 13.1 mg/L) was apparently lower than that of healthy subjects (74.1 ± 23.2 mg/L) (Table 1).only after these stones are formed; as such, the presence or occurrence of stones cannot be successfully predicted or early diagnosis cannot be performed.

Discussion
Therefore, the following aspects should be considered: (a) the occurrence of urinary stones should be detected rapidly and conveniently before these stones are formed, thereby preventing stone formation, and (b) the type of stone should be determined (according to the main component) before stones are removed and treated.For example, clinicians can choose different shock frequencies and shock time in extracorporeal shock wave lithotripsy (ESWL) according to the type of urinary stones because the hardness of stones varies in terms of different components.The relative hardness of different types of stones is listed as follows: COM (4 to 5) > cystine, apatite (3 to 5) > uric acid (2.5) > calcium hydrophosphate, magnesium ammonium phosphate, or COD (2.0).
Different therapeutic methods can be used to treat different types of stones.(a) For acidic stones (e.g., uric acid and cystine), alkaline medicines, such as tris(hydroxymethyl)aminomethane (THAM), are used to alkalize urine (pH = 6.5 to 6.8).Potassium citrate also increases urine pH.(b) For alkaline stones (e.g., struvite), L-methionine can be used to acidify urine.Trimethoprim-sulfamethoxazole can be used to prevent urinary tract infection or acetohydroxamic acid can be administered to control the concentration of urease.(c) For neutral stones (e.g., calcium oxalate), hydrogen sodium potassium citrate, potassium citrate, or dihydrochlorothiazide can be used to carry out defensive treatment.
Urinary nanocrystallites highly indicate stone diseases and can be used to predict stone recurrence; their physicochemical properties can also be evaluated in terms of potential clinical values because crystalluria precipitation results from diverse factors, which act in urine to trigger crystal formation, including inhibitors and promoters, as well as measured and unmeasured trigger factors [8][9][10].Urinary crystallites exhibit a higher accuracy of predicting stone diseases than daily urine volume, 24-hour calcium excretion, or urine calcium or oxalate concentration.However, it was also reported that the presence of crystalluria did not enable an efficient characterization of recurrent stone formers because crystals are found in the urine of healthy men and lithogenic patients [27][28][29][30].The particle size of the former is not always smaller than that of the latter; the pathogenesis of male and female patients may differ.
However, all of the investigated crystals were at a micron level; most of these crystals are frequently affected by food metabolism.Moreover, the majority of tissue debris and apoptotic cells in urine are in the same order of magnitude as urinary crystals [31]; thus, clinical application of crystalluria determination is limited to a high extent.Therefore, this study focused on the properties of urine nanocrystallites, expecting to perform early diagnosis for MAP stones.

Urinary Nanocrystallite Components and Stone Formation.
The results of this study showed that urinary nanocrystallites could indicate stone diseases.The results of HRTEM (Figure 1), SAED (Figure 2), and EDS (Figure 3) showed that the main components of urinary nanocrystallites in patients with MAP stones were MAP⋅H 2 O, MgHPO 4 ⋅3H 2 O, and small amounts of CaP or COM, which significantly differed from those in healthy subjects.Therefore, the properties of urinary crystallites should be detected to help predict the formation of stone and certain types of urinary stone; as such, relevant information could be used as basis for appropriate remedies and personalized treatments.
MAP is a common component in the urine of healthy subjects but plays an insignificant role for its unsaturation in healthy subjects' urine [32,33].However, compared with the urine of controls and patients with other stones, the proportions of MAP⋅6H 2 O and MgHPO 4 ⋅3H 2 O crystals increased in the urine of patients with MAP stones.We have detected the concentration of Mg 2+ and phosphate in the urine of patients with MAP stones compared with those of the control subjects and patients with CaOx stones.The respective concentrations of Mg 2+ were 74.1 ± 23.2, 58.8 ± 13.1, and 62.1 ± 35 mg/L.The lowest Mg 2+ concentration was found in the urine of patients with MAP stones.He et al. [34] also reported that the percentage of hypomagnesiuria in patients with infectious stones was 55.6%, which is higher than that in CaOx group (40.3%) and UA group (36.7%).The respective phosphate concentration is 441 ± 59, 550 ± 72, and 481 ± 89 mg/L.Srinivasan et al. [35] also found that the concentration of urine phosphate was 919 ± 67, 801 ± 57, 779 ± 81, and 754 ± 114 mg/24 h, respectively, in urine of patients with MAP, CaOx, and UA stones, and in urine of healthy controls.The highest phosphate concentration was found in the urine of patients with MAP stones.
The main components of stones were MAP⋅6H  urease produced from bacteria, leading to a significant increase in urine pH [36]; as a result, NH 4 + , CO 3 2− , and PO 4 3− concentrations in urine rapidly increase.
The average value of urine pH of the six patients with MAP stones was 6.5 ± 0.4, which was higher than that of patients with uric acid stones (pH = 5.3 ± 0.3) and the healthy controls (pH = 6.0 ± 0.3) [2].At urine pH 6.8, PO 4 3− , CO 3 2− , and NH 4 + combine with calcium ions in urine, and calcium phosphate and carbon-apatite are formed.At urine pH 7.2, these ions interact with Mg 2+ ions adsorbed on the biofilm surface of bacteria.Struvite-calcium phosphate precipitates are formed, providing a nidus for crystal nucleation, growth, and aggregation.As MAP saturation in urine increases, crystals constantly precipitate on bacteria, forming struvite stones [39,40].Furthermore, calcium phosphates induce the development of calcium oxalate monohydrate crystals through heterogeneous nucleation [41,42].Lee et al. [43] performed TGA and EDS to identify the components on surface and interior layer of MAP calculi; the results show that the interior layer is composed of 64% MAP and 36% apatite, whereas the surface is composed of calcium oxalate (CaOx).
Based on these findings, a model diagram of MAP stone formation is shown in Figure 4.The formation of MAP stones was closely related not only to high concentrations of phosphate and pH in urine but also to the properties of urinary nanocrystallites.Urinary tract infections cause high urinary pH, high NH 4 + , CO For instance, a large sample quantity was required for XRD and FT-IR analyses; the spectral baseline was very high when a small amount of samples was used.XRD and FT-IR detection results provided comprehensive data of all crystallites; thus, a component of <5% was difficult to be detected.The absorption peaks of such low contents (<5%) were not visible when the content of one component in the crystallites was extremely high.This low content may act as a nidus to induce crystal growth in renal stone formation.By contrast, the combination of HRTEM, FFT, SAED, and EDS can be applied to accurately analyze the components of a single nanocrystallite in urine, even small crystallites with the size of several nanometers.Thus, these methods can provide information on initial renal stone formation.

Conclusions
Figure 1 shows the HRTEM images obtained in different areas of the urinary nanocrystallites.To analyze the clear lattice fringes in random selection, we detected the spacing of lattice fringe at  = 4.72 Å, which was assigned to (110) plane of MAP⋅H 2 O in Figure 1(a).In Figure 1(b) we also detected the lattice fringes at  = 4.61 and 4.49 Å, which were assigned to (210) and (102) planes of MgHPO 4 ⋅3H 2 O, respectively.Namely, the main components of urinary nanocrystallites in patients with MAP stones were MAP⋅H 2 O and MgHPO 4 ⋅3H 2 O.

Figure 4 :
Figure 4: Model diagram of MAP stone formation.

A
combination of HRTEM, SAED, EDS, and XRD was performed to detect the components of urinary nanocrystallites in six patients with MAP stones.The components of urinary nanocrystallites were compared with stone components.Stones mainly comprised MAP⋅6H 2 O, MgHPO 4 ⋅3H 2 O, and a small amount of CaP; urinary nanocrystallites mainly comprised MgHPO 4 ⋅3H 2 O, MAP⋅H 2 O, and CaP.The formation of MAP stones was closely related to the presence of high amounts of MgHPO 4 ⋅3H 2 O and MAP⋅H 2 O crystals in urine.MAP⋅H 2 O induced the formation of MAP⋅6H 2 O as seed crystals.MgHPO 4 ⋅3H 2 O was often accompanied by the precipitation of MAP⋅6H 2 O.The combination of HRTEM, SAED, EDS, and XRD can be applied to accurately analyze the components of urinary nanocrystallites.This study provided insights into the formation mechanism of magnesium ammonium phosphate stones.

Table 1 :
Statistical analysis of the components of stones, urinary nanocrystallites, and urine of six patients with MAP stones compared with six healthy subjects.
Notes.*1Stone composition was detected by XRD and FT-IR spectra.MAP: magnesium ammonium phosphate; MgHPO 4 ⋅3H 2 O: magnesium hydrogen phosphate trihydrate; CaP: calcium phosphate.UA: uric acid; COM: calcium oxalate monohydrate; COD: calcium oxalate dihydrate.* 2 Crystallite composition was detected by HRTEM, SAED, EDS, and XRD and was arranged in decreasing order.stones were simultaneously analyzed through a combination of HRTEM, SAED, EDS, and XRD.These nanocrystallites mainly comprised MAP⋅H 2 O, MgHPO 4 ⋅3H 2 O, and CaP.Namely, MAP⋅H 2 O was the main component of urinary nanocrystallites and MAP⋅6H 2 O was the main component of stones.
2 O and MgHPO 4 ⋅3H 2 O, whereas those of urinary nanocrystallites were MgHPO 4 ⋅3H 2 O and MAP⋅H 2 O.The reasons are as follows.
4.3.The Formation Mechanism of MAP Stones.After urine was infected by bacteria, urea in urine is decomposed by 3 2− , and PO 4 3− concentrations, as well as the presence of Mg 2+ , resulting in the appearance of nanocrystallites of MAP⋅H 2 O, MgHPO 4 ⋅3H 2 O, and CaP, among others; thus, MAP stones composed of MAP⋅6H 2 O and MgHPO 4 ⋅3H 2 O are formed.4.4.The Advantage of HRTEM in Detection of Urinary Nanocrystallites.X-ray diffraction (XRD) is a commonly used tool to identify material components.Previous studies [1, 2] determined the components of urinary nanocrystallites in patients with CaOx stones and uric acid stones through XRD and Fourier-transform infrared (FT-IR) spectrometry.However, XRD and FT-IR analyses exhibit disadvantages.