The composition and morphology of nanocrystals in urines of healthy persons and lithogenic patients were comparatively investigated by means of X-ray diffraction (XRD) and transmission electron microscopy (TEM). It was shown that the main composition of urinary nanocrystals in healthy persons were calcium oxalate dihydrate (COD), uric acid, and ammonium magnesium phosphate (struvite). However, the main compositions of urinary nanocrystals in lithogenic patients were struvite,
Urolithiasis, which occurs in 1
The first step of urinary stone formation is the nucleation of particles in supersaturated urine. Then the formed nuclei (generally less than 10 nm) grow or/and aggregate to a pathological size (several tens micron). After these crystallites are retained on the urinary tract (free-particle theory) or fixed by the urinary tract organization (fixed-particle theory), urinary stones ultimately formed [
According to the chemical properties, urinary stone can be classified into acidic stones (such as uric acid, cystine), alkaline stones (such as struvite), and neutral stones (such as calcium oxalate, calcium phosphate). However, clinical practices neither can be predicted before stone formation nor can be individually treated by taking different medicine and different treatment methods for different types of urinary stones. Thus it results in a low treatment ratio. Thereby, if the occurrence of urinary stones can be predicted or the type of stones can be estimated before treatment by the determination of urinary composition and urinary nanocrystals, it will provide evidence for a suitable remedy and personalized treatment in clinic.
XRD has advantages of reliability in qualitative analysis and accuracy in quantitative analysis. It operates simply and has a high sensitivity. Based on XRD diffraction data, the multicomponents in a sample can be measured simultaneously, and the approximate size of the nanoparticles can also be calculated by means of Scherer formula. For example, Chen et al. [
Based on the discussed above, the composition, size, morphology, and aggregation of the nanocrystals of less than 100 nm in healthy urines and in lithogenic urines are comparatively investigated by XRD and TEM. We hope it would provide enlightenment on diagnosing and treating urinary stone.
Anhydrous alcohol and sodium azide were reagent-grade chemicals. All the glasswares were cleaned with the ultrapure water from the Millipore-Q system; the resistance was 18.2 MΩ cm. The fresh morning urine samples were collected from both patients who had urinary stones and healthy persons with no prior history of urinary stones.
TEM was carried out by PHILIPS TECNAI-10 transmission electron microscope at an operating voltage of 100 kV. X-ray diffraction (XRD) results were recorded on a D/max 2400
X-ray diffractometer (Rigaku, Japan) used graphite monochromator, Ni-filtered Cu K
The participants in the study included sixteen lithogenic patients (9 men and 7 women) with a mean age of 44.7 years (23
Urinary stone risk profile in lithogenic patients and in normal subjects.
Urine parameter | Lithogenic patients ( | Normal subjects ( |
---|---|---|
Volume, L | ||
pH | ||
Calcium, mmol/L | ||
Oxalate, mmol/L | ||
Citrate, mmol/L | ||
Phosphorus, mmol/L |
XRD patterns of three representative urinary stones (
After the fresh morning urine was collected, 2% (weight/volume ratio)
The filtrate was added on a copper mesh for TEM detection and on clean glass tablet for XRD characterization simultaneously. For the former, the copper mesh was put in a desiccators, and for the latter, the glass tablet was put in a no dust incubatorat at 50°C for 4 hours to make urine evaporation.
XRD was used to investigate the composition of urinary nanocrystals of 16 lithogenic patients and 17 healthy persons. Figures
Main diffraction peaks (
Nanocrystal in urine | ASTM card [ | |
---|---|---|
8.79(010), 2.80(121), 4.73(110), 4.20(011), 3.24(012) | 36-1491 | |
5.60(020), 3.66(121), 3.29(130), 2.80(040), 2.01(151) | 15-762 | |
4.91(210), 3.86 | 31-1982 | |
4.06(024), 4.00(116), 2.88(217), 2.20(404), 1.81(210) | 9-169 | |
5.93 | 20-231 | |
6.18(200), 4.42(211), 3.91(310), 2.78(411), 2.34(501), | 17-541 | |
2.24(213), 2.00(323), 1.62(730) |
XRD patterns of typical urinary crystals of three health persons (H1, H2, H3). (
XRD patterns of typical urinary crystals of three lithogenic patients (L1, L2, L3). (
In the three XRD patterns of urinary nanocrystals of lithogenic patients (see Figure
All the XRD results showed that the urinary nanocrystals were mainly COD, uric acid and magnesium ammonium phosphate in healthy urine (see Figure
It could be seen that the peak intensities of struvite crystals in urine of lithogenic patients (see Figure
It could also be seen that the peak intensities of (
The differentiation of COD crystals in the two kinds of urine was attributed to the difference of urinary inhibitors. These urinary inhibitors could inhibit the formation of COM crystals and induce the growth of COD crystals [
Figure
TEM images of typical urinary nanocrystals of two health persons (a), (b) and two lithogenic patients (c), (d). The bar is 100 nm.
Based on the XRD diffraction data, not only could the components of samples be measured, but also the approximate size of the nanoparticles could be calculated using Scherer formula [
Sizes of urinary nanocrystals in three healthy persons (H) and in three lithogenic patients (L) calculated using scherer formula (nm).
Urine nanocrystals | H1 | H2 | H3 | L1 | L2 | L3 |
---|---|---|---|---|---|---|
COD | 41 | 37 | 45 | — | — | 36 |
struvite | 32 | 40 | — | — | — | — |
uric acid | 23 | 72 | — | — | — | 27 |
— | — | — | 29 | 118 | — | |
struvite | — | — | — | 51 | 12 | — |
For the urinary nanocrystals in lithogenic urine sample L1 (Figure
That is, the size of nanocrystals in the healthy persons urine ranged from 23 nm to 72 nm, and those in lithogenic patients urine ranged from 12 nm to 118 nm. The results obtained from Scherer formula were nearly consistent with those observed from the TEM method.
Since there was a different ratio of volume to surface area for the particles with different size, it led to the difference in their solubility. According to the mechanism of Ostwald ripening [
Although the formation of urinary stones correlated with many factors, it necessarily went through the following processes: the formation of crystal nucleus in urine, the growth and/or aggregation of the crystallites, and the adhesion of crystallites on renal epithelial cell, as shown in Figure
Schematic diagram of urinary stone formation.
Since the growth, aggregation and the final formation of urinary stones were apparently affected by the components, size, and uniformity of urinary nanocrystals, therefore, urinary stone formation may be prevented by diminishing the size differentiation and the aggregation of nanocrystals in urine through physical or chemical methods. The investigatation about the existing state of urinary nanocrystals and the detection of the components and properties of nanocrystals in urine would be beneficial to find out the connection between stone formation and urinary nanocrystals and then to provide evidences for a suitable remedy and personalized treatment to urolithiasis in clinic.
The composition and morphology of nanocrystals in urines of healthy persons and lithogenic patients were comparatively investigated by means of XRD and TEM. XRD patterns showed that the urinary nanocrystals mainly were COD, uric acid, and magnesium ammonium phosphate in healthy persons; but mainly were magnesium ammonium phosphate,
This research work was granted by the Natural Science Foundation of China (30672103) and the Key Project of Guangdong Province (2009B030801236). The authors are grateful to Dr. Kuang Li and Dr. Liang Wei-Bo of the first Affiliated Hospital of Medical College of Jinan University for providing the urine samples of lithogenic patients.