Morphology, Particle Size And Size Distribution Of Nanocrystallites In Urine And Their Relation To Stone Formation

As supersaturation of urinary mineral, the precipitation of crystallites in urine was recognized as the initial step in urinary stone formation. Urinary nanocrystallites were correlated tightly with stone formation. In our work, varieties of modern instruments were employed to comparatively investigate the physicochemical properties of nanocrystallites in urine of lithogenic patients and healthy subjects. These properties include average particle size, size distribution, morphology, composition, intensity-autocorrelation function, Zeta potential, aggregation, et al. The results from this work may shed light on the formation mechanism of urinary stone, and provide the important implications for the clinic diagnosis and therapy of urolithiasis, as well as their prevention.(1) Different types of calculi, such as calcium oxalate (CaOxa), uric acid, calcium phosphate (CaP) and magnesium ammonium phosphate calculi (struvite) were determined by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and Thermoluminescence spectrometer (TLD). The results showed that among more than 300 calculi determined by our group,75% calculi corresponded to CaOxa calculi,12% to uric acid calculi, 8% to CaP calculi and 5% to struvite; In addition, more or less calcium oxalate monohydrate (COM) was always contained in calcium phosphate calculi, which was attributed to that CaP crystal can induce the nucleation of CaOxa crystal and promote their growth. The stone-forming was facilitated by the coexistence of CaP and CaOxa crystals in urine. There were significant differences among the thermoluminescence spectrums of above calculi, which can conveniently provide the convincing evidence for the clinic diagnosis of urinary stone types.(2) The experimental conditions for sample preparation of urinary nanocrystallites were optimized by XRD to determine the urinary nanocrystallites of healthy persons. The optimized conditions were as follows:removing protein from urine by ethanol (Vurine:Vethanol= 5:3); membrane pore size,1.2μm; washing time,1 min; sample volume,2 ml (for the slide with the size of 25 mm×12 mm). Then, under the optimum conditions, the urinary nanocrystallites from 3 healthy subjects and 3 patients with CaP calculi were determined by Nanoparticle size analyzer, XRD and FT-IR. The results showed that the range of particle size distribution of nanocrystallites in lithogenic urine was 2-1000nm, while that of healthy subjects was 30-400 nm; the main composition of nanocrystallites in the urines of CaP stone-formers was CaP and uric acid, while that of healthy subjects was uric acid; between them the difference was significant. Simultaneously, a research method to eliminate the influence of soluble components in urine on the determined results of urinary crystallites by XRD and FT-IR was present in this paper.(3) With transmission electron microscopy (TEM) and nanoparticle size analyzer, comparative studies were conducted on the property variation of nanocrystallites in the urine of 5 lithogenic patients and 5 healthy subjects following the placement time (t1). These properties include average particle size (d), size distribution, intensity-autocorrelation function, Zeta potential (Q and aggregation state. With the prolongation of t1 from 0 h to 4 h, d value of the nanocrystallites in urine of lithogenic patients increased from 742 nm to 1667 nm, the autocorrelation time increased from 7.68 ms to 1050 ms andζdecreased from-1.52 mV to-4.44 mV, respectively; the autocorrelation curves were of fluctuating and unsmooth, and TEM showed that most of the patient's urinary nanocrystallites were in aggregation state and three types of agglomeration were observed. However, for nanocrystallites in urine of healthy subjects, there was little variation in the above properties within 4 h:d increased only from 140 nm to 251 nm, Ta increased from 4.09 ms to 6.65 ms and the average value ofζdecreased slightly from-10.24 mV to-10.78 mV; the autocorrelation curves were of regular and smooth, and TEM showed that healthy urinary nanocrystallites were well-dispersed. The above results showed that the nanocrystallites in urine of healthy subjects can keep stability, whereas those of lithogenic patients were easier to agglomerate gradually; and the agglomeration of urinary nanocrystallites is the key factor to stone formation.(4) By means of nanoparticle size analyzer and transmission electron microscopy (TEM), the effect of membrane pore size on the properties of nanocrystallites in urine of 5 lithogenic patients and 5 healthy subjects were comparatively investigated after urines were filtrated through microporous membrane with different pore size (0.22,0.45,1.2,3.0,10.0μm). These properties include average particle size (d), size distribution, intensity-autocorrelation function, Zeta potential (ζ) and aggregation state. With the increase of pore size from 0.22μm to 10.0 um, dvalue of the nanocrystallites in urine of lithogenic patients increased from 182 nm to 3753 nm, the range of size distribution became wider from 5-190 nm to 2700-4800 nm, the autocorrelation time (Ta) increased from 1.92 ms to 2100 ms, the average value ofζdecreased from-2.55 mV to-9.56 mV, respectively; and the autocorrelation curves were of fluctuating and unsmooth, TEM showed that the number of large-size crystallites increased gradually, the less small-size crystallites were observed and most of the patient's urinary nanocrystallites were in aggregation state. However, for nanocrystallites in urine of healthy subjects, there was little variation in the above properties:dincreased only from 286 nm to 1010 nm, the range of size distribution changed slightly from 40-350 nm to 40-700 nm, Ta increased from 1.40 ms to 4.60 ms and the average value ofζdecreased from-5.02 mV to-6.42 mV, the autocorrelation curves were of regular and smooth, TEM showed that the small-size crystallites were much more than that of patients, and urinary nanocrystallites were well-dispersed. The above results demonstrated that the formation process of the nanocrystallites in urine of lithogenic patients belonged to the growth-controlled process, whereas that of healthy subjects belonged to the nucleation-controlled process; the urinary nanocrystallites of lithogenic patients are easier to agglomerate than those of healthy subjects.