尿液中纳米微晶的检测及其影响因素
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摘要
本文采用X射线粉末衍射仪(XRD)和傅立叶变换红外光谱仪(FT-IR)研究了尿液pH变化与尿液中纳米微晶组分的关系。通过比较研究稀释、离心、过滤和去除蛋白后对测量结果的影响,得出适于纳米检测的尿液处理方法。为了比较尿石症患者尿液中的纳米微晶是否与健康对照者存在差异,采用纳米粒度仪测定了70例健康对照者尿液和70例尿石症患者尿液中1000 nm以下微晶的Zeta电位、光强自相关曲线、粒径及其分布。
不但尿pH差异大的不同人尿液中微晶组分存在差异,而且,同一人的尿pH发生变化时,其尿微晶亦发生变化。尿pH值较低(如pH＜5.8)时,主要为尿酸、酸式磷酸盐和草酸钙等;尿pH值较高(如pH＞6.2)时,主要为尿酸盐、磷酸盐、磷酸铵镁和草酸钙等。联合运用XRD和FT-IR两种方法,可以更好地检测尿液中晶体组分,有助于了解尿石症的成因。
比较了对尿液进行稀释、离心、过滤和去除蛋白后对测量结果的影响,得到了可用于纳米检测的最佳尿液处理方法为:尿液用甲醛防腐和凝固蛋白后,稀释50%,再用孔径3μm的微孔滤膜过滤,然后滤液在转速4000 r/min下离心15min。该方法可以去除尿液中的细胞碎片和大分子物质,并且不影响1000 nm以下微晶的检测,其结果与透射电子显微镜(TEM)观察结果一致。
通过比较二类尿微晶的光强自相关曲线、多分散系数(PDI)、Zeta电位和平均粒径相对误差,表明健康对照者的尿液微晶比尿石症患者的稳定性好,正常人尿液中纳米微粒的Zeta电位、平均粒径和粒径分布均具有相似性和一致性,光强自相关曲线显示均为稳定体系,TEM也显示粒子分布均匀,无聚集现象;患者尿液中纳米微粒则显示彼此间较大差异,Zeta电位、平均粒径和粒径分布都在一个较宽的范围,光强自相关曲线显示体系不稳定,容易发生聚集和聚沉现象,TEM表征也证实了这一点。从尿液中抑制剂差异、尿晶体组分差异、尿浓度等方面对上述结果进行分析讨论,对成石原因进行了探讨,并且从物理和化学角度对结石病的预防和治疗提供了可行性的建议。
The effect of urinary pH values on composition of urinary nanocrystallites were investigated by X-ray diffraction(XRD) and Fourier transform infrared(FT-IR) spectroscopy. By comparing the effect of dilution,centrifugalization,filtration,and protein remove on detection results of urinary nanocrystallites,a good treatment method for urinary nanocrystallites detection was found.The zeta potential,intensity-autocorrelation curve,mean size and distribution of nanocrystallites with a size less than 1000 nm in urines of 70 lithogenic patients and 70 healthy people were investigated by nanoparticle size analyzer.
The differentiation of composition of urinary nanocrystals not only occurred between the people with different urinary pH,but also occurred in the same person when his urinary pH changed.At lower urinary pH(such as pH＜5.8),most of the urinary components are uric acid, acidic phosphate and calcium oxalate,etc.However,most of these components are urate, phosphate,magnesium ammonium phosphate,and calcium oxalate etc.at higher urinary pH (such as pH＞6.2).Combined XRD and FT-IR methods,the compositions of urinary nanocrystals can be determined accurately.It is ehelpful to explore the cause of urinary stones.
By comparing the effect of dilution,centrifugalization,filtration,and protein remove on detection results of urinary nanocrystallites,a good treatment method for urinary nanocrystallites detection was found:antisepticising and protein-coagulated with formaldehyde,the urine was diluted with distilled water of the same volume;then filtrated through 3μm micropore film,and the filtrate was centrifugalized under 4000 r/min for 15 min.This method could remove the cell fragment and the macromolecular substances and did not affect the detection of the urinary nanocrystallites below 1000 nm.The results were consistent to those obtained by transmission electron microscope(TEM).
By comparing the intensity-autocorrelation curve,polydispersity index(PDI),zeta potential and relative error of average diameter of urinary nanocrystalllites in two kinds of urine,it was concluded that the nanocrystallites in healthy urine were more stable than those in lithogenic urine.The zeta potential,mean diameter and size distribution of nanocrystallites in healthy urines were comparability and consistency,healthy urines were stable system which were revealed by their intensity-autocorrelation curves;the TEM images showed well-proportioned size distribution and little aggregation of healthy urinary nanocrystallites,too.The zeta potential, mean diameter and size distribution of nanocrystallites in lithogenic urines were observably different from each other,whose zeta potential,mean size and size distribution were in a wide range;lithogenic urines were unstable system which were revealed by their intensityautocorrelation curves,aggregation and deposition were incidental;The results were consistent to those obtained by transmission electron microscope(TEM).The difference of urinary inhibitor, urinary nanocrystaiiltes component and urinary concentration between healthy and lithogenic urines were discussed,the reason of stone formation was discussed,too.Then the feasible suggeation was adviced base on physics and chemistry.
不但尿pH差异大的不同人尿液中微晶组分存在差异,而且,同一人的尿pH发生变化时,其尿微晶亦发生变化。尿pH值较低(如pH＜5.8)时,主要为尿酸、酸式磷酸盐和草酸钙等;尿pH值较高(如pH＞6.2)时,主要为尿酸盐、磷酸盐、磷酸铵镁和草酸钙等。联合运用XRD和FT-IR两种方法,可以更好地检测尿液中晶体组分,有助于了解尿石症的成因。
比较了对尿液进行稀释、离心、过滤和去除蛋白后对测量结果的影响,得到了可用于纳米检测的最佳尿液处理方法为:尿液用甲醛防腐和凝固蛋白后,稀释50%,再用孔径3μm的微孔滤膜过滤,然后滤液在转速4000 r/min下离心15min。该方法可以去除尿液中的细胞碎片和大分子物质,并且不影响1000 nm以下微晶的检测,其结果与透射电子显微镜(TEM)观察结果一致。
通过比较二类尿微晶的光强自相关曲线、多分散系数(PDI)、Zeta电位和平均粒径相对误差,表明健康对照者的尿液微晶比尿石症患者的稳定性好,正常人尿液中纳米微粒的Zeta电位、平均粒径和粒径分布均具有相似性和一致性,光强自相关曲线显示均为稳定体系,TEM也显示粒子分布均匀,无聚集现象;患者尿液中纳米微粒则显示彼此间较大差异,Zeta电位、平均粒径和粒径分布都在一个较宽的范围,光强自相关曲线显示体系不稳定,容易发生聚集和聚沉现象,TEM表征也证实了这一点。从尿液中抑制剂差异、尿晶体组分差异、尿浓度等方面对上述结果进行分析讨论,对成石原因进行了探讨,并且从物理和化学角度对结石病的预防和治疗提供了可行性的建议。
The effect of urinary pH values on composition of urinary nanocrystallites were investigated by X-ray diffraction(XRD) and Fourier transform infrared(FT-IR) spectroscopy. By comparing the effect of dilution,centrifugalization,filtration,and protein remove on detection results of urinary nanocrystallites,a good treatment method for urinary nanocrystallites detection was found.The zeta potential,intensity-autocorrelation curve,mean size and distribution of nanocrystallites with a size less than 1000 nm in urines of 70 lithogenic patients and 70 healthy people were investigated by nanoparticle size analyzer.
The differentiation of composition of urinary nanocrystals not only occurred between the people with different urinary pH,but also occurred in the same person when his urinary pH changed.At lower urinary pH(such as pH＜5.8),most of the urinary components are uric acid, acidic phosphate and calcium oxalate,etc.However,most of these components are urate, phosphate,magnesium ammonium phosphate,and calcium oxalate etc.at higher urinary pH (such as pH＞6.2).Combined XRD and FT-IR methods,the compositions of urinary nanocrystals can be determined accurately.It is ehelpful to explore the cause of urinary stones.
By comparing the effect of dilution,centrifugalization,filtration,and protein remove on detection results of urinary nanocrystallites,a good treatment method for urinary nanocrystallites detection was found:antisepticising and protein-coagulated with formaldehyde,the urine was diluted with distilled water of the same volume;then filtrated through 3μm micropore film,and the filtrate was centrifugalized under 4000 r/min for 15 min.This method could remove the cell fragment and the macromolecular substances and did not affect the detection of the urinary nanocrystallites below 1000 nm.The results were consistent to those obtained by transmission electron microscope(TEM).
By comparing the intensity-autocorrelation curve,polydispersity index(PDI),zeta potential and relative error of average diameter of urinary nanocrystalllites in two kinds of urine,it was concluded that the nanocrystallites in healthy urine were more stable than those in lithogenic urine.The zeta potential,mean diameter and size distribution of nanocrystallites in healthy urines were comparability and consistency,healthy urines were stable system which were revealed by their intensity-autocorrelation curves;the TEM images showed well-proportioned size distribution and little aggregation of healthy urinary nanocrystallites,too.The zeta potential, mean diameter and size distribution of nanocrystallites in lithogenic urines were observably different from each other,whose zeta potential,mean size and size distribution were in a wide range;lithogenic urines were unstable system which were revealed by their intensityautocorrelation curves,aggregation and deposition were incidental;The results were consistent to those obtained by transmission electron microscope(TEM).The difference of urinary inhibitor, urinary nanocrystaiiltes component and urinary concentration between healthy and lithogenic urines were discussed,the reason of stone formation was discussed,too.Then the feasible suggeation was adviced base on physics and chemistry.
引文
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